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Inhibition of human complement by extracellular lipoteichoic acid from Streptococcus Mutans BHT

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Title:
Inhibition of human complement by extracellular lipoteichoic acid from Streptococcus Mutans BHT
Creator:
Silvestri, Louis Joseph, 1952-
Publication Date:
Language:
English
Physical Description:
x, 132 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Antibodies ( jstor )
Cell membranes ( jstor )
Elution ( jstor )
Gels ( jstor )
Incubation ( jstor )
Ions ( jstor )
Molecules ( jstor )
Purification ( jstor )
Sheep ( jstor )
Titration ( jstor )
Dissertations, Academic -- Microbiology and Cell Science -- UF
Lipoteichoic acids ( lcsh )
Microbiology and Cell Science thesis Ph. D
Pathogenic bacteria ( lcsh )
Streptococcus ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis--University of Florida.
Bibliography:
Bibliography: leaves 120-131.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Louis Joseph Silvestri.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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026220234 ( ALEPH )
03928986 ( OCLC )

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INHIBITION OF HUMAN COMPLEMENT BY EXTRACELLLLAR LIPOTEICHOIC ACID FROM STREPTOCOCCUS MUTANS AIHT










By

LOUIS JOSEPH SIVESTRI

















A DI SSERTAT ION PR1, SENTED TO THE
GRADUATE COUNCIl OF THE UNIVERSITY OF FOR IDA
IN PARTIAL FULFILMENT OF THIE REQUIREMENTS FOR THE
DECREE OF DOCTOR OF P I LOSOPIY











UNIVERSITY OF FIORID;\ 19 77












ACKNOWLEDGEMENTS



In all sincerity, no occasion or project thus far undertaken has had a more humbling effect on my life than the completion of this Ph.D.dissertation. It is unfortunate that only now in retrospect can I clearly see the tremendous debt I owe for the help, patience, understanding, and knowledge so generously contributed by my mentor, my friends and wife.

If I were to formally thank everyone who contributed in someway to the successful completion of my Ph.D. dissertation, it is likely that my acknowledgements would read like the listings of a telephone book. I do however

feel compelled to thank a Few very special people.

First of all I would like to thank my mentor and friend, Dr. E.M.

Hoffmann. Ed possesses the rare ability to earn respect rather than having to demand it. His tolerance for my idiosyncrasies was unlimited (almost). He gave me direction yet lef. me with alternatives; hb challenged my intellect, vet never made me feel ignorant; he provided the foundation on which I am still building my scientific character. Most of all, he was (and is) a friend.

I would also like to thank the members of our laboratory familyy" (Suzanne, Jean, Bert and Tom) for their help, patience and tolerance during these difficult last days. One cannot help but reflect upon the many events that shape the complex web of friendships within the laboratory. All of you will always he regarded as the closes; t of friends.

I wish to express my gratitude to Ron Craig not only for his friendship, but also for the professional technical assistance that he afforded.

I would also like to expres my appreciat ion to the employees of 'eachin; Resources, for dedication above and beyond the call of duty. I would











especially like to acknowledge the professional artistic assistance of Margie Summers, Margie Nibl.ack and John Knaub.

I would like to thank Steve Hurst for being there when I needed a friend and for helping with some of the last minute photography.

The typist, Joanne Hall, deserves a partL cularlyv pecial mention.

If not for her personal concern and dedication the deadlines would never have been met. She worked on this dissertation under conditions for which no degree of monetary reinbursement could possibly compensate. I thank you Joanne and I sincerely hope you never have to go through that again!

Finally, and most importantly, I wish to thank my wife Lvn for her infinite patience and encouragement, Her attitudes, her ideals, her "being" is so much a part of me that it would be hopelessly futile to list all the things for which I am indebted to her. She is a friend and lover, a typist and an occasional laborato ry techniiLnn. S he is ': driving force in life and rightfully so, I dedicate this dissertation to her.




And t ith onie ou oCot i ao to est t he jumped at Athe' orc '








-- have I I'



e(t o d Ato he "T Ond' ag at K O e ba AaA. Scrud
struggle got &L head ito dau igh nl adsi cetul



















TABLE OF CO':TENTS


PAGE

ACKNOWLEDGMENTS ........................................ i

LIST OF TABLES ......................................... v

LIST OF FIGURES......................................... v

GLOSSARY OF ABBREVIATIONS.............................. viii

ABSTRACT............................ ................... ix

INTRODUCTION ........................ ................... I

MATERIALS AND METHODS................................... 17

RESULTS. .............. .................................. 11

DISCUSSION. ............................................. 1

LITERATURE CITED .................................. .....- n

BIOGRAPHICAL SKETCH.... ........................ .......... 1




























iv


















I,TST OF TABLES



TABLE PA\;E

1. Partial Puriffication of LTA byv AS-M
Gel Filtratio n ................................... 57

2. I. Results from Partial Purification
of LTA .......................................... 62

3. TI. Results from Partial Purification
of LTA ...... ....................... ............... 63

4. Percent Recovery of LTA During Octyl
Sepharose Purification .......................... 70
1 4
5. I) distribution of C-Phosphatidvl Choline
During PCV Purification of LTA .................. 72

6. Percent Reucoverv (of ,ITA from ;ario us,
Steps of PCV Puriffication........................ 73

7. Summairized Chtumicni Comnposition ,f Vairiouis
LTA Containing Sources ................ .... ........ 7

8. Specific Activitv Determinations of Puri filed
LTA............................................. 70

9. Effect of LTApcx on th Abiliti of Cls tc
Consume (' s ;rc (: 2 A ctivitv ....................... 07

10. C(ompa rison cf thb Re I t. ive Numbeors o f Ef fect ie C1 Mol I ,cul ,s Capable o f Tran; i 'r
from EACl TI 'r acted with L T'Apc. ........... ......... 1(1

11. he Inhilbition on f FA I ysi s bvy Iip(t( ,ic hol ic A\c ids from S,,vo ril Bictori i l Snurco, ...... ..... 7...7













V


















L IST iOF FIGURES



FIGURE PAGE

1. Titration of whole human complement
after incubation with crude extracellular lipoteichoic acid (LTAcx).............. 33

2. Dose response inhibition of whole
human complement after iticuhat on
with varying concentrtions of LTAcx ............ 35

3. Titration of (C3 in whole human scrum
after treatment with LTAcx....................... 18

4. Complement cmpl)onent titration of
whole human srra after treatment
w ith LTAcx ...................................... n0

5. Inhihition of complemen t med it d
lysis of FA treated with varvi tng
concentrations of T,TAcx ......................... .3

6. Passive heminllgg t nation (PlA\) of EA
t reated wi th varying concon t rat i ons;
of LTAcx .................................. ...... 45

7. Effects of ,TAcx treatment on the
SYsis of var ous comp element component
intermediates ............ ...................... 48

8. PIIA of vnriouis LITArx tr heated complement
romponul nt ii te rm ed ilt i s ......................... 0)

9. EIffrct of 'lTAcx t r atm'nt o1n 1 it, 0 ve i


10. Effect of LAcx on hemolvt ic ant ibodv t it rat ion . ........ ..... ..................... .

11. Partial p; i rif cait ion o f TA hv A5-M gel filtr.ition....... ........................... 59

12. Partial. purificition of ITA by AS-M pI, 1 iltr ti o it i1 TA I I nri h( d
start in g materi I al............................... 1











13. Purification of ITA by ('ty. Sopharoso
hydrophobic gel chromatography ..................

14. Simultaneous removal of ,salt and propano l
from LTAosx by LI1-20 g.l, chromatography ......... 69

15. Carbohydrate analysis of TA containing
preparations by gas liquid chromatography ....... 76

16. Passive hemagglut i 'tin tton (PHIA) t itrati ion
and inhibition of complement mediated lysis
of EA treated with varying concentrations
of LTApcx........................................ 81

17. Effect of LTApcx on the complement
mediated lysis of various cellular
complement component intermediates ............... 83

18. Effect of LTAcx and LTApcx on functionally purified human C1 ........................... Q

19. Immunodiffusion and precipitation analysis
of various steps in the purification oFi
human Clq.........................................

20. Disc gel electrophoresis of purified
human Clq .......................................

21. DEAE elution profile of human C(ls............... 03

22. Immunoelec trophoresis of human Cls and
C ls ............................................... 95

23. Effect of LTApcx on the ability of C1:
to hydrolize TAMe ............................... 99

24. Difference in complement medilated yvtic
susceptability of ITApx treated EAC4 versus
EAC( 4........................................... 103



















vi i

















GLOSSARY OF ABBREVIATIONS



A: Antibody C: Complement Cl, C2---C9:a Complement components. Horizontal bars
above the component des ignation denotes
a biologically active state. CVF: Cobra venom factor E: Erythrocyte EDTA: (Disodium) Ethylenediamine tetraactic acid ECTA: Ethyleneglycol-bis (f Amino Ethyl Ether) N,N tetra
acetic acid

LTAcx: Crude extracelular lipoteichoic acid LTApcx: Extracellular LTA purified via phosphatidyl-choline
vesicle adsorbtion

LTAppx: Partially purified extracellular LTA LTAosx: Extracellular LTA purified via Octyl Sepharose gol.
column adsorbtton

LPS: L ipopolysncc hna ride PHA: Passive hemagg 1 utination PHAg: Passive humagglutination (modlifted method) TA: Teichoic acid TANE: p-Tosyl-1-arginine methvlester






All complement nom~,n I;ltur, follows the WHOfI recommendations

(Bull. Wld. Hlth. Org. 39:939, 1968).


Vi i i.











Abstract of Dissertation Presented to ithe
Graduate Counci of the UlniversiLv of Florida
in Partial Fulfit lment of the Requirements for the
Degree of Doctor of Philosophv


INHIBITION OF HUMAN COMPLEMENT BY EXTRACELLULAR LIPOTEICHOIC ACID FROM STREPTOCOCCUS MUTANS BHT By

Louis Joseph Silvestri

December, 1977


Chairman: Edward M. Hoffmann
Major Department: Microbio logy and Ce ll Science


A number of biological and chemical similarities exist between the lipopolysaccharides (LPS) of gram negative microorganisms and the lipoteichoc acids (LTA) of gram positive organisms. The potent affects of LPS on the complement system are well documented: however, the effect of LTA on this host defense system has not been adequately studied. Furthermore, all studies thus far conducted have ben limited to the interaction of LTA with whole fluid phase compcloent. In this investigation it was demonstrated that extracellulr LTA from the cariogen ic mi croorgan ism Sutreptococcu as mrt;ns BIT was not only capable 1 of spontaneously binding to sheep erythrocvte target cells but was also capable of rendering them refractory to complriment mediated Iv i:s. Purification of the I,'A to homogenoittv was achieved by a combination of go[ filtration and adsorhtion to phospholipid chol ine vesicles (artificint membranes). By utilizing v;ritous cellular complement component intermediate complexes and firnctiona llv purif ied complement components. experiments were conducted to define the site and mechanism of Inhibition




ix











by LTA. The site of inhiiliton "as determined to ocrur between the formation of the SAC1 and SAC142 Compllex. Bcaus C Cl is no 1 longer necessary after formation of the C3 convertase (SAC42), lack of inhibition after this step implies a direct effect on Cl activity. Although experimental data derived from utilizing CI, Clq, Cls, and C1s were suggestive, data did not unequivocally establish this as the precise mechanism of inhibition. No evidence for fluid phase consumption of hemolysin Ab, CI, C4, or C2 by ,TA could be demonstrated. Evidence for the inhibitory activity of LTA from severe unrelated genera is presented and the possible role of LTA in periodontal disease is discu.I







































x













I NTRODUCT ON


As reviewed by Wlicken and Knox ( L,2), a number of chemi c;1 and

biological similarities exis:t between the l ipopolysa chcarides (LPS) of gram negative bacteria and the lipoteichoic acids (ILTA) of gram positive organisms. Because of these similitudes ou.r laboratory began to investigate whether LTA possessed anti.complementary activity analogous to that associated with the LPS endotoxin (3-8). Although there have been concentrated efforts to define the site and mechanism of LPS inhibition of complement, very few investigators have reported data on the possible effects of LTA on the complement system (9). This is somewhat surprising since the interaction of LTA, LPS, and complement almost certainly plai a significant role in the etiology of periodontal dis:eses~5. Bacterial products and sernm components in the gingival crevices of the oral cnvitv have been shown to act ivae complement by both the classical (10, 11) and the alternative pathways (12,13). In fact recent evidence suggests that bone loss (a major clinical manifestation of acute periodontal disease) may occur via osteoclast activation due to the interaction of complement and prostaglandin E (14). Prostagla dins are naturally occurring cyclized derivatives of unsaturated long chain fatty acids (15) and their conc:ntrations are dramatically ly elevated in inflamed gingival tissues (16). It is of interest to note that both LTA and LPS are also capable of initiating osteoclast mediated boue resorbh t inn (17) and this activity prrc,,ed.s without the contribution of complement t (or prostgland ni The potential fo syvnergism cannot be overlooked, and indeed t.'S endotoxins hayv lIong hoon implicated ;a partiriplnrs in tlho development of peri dental lo sions (,-R). Analogous LTA activity could be of significant clinical, import especially


1'










in light of the fact that gram positive bacteria represent the majinr cellular constituent of dutntal plaque nat the early stages of plaque formation (18). Most of the gram positive organisms found in dental plaque have been isolated, cultured, and identified. The production of copious amounts of extracellular LTA by several of thpce organisms has been well established (19,20). In fact, growing urender conditions estimated to reflect the growth rate in the oral cavity, Wicken and Knox have shown that the cariogenic bacterium Streptococcus m-tans BHT produlc s some eleven fold greater amount of extracellular ITA in tlhe culture fluid than that contained within the cells themselves (1,2). Therefore, if an effect on complement by LTA can be demonstrated in vitro, an in vive model can be readily envisioned. Preliminary experimentation with a crude LTA containing extract from S. mutans RBT did indeed indicate that complement activity was consumed However, consumption or alteration of complement activity can he due to a number of specific or non-specific factors. Because of the complexity of thi; system, n thorough understanding of the possible interactions is necessary before any mod 1 attempting to define a site and mechanism -f inhibition can he elucidated.

The complement system of vertebrates is comprised of at least

eighteen discrete plasma proteins capable of interacting in a specific and sequential fashion. There are two pathways by whtch this hiochemical cascade may b( initited and they a rc eferrvd to as the ci -a sical and the altern;ltive pathwa;vs ol complement activat ion. low'ver, regardless of how the activation scheme is init I tc td, the h io ogii consoquerlnces of act ivat ion are the same for both pathwayvs:




Silvestri et al. 1 q76 Ahst. Ann. Meet!in ASM, p77.










1). philogogenic activity modinted via complomant reaction b'y-prodr,:tc 2). increased opsonic susr cptibility of fnreigri s l .tmliu es 3). irreversible physinchemial membrane damn ge--a d ultimately, c 'ytol.vsis--of susceptahle target cells. A,]thoiugh the importance f coimplement as a component of the hIost defense system has been suspected for quite some time, only recently has its biomedical significance been firmly established. Indeed, the participation of complement in host resistance to infections and in several disease mechanisms is a topic which has generated considerable research interest in recent years (21,22).

The classical pathway of complement contains elven discrete glv;aproteins representing nine distinct components referred to sequentially as Cl through C(9. Cl is actually a multimolecular complex of three distinct proteins (Clq, Clr, and (Ils) and the aggregate is held together h the divalent calcium ions (23). Removal of caliilm inns hv chlelating agents such as ethylenediaminetetrnacetic naid (EDITA) results in the disassociation oE Cl into its subcomponents with concomi tnnt loss of acti ity

(24). Activation of the classical pathway is character ized by a dependence on IgG or igM antibodies complexed with antigenss. The classical pathway also specifically requires; the components Cl, (2, and C7 ans well. as the divalent cations ca;lciumi and magnesium. Although the componentCl3. and C5 through C9 are us iylv considered part of the classira syvsterm, they are :;shirrod by tlh' alt'runti\v' patlhwav ;ind li,: .thu re n ti cllon idr' d .As unique components of the clawsicnl1 system per so.

Thie rong'm nition .unl init iat ion f unct ion within ros 't to i mu oglobul) ins r s ides with the sui omr ponent (25,2 ). Clrq itself i; rather peoculir protein consisting of three different polypeptide -,h ins

(17). Chemically, Clq Contains npproximatel 10' I cirb h'dr"ite, 3K,











hdroxyproline, 27 hlvdromxvlvsin and 18 W yvrine. Tihi unusml colagenlike composition makes it unlike any plasan protein yet described (28,29).

When complement is activated by ant:ii ody-antigen complexes such as exists on the surface of an antibody sensitized erythrocyte (EA), it undergoes a self assembly process sequentially depositing the entire fluid phase cascade onto the surface of the target. Specifically, Clq recognizes a previously sequestered binding site located in the Fc fragment of IgG and IgM (30,31). The three polypeptidc chains of Clq are physically arranged in a manner perhaps analagous to a six headed mace or bola with each "head" representing a binding site for an igG molecule

(32). Thus each Clq molecule has six bind ing sites for IgG (and presumably the same number for gM). Internal activation of Cl probably is the result of a conformational change in Clq w:ich in turn induces a change in the pronzvme Clr (33). Once CI.r is act ivated to Clr it is endowed with enzymnti.c activity through which the pranzvm, ('1Cs i' converted to CL esternso. (Cls)(34,35, 3,) C(s is a sericn esternse and is inhibited by diisapropylphosphofl luoridate (DFP) (371. This estrs act ivitv call he used to hydrolvyze the svnthetiic subscrates p-Tosyl-l rginilne methvlester (TAMe) and N-actyl-l---tyrosnine ethv!oster (ATiC) (38). ecrentlyv Toos and Raepple have demonst rated thit rn, polyanion were cahpahle of inhibiLtLng the activity of Cl either hv ip rF ering with Clq hiding to the antLinbody-antigen complex x, or 1by pVrvent iIng int.era'ctL iO ol i itl with C; ( q,40(). Altho gh hindin, of '1 .iia ll lI OAdIs to .ACt iC t iOn, the two processes are noLt integral--tl" with modific'd trvptophl n ('1) and the human immunogloblul in s-;ubcltas 1qGk (A2)--ho th behind (Cliq b t d, not activate Cl.

After active at ion, Cls enzymatical y cleaves (CA into a large (CMbl










and small frament (C4a) (63). The cleava; of (4 expose s a rembrne attachment site on the CAh molecule and it will attach to the antihbodvantigen complex at a site juxtaposed to the C1-antibodv complex (44,45). Cls then cleaves C2 into C2a and C2h (46) with C2a attaching to the C4b site and C2 being released into the fluid phase. Thus, the molecular complex C.b2a is formed and is referred to as C3 convertase because it is capable of splitting and activating C3 (47,48). (C3 convertase is also an esterase, and although C3 is its natural substrate, it nlso hydrolyzes the ester bond of acetyl-glycl-lysine methyl ester (49). The catalytic site of C3 convertase is believed to reside in the C2a subunit and even after release from the CA4 complex, cvtol yticaly inactive C2a retains esterase activity, but is no ]clnger capable of cleaving C3

(49). The enzymatic half-life of CAb2a is quite ephemeral--nly 10 minutes at 37'. However, if the C2 is first oxidized by troltmernt With iodine (applicable to human but not guinea pig C2) not only is the binding of C2a to C4b enhanced, but the half life of the birolecular complex is increased 20) fold (50). No doUt the tLins int association of C2a with the C2 and (A3 complex pla s a vital role in controlling the comple-ent react ion by tomporarilv l in g tiH the functional assocr iation of these complex enzymes.

Once C3 is cleavd into C3l and (C3, the small (:a fram:nn t is

released into tihe fluid phase and C l becomes as--s int tedi wi th the C4b2a complex anj with other non-h mnol\t i r itr; on th .irg,'t membrane ( '7). The associ tion of (3b with he (, convertasc modul.to" its activity so that now M, becomes the natural substratc of this triinoiculr ormpleox. The C423b complex is ref.rrd to as (*5 conlvertase (l1) and like C '2, is a hihIv s"ecialimed protease. lust as (l is the onylv knon pret in










sul)strat for C42, C5 is the only known substrate for C1423.

Once CS is cleaved into C5a and (C51, C n in; r e l-,r sed inii tht: fl iid phase and C5h transiently acquires the abit it v to I id one O Inl cii each of C(6 and C7 (52,53). With this, a self-assemblv process is initiated and results, without any further enzymatic activity, in the formation of the stable C5b-9 comp].ex (54). It should be noted that the small by-product fragments C3a and C3a are endowed with marked philogogenic activity (55,56,57). Some of these activities include release of histamine from mast cells, contraction of smooth muscle tissue, directed chemotaxis of polymorphonuclear Icukocytes,and vasoldiation both in conjunction and independent of histamine activity (58). Such potent pharmacological activities obviously play a major role in the normal course of the inflammatory response.

Once the C5b67 complex is formed, it too can hind inonspecificallv to areas on the membrane other than at the location iF the CS convertoise

(52). The trirmolecular association of C567 provide s tire molecular rran emernt for the adsorptivle binding of one mol cutlc of C(:R which in tLurn provides a binding region for up to six molecules of () (54). A Low grade lesion of the target membrane occurs :.ith cnly t:he, additionn of C(:8 L ti t complex (+9); ,ut with the binding of :9, a ten component macroi olcr!.lr complex is formed which greatliv 'enhlli; ces the rate of tll):c 'et 1 "tlvsis (54). It should h" noted that the CIh 7 compl x ,r even the C b complex r;irl attach to lOln-se5ll ti z e'd "i 111m nno lit Iby-st Illid'" r l Is rnd t uIs promot )t ( 1 feormirnal vI tol vt i event. 'hi phi om,, n hen termed "reactve ivysis" (60) and is controlledd iby the rnp id dcnv of thie 11111unbund omple recis (1t m62)

The precise merch:ni:m by which complement mediaites cvtol,sis of











susceptible target cel is is not clearly understood. (One h 'octis, in light of the newly disrcvred tributvrini n' activitv of C7. is tOat the lytic event is caiised by an enzymatic attack on the lmlrhrao ne (631. Ilowever, no enzymatic degradation products have ever been disrnvere>d in either lysed cell membrnnus or in ruptured svnthet:ic lipid hil;aers (64). The two most favored models taree the "doughnuilt" insertion hvoerihis (05) and the C8 insertion model (29). The former model purports that the C5b-9 complex inserts inself into the membrane as a "prefabricated h e'le" allowing the exchange of inLra- and extracellular material vi rn an internal hydrophilic channel (63). HIowever, the model fails to explain how: the hydrophilic complement components enter the hydrop obic expanses of the membrane. In addition, :lthlrogh electron microscpv has revealed apparent ultrastructure doughnut shaped "les ons"' n the surface of i: s lysed by complement (66), freeze etchlin g tLchniques have shown that the ultrastructure alterations are confined to the neteorleaflot of the mmlmbrane, i.e. the lesion does not penetrate the mTmbrane (67). The (' insertion model embraces most of the sali nt features of the2 do..ughnut ::odeli but in addition postulates that thie a and y chai ins of Ci ;ire inserted into the channel formed by the surface macromolecular complex. h c -, d y chains thus extend into the membrane hilaver causing disruption if orderlv structure.

In add ition to the rentraints placed on the ncompl lent icasnl diTin to the rapid decay of several of the i.termdiatos, there are two naturally o rcurring inhibi tors of complement present in the icr of main and. probably in all vrtebr t s. The first inhibitor is r ief rre'd Ito is Cis inhibitor and, as the name implies, it directly abrogates the her,lvtic and sternlvti .:at ivity of CL (8,fM ). The sn cond inhibitor i










referred to as C3b inactive tor and cleaves both soluble and rill bound C3b into two antigentally distinct fragments, C3c and C3d (70). As a result, C423 loses C5 convertase activity, and C3h activation of both the alternative pathway and the immune adherence phenonenon is abolished (71,72,73). This latter activity can be visualized by the clustering of cells bearing C3b on their surface around other cells displaying C3b receptors. Such receptors have been shown to be present on human erythrmcytes, polymorphonuclear leukocytes, platelets, macrophages, and on lymphocytes (74,75). The attachment of C3b not only plays a direct roie in the increased opsonization of target cells (76), but C3b binding to B lymphocytes has been postulated to play a role in B-cell activation as well (77).

The second pathway by which complement may be activated is referred to as the alternative or properdin pathway. llistoricallyv, the existence of this pathway had been suggested as enrlv as 1954. At th at time, Pillemer and his associates reported the discovery of a; new protein in normal human sera (78). Properdin, as it was called, was c capable of reacting non-specifically ith diverse nnaturally occurring polysn'charides and l ipopo I ysaccharides ultimately resulting in the activation of complcnimnt. This process ostens bihlv occurred without the interaction of antibody and was proposed as a m:ajo pathwayv hv which s lsceprtibl hauci;itora and viruses werp destroyed. However, th s provocative hypothesis was discarded as opocryphaLn and the described octtviiLs were attributed to the presence ofI atral annti Lbodie (70). lThe controversy remained utnresolved until recent vears whIen igorous immunochmical techniques wore employed in the isolation, puriFication, and determi nation nf function of many of these components.. The mn~inticip ted compl'xitv of the properdin











system has spawned a multiplicity of modolq attempting to eliucidate its precise mode of initiation and function. Clearly, a plethrn of diverse stimuli are capable of activating this pathway, and this fact alone imposes a formidable constraint on any molecular model. Some of the more common naturally occurring activators of the alternative pathway include bacterial and fungal cell wall 1 constituent s such as ILpopolschiaride, zymosan, and inulin (a polyfructose) (71,80-83). In addition, aggrega tes of some immunoglobulin classes (84,85), some types of animal cell membrane constituents (86,87), and antibody-coated budding virus infected cells (88,89) also stimulate this pathway. The alternative pathway can even be activated by substances of relatively defined chemical nature such as benzyl-B-D-frc topyranoside (90), rolyglu coso with rapititious a 1-3 and branched a 1-6 linkages (91), din it rophnylatod albu- in (92), and many polyanionic substances. Cobra venom factor (a non-lipolytic, non-hemolytic glycoprotein isolated from tihe venom of the cohra Naga naja) is also a potent activator of complement cvtolvtic potential, but it appears to act as a (3b analog and is thus unique in its mode of alternative pathway activation (93,94,95). Potentiat ion of this system requires devalent magnesium ions and the interact ion of at Least five novel serum proteins. By convention, the names of these proteins are TF (or initiating factor), P or F (properdin). Factor B (Cl pr:n tivator), Factor B (C3 activator), and Factor ID or I ((:3 proctivator convert:-se). To date, all of the above components have been isolated, puri f ied, and character: ,d as to molecular weight, cl oct rophoretic mobility, and sodimentation coefficients (83,96-8S). C3b (t the classical pathwav) plays an intregal role in the a lternative pathway (71,96,99), and thus it in essence forms the luncrtion point of the two systems. Because all terminal












components (C3, C5-9) are shared, the biological consequences o ativation encompass all the processes previous ly described (immune adherence, opsonic activity, annphylatoxin production, membrane attack complexes, etc.).

There are similarities between some of the more salient features

of the classical pathway compared with those of the alternative pathway. Analogous to Cl1q, IF seems to function as the recognition unit for the properdin pathway, but its relationship to another factor (referred to as a C3 nephritic factor from the sera of patients with membranoproliferative glomerulonephritis (100) and its mode of activation is poorly understood (96). Factor D is capable of enzymnaLtcally cleaving Factor B into Ba and l-b (29,94). u the presence of C3h, a bmolecular complex CIhBb is formed (29) which is endowed with C3 splitting activity similar to the C3 convertase (C4hb2a) of the classical pathway. Furthermore, just as CAb anchors the classical convertasc to the memhr:ine allowing Cn to exert its enzvmatin activity, so too cytophilic C3b niiichors: tlhe (ibBb complex to the membrane allowing the enzyma tic activity ,of Factor Bb to he expressed (83). Both complexes merely gain addit ional C3( to modulate C5 cleaving activity (99). Thus, the prescec of (C3 not onlv prevents aln "abort" due to rapid decay of either convtrtase, but because C(b is utilized as part of the alternative pathway convertasc, it participates in a type of amplification loop. Tn other words, the more C3b that i formed from either pathway, the more C3 c;leaving potential is endowed upon the proprdin C3 conlvertaso. Properdin (P) se'ms to st ilize rthe fragile C3hBlb complex hut its possible role in stahil izinlg the classical C3 convertase has not been investigated (on). Netcworthv, however, is the potent effect pro prdin exertS on the C3b inhibitor (9). By











modulating the action of this enzyme, propordin at least inlrirectly plays a role in stabilizing the classical pathway sequence.

The recognition of foreign substance; by a host usually leads to

the neutralization and eradication of these substances by immune lymphocytes, phagocytic cells, specific antibodies, complement, or an amalgamation of these factors. However, in instances where antigenic substances interact directly with host tissue, the reactions of the host's immunological defense system could sometimes result in a considerable amount of autodestruction. LTA represents a class of antigens that are capable of spontaneous cytophilic binding to mammalian tissue (101,102,103). As a result, host tissue acquires a new "anitgenic face" and may now react with natural or induced antibodies to the LTA. Furthermore, antibodies directed primarily at LTA determinants may cross react with similar determinants of the host's tissue. Such a mechanism has been proposed for the high incidence of rheumatic fever and glomerulonephr itis in patients recovering from post streptococcal infections (l(.1(13). Recently, acyl.ated heteropnlysaccharides (LA) isolated from the cell membranes of several lactobacillus species were shown to replace pigeon excreta antigens in complement consumption tests d iagnostic for pigeon breeders disease (9,1(16). Thus, precedence may already be established for LTA's role in the manifestation of several clinical maladies. In addition, the chemical and biological similarities between LTA and .PS (1,2) plus the ability of LTA to stimulaLe bone resorbtion (17) make LTA a likely candidate for a role i.n periodontal disease. n tho other hand, LTA lacks some of the biological nctiviti is 5 ssociatod with ILPS such as pyrogenicity in rabbhits (2,107) and a mitgenic effect on B-cclls

(2). Since theso activities have been slown to reside with the compl x











Lipid A of LPS (108,109) and since the unique sugars and hydroxvacy esters of Lipid A are absent in LTA, it i s not surprLsing that :asscla.:t activities are absent as well. As a class. tcichic and lipoteichoic acids are wall and membrane components of gramn positive bacteria (107,108)i LTA is typically membrane associated and consists of a glycolipid covalently linked to a polyg lycero]phosphate halckhone which may carry cnrbn-hydrate and D-alanine substi.tuents (2). Teichoic acids (TA), however, are never associated with cell membranes; they lack the terminal glvcolipid coupling, and they may have a backbone nf either poly\tlcerolphosphate or polyribitol phosphate (2). LTA may be converted functionally to polyglyceroal TA by spontaneous deacylation in an aqueous environ1
ment, or mil.d alkaline, or acidic hydrolysis (107). The molecular weight of LTA (93) is probably between 3000-12000 but be cause of its tendency to form micelles in an aqueous environment, the apparent: molecular weight as determined by gel. filtration is approximately four million (110).2 Because LTA possess the glyly ipid moiet v, they are amphipathic molecules exhibitirng a propensity to spontaneously :ssociate with proteins and biological membranes (103). Mammalian red blood cells can be "coated" by spontaneous adsorbtion with an 1.TA contaniing extract and the cells can subsequently be agg:luinnted with an anti-TA serum. Passive henagglutination (P'HA) performed in this manner with sheep red blood cells has previously been reported by many invest i-.or4 s who discovered



SPersonal comn inicat ions from R. Crnic, Qpt. of rVS, 'niv. of rl.; K. Knox and A. J. Wickun, Institute for l)Dent l R research, Sydney, Australia; and personal Iunpublished data.

SD.ta supported hv prsonal oexp rience (soe Figuinron 11 and 12), and personal communication from R. Craig.










ervthrocyte--sensit izing antiens in cell free sa line wnshings or spent culture fluid from several gram positive organimn (I101.02). l. These so called "Rantz antigens" were recently shown to possess properties associated with LTA (11). Because only acylated LTA wil hind to erythrocytes, PHA provides a means of quantitating the amount of LTA in a preparation without having to contend with deacylated TA contamination.

The biological role of TA and LTA to the microorganism has been a subject of considerable disputation by several investigators in recent years. Thus far, at least three roles have been tentatively assigned: i). TA and LTA seem to fEunction as "carrier" molecules for membrane and cell wall. components, i.e. amphipathic LTA may be used by the cell to transport needed hydrophobic molecules through hydrophilic zones which would otherwise pose an almost impenetrable harrier. Fielder and Glaser have established that incracellular LTA serves as a lipid carrier for the biosynthesis of cell '.all ribitol tLichoic acid in Staphylococcus aureus (112,113). Chat rj ce and Won g (114) have demonstrated that LTA may serve as the acceptor in which nascent peptidoglyran polymers are synthesized. 2). LTA seems to be involved in cell wall division and regulation. Holtje and Tomasz have reported that ITA exhibits an inhibitory effect on the function of ,nutolvtic cmnzymos during the division cycle of pneumococcus (115). It is intoresttilg to note that !;imilar functions have been describe d vby Cleveland, et al. working w ith a strain of Streptooccus faccalis (11,!.17,118). In these sy;vtems. 17TA is dencylated and released iito the enviroricnt as 'TA. Once the c.'ncmontration of LTA is sufficient lv lwered, or the concentration of auitolvtic: enzymes is sufficiently e lcvatd I, cel wall auteol\ysis begins at the division zone. This autolvtic .ctivitv theni allows for insertion of additional











cell wall material. 3). [,TA or TA may contribute to the nvorrall electrostatic charge of gram positive nrganismn. Al thth membra ne localized, the long polar tails of many ['LTA penetrnt:e the thick peptidoglycan layer and become externalized (107). These, together with ,he TA which are covalentlv linked to the ce.ll wall (Ir8) present a myriad of antigenic faces to the external environment (1i10,12). This antigenic presentation is of serological import since these antigens are often genus, species, group, or type specific (103,120). In addition, these polar tails generate a net negative charge by exposing the phosphate groups of the polyglycerol or polyribitol backbone. This net negative charge has been teleologicaIly assigned the functinn of maintaining electrostatic repulsion and dispersion of the bacterial cell (121). Since LTA has been shown to sequester certain cations such as magnesium (122), an additional function as a site of divalent caionic c on \ver r nce has also been postulated. The association with ma gnesium ions appears to be more than casual since protopL.asts of Lactobacil us casei placed in a magnesium ion free or chelated medium rapidly lose their 1,TA from the cell membrane.

Anti-LTA titers (of both the Igi and Ig, 'asses) have been reguLarl v reported in mice, rabbits, and man (2,123). Several clinical studies have reported increases in anti-LTA titer-- incl uding r: t ibhodi es of the class [gA--after acute gram positive infections (121.125). P gs, guinea pigs, and r;at exhibit a low level of natural inmuonitv t, 1,TA and rerontly', there have been reports of silivary [:A production as a result of gastric intuhation of monkeys with Stre pt,oc..cus mnutalns h715 s;ro t \po C. There is no doubt that TA and ITA of a 1I grnm positive ganera thus f;r investigated contain antige nic mniorit:is and that nmdcr i'rtii n circumst-ances,







15



LTA can be immunogenic (2). of particular interest is the fact that the attachment of streptococc-al LTA to erythrorvtes could he revc rsWiblv transferred from the Crvthrocvtrs to other t[ssu: cells (10(.l ,126). The possible significance of this "transfernhilitv" in relation to rheun tci fever and glomerulonephritis and pigeon breeders disease has been previously discussed (9,104-106). However. despite this precedence the significance of the binding of LTA to oral epithelial cells in gingival pockets has not yet been investigated. Not only os ITA mediate bone resorbtion as previously indicated, but spontaneous hybrid micells of LPS and LTA are known to occur, thus compounding the possibility of in situ immunological modulat ion. There is little doubt of the availabitit of extracellular LTA in this environment--Streptococcus mutans BUT alone has been reported to produce excess of 50 u~ of l'TA/ml in culture media

(21)). Recently, Wicken and Knox have studied the cxcrtion ofr xtracellular LTA from this organism in a chemostat under steady state logarithmic growth conditions. Results indicated that a genera tin time of 10-14 hours (estimated to reflect that actual in vive growth rate of this organism in the oral cavity) i)rodiced the maximal amount of 'xtranellular LTA (I). Considering its ud iquity and the cariogenic nature of Strepto-C coccus mu tans BIlT (127-130), the secretion of cop ious amounts of hioloj irally active LTA into the oral rnvitv has the potcnial of considerable influence on the host-parnsite relationship.

Th'L objectives of the project were than defined ans follow:

(L) To establish if an LTA-containing extraccelular extract of







Pers ona l communication o f A. 1 icken.







1.6


Streptococcus mutans BuT was capable of inhibiting complement mediated cytolysis of target sheep erythrocytes.

(2) To purify the extracellutar LTA of S. mrtans BilT to homogenity.

(3) To describe tihe nature of any anti-complementary activity that purified extracellular LTA may exhibit.

(4) To determine the site of action of any such inhibition.

(5) To determine the mechanism by which purified extracellular LTA may enhibit anti-complementary activity.

(6) To determine if the I,TA from other gram positive genera and species can be shown to demonstrate anti-complementary activity.
















MATERIALS AND METIHODS



Crude extracellular LTA (LTAcx). The initial. studies were carried out utilizing LTAcx prepared in Australia by the method of Wicken and Knox (110). Streptococcus mutans BHIT was grown to late stationary phase in a New Brunswick Microfirm fermenter at 370C, under anerobic conditions (95' N2 and 5 CO ) in a complex medium.

Later experiments utilized LTAcx prepared at Cainesville,

Florida. The original method was modified as follows. A Pellicon
2
Cassette system (Millipore Corp., Bedford, MA) equipped with 5.0 ft of PTGC filter materiaJ was used to dinlvzc Todd-Hlw itt broth (Di fo Laboratories, Detroit, MN). A 100 ml culture of early log pha:e S. mutans BHT was inoculated into 10 liters of dialyzed medium and incubated at 370 for 24 hours. The cells were harvested using a Delaval Cyrotester (Po'ighkeepsie, NY). The superna:t was pa ss through the Pellicon Cassette system (loaded with 1.0 ft of 0.45 1 microporous membrane) to remove remaining c lls and debris. The cellFree spent fluid was then frac tionnted and concentrated by passage through 5.n ft PTCC membrane (nominal molecular weight exclusion limit of 10,0(0). The filter retentatc was washed in situ with syvcral liters of water, collpIcted and Ivophilized. The freeze-dried retenrate, designated as LTAcx. w~as stored in a de si'ator at -20"C.

Solutions for mpl-mnt ,j;i ypvs. Isot nic Ver.nal huffured sodium chloride (V.;S), dextrose celatin Veronal bu"ffr with added












CaC12 and NgC12 (DGVB), EDTA containing Veronal buffer (O.0'W : EDTA-GVB) and gelatin Ve rnal I huf fer with added CaiUl, and IMgC( (GVB) were prepared as previously described by Hoffmann (131).

Human complement (HuC). Fresh human blood samples were obtained from the Gainesville Plasma Corp., Gainesville, F L. The blood was allowed to clot at room temperature for about 60 minutes, and the serum was separated by centrifugation at 500 X at O'C. The serum was collected and stored at -700C.

Guinea pig complement_ (GC). Fresh frozen uine pig complement was purchased from Pel Freeze Laboratories (Rogers, AR). The serum was shipped in dry ice and it was stored at -70'C after arrival in the laboratory.

_Comjlement components. Purified guinea npi5 Cl and C2 were prepared according to Nelson et al. (132) and Ruddv and Austin (133,134). Functionally purified guinea pig C3, CS and C9 and human C1, C5, C6 and C7 were purchased from Cordis Laboratories (Miami, FL).

Erythjrocytes (E) Sheep blood was taken by vCni ncture from a single animal maintained at the animal research laboratory of the J. Hillis miller Health Center ((;;G inesville, FL). One hundred milliliter volumes of blood were collected in cqual volumes of sterile Al:aever's solution (135) and the blood was tored at /4" for up to three weeks.

Antibody sensit imod ;he ._rythrocytes (E.A). Rabbit anti

sheep E stromata was obtained from Cordis laboratories (Miami, FL). Sensitization of washed sheep E was performed as rcccmmended by the supplier.











Complement component intermediate complexes. Sheep E in various stages of complement fixation were used in this study. EAC1, EAC14 and EACi42 were prepared by methods described by Borsos and Rapp (136). EAC1423567 were prepared by the procedure described by Hoffmann (137). Unless otherwise indicated, guinea pig Cl, C8 and C9 were used in all instances, and the remaining C components

were from human serum.

Treatment of cells and cellular intermediates with LTAcx.

Unless otherwise indicated, cells were washed and suspended in VBS at a concentration of 10'/mi. Equal volumes of these cells and LTAcx were mixed and incubated at 37' for 20 minutes with continuous shaking. The mixture was then placed in an ice bath for 10 minutes. At the end of incubation DGVB was added to the mixture and it was centrifuged at 500 g for five minutes. The supernate ,was discarded and the cells were suspended and washed thrice with DGVB (00 for 10 minutes at 500 g) to remove any unlound material. The cells were then resuspended in DGVB at a concentration of 10i/mI. A sample of the cells were tested for cell-bound ILTA using passive hemagglutinn tion with rabihit anti-lTA. The rema ining, cells were used in experiments to detct acqu ired rosist:nce to homolvsis.

Passive hemaggutinat ion (PlA). Passive hemalgl ut inat ion was carried out using a micrt. itration system. F[ft-y i. of a VBS dilution of anti-TA readded to the first row of wells of a round bottom microtiter plate (Cook Engineering Co., Alexandria. VA) and 2D iii (one lron From the calibrated pipetes supplied with the system) of VIS were added to the other wells on the plate. The anti-serum was serinllv dit.-ed in situ and one drop of ILTAcx







20



treated cells was added to each well. Controls frr spontaneouss or nonspecific agglutination consisted of wells that contined .anriserum and sheep E which had never been exposed to lTAcx. Treated sheep E plus VBS constituted another control. The microtiter plate was incubated at 370C on a Cordis Micromixer (Cordis Laboratories, Miami, FL) for 15 minutes. The plates were removed from the mixer and the cells were allowed to settle for two hours at 370C, followed by three hours at room temperature.

Modified Lassive hemagglutination (PHAg). A modification of the above technique was used to semi-quantLtate the amounts of LTA present in various preparations. The same apparati were used, but instead of antibody, LTA-containing extracts were added to the bottom wells and serially diluted in situ as described. After each LTA source was diluted, one drop of sheep ervthrocvtes (1in/ml in VBS) was added to each well and the plate was then incubated at 370C for 20 minutes and at 00C for 10 minutes. The cells were kept in suspension by vibrating the plate on a Cordis Micromixer during both incubation periods. One drop of CVB was then added to each well and the plate was centrifuged at 200 g for 5 minutes. The entire plate was then abruptly inverted over absorbant paper towels and allowed to drain for approximately one minute. One drop of (CVB was again aldded o each well and the plate was \'ibr ted at O0C for 5 minutes to resu spend the pellet. An additional drop of GVB was added per well and the plate was again centrifuged at 200 g for 5 minutes. This washing procedure was repeated thrae times and the cells were then finally resuspended in one drop of cGV. One cop of anti-LA (diluted 1:1000 in VBS) was then











added to each well and the plate was incubated at 370C for 15 minutes on a Cordis M!icromixer. The plate was removed from the mixer and the cells were allowed to settle for two hours at 370C, followed by three hours at room temperature.

Inhibition of complement mediated lv__s. EA coated with

LTA (EALTA) were tested by mixing 0.1 ml of EAITA (10 rells/ml) in DGVB and 0.4 ml of DGVB diluted HuC. The HuC was diluted so that a maximum of 80 percent lysis was produced in EA which had not been treated with LTAcx. The mixture was incubated at 370C with continuous shaking for 60 minutes. One milliliter of ice cold EDTA-GVB was added, the mixture was centrifuged for 5 minutes at 500 g at OC and the supernatent fluid was recovered. The optical density of the supernntent fluid was determined at a wave length of 414 nm. Inhib ition of hemolysis was calculated for each concentration of LTAcx used by comparing the extent of lysis in each assay with a control reaction mixture which cont lined EA that had not been treated with lTAcx.

Effect of LTAcx on the titer of antibodies specific for sheet

ervthrocvte stromata. 3ecaue ITA associate with some proteins (138S it was necessary to perform a hemolytic nntihody titration to determine if the ability of the immunog lobul ins to fix complement at the cell surface wa heing affected by lTAcx treatment. The possibility of similar it igens in LTAcx and sheep ervthrocvte stromltna was al;o considered. Equal volumes of LTAcx (00 o g/ml in VBS) and rabbit anti-sheep E stromata were inciuhated together at 37C for 20 minutes. A control consisted of incubating an











equal volume mixture of VRS and anti-sheep erythror:te stromata for the same time at the same temperature. The ant ibodies were then titrated using limiting amounts of complement (135).

C1 fixation and transfer. The number of Ci molecules bound to an antigen-antibody complex can be measured by the CL fixation and transfer test described by Borsos and Rapp (130). In a modification of this procedure, an attempt was made to quantitate the number of C1 molecules fixed to EA which had previously been treated with LTApcx. Buffer controls and EALTA were prepared as previously described, and after washing were resuspended at 108 cells/ml in DGVB. Equal volumes of EA and EA were inLTA VBS
cubated with Cl at 30C for 15 minutes. The cell mixtures were washed twice with DGVB, and resuspended in CVB at a cell concentration of 1 X 10 8/ml, 5 X 10 7/m, and 1 X 10 /ml. One volume of each cell concentration was added to one volume of EAC4 (at

1 X 10 cells/ml) to permit transfer of Cl from EA C1 to EAC4. The cells were incubated at 300C for 15 minutes. and then C2 and C-EDTA were added in relative excess as described previously.

A variation of the C( transfer assay was performed by treating preformed EACI with LTA or buffer control as described. The resulting EACI were resuspended to 1 X 10 cells/ml in GVB and
x
the amount of Cl capable of transfer was measured as described above.

Gel Filtration. LTrAcx was fractionated on a 2.5 m X 100.0 cm column of Bio-Gel A-5M, 200-40/ mesh (Biorid Laboratories, Richmond,



In this instance, "x" represent s LTA or the appropriate b,"ffer treated control.










CA) using a modification of the method described by Wic:ken and Knox (110). The column was equilibrated and eluted using 0.01 'I Tris carbonate (Sigma Chemical Co., St. Louis, MO), pl 6.8.

Hydrophobic Affinity Column chromatography. Because of the hydrophobic nature of the fatty acid moieties of lipoteichoic acid, adsorbtion to a stationary phase of a chromatographic column was used in an attempt to further purify the LTA. LTAppx in buffer A (0.01 M Tris carbonate pH 6.8, 1.0 M Nacl was loaded on a 25.0 X 2.25 cm column packed with Octyl Sepharose (Pharmacia Fine Chemicals, Piscataway, NJ) and equilibrated in the same buffer. After eluting with 150 m! of starting buffer A, the reservoir was then changed to buffer B (0.01 M Tris carbonate pH 6.8) and another 100 ml were eluted. Buffer C consisted of 250 ml of a gradient ranging from 10-70 % propanol (by volume) in 0.01 M Tris carbonate, pH 6.8.

Octyl Sepharose is a derivative of the cross linked agarose Sepharose CL-4B. The terminal n-octyl groups of this agarose gel. confer a hydrophobicity to the matrix. By exploiting this property it was hoped that polar or neutral non-interacting components would be removed by elution with solutions of high ionic strength. The lipoteichoic acid would then be eluted from the matrix with an organic solvent such as propanol. (It is imperative that all tubing, connections and gaskets used throughout the column he constructed of a material that is resistant to organic solvents).



1This method represents a nodific:ltion of a procedure described by A... Wicken and K. Knox (Svdney, Australia) via personal communication.











Removal of salt and ronl From LTA conta in ig extracts.

Removal of salts and/or propanol from various preparations was rapidly and quantitatively accomplished by gel filtration utilizing 1,11:20 (Pharmacia Fine Chemicals, Piscatawav, NJ) as the solid nhase support matrix. The most commonly employed column was 50.0 cm X 2.5 cm but a larger 65.0 cm X 3.0 cm column was sometimes utilized. The column was packed and equilibrated with deionized water. Sample preparations usually involved rotary flash--evaporation (Buchler Instruments. Fort Lee, NJ) in order to reduce the volume of sample to 15-20 ml. Elution of product was carried out at a pressure head of aproximatel 50 cm water and approximately 4.0 ml effluent were collected per tube.

Phosphatidyl choline vesicle (PCV) _urification of LTA -(a) Preparation of PCV. Although reported as the method of choice by other invest igators, in our hands POctyl Sepharose u purificat io, of LTA from Streptococcus mutans BHT resulted in a product still highly contaminated with polysaccharides In an attempt to achieve homogeneous purification of LTA, a modification of the above ment-ioned hydrophobic adsorbtion principle wa;s employed. In this procedure, artificial membrane vesicles were prepared with 1),nhosphatidyl choline dipalmitoyl (1PC) (Sigma Chemical Co.) as the sole constituent via a modified method of Hli.. (1.40). In brief, '0. 0 m of PC was placed in each of several 10 ml high speed glas.- Corex (cntriffuge tubes (Corning Glass iJWorks, Corniiig, NY') and (1dLssolved with one mti chloroform. The solvent was gently evaporated in a 500C water bath while rotation ng the tubes (so as t.o coat the bottom 5 or 6 cm of the tube with PC. Once dry, rhe tube were pL.iced in a Ivophiliat ion flask and any residiln solvTnt was removed in \la.cuo.. ()ne milliliter Wichen, A.J.., and Knox, K.--Personal communic.-tion.










of 0.01 M Tris carbonate pH 6.8 was then added to each tube and they were placed in a 500C water bath. Once warmed, the tubes were vigorously vortexed (Vortex Genie Mixer, Scientific Industries Inc., Bohemia, NY) and the cycle of warming and vortexing was continued until a milky emulsion was formed. Fifteen milliliters of 0.01 M Tris carbonate were then added to each tube and the tubes were centrifuged at 27,000 g for 30 minutes. The supernatent fluids were then decanted, the pellets were resuspended in 1.0 ml Tris carbonate buffer and warmed to 500C in a water bath. The tubes were gently swirled (butnot aggitated) to dissolve and resuspend the pellet The resulting phosphatidyl choline vesicles (PCV), devoid of very small vesicles, were then used to adsorb LTA from LTAppx.

(b) Prearation of PCV-LTA. Two milliliters of TAppx at a concentration of 1.5 mg/ml in 0.01 M Tris carbonate, pH 6.8 were added to each centrifuge tube containing 1.0 ml of PCV. The tubes were covered with parafilm (American Can Co., Neehaw, iS) and incubated for 90 minutes in a 370 shaker water bath. Thirteen milliliters of

0.01 M Tris carbonate were then added to each test tube and they were centrifuged at 27,000 g for 45 minutes. The supernates were discarded and the pellets were gently resuspended in 1.0 ml of Tris carbonate buffer at 500C as previously described.

Fifteen milliliters of buffer were tlhn added to anch pellet, the tubes were gentlv swirled and then centrifuged as described. The pellets were washed three times in this manner. The final pellet was drained and then dissolved in 5.0 ml of chloroform/methanol (3 + I v/v). The tubes were then covered with aluminum foil and allowed to sit at room temperature for 60 minutes.











A Millipore 15 m analytical filter holder (Millipore Corp.,

Bedford, MA) was loaded with a 3.0 w fluoropore membrane (Millipore Corp.) and washed with several volumes of the chloroform/methnnol solvent. The test tubes were all sequentially decanted into the apparatus and the contents were allowed to filter by gravity through the membrane. Each test tube was washed with several volumes of warmed chloroform and decanted into the filtering apparatus. Finally, the barrel and filter were washed in situ with warm chloroform. The filter was removed after air drying in situ and placed in 10.0 ml. of deionized water warmed to approximately 40'C. All centrifuge tubes and the barrel of the filtering apparatus were washed with warm deionized water and all products were combined. The resulting product was passed through a 25 mm Swinne> filter (Millipore (Corp.) loaded with a 5 o microporous membrane (Millinore (Corp.) to remove particulate debris. The membrane was washed in situ with several volumes of warm deionized water. The filtrate was collected directly into a lyophilization flask and was then shell frozen and lyophilized. The final product was stored in a dessicator at -20(C.
14
C Phosphatidyl choline analysis. In order to detect any phospholipid contamination of the LTA throughout the previously described PCV purification, radioactive PC was used to label the phospholipids in the vesi.les. Approximatelyv 2. 3 Ci. (3 X 10 6 D M) of C labeled phosphatidyL choline (Amerslham Searle Corp, Arlington Heights, TI,) were added to 4(0 mg of phosphntidvl choline dipnlmitoyl in a 30 ml Corex centrifuge tube. Phosphatldvl cRholine vehicles were prepared from this and the non-labeled contents of three other tubes bv the methods previo uyiiv described. Fifty microliter







27


14
samples from the C containing test tube were taken at each ' PPO (2,5 diphenyloxazole), and 0.01% POPOP (1,4-di (2-(5-phenyvoxazolyl)benzene) were added to each vial. The degree of 14C-PCV contamination of the final product was determined by placing the entire LTA-containing-fluoropore filter in a scintillation vial with 5.0 ml scintillation fluid. The possible influence of quenching by the fluoropore filter was investigated by adding equal aliquots of 14
C-PC to two scintillation vials one of which contained a fluoropore filter in addition to scintillation fluid. No npprociabtle difference in CPM was observed. Disintegrations per minutee (1)PM) values were calculated from a standard quench curve constructed for use with chloroform. Standard ratios were determined for each sample and percent efficiencies were extrapolated from the standard quench curve. This volume was then used to correct counts per minute (CPM) to PPM. Unless otherwise indicated, the samples were counted for 10 minutes in a Beckman LS-733 liquid scintillation counter (Bockman Instruments, Fullerton, (CA).

Colormetric assays. Phosphorous was determined by the method of Lowry et al. (141) with absorbancies measured at R20 nm. .t al carbohydrate was measured hy the phenol sulfuric acid assay as described by Duboijs et al. (142). Total protein was performed on o;amples using the Bio-Rad Protein Assayv (Bio-Rad Laboratories, Rockville Center, NY). Samples and the standard curve were prepared ftlltowing the manufacturer's recommndat ions.







28


Gas Iiquid Chromatography. Carbohydrate analvsis was performed after treatment of the samples with 1.0 N P ,s in sealed ampules for 8 hours at 105C. Upon cooling, the sent was broken and exactly

0.2 ml of mannitol (either at 5.0 mg/mi or 1.0 mg/ml depending on carbohydrate concentration of the sample) was added as an internal standard. The contents of each vial were quantitatively transferred to 15 ml centrifuge tubes (Corning Glass Works) containing 0.3 g BaCO3. Each centrifuge tube was heated in a boiling water bath and alternately vortexed until the pH approached neutrality as indicated by full-range pH paper (Micro Essential Laboratory, Brooklyn, NY). All tubes were centriufged at 500 g for 5 minutes and the supernates were removed and collected in appropriately labeled 13 mm screw cap tubes fitted with teflnn lined lids. The centrifuge tubes containing BaCO3 were washed once with one ml of deionizced water and the supernates were appropriately pooled.

After lyophilization, the hydrolyzed carbohydrates were converted to trimethylsilyl ester (TMS) derivatives by the addition of 0.2 or 1.0 ml (depending upon carbohydrate concentration) of TRI SIL Z (Pierce Chemical Co.). Samples were warmed to approximately 600C in a water bath for 15-3() minutes before use and assayed using a Packard 800 series gas chiromatograph equipped with a flame ioniza t ion detector. 'The gas chromant graplic column (153 cm X

4 cm) was packed with SE-40 1UTRAPHIASE 37: on Chromosorh W (1P) 80/[00 mesh matrix (Pierce Chemical Co., Rockford, IL). Column and detccror temperatures were set at 16(0'C and 1S C, repr tively. The N. carrier gas was set at approximately 30 cc/minute.










Amino acid analysis. Amino acids and amino sugi:rs were measured on a JEOL model JLC-6AH automated amino ac Ld ana lyser (JIEOl, Inc.. Cranford, NJ). Sample hydrolysates were prepared as described by Grabar and Burtin (143).

Clq Cs, and Cls purification. Highly purified human Clq

was prepared from whole human sera by the method of Yonemasu nnd Stroud (144). Highly purified human Cis and Cls were prepared by a minor modification of the method described by Sakai and Stroud (35). For the final resolution step, Bio-Rad Cellex-D DEAE with binding capacity of 1.07 meq/g (Cellex-D, Bio-Rad Laboratories, Rockville Center, NY) was substituted for fibrous DEAE cellulose Whatman DE-23. The DEAE was washed and prepared according to the manufacturer's specifications. Final elution of the product was accomplished with the use of the same elut ing buffer is described, but instead of a stepwise elution of the column, an ionic gradient from

0.2 0.4 RSC (relative sodium chloride concentration) was utilized.

Disc acrylamide gel electrophoresis of Cfl, Cis and Cs s. This was

carried out essentially as described by Yonemasu and Stroud (i.4 ) but without the use of sodium dodecyl sulfate (SDS).

CIs inhibition assays. The ability of Cls to consume C2 activity was assayed by a modification of the method described by Sakai and Stroud (35). Briefly, 0.1 ml of CLs (approximately 8.0 X 107 site forming unit SFU/ml) plus 0.1 ml LTApcx (100 ig/ml in D)VB) were ircubated at 30" For L5 minutes. One tenth millii liter of (2 was then added at a concept ration of appreximately 9.0 X 10 effective moleculc;a/ml andt inclhatd at 37" for 10 minutes. At the end of the incubation, 9.7 ml cold I)(OVR were added to the mixture resulting in a 1:1.)0 dilution of the (C. The (2 was then serially diluted and .! ml aliquots from each dilution were added to 0.1 ml of







3)0



EAC14 (10 cells/ml in DGVB). The mixture was incubated at 130 for .Io minutes and cooled to O0C in an ice bath for 2.0 minutes. Three tenths of a milliliter of C-EDTA (1:37.5 in 0.04 M EDTA-CVB ) were then added to each test tube and the mixtures were incubated at 370C for 60 minutes. At the end of the incubation period, 1.0 ml of cold EDTA-GVB was added, the tubes were centrifuged, and the supernntes read for release of nxvhemaglobin at a wave length of 414 nm. External controls consisted of C2 with no Cis nor LTApcx, C2 with Cis but not LTApcx, and C2 with LTApcx but no Cis. The usual internal controls (spontaneous lysis, color correction, no C2, and total ]ysis) were included at all times. Results were expressed as percent inhibition of C2 consuming ability compared with a control containing only Cls and C2.

The ability of Cls to hydrolize the synthetic substrate p-Tosyl-larginine methylester (TAMe) was determined as described by Nagaki and Stroud (38). Inhibition assays were performed by incubating equal volume,7
of CIs (approximately 8.0 X 10 SFU/ml) and LTApcx (npproximtelv 101 ,a.i at 370 for 10 minutes. Residual Cls activity was then determined as described (38-40).

C1q inhibition assays. The effect of LTApcx on the ability of purified Clq to bind to antibody sensitized sheep erythrocytes was determined by methods described by Loos et al (39) and Raepp e et al. (40). Fqual volumes of (Alq (approxim;leclv 1.5 X 1()0 SFU/mi.) and 'Apex (10) g/ml) were incubated at 37 for 1() minutes. Residual Clq activity was then determined as indicated nbove.

















Inhibition of whole human complemen-t by crude extracellui.ar

lipoteichoic acid (LTAcx). To determine whether L,TAcx had any effect on whole human complement, equal volumes of LTAcx nnd whole human complement were preincubated at 370/30 minutes. After preincuahtion, the complement source was serially diluted in DGVB and the residual hemolytic activity was titrated. As shown in Figure 1, approximately 50'O of the whol-e complement hemolytic activity (measured in CH50 units) was consumed. Furthermore, as seen in Figure 2, this consumption was dependent on the concentration of the LTAcx used.

Titration of complement components in whole human sera after treatment with LTAcx. One mechanism for fluid phase consumption of whole complement could have been the interaction of natural antibodies in the human sera with LTA or some other antigenic substance in the crude extract. The result would be the fixation of Cl and subsequent activation of (4 and C2 via classical pathway. Another explanation for decreased hemolytic activity could have been the activation of the alternative pathway in a manner analogous to LPS. To differentiate between these two modes of activation, individual component titrat ions were performed on huma;in scra incihatted with lTAcx. Tn addition, (: titrations were carried out in the presence of oetyleneglycol-his (i Amino E:thyl Etlher) N,N totra;iectic acid (EC.\A) and Mg ions. This c l, t inlg agent preferentiallv hinds Ca ins (1 I,1 f and by reinforcing the E(I'A buffer with Mg ions one can effectively deplete the available Ca ions yet inninta in relatively high levels of Mg ions. Thus, the Ca ion dependent classical pathway is blocked, hut the alternat ive pathway can function relat ivel-v unimpai red (145, 157).



































Figure 1. Titration of whole human complement after incubat.iln
with crude extracellular lipoteichoic acid (LTAcx).
Symbols: (o) Non-treated control: (e) Secrum triatcd
with LTAcx at 500 ig/ml.







33



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00

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0 0




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Figure 2. iose response inhilbition of whole humin compl ement
n after incubh;tion with varving concentrate ions oF
I'TAcx. The non-treated control is ibh reviaLoted
,s NTC.















LL I oD aO to o z



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A typical component Litration in serum trrotead with L.TA'x is depicted in Figure 3. In this example, the LTAcx treaonted sirulm was serially diluted in DGVB. Next EAC142, C5, 6, 7, and CS-9 were added sequentially to the dilutions. Since all components were added in excess, C3 became the limiting factor in contributing to the hemolvsis of the target cells. Percent lysis in each test tube was mathematically converted to Z (the average number of SAC1423 sites per cell) and this was plotted against the reciprocal of the serum dilution. Percent inhibiticn of site forming units (SFU) was then calculated from 7=1 values or percent inhibition of CH150 units was determined from value es associated with Z= 09. Figure 4 represents a composite of multiple component titrations from whole human sera treated with LTAcx. As can he seen in this figure, CL and C4 activities were consumed to omie degree, however, more than 501 inhibition of C2 activity was observed. As indicated, C3 activity was also consumed during preincuibation of complement with LTAcs, but incubation with purified [iHU prordu d no inhibiticn of C3 hemolytic potential. No C3 consumption occurred if the incubation was performed in the presence of the chelator ethvlenediamine tetra acetic acid ( EDTA) and Less than 7" if incubaited in the presence of EcTA-Mg ions. The above results indicated the necessity for divalent cations as cofactors mediating the consumplhtion of C3 in the presence of LTAcx. In addition, there appeared to he a requ irpment for other setum factors (possibly natural AB and/or components of the alternative pathwavt since purified C3 activity remain d unaffected ,v inieiition wirh LTAcx.




































Figure 3. Titration of C3 in whole human serum :EFter trentnont
with LTAcx. Symhols: (a) Non-treated control; (0) Serum trreatd w ith lAcrx it a concentrate n of 2 T0
lg/ml. After incubation, sera were t itrnted for
residual C3 activity according to procedures
described in Material.s and Methods.
























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Figure 4. Complement component titration of whole human sra
after treatment with LTAcx. The sern were incubated
with the LTAcx (50(0 ug/ml) then titrated for residual activity of the components indic ated as described
in Materials and Methods.























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Inhibition of complement Lysis of LTAcx treated EA. During an experiment in which EA treated with LTAcx were testeded for reactive lysis, it was discovered that the LTAcx treated cells exhibited Lesq hemolysis than even the buffer treated controls. This serendipitous observation led to the discovery that LTA treated EA were refractory to complement mediated lysis. To confirm these results, various concentrations of LTAcx were used to treat EA. After tlhe treated cells were extensively washed they were tested for their suseptibility to lysis by complement. The same cells were also tested for the presence of cell-bound LTA using the passive hemagglutination technique (PH.A) w th anti-LTA. The results shown in Figures 5 and 6 indicated that both the extent of inhibition of hemolysis and PHA titers were LTAcx dose dependent. There was a decline in both activities n nlv after the iTAc:. had been diluted to a concentration of 62. 5 ig/ml. The decrease in titer below this concentration indicated that the test cells were no longer saturated with LTA. There was a conco('mitannt drop in inhibition of lysis at 62.5 pg/ml. EA which were treated with uninoculated culture medium (dialyzed Todd-llewitt broth) were unanI fec ted when compleemnt was added.

Effect of LTAcx on lysis of sheeP E and sheep E: cellul-ar

intermediates. The tr(atm(nt of EA with LTAcx caused the cells to become relatively resistant to complement mcdi ated Isis. This could have been dUo to an cI c ct ion te ant ;libody nlo ls al' : In effect on on or more of the complimonut componuits., or an al. rat iion of the c'll membrane.

To further invest-i ~tc the nature of the comp lemnt inhhiit inn

associated with LTAc, hep E. sheep E., and various ; sheep F rnmc, o lent



































Figure 5. Inhibition of complement mneditd lysis of EA
treated with varving concentrations of LITArx.










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Figure 6. Passive hcmagglutination (PlIA) of LA treated with
varying concentrations of LTA\x.

















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component intermediatesn were treated wi. th LTAcx and analyzed for suIsceptibil[ty to complement mediated Ivsis. The LTAcx treated cells were also tested for bound LTA using PRIA with ant-'LTA. Results indicated that E, EA, and EACI4 were al.l refractory to complement medinted lvsis and that LTA was detectable on the surfaces of the cells (Figures 7 and 8). However, EACL423567 which had been treated with LTAcx were not resistant to lysis despite the fact that LTA was detectable on t:he cells (Figure 8). Thus, the inhibitor appeared to affect a complement component required for lysis os EACI4, but which was unnecessary for Lysis of EAC1423567.

In an attempt of focus on the site of inhibition, the ability of LTAcx to affect the hemolytic susceptibility of EACL42 was examined. This intermediate possesses C3 convertase activity (C42) which is involved in the generation of SAC423 and SAC,\4235. However, C( is not required for lysis of the intermediate once SAC142 have been formed (148). Failure of LTAcx to inhibit this intermediate would indicate that C3 convertase was not the step in thie complement sequence affected by the LTAcx.

Sheep EACi42 were treated with LTAex according to the protocol.

that has been described. For this experiment, tire different amounts of C2 were used to generate EAC1.42 from EAC.4. The results clearly indicated that there was no inhibition of the intermediate comply I'x EAC14'2 (Figure 9). testing by PHA with antibodies specific for ILTA confirmed the presence of LTA on the surfaces of the cells at the samo relative conlcen trations found when the other intermediate complexes were tested.

Effect of LTAcx on anti-sheep ervthrocvte antibodies. Some



































Figure 7. Effect of LTAcx treatment on the lysis of various
complement component intermediates. Each cellu Nr
intermidLate was prepared and then treated with
LTAcx (125 pg/mi). Lysis was developed using procedures described in Materials and Methods.
Percent inhibition was calculated by comparison
against buffer treated controls.










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Figure 8. PHA of various LTAcx treated complement component
intermediates.



















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Figure 9. Effect of LTAcx treatment on the lysis of EAC142.
Various limiting concentrations of C2 were used to
prepare EAC142 cellular intermediates. The cells were then treated with LTAcx (250 Vg/ml) and lysis
was developed using procedures described in Materials
and Methods. Symbols: (o) EAC142 incubated with
LTAcx; (e) EAC142 incubated with buffer.





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substances in LTAcx might be capable of interacting with the ant ibodies used to sensitize sheep E. This internctinn could then lead to an impairment of C1 activation and result in reduced lysis. Such a mechanism might be the reason why E and EA become resistant to lysis after treatment with LTAcx. Therefore, antibodies to sheep erythrocyte stromata were incubated with LTAcx. The mixture was then diluted to the point where the LTAcx:-related inhibition could not be detected and the antibodies in the mixture were titrated (135). It was found that antibodies that had been preincubated with LTAcx had the same titer as antibodies that were incubated for the same time and temperature with VBS (Figure 10).

Partial purification of LTA. Partial purificat Lon of LTA and

the complement inhibitor was acc mplished by gel lil trntion n the LTA: through an A-5M Biogel column. The results of a typical experiment arc, shown in Figure 11. Arens of antigenicity were resolved by immnodiFfusion in an agarose gel utilizing ann nti-sernm specific for the IAl backbone. Fractions were pooled as indicated (A-F), and each pool was dialyzed against water and subsequently lvophlized. Note that po ls B, C, and E contained high levels of phosphorus and tit the zones of antigenicity were also located in these areas. Utilizing extra ca e I cl(ar extracts from S. mutans and other microorgan isnms, similar frActi onat ion profiles under comparable lc con ditions were obtained by Wicken and Knox (110) and ilewiuis and Craig. Analysis by these workers revealed that the second phosphorouns contain ing pvak (peak 11) conta ined LA\ whereas the trailing phosphorus peak contained deyiclated TA. and wall teichoic



1.
Personal commun ica t ion.






































Figure I). Effect of ITArx on homovltic antihody titration.
Antibodies to shoep red hlood cell stromaeta (Ah) were
incuhbated with ITAcx (95OO ug/ml) and residual hiemnal-svin
activity was titrated y procedures described in
Materials and Methods. LUsis nf cells was developed with whole guinea pig complement. Symbols: (o) Ab incubated with LTAcx; (e) Ab incubated with bnffer.




















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acids. Because peak 11 represented partinl ly puri [i' d ext ran:ciliLliar lipoteichoic acid, the recovered material was designated LTAppx.

A sample of each pool was rehydrated to 50 ye/ml and reacted with EA, according to standard procedures (Materii!s and i:thods). Fach FA preparation was analyzed using the complement inhibition nssav and tested for bound LTA by PHA. Onlv the pools contaiini; n LTA (as demonstrated by PHA) caused inhibition of complement mediated 1vsis (Table 1).

Despite the excellent separation of LITA from most of the material that absorbed light at a wave length of 260() nm and p resumably from all deacylated LTA or TA, two persistent problems arose with this purification procedure:

1). Polysacchar le contamina tion accounted for a major portion Vf the mass recovered in peak I1I, and

2). The total mass of lTAppx under peak II was anlmos t immcasrai small.

In an attempt to at least increase the yield of peak 11 material, a Millipore Cassette system was emp Loyed to both concentrate and frtnctionate the spent culture supernate (Materials nnd Methods). This method of LTA enrichment proved highLlv successful as evidenced bv lhe results in Figure 12. Even after va;llues are corrected for the greater mass of crude extract applied on the latter column the mans vield ,f I ,\Apx was some fifteen fold grestut r than thnt obtained with previously employed procedures (F igur.e 11).

An analysis of result tracing th' partial puri ication of LTA is summarized in Tables 2 and 3. It ;houtl he noted that the total amount of Pi, mass, protein, and A 260 absorbing mat oral decreased several thousand fold in the purificnt s n process, whereas the total



















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Figure 11. Partial purification of LTA by A5-M gel filtr-ition.
Symbols: (e) A 26I0 aorbance (m'xmql1 :Ibsorh;lnce
wavelength for nuclcic acids): (*:) A,. absorhbnce (maximal absorhance wavelength for carbohydrates ns
determined by the Pheno S lIfuri c A:id assayv ); (A)
Pi concentration in n-moles As dctermind by the Lowry PL nssay; (+) Antigenicity as dctcrmined by
Ouchterlony gel di ffusios n 1sinC a nntiscra
directed against LTA backhone.
















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Figure 12. Partial puWrification of LTA b\ A5-M gel filtration
with LTA enriched starting -mterial. Svmbol.s: (e)
A absorhance: (o) Pi concentration in u-mmlu's/mli
as determined by the Lowry i assay: (+) Antig&"nicity
as determined by PHA using ;ntiqern directed against
LTA backbone.

















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amount of LTA in the sample, PHA titer, and percent lytic inhibition of EA remained relatively unchanged or increased in value.
Purification of LTA by hVdrophobic interaction gel chromatogra hy. A 25.0 X 2.25 cm column packed with Octyl Sepharose ;ind equilhrated in buffer A was prepared as described in Naterials and Methods. Approximately 6.0 mg of LTAppx dissolved in 10.0 ml of buffer A were applied to the column. As can be seen in Figure 13, a small amount of phosphate containing material passed unimpeded through the column. A slightly greater mass of polysarcharide was also excluded without binding No additional material eluted from the column with buffer B. Point C on the graph marks the location where a 10-70% propnnol gradient was begun. Point D represents the point where a signficant volume decrease per test tube was observed. Since fractions were collc:ct.d n a "Idrops per tube" basis, the presence of propanol in che effluent causes a change in surface tension of the drop resuLting in decreased volume per drop. The ultimate result is a decrease in the volume per tube. This, test tube volume provided a convenient means o, monitoring the progress of the propnnol gradient.

It should he noted that despite the use of a grncient (the original procedure called for a single step-wise clution with 50" propnlol) significant amounts ,f carbnWhydrnte eiteld with the LIA. As indicated on the the graph, all areas containing phosphates a lso contained L'TA as detetced using PHA. The Iact thAt a smAill ount of LTA pa ssd inhound through the column sugp pests- that either the column binding capacitv was exceeded, r perhaps the ITA was onlv part inllv n'viated and not capable of tenacious hvdrophhir hindin .




































Figure 13. Purification of LTA by Octyl Sepharose hydrophobic gel
chromatography. Symbols: (o) A260 :ibsorbance; (A)
concentration of carbohydrate (n-mole /mt) as dprermined by the Phenol-Sulfuric Acid assay. (Concentrati,,ns were
determined using glucose as a standard carbohydrate.
(o) Concentration of Pi (n-moles/mi) as determined by
the Lowry Pi assay; (+) zones of antigencity as
determined by PHA using antisera directed against LTA backbone; (A) elution with buffer A (1.OM NaCI, 0.01 M Tris-carbonate pH 6.8); (R) elution with buffer B (0.01 M Tris-carbonate, pH 6.8); (C) elution with a 10-70' gradient of a propanol-buffer B mixture; (D)
elution volume at which significant reductions of volume/tube were observed, indicating elution of propanol.














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67



All test tubes containing greater than 25.0 n-moles Pi/ml were pooled. The entire peak (approximately 22 ml) was loaded on to a 65.0 cm X 3.0 cm column packed with LII-20 equilibrated with deionized water. Four and two tenths milliliter of effluent were collected per test tube a a flow rate of approximately 30.0 ml/hour. The results of this procedure, which simultaneously removed salt and propanol, nre shown in Figure 14. The column effluent was monitored at a wave length of 220 nm and was also screened for LTA by PHA (++++) using a single dilution sample. In addition column fractions were tested for the presence of chloride ions by placing one drop of a saturated AgNO3 solution on a coverslip containing one drop from each test tube. Any resulting precipitation was evaluated on a +1 to +5 basis and plotted accordingly. It was empirically determined that not only Cl reacted with the AgNO3 resulting in insoluble AgC1, but the NaN3 and tries carbonate in the buffers reacted as well. The presence of propanol was monitored indirectly by changes in test tube volume. Since LTA. azide, and tris carbonate all absorb at a wave length of 220 nm the combination of ultraviolet light screening, the AgNO3 precipitation test, and visual inspection of volume changes per test tube proved to be invaluable for rapidly discerning the location and separation of LTA from contaminating salts and solvents. The entire contents of peak I were pooled, frozen, and lyophilized. The Final product was referred to as LTAosx (extracellolar l ipoLeichoic acid purified by OcLvl. Spharose hydrophobic a ffinityv gel chromatography). The typicAl mass yield from s!(h a procedure was about 60-700. Prcent recovery of LTA at var Ins points in the procedure is summarized in Table 4.




































Figure 14. Simultaneous removal of salt and propnot free LTAosx
by LH-20 gel. chromatography. Symbols: (o) A220 :Ibsorbance; (o) Volume/test tube: (+) Antigenicity as determined by PItA; (Shaded Area) relative degree of precipitation of salt and other low molecutiar weight materials
as determined by AgNO3 test.







69




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80

O




C
li a.n 0 u to
O 4 0








>-J cl cf 00 "
O rd <




41 cn m o J_- L~
WO




o










I- 0
'OH
i to ON Nio

4 o




O 0












ct vi a
H w 0 C
3 EE










4-1 w
o a r- Q -4 COC
o 0i 0 <1 '-0, 04
























cco
UHO O) (0 C! 0~ 1'- 1-4 C)a o N


0 F-1 4u 0

QH (-4
-1

4-4 4

-.J u















0 0
CO _r


o C


















uN 4a -4a
t4-4 r-u

C: m 0H- 0j V40 0 IJ .14 rd i
0 t 0 0





















3 ~ t~ Q. Ol O
-~Q -4U~ 0 0 0
Cu--4
4-1 -4 aa~

0d c'o C
i-(-H




















O3C 0 ) I a 3 C
'H fl 0
a
U ~~~ aa(-t
o~ Uu


I- u 0 N N1 0 0 C CO-A 0 0 0 '0 U 0) (-C '1 r-- -af 04

04I U-H 4U

tnC '-- C) E O 040

4-,

c~o to CO CO.- 04 O 0 '-I~~e e
u-i)C i -JO u Z WN .-u I-- ---4


lc OOC C COP C)0

CO 0:n ) QI C ) P .
HUd HO-d 0 3 04



U U- C ) -1
a --0 4-C, -'0 04uiu a
o3 X 4' '0( -4to 'HM '0 0) U 1!) 0. -C,) Ud CC U r 0.: -1: -i1C Om CO 04 (0r r o
-r '~ ( O0'0 ~m Cofl
.2 r 0. 0. 0C4-C C'. C' 2










Phosphatidyl choline vesicle (PVC) purification of LTA using C labelled phosphatidyv choline. Approximately 5 X 10 DPM of C labelled phosphatidyl choline were added to 40 mg of phosphatidyl choline dipalmitoyl. Phosphatidyl choline vesicles (PVC) were prepared as described in Materials and Methods. Three test tubes containing identical volumes and concentrations of non-labelled PCV were prepared simultaneously and 2.0 ml of LTAppx (1.5 mg/ml) were added to each test tube. Fifty microliter samples from the C containing test tube were removed at various steps during the purification process and analyzed as described (Materials and Methods). A standard chloroform quench curve was constructed and all reported counts represent corrected DPM values. Table 5 depicts the distribution of 14C counts at various steps in the purification procedure. Utilizing this procedure as described, essentially no contaminating phospholipid could be detected in the final product. The typical mass yield of product via PVC purification was about 10-15%. Percent recovery of LTA at various steps in the procedure is summarized in Table 6.

Comparison and summary of LTApcx versus LTAosx. As indicated in Table 7, both methods of LTA purification removed the majority of protein as compared to the total amount abailablo in the LTAppx. Both methods ostensibly recovered > 80% of the original ITA. However, the major difference between the two products is reflected in the percent total, mass recovery and the concomitant increase in percent carbohydrate in the final LTAosx product. This latter difference can he most readily discerned by observing the composite gas chiromatograph tracings in Figure 15. The carbohydrate standard (CHO-STD) depicts the typical chromatograph of glucnse and ;glactoe after preparing trimthylsillvl











>r



0
Ca
c o O O" r-. 'a -o C 7 -i O C >
-4 ao l- a < o 0 C C CD 0 m O O C -O c i O






0 S W 4 C 00


<- -0

Sa a c




4-1O 4- N 4o t o 4 3
O 0 4 O e-1 C "O C C1 0i O 00 01 m try ,-4 4 i a ,-a I



C) :)L 0 CL -41 c-0 co c _r Ln CD 0 C o a oa CD c cc o c)-a ca -- -- a a-> O a C) 0.0 0000 000C o a u L c. V1 *I
-,4













O .M
U Ga













--4C) 3, C o -. -T C) C) -4 Lc- O] C .;














- a c O #O O- O CO --. H- . r 4 r 0 C) C- c-i fq ? ) 0 N c CD O i .r4 m









-W 0 r. I) Hn


















O t C) C : i - c- ) O O -4 C O

















",Inc a c ..- o O .0 0 -) Oc C2c --a

-W


L) Li










S c C) C C) C) c- C C) c --4
I0 C Cc > CC 1 0 co c- w Li r r. r L i C
a w- c.:. ca )x Li) Li

w a





















O7






o
0 0
t4.

















w o D C C: .








o co O
U










o a on 00 0 0 0





c o-' o oo 00

Ooic4 >0 w o 0 0 c .. $o uc

SHO 0 o




- on -0 0 "T L. CO C) 0








O cd c o -4 0 a 0 0 c





C) 0
CC





-4 T- 7 1













.2 D .-4 N (l 2 m .














c 0- r- .c Oc 0 r. C Z C




C~ OC



CC1

f O CL
ID .-.

N C>': -- .-ac L




,,, ... 41 ,
CS 0


CD, Li 3 C i <- .. U ,/














C 0 1
Lr n
0

Ui LC C








0 n






4 C: .,q .-,4- 4 0- C '2 Q rU C 0 Li C C C C Li ,- L .i








o.0 .-,0- a





< C'-, ,nLi 0C Li CC S i 0 C .: l) u C- C C) C-, *H-) C) C C










-'- .H U -u 0Ti Li--* n a














-"C 2 4 4 LC i C CC
C- 0 c L i Cr C 1(. C -4




3) I Li C- 3 C.a




































Figure 15. Carbohydrnte analysis of ITA containing preparalt-o,ns
by gas liquid chromatography. Abbreviations: (MAN) Mannitol; (GLC) Glucose; (CAL) Galactose. Mannitol
was incorporated as an internl standard with all
samples.
















CHO STD LTA ppx










MAN
MAN



GAL
GLC
GAL GLC






G P2 CARDIOLIPIN








MAN

MAN













LIA osx LTA p~x








MAN

MAN










ester (TMS) derivatives as described in Miterial and Methods. An internal mannito standard is included with all samples. The LIAppx chrnmatograph represents the typical carbohvdrnte profile achieved with partially purified LTA. The tracings for LTAosx and LTAppx contrast the qualitative and quantitative differences in carbohydrate content. The second two chromatograms,dencylated cardiolip in ((3 P2) and cardiolipin, were included as a comparison of how a naked polyglycerol phosphate backbone might be expected to react under the described conditions. The base line instability of the G3 P2 looks remarkably similar to the profile of the purified LTApcx. The procedure for purifying deacylated cardiolipin requires passage through Sephadex columns. It is quite conceivable that the minute quantities of unidentified carbohydrates which are indicated may be due to dextran contamination from the column. However, it would be difficult t to account for the same source of contamination for the LTApcx since gel. chromatography was not used in the final purification. On the other hand, the similarity of the indicated chromatogram tracings may be more than more coincidence and may ref lect a:ctun! reactions of the derivatizing agent with the pol.:yglycerol phosphate backbone. This latter hypothesiis s suipprted by the fact that an unidentified trailing "carbohyvdrato" peak of sign i i.cnnt mass appears in both the cardiotipin and C3 2 chromatographs. The RE value of this peak is similar (huitv t s,,sp iciously d isparate) to the rcat: tion t ine normally observed for N-glucIse. However, if inded this peak does represent B-glucose, one is hard pressed to, ration alize why a corresponding n-glucose peak does not occur as wel. In either case, it is




Dencyl:ited cardioL ipin was prepared by the method of Wi lkinson (14 q) and was kindly provided by R. Craig, University of Florida.










apparent that the LTAosx st ill contains if icant nmounts of carbohydrate contamination in contrast to the i noer vilid, but high!: prified LTApcx.

A summarv of specific activities relative to PH;A activist is pr.sented in Table 3.

Inhibition of complement mediated l':sis of LTApcx treated EA\.

Once a highly purified preparation of LTA was obtained, it was necessary to confirm the results that had been previously established with LTAcx. As can be seen in Figure 16, not only were LTApcx treated EA refractory to complement mediated lysis, but in addition the general profile was remarkably similar to LTAcx treated EA. As indicated, LTApcx was used in concentrations five-fold to ten-Fold less than those used with LTAcx to achieve comparable degrees of inhibition. PHA titers typically indicated LTA saturation of the cells.

Effect of LTApcx on_ the .vsis of various cel Lular complement

component intermediates. Sheep E, EA and various complement intermediates were treated with LTApcx (100 c /ml in DVR) and washed extensively in DGVR. Percent lysis and inhibition of CH5 units were determined as described in Miaterials and Metihods. The results of these experiments are summarized in Figure 17. As with LTAcx treated cells. E, EA, and EAC1 were al 1 ref ractorv to lvsis 1by n mln ment. Li kewise, EACI42 and 'ACI-7 inritWrmediates were total llyv iif:iIfe. etd by the presence of LTApex on their cell s rfaceq. The on lv observahl, diffr'nce in the activity of LTApcx on ce:tlllalr intermedi teos versus LIArx was that E.\:li" cells were inhibited to a l esser degree with LTAp'x than TArcx.

Effect of LTAcx and LIApcx on fluid_ phase Cl. As previo us v

discussed, CA is not ronuired for yIvsis once SAC42 are formed but it is
























t"1 sq 4m 1 1 O I n
r co cs n
-0 cn I








Z) CC
U
<"1 C LO N- M








Icr
N0 .

4-44








0O
Otf a- l






S.4
oo : 0 co










,4 CL : t





















c-4
-siv e-1 e
Cs ..o r- CO Cs .I * O 2.CO 0 -Z n- Cl C
N-tf- 0 0
c I0



















C OO
Q)




JO C O D C U 4O4 C r' .) 0 C) CI -4l C '] C 0 "O
*,- ..- .. -. cC O tt O



































ca~O 0 0
Co 0 \ O~ O '0 0
CC)
0 4 0 ,l -- 0









































a4 .. ro a 7*
o C CO






















m~ 0) MOi
41n -> 'H

























&-4 & H -O U ** U






















U M L W O O O
-n o rt a C -'























GO 4HOC H H HH-GO 0410 H ) .? f r 1 : U U 0 3-4 04 --t .- C
09) .0 Cs. '-0



U 54



C. 4. C



09 U 0 CC .


410 0 *- ~ ~ 3 iV
0 Ls 04 0 0-v (
W I L-. -. 0 jOC U ~ ~ 4-O -r CO 'CU 0i 0.tl U3 Cs C CCU
'-t C:. 400 a


Q^ CJU




c C C *l U

C4. 04 cl Co Cl r C
'TCs .0 C: 0.)r:c,


-]~~- C) oSi" -3 C




































Figure 16. Passive hemagglutination (PH1A) titration and inhibhit ion
of complement mediated lysis of EA treated with varying
concentrations of L'TApcx.














z
0
!
] TITER 1600 3200 3200 3200 3200 C 6400 S3200

S1600 Z 800
uLL 400
0
1 200

o
CL 6.25 12.5 25 50 I00

:: 0 % PHA
50 % PHA S100 % PHA






Un
> 70

0
50
Z
O
- 40

30
z
20
I
z 10


w 6 25 12.5 25 50 100

LTAcx (ug/mil) USED IN EALTApcx PREPARATION




































Figure 17. Effect of ITApcx on the complement mediated 1vsi~ of
various cellular complement components intermediate.







83








*



C
I o






W






x
w~lU O O-- O rcr
< <0 w- 32








LLJ -D LU






n O O N O


SISAk1 -JO NOILIIHNI IN3O31d











essential until that point is reached (148). In addition, mnny polyanionic substances are known to directly affect Cl by interfering with Clq binding or Cl esterase (CIs) activity (39,40). Because LTA is polyanionic due to the polyglycerol phosphate backbone and because cellular intermediates beyond the EACL42 step were no longer inhibited, it seemed reasonable to hypothesize that LTA was behaving like a polyanion and directly affecting Cl.

To test this hypothesis, LTAcx and LTApcx were preincubated with functionally purified human Cl at 300/15 minutes. Residual Cl activity was titrated as described by Rapp and Borsos (139) and activity was compared against buffer treated controls. The results shown in Figure 18 indicate that although LTAcx consumed C1 activity, purified LTApcx did not.

Purification of human C1l, Cls and C1s. In an attempt to further eludicate a possible site and mechanism of C inhibition, human C1 subcomponents were purified by the methods and modifications previously described (Materials and Methods). Although homogeneity beyond functional purity was not essential, the methods employed yielded highly purified products. Figure 19 demonstrates Clq homogeneity by immunodiffusion against several monospecific antisera. Precipitation bands of identity were observed in adjacent wells containing the whole human serum starting material, the purified Clq final product, and a hi ghlyl enriched C1lq prior to final precipitation (Figure L9, plate 5, we11 numbers A. (, and E). As can be observed in Figure 20, disc gel electrophorests of the final product revealed a single dark staining band which barely migrated into the separation gel. These results are consistant with the observations of other investigators (144).




































Figure 18. Effect of LTAcx and LTApcx on functionally purified
human Cl. The upper graph represents a residual Cl
titration after incubation with LTAcx (500 vg/mi).
The lower graph represents the restIlts from an analogous experiment using LTApcx (500 ug/ml) instead of LTApcx in the incubation mixture. Symbols: (0) C
incubated with biiffer; (o) Cl incubNaited w ith the
appropriate LTA containing extract.







8











2.2

1.8 1.4

. I .0
_J 00.6 U0.2 to L. I 6,000 8,000 4,000 2,000
0

Li
aD

Z2.2 w2
Li

I.8




06 02

16,000 8,000 4,000 2,000
RECIPROCAL OF HUMAN Cl DILUTION




































Figure 19. Immunodiffusion and precipitation analysis of various
steps in the purification of human Clq. Purification was achieved by repeated fractional precipitations of whole human sera in buffers varying in ionic strength,
pH, and concentrations EGTA or EDTA (144).
Well designations:
(A) Whole human sera (starting material);
(B) Supernate from first precipitation;
(C) Supernate from second precipitation;
(D) Supernate from third precipitation;
(E) Material from resuspended pellet prior to final precipitation.
(F) Final product (purified Clq).
Plate designation: (1) Center well contains anti-IG(;
(2) Center well contains Anti-IgA; (3) Center well
contains anti-whole human scra: (4) Center well
contains anti-IgM: (5) Center well contains anti-Clq.























'. ia




ownngr

















~LB~lg lldQQ ~J






































Figure 20. Disc gel electrophoresis of piirified human C1q.
Cathode was at the top.







9 ) E.




Full Text
131
151. Bladen, II. G. Hagenge, R. Harr, and F. Pol.lurk. 1972. l.ysls of
certain organisms by the synergistic action of complement and
lysozyme. J. Dent. Res. 52:371.
152. Mill ler-Eberhard, H. J., M. J. Policy, and M. A. Calcott. 1967.
Formation and functional, significance of a molecular complex
derived from the second and the fourth component human complement.
J. Exp. Med. 125:359.
153. Kingsley, G. R. 1939. The determination of serum total protein,
albumin and globulin by the biuret reaction. J. Biol. Chem. 1_31:19
154. Lowry, 0. H., N. J. Rosegrough, A. L. Farr, and R. .1. Randall. 1951.
Protein measurement with the folin phenol reagent. J. Biol. Chem.
193:265.
155. Agnello, V. R. J. Winchester, and 11. G. Kunkel. 1970. Precipitin
reactions of the Clq component of complement with aggregated y
globulin and immune complexes in gel diffusion. Immunology. 1.9: in11
156. Agnello, V. R, 1. Carr, D. Doffler, and H. G. Kunkel. 1969. Gel
diffusion reactions of Clq with aggregated y globulin, DNA, and
various anionic substances. Fed. Proc. 23:696.
157. Borsos, T., H. J. Rapp, and C. J. Crisler. 1965. The interaction
between carrageenan and the first component of complement. J.
Immunol. 94:662.
158. Harhoe, M. 1964. interactions between trace labeled cold agglu
tinin, complement, and red cells. Brit. J. Hnemat. 10:339.
159. Harboe, M. 11. .1. MU l ler-Eberhard, 11. Fudenherg, M. J. Policy,
P. L. Mollison. 1963. Identification oc components of complement
participating in the antiglobulin reaction. Immunology 6:412.
160. Sweet, C., and J. K. Full. 1970. The binding of serum albumin to
phospholipid liposomes. BBA. 219:253.
161. Weissmann, G., A. Brand, and F. C. Franklin. 1974. Interaction of
immunoglobulins. .1. C.lin. Invest. 4:V3G.
162. Shin, M. L., W. A. Paznekas, A. S. Ahranevits. and M. M. Mayer.
1977. On the mechanism of membrane damage bv complement: Expo
sure of hydrophobic sites on activated complement. .1. Immunol.
119:L 358.


substances in LTAcx might be capable of interacting with the anti
bodies used to sensitize sheep E. This interaction could then lead to
an impairment of Cl activation and result in reduced lysis. Such a
mechanism might be the reason why E and EA become resistant to lysis
after treatment with LTAcx. Therefore, antibodies to sheep erythro
cyte stromata were incubated with LTAcx. The mixture was then diluted
to the point where the LTAcx-related inhibition could not be detected
and the antibodies in the mixture were titrated (135). It was found
that antibodies that had. been preincubated with LTAcx had the same
titer as antibodies that were incubated for the same time and temperature
with VBS (Figure 10).
Partial purification of LTA. Partial purification of LTA and
the complement inhibitor was accomplished by gel filtration of the LTAcx
through an A-5M Biogel column. The results of a typical experiment arc
shown in Figure 11. Areas of antigenicity were resolved hv immunodif
fusion in an agarose gel utilizing an anti-serum specific for the LTA
backbone. Fractions were pooled as indicated (A-F), and each pool was
dialyzed against water and subsequently lvophilized. Note that pools B,
C, and E contained high levels of phosphorus and that the zones of anti
genicity were also located in these areas. Utilizing extracellular
extracts from S. rnutans and other microorganisms, similar fractionation
profiles under comparable conditions were obtained by Ktoken and Knox
(lit)) and Blewieis and (iraig. Analysis bv these workers revealed that
the second phosphorous containing peak (peak II) contained LTA whereas
the trailing phosphorus peak contained deucvlatcd LTA and wall teichoic
Personal communication.


27
samples from the C containing test tube were taken at each step
of the purification and placed in empty glass scintillation rials.
The samples were heated to 50C in a drying oven to remove the
solvent from the sample. Once dry, 50 ul of chloroform were used to
redissolve all samples and then 5.0 ml scintiJlation fluid containing
toluene (scintillation grade, Mal 1 inckrod t, St. Louis, MO), 0.4/' PPO
(2,5 diphenyloxazole), and 0.01% POPOP (1,4-di (2- (5-phenyloxazolv 1)-
benzene) were added to each vial. The degree of ^C-PCV contam
ination of the final product was determined by placing the entire
LTA-containing-fluoropore filter .in a scintillation vial with 5.0 ml
scintillation fluid. The possible influence of quenching by the
fluoropore filter was investigated by adding equal aliquots of
14
C-PC to two scintillation vials one of which contained a fluoropore
filter in addition to scintillation fluid. No appreciable difference
in CPM was observed. Disintegrations per minute (DPM) values were
calculated from a standard quench curve constructed for use with chloro
form. Standard ratios were determined for each sample and percent
efficiencies were extrapolated from the standard quench curve. This
volume was then used to correct counts per minute (CPM) to DPM. Unless
otherwise indicated, the samples were counted for 10 minutes in a Beckman
LS-.133 liquid scintillation counter (Beckman Instruments, Fullerton, CA).
Col orine trie assays. Phosphorous was determined hv the method
of Lowry et al. (141) with absorbancies measured at 820 nm. Total
carbohydrate was measured by the phenol sulfuric acid assay as des
cribed by Dubois ct al. (142). Total protein was performed on samples
using the Bio-Rad Protein Assav (Bio-Rad Laboratories, Rockville Center,
NY). Samples and the standard curve were prepared following the
manufacturers recommrndations.


121
41. Allan, R., and H. IslLker. 1974. Studies on the complement-binding
site of rabbit immunoglobulin GModification of tryptophan resi
dues and their role in anticomplementary activity of rabbit IgG.
Immunochemistry. 11:175.
42. Wilder, R. L., G. Green, and V. N. Sehumaker. Bivalent hapten-anti
body interactions. Immunochemistry. 12:54.
43. Patrick, R. A., S. B. Taubman, and I. H. Lepow. 1970. Cleavage of
the fourth component of human complement (C4) by activated Cls.
Immunochemistry. 7:217.
44. Schreiber, R. D., and H. J. M 1 ler-Eberhard. 1974. Fourth component
of human complement: Description of a three polypeptide chain
structure. J. Exp. Med. 140:1324.
45. Mller-Eberhard, H. .1., and I. H. Lepow. 1965. Cl esterase effect on
activity and physicochemical properties of the fourth component of
complement. J. Exp. Med. 121:819.
46. Policy, M. J., and H. J. MU 1. ler-Eberhard. 1968. The second component
of human complement: Its isolation, fragmentation by C'l esterase,
and incorporation into C'3 convertase. .J. Exp. Med. 128:533.
47. Mller-Eberhard, H. J., A. P. Dalmasso, and M. A. Cnlrott. 1966.
The reaction mechanism of Blc-globulin (C'3) in immune hemolysis.
J. Exp. Med. 1_2J3:33.
48. Shin, H. S., and 11. M. Mayer. 1968. The third component of the guinea
pig complement system. II. Kinetic study of the reaction of EAC'4,2
with guinea pig C'3. Enzymatic nature of fixation, and hemolytic
titration of C'3. Biochemistry. 7:2997.
49. Cooper, N. R. 1971. Enzymes of the complement system. Prog. Immunol.
JL: 5 6 7.
50. Policy, M. J., and I!. J. MU 11er-Ehorhard. 1967. Enhancement of the
hemolytic activity of the second component of human complement
by oxidation. J. Exp. Med. 126:1013.
51. Gold lust, M. B. H. S. Shin, C. 11. Hammer, and M. M. Mayer. 1974.
Studies of complement complex C5b,6 eluted from EAC-6: Reaction
of C5b,6 with EAC4b,3b and evidence on the role of C2a and C3b
in the activation of C5. J. Immunol. II3:998.
52. Arroyave, C. M. and li. J. MU 1 1 cr-F.be rhard. 1973. Interact ions
between human C5, C6, and C7 and their fimet ional significance
in complement dependent cytolysis. J. Immunol, ill:536.
53. Lachmann, P. J., and R. A. Thompson. 1970. Reactive lysis, (ho
complement mediated lysis of unsensitized colls. II. The charac
terization of activated reactor as C56 and the participation of
C8 and C.9. Exp. Med. 1 31 : 643.


The procedures for Cls and Cls purification were modified only in
that an ionic gradient was used in the final, purification step of both
reagents rather than the stepwise elution utilized by Sakai and Stroud
(35). The rationalization for this modification was that a difference
in binding capacities of the DEAE matrix could have deleteriously
effected the elution characteristics of the Cls (Cls) at a fixed ionic
strength. The elution profile of Cls is shown in Figure 21. It should
be noted that two peaks of material which absorbed light at a wave length
of 280 nm were resolved during the gradient elution. Both peak II and
and peak III reacted with monospecific antisera to Cls, however, only
peak III contained Cls activity. Peak II presumably represents an in
active form of either Cls or Cls. No such ext raucous peak was resolved
during DEAE chromatography of cls.
Iminunoelectrophoretic analysis of purified human Cls and Cls on
1% Noble Agar is depicted in Figure 22. Results indicate a difference
in electrophoretic mobility of Cls and Cls which is consistent with the
observations of previous investigators (35). Also, there was a "gull
wing" pattern displayed bv Cls apparently representing microhetero-
geneity of the activated proesterase. This too has been observed In
previous investigators (35) .
Effect of LTApe.x on the ability _o_f Cls to consume C4 and C2 activity.
As previously discussed, activated Cl esterase (Cls) is capable of
cleaving C't into C4a and C4b (43) as well as cleaving C2 into C2n and
C2h (45). In either case, the active fragments rapidly decay and it not
quickly attached to membrane sites, lose their ability to do so. The
ephemeral nature of these active fragments can he used as sensitive in
dices of Cls activity. As described in Materials and Methods, equal


RECIPROCAL OF ANTI-LTA DILUTION
0 % PH A
~50 % PH A
IOO % PH A
LTAcx (|jg/ml) USED IN EAltac* PREPARATION


1 h
5). The attachment of LTA to the cell membrane prevents the subse
quent attachment of the C4 or C2 active fragments (i.e. C4b or C2n
respectively). Thus, the activities of all complement components
would remain intact and no observable dysfunction should be observed.
However, if C4b or C2a were in the least impeded in their attachment
to the cell membrane, these active fragments would rapidly decay and
lose their ability to do so.
If the first model accurately portrayed the mechanism of inhibi
tion, one would predict a decrease in Cl uptake by EA This predic
tion was not corroborated by experimental results. In addition, this
model would not explain the high degree of inhibition of cells in the
EAC1 state where Cl is already attached.
If the second model were true, one would predict a decreased
consumption of fluid phase C4 or C2 after pre incubation with EA01 ...
L I A
Again, such was not the case. Neither residual C-'t activity when in
cubated with EAC1 nor residual C2 activity when incubated with
1-1 X
EACl4[TA was appreciably different from their buffer treated controls.
Model three would predict a decrease in fluid phase activity of
C4 or C2 when preincubated with LTA. As demonstrated in Table 8, no
such decrease in activity was observed.
Model, four maintains that the attachment of LTA would somehow
alter the membrane such that loosely attached components would he
released more readily. The first problem with this model is that the
attachment of the enrlv complement components to the membrane is quite
tenacious. In fact, some evidence suggests that membrane attachment
of cytophillis C4b is accompanied bv the formation of covalent bonds
(158,1V)). Once attached, it seems unlikely that (14b would be readily


DISCUSSION
Evidence has been provided for the inhibition of complement
mediated lysis of target cells by an extracellular material obtained
from Streptococcus mutans BHT. This material has been identified a;
lipoteiehoic acid (LTA) and is a plasma membrane constituent of most
gram positive bacteria (107,108).' Various gram positive bacteria
isolated from the oral cavity differ in the amount of LTA they excrete
into the external environment. S. mutans BHT is an example of a carin
genie streptococcus that not only produces copious amounts of LTA (1,20),
but its ubiquitous nature provides for a constant inundation of LTA
and other metabolites into the gingiva] crevices of the oral cavity.
The presence of a complement reactive component in the microenviron
ment of the gingival crevices could result in any number of biological
effects. Direct activation of the complement system (cither classical
or alternative) may result in the destruction of nearby "innocent by
stander" cells. This is particularly true if the activator is evto-
philie and thus capable of "sensitizing" nearby host cells. Activation
of complement in the gingival crevices can also result in osteoclast-
mediated bone resorbtion (14). This phenomenon is further complicated
by the fact LTA and LIS (and ostensibly hvdrid micolls of the two) are
1 Some bacteria are known to lack LTA in the i r membranes but in these
cases "LTA-like" molecules are inserted instead. Examples are the
lipomannan of Hicrococcus lysodeikti cus (150) and the F-antigen of
D iploeoeens pneumoniae (115).
108


PERCENT INHIBITION OF LYSIS
80-1
70-
3
.25
LTAcx (/xg/ml) USED IN EAltacx PREPARATION


1 2 6
84. Sandberg, A. L. A. G. Osier, H. Shin, and B. Oliveira. L970. The.
biologic activities of guinea pig antibodies, il. Modes of com
plement interaction with yl and y2 immunoglobulins. J. Immunol.
104:329.
85. Sandberg, A. L. 0. Gdtze, H. J. Miiller-F.berhard, and A. G. Osier.
1971. Complement utilization by guinea pig y 1 and y2 immunoglo
bulins through the C3 activator system. J. Immunol. 107:920.
86. Platts-Milis, T. A. E. and K. Tshizaka. 1974. Activation of the
alternate pathway of human complement by rabbit cells. J. Immunol.
113:348.
87. Poskitt, T. R. II. P. Fortwengler, ,Ir. and B. J. Funskis. 1973.
Activation of the alternate complement pathway by autologous red
stroma. J. Exp. Med. 138:715.
88. Joseph, B. S., N. R. Cooper, and M. B. A. Oldstone. 1975. Immunologic
injury of cultured cells infected with measles virus. I. Role of
TgG antibody and the alternative complement pathway, d. Exp. Med.
141:761.
89. Perrin, L. H., B. S. Joseph, N. R. Cooper, and M. B. A. Oldstone.
1976. Mechanism of injury of virus-infected cells by antiviral
antibody and complement: Participation of IgO, F(as'), and the
alternative complement pathway, d. Exp. Med. 14 3: 1 0271
90. Leon, M. A. 196], Inhibition in the properdin-dextran system. In
Immunochem i ca 1 Approaches to Problems in Mie rob i o .logy. M.
He ideiberger, O. d. Pleseia, and R. A. Day, ed. Rutgers-Un iver-
sity Press, New Brunswick, N. J. p 304.
91. Itiai, S., S. Ehisu, K. Kato, and S. Kotani. 1976. Activation of
complement through the alternate pathway hv microbial glucans.
J. Immunol. 116:1737.
92. Konig, W., D. B i t Ler-Siiennann, M. Piorich, M. I.imhert, i!. I'.
Sclior 1 emmer and II. lladding. 1974. DND-autigens activate the
alternate patiiwav of the complement system, d. Immunol. l_13:501.
93. Alper, C. A., and D. Bnlnvitch. 1976. Cobra venom factor: Evidence
for its being altered cobra C3. Science. 191:1275.
94. Hunsicker, 1.. f.. S. Ruddy, and K. F. Austen. 197!. Alternate C'
pathway: Factors Involved in CVF act tvaliou of (''!*". d. Immunol.
1 Ml: 128.
95. Cooper, N. R. 1973. Formation and Function of a complex of the C3
proactivator with a protein from cobra venom, d. Exp. Med. 137:451.
96. Sehreiher, R. D. 0. Gotxe, and H. d. Mill ier-Eberltard. 1976. Alter
native pathway of comp 1ement: Demonstrat ion and characterization
of initiating factor and its properd in-independent function, d.
Exp. Med. 144:1062.


Figure 10.
Effect: of LTAcx on hemolytic antibody titration.
Antibodies to sheep red blood coll stromata (Ah) were
incubated with LTAcx (SOD ug/ml) and residual hemolys
activity was titrated by procedures described in
Materials and Methods. Lysis of cells was developed
with whole guinea pig complement. Symbols: (o) Ab
incubated with LTAcx; () Ah incubated with buffer.


in light of the fact that gram positive bacteria represent the major
cellular constituent of dental plaque at fhe early stages of plaque
formation (18). Most of the gram positive organisms found in dental
plaque have been isolated, cultured, and identified. The production of
copious amounts of extracellular LTA by several of these organisms has
been well established (19,20). In fact, growing under conditions esti
mated to reflect the growth rate in the oral cavity, Wicken and Knox have
shown that the cariogenic bacterium Streptococcus mutans BUT produces
some eleven fold greater amount of extracellular LTA in the culture fluid
than that contained within the cells themselves (1,2). Therefore, if an
effect on complement by LTA can be demonstrated in vitro, an in vivo
model can be readily envisioned. Preliminary experimentation with a
crude LTA containing extract from S. mutans BUT did indeed indicate that
complement activity was consumed. However, consumption or alteration
of complement activity can he due to a number of specific or non-specific
factors. Because of the complexity of this system, a thorough under
standing of the possible interactions is necessary before anv model
attempting to define a site and mechanism of inhibition can he elucidated.
The complement system of vertebrates is comprised of at least-
eighteen discrete plasma proteins capable of interacting in a specific
and sequential fashion. There are two pathways by which this biochem
ical cascade may he initiated and they arc referred to as t lie classical
and the alternative pathways of complement activation. However, regard
less of how the activation scheme is initiated, the biological consequen
ces of activation are the same for both pathways:
1
Silvestri et al. 1.97b. Abst. Ann. Meeting, ASM, p77.


Che cells and being expressed In Che C2 deration. What anpenred to
be consumption of C2 activity was actual Iv the inability or the comple
ment system to lyse resistant cells. Because of the greater extent of
dilution, the same phenomenon did not influence Cl, C4 and C.3 titrations.
Because the EAC142 and EAC.l423.i67 intermediates were not effected
by LTA, some component no longer necessary for their stability was a
likely site of inhibition. C4 was probably not the site of attack
since this component is a necessary part of the C'3 convertase (152),
and EAC142 were not inhibited. Only C.1 is expendable after the EAC142
complex is formed and thus Cl seemed to be the most likely candidate
for the site of inhibition.
The. first consideration was the possibility that LTA was causing
inhibition of complement mediated lysis by blocking fixation of Cl to
antibodies specific for sheep erythrocytes or by blocking the site of
antibody attachment. The fact that the inhibitor functioned equai.lv
well when it was presented either before or after the addition of speci
fic antibodies to the cells indicated that blockage of antigenic sites
was not the mechanism of inhibition. This experiment did not rule out
the possibility that the inhibitory subsLanec could react with the Cl
fixation sites on immunoglobulin molecules. However, Figure 10 shows
that prelncuhation of LTAcx with anti-sheep L, hemolysins did not
decrease the hemolytic antibody titer of the serum. if LTA were capable
of binding or inactivating immunoglobulin molecules (either specifically
or non-spec if icaily) then the titer of the antiserum should have, been
reduced as a result of treatment with the bacterial extract.
There was some speculation that LTA might inhibit complement medi
ated lysis by inducing some alteration in the structure of the target


12.
121
Attstrom, R. A., R. Laurel 1, !!. Larsson, and A. Sjoholm. 1975.
Complement factors in gingival crevice material from healthy and
inflamed gingiva in humans. J. Periodontal Res. 10:19.
13. Allison, A. C., and II. U. Schorlemmor. 1970. Activation of comple
ment by the alternative pathway as a factor in the pathogenesis
of periodontal disease. Lancet. 2:1001.
14. Raisz, L. G., A. L. Sandburg, J. M. Goodson, II. A. Simmons, and S.
Mergenhagen. 1974. Complement-dependent stimulation of prosta
glandin synthesis and bone resorption. Science. 185:789.
15. Dietrich, J. W., and L. G. Raisz. 1975. Prostaglandin in calcium
and bone metabolism. Clinical Orthopaedics and Related Research.
111:228.
16. Goodson, J. M. F. Dewhirst, and A. Brunotti. Prostaglandin levels
in human gingival tissue. J. Dent. Res. 5_2 (special issue) :182.
17. Hausmann, E., 0. Luderitz, K. Knox, and N. Weinfeld. 1975. Structural
requirements for hone resorption hv endotoxin and lipoteiehoic acid.
J. Dent. Res. B54 (special issue):94.
18. Carlsson, J. 1967. Presence of various types of non-hemolvtic strep
tococci in dental plaque and in other sites of the oral cavity in
man. Odontol. Revy. 18:55.
19. Joseph, R. and G. D. Shocktnan. 1975. Synthesis and excretion of
glycerol teichnie acid during growth of two streptococcal species.
Infect. Immun. 12:333.
20. Markham, J. L., K. W. Knox, A. J. Uicken, and M. J. lleve tt. 197".
Formation of extracellular lipoteiehoic acid by oral streptococci
and 1actobacilli. Infect, immun. 12:378.
21. Mll er-Eherhard, H. J. 1975. Textbook of_ I miminop.tt ho l.ogv ed P. A.
Micsehea, H. J. Mil 1 ler-Eberhard. Grue and Stratton, N. Y., N. Y.
2nd ed.
22. Austen, K. F. 1 974. (.hem is try and biologic activity of the comple
ment system. Trnnsp l.ant. Proc. 6:1.
23. Naff, (?. B. J. Pen sky, and I. 11. Lepov. 1964. The mae romo 1 ecu lar
nature of the 1st component of human J. Exp. ilc?d. !1_9: 593.
24. Lepow, [. H. G. B. Na f T, E. W. Todd, .1. Ienskv, and C. V. llinz, Jr.
Chromatographic resolution of the first component or human comple
ment into 3 activities. J. Exp. Med. H7:983.
25. Mii 1 1 er-KUerha rd, II. J. 1 *72 The molecular basis of
activities of G. Harvov Lec.t. 66:75.
the biological


1.3
erythrocyte-sensitizing antigens in cell free saline washings or spent
culture fluid from several gran positive organisms (101.102). These so
called "Rantz antigens" were recently shown to possess properties asso
ciated with LTA (111). Because only acylated LTA will hind to erythro
cytes, PHA provides a means of quantitating the amount of LTA in a
preparation without having to contend with deacylated TA contamination.
The biological role of TA and LTA to the microorganism has been a
subject of considerable disputation by several investigators in recent
years. Thus far, at least three roles have been tentatively assigned:
1). TA and LTA seem to function as "carrier" molecules for membrane
and cell wall components, i.e. amphipathic LTA may be used by the cell
to transport needed hydrophobic molecules through hydrophilic zones
which would otherwise pose an almost impenetrable harrier. Fielder and
Glaser have established that intracellular LTA servos as a lipid carrier
for the biosynthesis of cell wall ribitol teichoic. acid in Staphylococcus
aureus (112,113). Chaterjee and Wong (114) have demons!'rated that LTA
may serve as the acceptor in which nascent peptidoglyran polymers are
synthesized. 2). LTA seems to be involved in ceLl wal.1 division and
regulation. Holtje and Tomasz have reported that LTA exhibits an inhi
bitory effect on the function of nutolytic enzymes during the division
cycle of pneumococcus (115). It is interesting to note that similar
functions have been described by Cleveland, et al. working with a strain
of Streptococcus faecal is (11 A.117,118). In these systems, LTA is
deacylated and released into the environment as TA. Once the concentra
tion of LTA is sufficiently lowered, or the concentration of nutolytic
enzymes is sufficiently elevated, cell wall autolysis begins at the divi
sion zone.
This nutolytic activity thou allows for insertion of additional


Figure 20.
Disc gel electrophoresis of purified human Clq.
Cathode was at the top.


Figure 21. DEAE elution profile of human Cls. Peak I contains
Cls activating proteins (functionally pure Clr); Peak
II contains nonfunctional Cls; Peak III contains
functional, non-activated Cls. Symbols: () Absor
bance at (maximal absorbance for most proteins;
(o) Relative salt concentration (RSC) as measured
by electroconductivity. Arrows indicate the addition
of high ionic strength Sodium Chloride buffer.


Removal of salt and prop an oJ_ from LTA contain in g e :< tract s .
Removal of salts and/or propanol from various preparations was rapidly
and quantitatively accomplished by gel filtration utilizing LH20
(Pharmacia Fine Chemicals, Piscatawav, NJ) as the solid phase support
matrix. The most commonly employed column was 50.0 cm X 2.5 cm but
a larger 65.0 cm X 3.0 cm column was sometimes utilized. The column
was packed and equilibrated with deionized water. Sample preparations
usually involved rotary flash-evaporation (Buchler Instruments. Fort
Lee, NJ) in order to reduce the volume of sample to 15-20 ml. Elution
of product was carried out at a pressure head of approximately 50 cm
water and approximately 4.0 ml effluent were collected per tube.
Phosphatidyl choline vesicle (PCV) purification of_LTA
(a) Preparation of PCV. Although reported as the method of choice by
other investigators,^ in our hands Octyl Sepharose purification
of LTA from Streptococcus mutans BUT resulted in a product still
highly contaminated with polysaccharides. in an attempt to achieve
homogeneous purification of LTA, a modification of the above mentioned
hydrophobic adsorbtion principle was employed. In this procedure,
artificial membrane vesicles were prepared with DL^phosphatidyl,
choline dipalmitoyl (PC) (Sigma Chemical Co.) as the sole constituent
via a modified method of Hill (1.40). In brief, 40.0 me, of PC was
placed in each of several 10 mL high speed glass Corex centrifuge
tubes (Corning Glass Works, Corning, NY) and dissolved with one ml
chloroform. The solvent was gently evaporated in a 50C water bath
while rotating the tubes so as to coat the bottom 5 or 6 cm of the
tube with PC. Once dry, the lubes were placed in a 1yophi1 i cat ton
flask and any residual solvent was removed in vacuo. One milliliter
'wichen, A.J.-, and Knox, K.Personal communication.


Amino acid analysis. Amino acids and amino sugars were measured
on a JEOL model JLC-6AH automated amino acid analyser (JEQL, Inc..
Cranford, NJ). Sample hydrolysates were prepared as described bv
Grabar and Burt in (143).
Clg, Cls, and Cls purification. Highly purified human Clq
was prepared from whole human sera by the method of Yonemasu and Stroud
(144). Highly purified human Cls and Cls were prepared by a minor modi
fication of the method described by Sakai and Stroud (35). For the final
resolution step, Bio-Rad Cellex-D DEAE with binding capacity of 1.07 meq/g
(Cellex-D, Bio-Rad Laboratories, Rockville Center, NY) was substituted for
fibrous DEAE cellulose Whatman DE-23. The DEAE was washed and prepared
according to the manufacturer's specifications. Final elution of the pro
duct was accomplished with the use of the same eluting buffer as described,
but instead of a stepwise elution of the column, an ionic gradient from
0.2 0.4 RSC (relative sodium chloride concentration) was utilized.
Disc acrylamide gel electrophoresis o f_C Lc^, Cls and Cls. This was
carried out essentially as described by Yonemasu and Stroud (1.44) but with
out the use of sodium dodecyl sulfate (SDS).
Cls Inhibition assays. The ability of Cls to consume C2 activity was
assayed by a modification of the method described by Sakai and Stroud (3.5).
Briefly, 0.1 ml of Cls (approximately 8.0 X 10^ site forming units, SFU/ml)
plus 0.1 ml LTApox (100 t|g/m! in DVB) were incubated at 30 for 15 minutes.
One tenth milliliter of C2 was then added at a concent ration of approxi
mately 9.0 X IQ7 effective mo I ecu 1es/m1 and incubated at 37^0 for 30 min
utes. At the end of the incubation, 9.7 ml cold DCVR were added to the
mixture resulting in a 1:1.00 dilution of the 0,2. The C2 was then serially
diluted and 0.1 ml aliquots from each dilution were added to 0.I mi ot


Figure 13. Purification of LTA by Octyl Sephnroso hydrophobic gel
chromatography. Symbols: ( concentration of carbohydrate (n-moles/ml) as determined
by the Phenol-Sulfurie Acid assay. Concentrations were
determined using glucose as a standard carbohydrate.
(o) Concentration of Pi. (n-moles/ml) as determined by
the Lowry Pi assay; (+) zones of antigenicity as
determined by PHA using antisera directed against LTA
backbone; (A) elution with buffer A (1.0M NaCl, 0.01
M Tris-carbonate pH 6.8); (R) elution with buffer B
(0.01 M Tris-carbonate, pH 6.8); (C) elution with a
10-70 gradient of a propane 1-buffer B mixture; (D)
elution volume at which significant reductions of vol
ume/tube were observed, indicating elution of propanol.


TABLE 2
I. Results from Partial Purification of LTA
Sample
Pi (p-moles)^
b
Percent Lytic
Inhibition by
LTA Treated EA
PHA T
Dialyzed, Non-Inoculated
Todd-Hewitt Broth
1.1x105
0
0
Supernate from Inoculated
but Lion-Fractionated Broth
l.OxlO5
ND
ND
PTGC Retntate Fraction
of Supernate (LTAcx)
6.9xl02
AS
3200
Peak II from A5M After
Desalting (LTAppx)
6.OxlO1
55
3200
Data are expressed in the units indicated and represent values extrapolated back to the
undilute sample times total volume.
EA were prepared with the LTA source at a concentration of 250pg/ml. Hemolysis was
developed as described in TabLe 1.
PILA titers were determined by methods described in Table 1.


Immunology, Univ. of Louisville) provided samples of LTA purified from
Bacillus subtil is strain gta B290. Purified (,TA from Lactobacillus
casei ATCC 7469 was obtained From the Institute of Dental Research,
Sydney, Australia, and Dr. A. S. Rleiweis (Dept, of Microbiology and
Cell Science, Univ. of Florida) provided a sample of partially purified
LTA from Streptococcus tiutans strain AHT. Each preparation was mixed
with £A; the cells were thoroughly washed and analyzed using the pre
viously described techniques of PHA and susceptibility to whole comple
ment lysis. As depicted in Table 11, all preparations contained material
that reacted with anti-LTA by PHA and all such cellsespecially those
prepared with the purified L. casei--were more resistant to the hemo
lytic action of complement than were untreated controls.


by LTA. The site of inhibition was determined to occur between the
formation of the SAC1 and SAcl42 complex. Be cause Cl. is no longer
necessary after formation of the C'l convertase (SAC42), lack of inhi
bition after this step implies a direct effect on Cl activity. Although
experimental data derived from utilizing Cl, Clq, Cls, and Cls were
suggestive, data did not. unequivocally establish this as the precise
mechanism of inhibition. No evidence for fluid phase consumption of
hemolysin Ab, Cl, C4, or C2 by LTA could be demonstrated. Evidence for
the inhibitory activity of LTA from several unrelated genera is pre
sented and the possible role of LTA in periodontal disease is discussed.
x


of bound LTA as well), over 92% of the label could bo accounted for in
the chloroform/methanol filtrate and the first filter washing. Only
0.004% of the label was present in the final product therefore elimin
ating phosphatidyl choline as a source of contamination. Figure 15 and
Table 7 indicate that less than 5% polysaccharide contamination can be.
detected in the final product by gas liquid chromatography. Considering
the unusual profiles obtained from the gas liquid chromatography of
both cardiolipin and deacylated eardiolipin (Figure 15), it is likely
that the percentage of contaminating polysaccharide in the final LTApcx
preparation is even Less than 5%. As can he seen in Table 6, approxi
mately 85% of the LTA in the original partially purified extract can be
accounted for in the final product and washings. However, it should be
cautioned that the method used for these determinations (PHAg) is semi-
quantitative at best and is only considered accurate to within one two
fold dilution.
Although the percent protein of all partially purified samples
was determined by amino acid analysis, unfortunateLy the tremendous
quantity of purified material required in analysis for < 5% sensitivity
in analysis, exceeded the total amount of purified material available.
In fact after allocating fixed quantities of purified product for the
various other quantitative and complement assays, the required 5-6 mg
of purified LTA needed for amino acid analysis far exceeded the poten
tial amount available from the LTAppx. For this reason, the Bio-Rad
Protein Assay was used to estimate, the total amount protein in each
sample. As can he seen in Table 7, there was a relatively close cor
relation between values determined by amino acid analysis and those
determined using the Bio-Rad Assay. It is therefore reasonable to


LO 4
In addition to the above, mentioned experiments, several other
assays to elucidate the mechanism of inhibition were attempted. Unfor
tunately none of these experiments led to results that were consistent
with any models attempting to explain how some complement cellular
intermediates became refractory to complement lysis when pretreated
with LTA. These experiments and their summarized data are presented
below:
Cl uptake by EA EA were prepared with LTApcx at a con-
LTA LTA
centration of 100 pg/ml using procedures described in the Materials
and Methods. Buffer treated EA were also prepared at the same time.
10
Human Cl (approximately 1.0 X 10 SFU/ml) was reacted with aliquots
from each cell preparation and incubated for 1.5 minutes at 30C. The
cells were pelleted by centrifugation and the supernates analyzed for
9
residual Cl activity. Approximately 6.5 X 10 SFU Cl/ml remained in the
9
supernate of the buffer treated controls whereas approximately 6.8 X 10
SFU Cl/ml were titrated in the supernate of the EA treated cells.
LTA
Because values fluctuated by 5-8 / from one experiment to the next, this
slight degree of enhancement was not considered significant.
HU HU
Residual C4 titration after preincubat.ion of C4 with EAC1
~~ 9 LTA
Human C4 (approximately 4.0 X 10 SFU/ml) was added in equal volumes to
EACl which had been preincubated with either LTApcx (100 iig/ml) or with
buffer. The mixture was incubated at 30 for 15 minutes and residual C-'t
activity was titrated as described in the Materials and Methods. EA
were incubated with the C4 reagent as a negative control. Results in
dicated that there was approximately a 30% decrease in residual C.4


14
Distribution of C-
Phosphatidyl Choline
During PCV Purification of LTA
Sample Source
Reciprocal of
Dilution Factor
DPM Aliquot'5
(x io'z)
Total DPM
in Sample
(X 10 )
Percent of
Total DPM
Calculated
Corresponding
Weight of PCV (mg)
14
C-PCV Suspended in Starting
Suf f er
60
830.00
4980.00
100.00
40.00
Supernate from Preliminary
Vesicle Washing (#1)
320
1.91
61.12
1.23
0.49
Supernate from Preliminary
Vesicle Washing (r2)
320
0.29
9.28
0.19
0.07
Decanting After Reaction of
PCV with LTAppx
320
2.95
94.40
1.90
0.76
1st Washing Supernate
320
2.82
90.24
1.81
0.72
2nd Washing Supernate
320
1.61
51.52
1.03
0.41
3rd Washing Supernate
320
3.44
110.08
2.21
0.88
Chloroform/Methanol Filtrate
72
590.00
4248.00
85.30
34 1 2
1st Chloroform/Methanol Washing
100
35.00
350.00
7.03
2.81
1st Chloroform Only Washing
' 60
1.09
6.54
0.13
0.05
2nd Chloroform Only Washing
60
0.58
3.45
0.07
0.03
Final Product (LTApcx)
1C
1.96
0.20
0.00
0.00
a Dilution factor was calculated
for analysis.
by dividing
t he
totaL volume of
the sample by the
volume of the
aliquot removed
^ DPM values were calculated from
CPMs and a
standard quench curve as described in
Materials and
Methods.
O contamination of che final product was determined by i icing the entire [,TA-conta in ing floroporo filter in
a scintillation vial and analyzing as described in Materi u _> and Methods.


1 2f)
124. Daugherty, !1. D. R. R. Martin, and A. White. 1969. Reaction of
sera and nasal secretions with staphylococcal antigens. .1.
Lab. Clin. Med. 73: LOU.
125. Markham, J. L., K. W. Knox, R. G. Schamschula, and A. .1. Wicken.
1973. Antibodies to teichoic acids in humans. Arch. Oral. Biol.
IS:313.
126. Jackson, R. W., and U. Muskowitz. 1966. Mature of a red cell
sensitizing substance from streptococci. J. Bncteriol. 91:2205.
127. Carlsson, J. 1967. Presence of various types of non-hemolvtic
streptococci in dental plaque and in other sites of the oral
cavity in man. Odontol. Revy. 18:55.
128. Zinner, D. D. J. M. Jablon, A. R. Aran, and If. S. Sas.lnw. 1965.
Experimental caries induced in animals by streptococci of human
origin. Proc. Soc. Exp. Biol. Med. 118:766.
129. Guggenheim, B. 1968. Streptococci of dental plaques. Caries Res.
2:147.
130.Fitzgerald, R. J. and P. H. Keyes, i960. Demonstration of the
etiological role of streptococci in experimental caries in the
hamster. J. A.mer. Dent. Asso. 61:9.
131.Hoffmann, E. M. 1969. Inhibition of complement by a substance
isolated from human erythrocytes. 1. Extraction from human
erythrocytes stromata. Immunochemis try. 6:30]..
132.Nelson, R. A., J. .Jensen, I. Gigli, and N. Tamura. 1966. Methods
for the separation, purification, and measurement of nine com
ponents of hemolytic complement in guinea pig serum. I inmuno-*
chemistry. 3:11.
1.33.
Ruddv, S., and K. F. Austen. 1967. A stoichiometric assay for the
Cl
fourth component of complement in whole human serum using EACl
and functionally pure human second component. J. Immunol. 99:1162.
134.Ruddy, S., and K. F. Austen. 1969. C3 inactivator of man. I. Hemo
lytic measurement bv the inactivation of cell-hound C3. J. Immunol.
102:533.
135.Rabat:, E. A., and M. M. Mayer. 1961. Comp lenient and complement fix
ation. In Kxper ¡mental f.mmnnochem ist rv. Charles C. Thomas,
Springfield, 111. p 149.
136.Bersos, T., and II. J. Rapp. 1967. Immune homolvsis: A simplified
method for preparation of F.AC4 with guinea pig or with human
complement. J. Immunol. 99:263.


RESULTS
Inhibition of whole human complement by erudo extracelLuIar
lipoteichoic acid (LTAcx) To determine whether LTAcx had any effect on
whole human complement, equal, volumes of LTAcx and whole human complement
were preincubated at 37/30 minutes. After pre tncuabt ion, tin.' complement
source was serially diluted in DGVR and the residual hemolytic activity
was titrated. As shown in Figure 1, approximately 50% of the whole com
plement hemolytic activity (measured in CH,.^ units) was consumed. Further
more, as seen in Figure 2, this consumption was dependent on the concen
tration of the LTAcx used.
Titration of complemcnt components in whole human sera after treat
ment with LTAcx. One mechanism for fluid phase consumption of whole com
plement could have been the interaction of natural antibodies in the human
sera with LTA or some other antigenic substance in the crude extract. The
result would be the fixation of Cl and subsequent .activation of C4 and C2
via classical pathway. Another explanation for decreased hemolytic activ
ity could have been the activation of the alternative pathway in a manner
analogous to LPS. To differentiate between these two modes of activation,
individual component titrations were performed on human sera incubated
with LTAcx. Tn addition, C3 titrations were carried out in the presence
of ethy Lenoglvcol-bis (b Amino Ethyl Ether) N,N totrnaectic acid (L(.IA)
and Mg ions. This chelating agent preferentially hinds Ca ions (1 4 5,1 4M .
and by reinforcing the F.OTA buffer with Mg ions one can effectively deplete
the available Ca ions yet maintain relatively high levels of Mg Ions.
Thus, the Ca ion dependent classical pathway is blocked, hut the
alternative pathway can function relatively unimpaired (145,147).


Inhibition of complement Lysis of LTAcx treated EA. During an
experiment in which EA treated with LTAcx were tested for reactive
lysis, it was discovered that the LTAcx treated cells exhibited Less
hemolysis than even the buffer treated controls. This serendipitous
observation led to the discovery that LTA treated EA were refractory
to complement mediated lysis. To confirm these results, various concen
trations of LTAcx were used to treat EA. After the treated cells were
extensively washed they were tested for their susceptibility to lysis
by complement. The same cells were also tested for the presence of
cell-bound LTA using the passive hemagglutination technique (PHA) with
anti-LTA. The results shown in Figures 5 and 6 indicated that both
the extent of inhibition of hemolysis and PHA titers were LTAc.x dose
dependent. There was a decline in both activities only after the LTAcx
had been diluted to a concentration of 62.5 pg/ml The decrease in
titer below this concentration indicated that the test cells wore no
longer saturated with LTA. There was a concomitant drop in inhibition
of lysis at 62.5 pg/ml. EA which were treated with uninoculated cul
ture medium (dialyzed Todd-IIewitt broth) were unaffected when comple-
emnt was added.
Effect of LTAcx on lysis of sheen E and sheep E cellular
intermediates The treatment of EA with LTAcx caused the cells to
become relatively resistant to complement mediated lysis. This could
have been due to an effect on the ant¡body molecules, an effect on one
or more of the complement components, or an alteration of the cell
membrane .
To further investigate the nature of the complement inhibition
associated with LTAcx, sheep E. sheep EA, and various sheep E complement


MATERIALS AND METHODS
Crude extracellular LTA (LTAcx) The initial studies were
carried out utilizing LTAcx prepared in Australia by the method
of Wicken and Knox (110). Streptococcus mutans RUT was grown
to late stationary phase in a New Brunswick Microfirm fermentar at
37C, under anerobic conditions (95 N and 5/- C.O^) in a complex
medium.
Later experiments utilized LTAcx prepared at Gainesville,
Florida. The original method was modified as follows. A Pell icon
2
Cassette system (Millipore Corp., Bedford, MA) equipped with 1.0 ft
of PTGC filter material was used to dialyze Todd-Howitt broth (Pifo.o
Laboratories, Detroit, Ml). A 100 mi culture of early log phase
_S. mutans BHT was inoculated into 10 liters of dialyzed medium
and incubated at 37 for 24 hours. The cells were harvested using
a Delaval Gyrotester (Poughkeepsie, NY). The supernote was passed
2
through the Pellicon Cassette system (loaded with 1.0 ft of 0.45 u
microporous membrane) to remove remaining cells and debris. The cell-
free spent fluid was then fractionated and eoncontrated by passage
through 5.0 ft PTGC membrane (nominal molecular weight' exclusion
limit of 10,000). The filter retentte was washed in s_itu with several
liters of water, collected and 1vophi 11 zed. The freeze-dried retentte,
designated as LTAcx. was stored in a dessicator at -20C.
So lut i ons for complement assays. Tsotonic Veronal buffered
sodium chloride (VRS), dextrose gelatin Veronal buffer with added




84
essential until that point is reached (143). In addition, many poly-
anionic substances are known to directly affect Cl by interfering with
Clq binding or Cl esterase (Cls) activity (19,40). Because LTA is
polyanionic. due to the polyglycerol phosphate backbone and because
cellular intermediates beyond the EAC142 step were no longer inhibited,
it seemed reasonable to hypothesize that LTA was behaving like a poiv-
anion and directly affecting Cl.
To test this hypothesis, LTAcx and LTApcx were preincubated with
functionally purified human Cl. at 30/13 minutes. Residual Cl activity
was titrated as described by Rapp and Borsos (139) and activity was com
pared against buffer treated controls. The results shown in Figure 1.8
indicate that although LTAcx consumed Cl activity, purified LTApcx did
not.
Purification of human Clq, Cls and Cls. In an attempt to further
eludicate a possible site and mechanism of C inhibition, human Cl sub
components were purified by the methods and modifications previously
described (Materials and Methods). Although homogeneity beyond func
tional purity was not essential, the methods employed yielded highly
purified products. Figure 19 demonstrates Clq homogeneity bv immuno
diffusion against several monospecific antisera. Precipitation bands
of identity were observed in adjacent wells containing the whole human
serum starting material, the purified Clq final product, and a highly
enriched Clq prior to final precipitation (Figure L9, plate 5, well
numbers A, G, and E). As can be observed in Figure 20, disc gel electro
phoresis of the final product revealed a single dark staining band which
barely migrated into the separation gel. These results are eonsistant
with the observations of other investigators (L44).


apparent: that the LTAosx stL.ll. contains stgni f leant amounts of carbo
hydrate contamination in contrast to the lower yield, hut highly puri
fied LTApcx.
A summary of specific activities relative to P11A activity is pre
sented in Table 3.
Inhibition of comp Iemeat mediated lysis of JL TApcx trented EA.
Once a highly purified preparation of LTA was obtained, it was necessary
to confirm the results that had been previously established with LTAcx.
As can be seen in Figure 16, not only were LTApcx treated EA refractory
to complement mediated lysis, but in addition the general profile was
remarkably similar to LTAcx treated EA. As indicated, LTApcx was used
in concentrations five-fold to ten-fold less than those, used with LTAcx
to achieve comparable degrees of inhibition. PHA titers typically Indi
cated LTA saturation of the cells.
Effect of LTApcx on the lysis of _var i nus j:_el^lulnr_ complemen t
component intermediates. Sheep E, EA and various complement Inter
mediates were treated with LTApcx (1.00 pe/ml in DVB) and washed exten
sively in DGVB. Percent lysis and inhibition of CH units were deter
mined as described in Materials and Methods. The results of these
experiments are summarized in Figure 17. As with LTAcx treated cells,
E, EA, and EACl were all refractory to lysis by complement. Likewise,
EAC142 and EACJ-7 intermediates wore totallv unaffected by the presence
of LTApcx on their cell surfaces. The onlv observable difference in the
activity of LTApcx on cellular intermediates versus LTAcx was that KALI'*
cells were inhibited to a lesser degree with LTApcx than LTAcx.
Effect of LTAcx and LTApcx on fluid pitase C 1 As previously
discussed, CL is not required for lysis once SAC¡42
are formed but it is




128
111. Rant 7,, L. A., E. R. Randall, and A. Z. Zurkertnnn. 1956. Hemolysis
and hemagglutination by normal serums of erythrocytes treated
with a non-species specific bacterial substance. Infect. Dis.
98:211.
112. Fiedler, F., and L. Glaser. 1974. The attachment of poly-
ribitol phosphate to lipoteichoic acid carrier. Carbohvdr.
Res. 37_:37.
113. Fiedler, F., J. Mauck, and L. Glaser. 1974. Problems in cell wall
assembly. Ann. N. Y. Acad. Sci. 2_3 5:198.
114. Chatterjee, A. N., and W. Wong. Isolation and characterization of
a mutant of Staphylococ.cus aureus deficient in autolyti.c activity.
J. BacterioL 125:961.
115. Holtje, J. W., and A. Tomasz. 1975. Lipoteichoic acid: A specific
inhibitor of autolysin activity in pneumococcus. Proe. Natl. Acad.
Sci. U. S. A. 72:1960.
116. Cleveland, R. F. .1. V. Holtje, A. J. Wicken, A. Thomasz, L. Dar.eo-
Moore, and G. I). Shockman. 1975. Inhibition of bacterial wall
lysins by lipoteichoic acids and related compounds. Biochem.
Biophys. Res. Comm. 67:1128.
117. Cleveland, R. F., A. J. Wicken, L. Daneo-Moore, and G. P. Shockman.
1976. Inhibition of wall nutoy Is is In St re p t ococcus facea Us by
lipoteichoic acids and lipids. .1. Bacteriol. 126:192.
118. Cleveland, R. F., L. Daneo-Moore. A. .1. Wicken, and G. D. Shockman.
1976. Effect of lipoteichoic acid and lipids on Ivsis of intact
cells of Streptococcus faecal is. .1. Bacteriol. 12_7:13S2.
119. Simmons, D. A. R. 1971. Immunochemistry of Shlgel la f le.xneri 0-
antigens: A study of structural and genetic aspects of the bio
synthesis of cell-surface antigens. Bacteriol. Rev. 3_5 : 11 7 .
120. Wicken, A. .1., and K. W. Knox. 1973. Characterization of group N
streptococcus lipoteichoic acid. Infect. Imimm. 11:973.
121. Doyle, R. J., A. N. Chatterjee, V. N. Streips, and F. K. Young.
1975. Soluble mneromoI ocular cenplexes involving bacterial
teichoic acids. .1. Bacteriol. 124:34 1 .
122. Driel, I). V., A. .1. Wicken, M. R. Dickson, and K. W. Knox. 19/3.
Cellular location of the lipoteichoic acids of l,ac_tobac_i_l 1 us
casei NCTC 6375. Ultra. Rsh. 43:483.
123. Frederick, G. T., and F. W. Chorponning. 1974. Characterization
of antibodies specific for polvglyceroL phosphate. -I. Immunol.
113:489.


12
54. Kolb, W. P. .1. A. Haxby, C. M. Arrovave, and 1!. .1. Mii I ] cr-Fberlmrd .
1972. Molecular ann Lysis of the monbrant attack mechanism of Cl .
J. Exp. Med. 1_35:549.
55. Jensen, J. A. 1967. Anuphylatoxin in its relation to the complement
system. Science. 1_55:1122 .
56. Dias Da Silva, W., J. W. Eisele, and !. H. Lepow. 1967. Complement
as a mediator of inflammation. J. Exp. Med. 126:1027.
57. Cochrane, C. G., and ii. J.. MU 1 ler-Eberhard. 1968. The derivation
of two distinct anaphylatoxin activities from the third and fifth
components of human complement. J. Exp. Med. 127:371.
58. Mahler, F., M. Intaglietta, T. E. Hugli, and A. R. Johnson. 1975.
Influences of C3a anaphylatoxin compared to other vasoactive
agents on the microcirculation of rabbit omentum. Microvasc. Res.
9: 345.
59. Stolfi, R. L. 1968. Immune lytic transformation: A state of irrever
sible damage generated as a result of the reaction of the eighth
component in the guinea pig complement system. J. Immunol. 100:46.
60. Thompson, R. A., and P. J. Lachmann. 1970. Reactive lysis: The com
plement-mediated lysis of unsensitized cells. .1. Exp. Med. 131:629
61. Kolb, W. P., and H. J. Mill ler-Eberha rd. 1973. The membrane attack
mechanism of complement verification of a stable C5-9 complex
in free solution. J. Exp. Med. 138:438.
62. Koethe, S. M., K. F. Austen, and I. Gigli. 1973. blocking of the
hemolytic expression of the classical C' sequence by products of
C1 activation via the alternate pathway. J. Immunol. 110:390.
63. Delage, J. M., G. Lenner-Netsch, and J. Simard. 1973. The tribu-
tyrinase activity of C7. Immunology. 24:671.
64.Inoue, K., and S. C. Kinsky. L970. Fate of phospholipids in lipo
somal model membranes damaged by antibody and complement.
Biochemistry. 9:4767.
65.
Mayer, M. M.
Nat. Acad.
1972. Mechanism of cytolvsis bv complement. Proc.
Sci. U. S. A. 69:2954.
66.
Humphrey, J.
membranes
!!. and R. R. Dotirmashkin.
caused bv complement. Ativan.
1969. The lesions in cell
10)1111111'' 1 11:75.
67.
lies, G. 11. ,
lesions in
P. Socman, 0. Naylor, and B
immune lysis: Surface rings
. Cinader. 1973. Mombram
, globule aggregates, am
transient openings. .1. Ge! I Biol. 56:528.
08. Gigli, 1., S. Ruddy, and K. F. Austen. 1968. The stoicbi ornotric
measurement of the serum inhibitor of the first component by the
inhibition of immune hemolysis. .1. Immunol. 100:1154.


amount of LTA in the sample, PHA titer, and percent lytic, inhibition of
EA remained relatively unchanged or increased in value.
Pur i f teat ion of LTA by hyd r o phobic, i n t e r ac. tion gel chroma tography .
A 25.0 X 2.25 cm column packed with Octyl Sepharose and equilibrated in
buffer A was prepared as described in Materials and Methods. Approxi
mately 6.0 mg of LTAppx dissolved in 10.0 ml of buffer A were applied
to the column. As can be seen in Figure 13, a small, amount of phosphate
containing material passed unimpeded through the column. A slightly
greater mass of polysaccharide was also excluded without binding. No
additional material eluted from the column with buffer B. Point C on
the graph marks the location where a 10-70 propanol gradient was begun.
Point D represents the point where a significant volume decrease per
test tube was observed. Since fractions were collected on a "drops per
tube" basis, the presence of propanol in the effluent causes a change in
surface tension of the drop resulting in a decreased volume per drop.
The ultimate result is a decrease in the volume per tube. This, test
tuhe volume provided a convenient means of monitoring the progress of
the propanol gradient.
It should be noLed that despite the use of a gradient (the original
procedure called for a single step-wise elution with 50 propanol)
significant amounts of carbohydrate eluted witli the I/LA. As indicated
on the the graph, all areas containing phosphates also contained LTA as
detected using PHA. The fact that a small amount of LTA passed unbound
through the column suggests that either the columns b i nding capacity was
exceeded, or perhaps the LTA was onlv partially aevlatcd and not capable
of tenacious hydrophobic binding.


r-H
acids. Because peak IT represented partially purified extracellular
lipoteichoic acid, the recovered material was designated LTAppx.
A sample of each pool was rehydrated to 50 pg/ml and reacted with
EA, according to standard procedures (Materials and Methods). Each EA
preparation was analyzed using the complement inhibition assay and
tested for bound LTA by PHA. Only the pools containing LTA (as demon
strated by PHA) caused inhibition of complement mediated lysis (Table 1).
Despite the excellent separation of LTA from most of the material
that absorbed light, at a wave length of 260 nm, and presumably from
all deacvlated LTA or TA, two persistent problems arose with this puri
fication procedure:
1). Polysaccharide contamination accounted for a major portion of
the mass recovered in peak LI, and
2). The total mass of LTAppx under peak II was almost immeasurable
smal1.
In an attempt to at least increase the yield of peak ¡I material,
a Millipore Cassette system was employed to both concentrate and frac
tionate the spent culture superante (Materials and Methods). This method
of LTA enrichment proved highly successful as evidenced by the. results
in Figure 12. Even after values are corrected for the greater mass of
crude extract applied on the latter column the mass yield of Ll'Anpx was
some fifteen fold greater than that obtained with previously employed
procedures (Figure 11).
An analysts of results tracing the partial purification of LTA
is summarized in Tables 2 and 3. It should he noted that the total
amount of Pi, mass, protein, and absorbing material decreased
several thousand fold in the purification process, whereas the total


treated cells was added to each well. Controls for spontaneous
or nonspecific agglutination consisted of wells that contained anti
serum and sheep E which had never been exposed to LTAcx. Treated
sheep E plus VBS constituted another control. The microtiter plate
was incubated at 37C on a Cordis Micromixer (Cordis Laboratories,
Miami, FL) for 15 minutes. The plates were removed from the mixer
and the cells were allowed to settle for two hours at 37C, followed
by three hours at room temperature.
Modified passive hemagglutination (PHAg). A modification of
the above technique was used to semi-quantitate the amounts of LTA
present in various preparations. The same apparati were used, hut
instead of antibody, LTA-containing extracts were added to the
bottom wells and serially diluted in situ as described. After
each LTA source was diluted, one drop of sheep erythrocvtes (10 /ml
in VBS) was added to each well and the plate was then incubated
at 37C for 20 minutes and at 0C for 10 minutes. The cells were
kept in suspension by vibrating the plate on a Cordis Micromixer
during both incubation periods. One drop of OVB was then added to
each well and the plate was centrifuged at 200 g for 5 minutes.
The entire plate was then abruptly inverted over absorbent paper
towels and allowed to drain for approximately one minute. One
drop of OVB was again added lo each well and the plate was vibrated
at 0C for 5 minutes to resuspend the pellet. An additional drop
of OVB was added per well and the plate was again centrifuged at
200 g for 5 minutes. This washing procedure was repeated three
times and the cells were then finally resuspended in one drop
of OVB. One drop of anti-ETA (diluted 1:1000 in VBS) was then


A 260
FRACTION NUMBER (5.0 ml)
i (n -moles)


Figure 9. Effect of LTAcx treatment on the lysis of E AC 142.
Various limiting concentrations of C2 were used to
prepare EAC142 cellular intermediates. ihe cells
were then treated with LTAcx (250 vig/ml) and lysis
was developed using procedures described in Material
and Methods. Symbols: (o) KAC 1.42 incubated with
LTAcx; () F.AC142 incubated with buffer.


equal volume mixture of VBS and anti-sheep erythroovte stromata
for the same time at the same temperature. The ant ihodies were,
then titrated using limiting amounts of complement (1.35).
Cl fixation and transfer. The number of Cl molecules hound
to an antigen-antibody complex can be measured by the Cl fixation
and transfer test described by Borsos and Rapp (139). In a mod
ification of this procedure, an attempt was made to quantitate
the number of Cl molecules fixed to EA which had previously been
treated with LTApcx. Buffer controls and EAt_a were prenared
LEA
as previously described, and after washing were resuspended at
g
10 cells/ml in DGVB. Equal volumes of EA^^^ and EA were in
cubated with Cl at 30C for 15 minutes. The cell mixtures were
washed twice with DGVB, and resuspended in GVB at a cell concen
tration of 1 X 10^/ml, 5 X 10^/ml, and 1 X lO'Vml. One volume
of each cell concentration was added to one volume of EAC4 (at
8 ~ 1
1 X 10 cells/ml) to permit transfer of Cl from EA Cl to EAC4.
The cells were incubated at 30C for 15 minutes, and then C2 and
C-EDTA were added In relative excess as described previously.
A variation of the Cl transfer assay was performed by treating
preformed EAC1 with ETA or buffer control .as described. The
resulting EACl^ were resuspended to 1 X 10* cells/ml in GVB and
the amount of Cl capable of transfer was measured as described
above.
Gel filtration. I.TAcx was fractionated on a 2.5 cm X 100.0 cm
column of Bio-Gel A-5M, 200-400 mesh (Biorad Laboratories, Richmond,
'in this; instance, "x" represents ETA or the appropriate buffer
treated control.


INTRODUCTION
As reviewed hv Wicken and Knox (1,2), a number o!" chemical and
biological similarities exist between the. 1 ipopo Lysncchar ides (bPS) of
gram negative bacteria and the lipoteichoic ac.ids (TLA) of gram positive
organisms. Because of these similitudes our laboratory began to inves
tigate whether LTA possessed anttcomplemenlary activity analogous to
that associated with the LPS endotoxin (3-8). Although there have been
concentrated efforts to define the site and mechanism of LPS inhibition of
complement, very few investigators have reported data on the possible
effects of LTA on the complement system (9). This is somewhat surprising
since the interaction of LTA, LPS, and complement almost certainly play a
significant role in the etiology of periodontal diseases. Bacterial pro
ducts and serum components in the gingival crevices of the oral cavitv
have been shown to activate complement by both the classical (10,11) and
the alternative pathways (12,13). in fact recent evidence suggests that
bone loss (a major clinical manifestation of acute periodontal disease)
may occur via osteoclast activation due to the interaction of como lenient
and prostaglandin E (14). Prostaglandins are naturally occurring cyclized
derivatives of unsaturated long chain fatty acids (15) and their concen
trations are dramatically elevated in inflamed gingival tissues (16). It
is of interest to note, that both LTA and LPS are also capable of initiating
osteoclast mediated bone resorbt ion (17) and this activity proceeds with
out the contribution of complement or prostaglandins. The potential for
svnergism cannot be overlooked, and indeed LPS endotoxins have long boon
implicated as participants in the development of periodontal lesions (4-8).
Analogous LTA activity could he of significant clinical import especially
1 '


especially like to acknowledge the professional artistic assistance of
Margie Summers, Margie Niblack and John Knaub.
I would like to thank Steve Hurst for being the.ro when T needed a
friend and for helping with some of the last minute photography.
The typist, Joanne Hall, deserves a particularly special mention.
If not for her personal concern and dedication the deadlines would never
have been met. She worked on this dissertation under conditions for whi
no degree of monetary reinbursement could possibly compensate. I thank
you Joanne and I sincerely hope you never have to go through that again!
Finally, and most importantly, I wish to thank my wife Lyn for her
infinite patience and encouragement. Her attitudes, her ideals, her
"being" is so much a part of me that it would be hopelessly futile to
list all the things for which X am indebted to her. She is a friend
and lover, a typist and an occasional laboratory technician. She is my
driving force in life and rightfully so, I dedicate this dissertation
to her.
" And leitk one loud ivotraieoriaieoi'iaierrraieo'i'ia he jumped at the
end 0^ the tablecloth, pulled it to the ground, mapped himself] up ti; it
three times, 'wiled to the other end of the loom, and aftci a ter'iiblc
struggle got lids head into dai/light again and said cheerfully
-- have 1 Hien?"
(tom "The IlmiSe at Pee/i Co'iml" bit A. A. M(fne)
i i t


1 18
when C4 was incubated with EAC1,_,,. This model is also consistant with
LTA
the fact no dysfunction of Cl, Clq, Cls, Cls, C4, or C2 could he demon
strated when incubated fluid phase with LTApcx.
This model would also predict that once C4b were attached to the
membrane, subsequent addition of LTA should have significantly less
impact on cascade disruption. As shown in Figure 17, this prediction
coincides well with the facts. Percent inhibition of lysis drops from
more than 65% in the case of EA treated with LTApcx (100 pg/nl) to less
than 20% in the case of EAC14 treated with the same concentration of
LTApcx. Furthermore, EAC142 are no longer inhibited as one would
LIA
expect if the C4b and C2a binding sites were already secured.
Although all data thus far presented are consistant with this
131
model, final proof would necessitate the 1 labelling of purified C4
and C2. Once labelled, one could determine if an excess of decayed CAn
and C2a fragments were released into the media after preincubation with
EACI.t, r EAC1.4 respectively.
L l A LTA
It is hoped that future research in this area may prove enlightening
not only in expanding upon the mechanism of inhibition but also upon
the specific role this extracellular metabolite, plays in the inflam
matory response of periodontal lesions.
It is apparent that the anti-complementarv activity of LTA is not
restricted to a single species or geneus (Table I!) and it may verv
well he that LTA plays ) significant role in protecting gram positive
organisms from immunologic destruction. If so, LTA could ho considered
a type of "virulence" factor and those organisms t li.it produce copious
amounts of extracellular LTA (such as S. mutans BUT) would not only
contribute to their own protection, but also to the protection of the


137. Hoffmann, E. M. 9 f3 9. Inhibition of complement by a substance
isolated from human erythrocytes. II. Studies on the site and
mechanism of action. Immunochemistry. 6:403.
138. Wic.kon, A. J., J. W. Gibbons, and K. W. Knox. 1973. Comparative
studies on the isolation of membrane lipoteichoic acids from
Lactobacillus fermenti 6991. J. Bacterio!. 113:365.
139. Rapp, J. J., and T. Borsos. 1970. Molecular Basis ojf Complement
Action. Appleton-Century-Crofts, N. Y., N. Y. p 105.
140. Hill, M. W. 1974. The effect of anaesthetic-1 ike molecules on
the phase transition in smectic mesophases of dipalmitoyl-
lecithn. I. The normal alcohol up to C--9 and three inhala
tion anaesthetics. Riochem. Biophys. Acta. 356:117.
141. Lowry, 0. H., N. R. Roberts, K. Y. Leiner, M. Wu, and A. L. Farr.
1954. The quantitative histochemistry of the lira in. J. Biol.
Chem. 207:1.
142. Dubois, M., K. A. Giles, J. K. Hamilton, P. A. Rebers, and F. Smith
1956. Colorimetric method for the determination of sugars and
related substances. Anal. Chem. 28:350.
143. Grabar, P., and P. Burt in. 1964. Immunoelec trophoretic Analys is.
Elsevier, N. Y., N. Y.
144. Yonemasu, K., R. M. Stroud. 1971. Clq: Rapid purification method for
preparation of monospecific antisera and for biochemical studies.
J. Immunol. 106:304.
145. Bryant, R. E., D. E. Jenkins, Jr. 1968. Calcium requirements for com
plement dependent hemolytic reactions. J. Immunol. 10_1:664.
146. Fine, D. P. 1977. Comparison of e.tliy lonegl yco 11 et rancet ic acid and
its magnesium salt as reagents for studying alternative comple
ment pathway function, rnfcct. Immun. 1_6:124.
147. Fine, D. P., S. R. Mamey, Jr., D. G. Colley, J. S. Sergent, and R.
M. Des Prez. 1972. G3 shunt activation in hitman serum chelated
with EGTA. J. Tmmunol. 109: <307.
148. Becker, E. L. I960. Concerning the mechanism of complement action.
V. The early steps in immune hemolysis. J. Immunol. 84:299.
149. Wilkinson, S. G. 1968. Clycosyl diglyeoridos from Pseudomonas
ruj 11 use e ns B BA. 1 6 4 : 14 8.
150. Schmit, A. S. D. D. Ploss, and W. I. Lennatz. 1974. Some aspects
of the chemistry and biochemistry of membranes of gram-positive
bacteria. Annals of the N. Y. Academy of Sciences. 235:91.


f
FÂ¥3 9 17 8. 0 3 8.5.
C' ^V\Cc_ /Jj/ / / J


ceil membrane. However, one Would expect all of the complement com
ponent intermediate cellular complexes to lie equally affected bv I.iA,
when in actuality this was not the case. Lt is possible that some of
the complement components could block the attachment of the Inhibitor
to cell membranes so that the material would have no opportunity to
cause membrane alteration. This is an unlikely possibility because
even EAC1423567 had LTA on their surfaces.
The highly purified LTA necessary for the final site of action
and mechanism studies proved to be considerably more difficult to ob
tain than previously anticipated. As suggested by the results in
Figures 13 and 15, and Tables 4 and 7, the Octyl Sophorose method of
LTA purification did not sufficiently resolve the I.TA from tenacious
polysaccharide contamination. This method yielded almost quantitative
recovery of LTA (as determined by PHAg) and also a significant portion
of the total mass which was applied to the column. However, considering
the contaminated nature of the final product even when an elution gra
dient waS utilized, it was determined that a significant percentage of
the mass was probably polysaccharide. Indeed, gas Liquid chromatography
of extracellular LTA purified by Octyl Scpharose (L'l'Aosx) indicated
that as much as 30% of the final weight was carbohydrate, presumably
existing as polysaccharide (Figure 15, Table 7).
Tn contrast, the somewhat more elaborate method ol purifying LTA
by adsorbtion to phosphatidy l choline vesicLes (PCV) yielded a product
that was virtually devoid of all nucleic acid, protein, and carbohy
drate contamination (Figure 15. Tables 5,6, and 7). table 5 indicates
1 4
that although approximately 7% of the radioactive C used to label the
PCV was lost during washing procedures (and ostensibly, a percentage


Figure 8.
PHA of various LTAcx treated complement component
intermediates.


of 0.01 M Tris carbonate pH 6.8 was then added to each tube and they
were placed in a 50C water bath. Once warmed, the tidies were
vigorously vortexed (Vortex Genie Mixer, Scientific Industries Inc.,
Bohemia, NY) and the cycle of warming and vortexing was continued
until a milky emulsion was formed. Fifteen milliliters of 0.01 M
Tris carbonate were then added to each tube and the tubes were centri
fuged at 27,000 g for 30 minutes. The supernatent fluids were then
decanted, the pellets were resuspended in 1.0 mi Tris carbonate
buffer and warmed to 50C in a water bath. The tubes were gently
swirled (but not aggitated) to dissolve and resuspend the pellet .
The resulting phosphatidyl choline vesicles (PCV), devoid of very small
vesicles, were then used to adsorb LTA from LTAppx.
(b) Preparation of PCV-LTA. Two milliliters of LTAppx at a concen
tration of 1.5 mg/ml in 0.01 M Iris carbonate, pH 6.8 were added to
each centrifuge tube containing 1.0 ml of PCV. The tubes were covered
with parafilm (American Can Co. Neehaw, I/S) and incubated for
90 minutes in a 37 shaker water bath. Thirteen milliliters of
0.01 M Tris carbonate were then added to each test tube and tbev were
centrifuged at 27,000 g for 45 minutes. The superoates were discarded
and the pellets were gently resuspended in 1.0 ml of Tris carbonate
buffer at 50C as previously described.
Fifteen milliliters of buffer were then added to end) pellet,
the tubes were gentlv swirled and then centrifuged as described.
The pellets were washed three times in this manner. The final pellet
was drained and then dissolved in 5.0 mi of chloroform/methanol.
(3+1 v/v). The tubes were then covered with aluminum foil and
allowed to sit at room temperature for 60 minutes.


Figure 24.
Differences in complement mediated lytic susceptibility
of LTAcx treated EAC4 versus EAC14. Upper graph: EAC4
were treated with buffer or with LTAcx (23(.) ug/ml).
Various limiting dilutions of human Cl worn then added
to aliquots of the cells and lysis was developed accordin
to procedures described in Materials and Methods. Lower
graph: EAC14 were prepared with various limiting dilu
tions of Cl. Aliquots of cells were then treated with
LTAcx (250 pg/ml) or with buffer. After extensive
washing, lysis was developed according to procedures
described in Materials and Methods. Symbols: ()
Buffer treated cells; (o') LTAcx treated cells.


DECIMAL EXPRESSION OF CHU DILUTION! MO'
ro




CA) using a modification of the method described by Wlcken and
Knox (110). The column was equilibrated and eluted using 0.01 M
Tris carbonate (Sigma Chemical Co., St. Louis, MO), pH 6.8.
Hydrophobic Affinity Column chromatography.' Because of the
hydrophobic nature of the fatty acid moieties of lipoteichoic acid,
adsorbtion to a stationary phase of a chromatographic coLumn was
used in an attempt to further purify the LTA. LTAppx in buffer A
(0.01 M Tris carbonate pH 6.8, 1.0 M Nad was loaded on a 25.0 X 2.25 cm
column packed with Octyl Sepharosc (Pharmacia Fine Chemicals,
Piscatawav, NJ) and equilibrated in the same buffer. After eluting
with 150 ml of starting buffer A, the reservoir was then changed
to buffer B (0.01 M Tris carbonate pH 6.8) and another 100 ml were
eluted. Buffer C consisted of 250 ml of a gradient ranging from
10-70 % propanol (by volume) in 0.01 M Tris carbonate, pH 6.8.
Octyl Sepharose is a derivative of the cross linked agarose
Sepharose CL-4B. The terminal, n-ortyl groups of this agarose gel
confer a hydrophobicity to the matrix. By exploiting this property
it was hoped that polar or neutral non-interacting components
would be removed by elution with solutions of high ionic strength.
The lipoteichoic acid would then he eluted from the matrix with
an organic solvent such as propanol. (It is imperative that all
tubing, connections and gaskets used throughout the column lie
constructed of a material that is resistant to organic solvents).
^This method represents a modification of a procedure described
by A..I. Wicken and K. Knox (Sydney, Australia) via personal commun-
ica tion.


AVERAGE NUMBER OF
16,000 8,000
4,000
2,000


system has spawned a multiplicity of models attempting to elucidate its
precise mode of initiation and function. Clearly, a plethrn of diverse
stimuli are capable of activating this pathway, and this fact alone im
poses a formidable constraint on any molecular model. Some of the more
common naturally occurring activators of the alternative pathway Include
bacterial and fungal cell wall constituents such as Iipopolysaccharide,
zymosan, and inulin (a poly fructose) (71,80-83). In addition, aggregates
of some immunoglobulin classes (84,85), some types of animal cell, mem
brane constituents (86,87), and antibody-coated budding virus infected
cells (88,89) also stimulate this pathway. The alternative pathway can
even be activated by substances of relatively defined chemical nature
such as benzyl-B-D-fruetopyranoside (90), polyglucose with repetitions
a 1-3 and branched a 1-6 linkages (91), d in itrophonylated albumin (92),
and many polyanionic substances. Cobra venom factor (a non-1ipolyt ic,
non-hemolytic glycoprotein isolated from the venom of the cobra Naga
naja) is also a potent activator of complement cytolytic potential, but
it appears to act as a C3b analog and is thus unique in its mode of
alternative pathway activation (93,94,95). Potentiation of this system
requires devalent magnesium ions and the interaction of at least five
novel serum proteins. By convention, the names of these proteins are IF
(or initiating factor), P or P (properdin). Factor B (C3 pnmit i vator) ,
Factor B (C3 activator), and Factor I) or 1) (C3 pronet i vator convurtaso).
To date, all of the above components have been isolated, purified, and
characterized ns to molecular weight, electrophoretic mobility, and sedi
mentation coefficients (83,96-98). CJb (of the classical pathway) plays
an intregal role in the alternative pathway (71,96,99), and thus it in
essence forms the junction point of the two systems. Because all terminal


components ((3, C5-9) are .shared, the biological consequences of acti
vation enc.ompass all the processes previously described (immune adherence
opsonic activity, anaphyIntoxin production, membrane attack complexes,
etc,.).
There are similarities between some of the more salient features
of the classical pathway compared with those of the alternative pathway.
Analogous to Clq, IF seems to function as the recognition unit for the
properdin pathway, but its relationship to another factor (referred to
as a C3 nephritic factor from the sera of patients with membranoprolifern
tive glomerulonephritis (1.00) and its mode of activation is poorly under
stood (96). Factor D is capable of enzymatically cleaving Factor B into
Ba and Bb (29,94). In the presence of C3h, a himolecular complex C3bBb
is formed (29) which is endowed with C3 splitting .activity similar to
the C3 convertase (C4h2a) of the classical pathway. Furthermore, just
as C4b anchors the classical convertase. to the membrane allowing C2a to
exert its enzymatic activity, so toe cytophilic C3h anchors the C3hBb
complex to the membrane allowing the enzymatic activity of Factor Bb to
be. expressed (83). Both complexes merelv gain additional C3b to modulate
05 cleaving activity (99). Thus, the presence of Cl nol oniv prevents an
"abort" due to rapid decay of either convertase, but because C3b is
utilized as part of the alternative pathway convertase, it participates
in a type of ampl ificat ion Loop. In other words, the more C3b that is
formed from either pathway, the more C3 leaving potential is endowed
upon the properdin C3 convertase. Froperdi.n (P) seems to stabilize the
fragile C 3 kill) complex but its possible rol' in stabilizing the classical
03 convertase has not been invest igated (Qr|). Noteworthy, however, is
the potent effect properdin exerts on tin* C3b inhibitor (99). By


Z(AVERAGE NUMBER OF SAC 1423 SITES/ml)
RECIPROCAL OF (HUMAN SERA) C3 DILUTION



Ht
. .
l&iSwSyt'S; *w8
ttHMi
^mMmsIwIIK
'' l
tt. S


in
titer of the supernates previously incubated with EACl versus the negative
control which consisted of C4 incubated with EA. However, both EACl
LIA
and EACl consumed identical amounts of C4 (residual supertate C4
buffer 9 9
activity was 2.71 X 10 SFU/ml and 2.79 X 10 SFU/ml respectively). There
fore, it was concluded that LTA had no apparent effect on C4 uptake by
Residual C2 titration after preincubation with EAC14 Guinea
TO LTA
pig C2 (approximately 1.5 X 10 SFU/ml) was added in equal volumes to
EAC14 which had been preincubated with either LTApcx (100 pg/ml) or
with buffer. The mixture was incubated at 30 for 12 minutes and resi
dual C2 activity was titrated as described in Materials and Methods. EA
were incubated with the C2 as a negative control. Results indicated that:
9
approximately 35% (5.3 X 10 SFU C2/mi) of the available C2 was utilized
9
by the EAC14 complexes and approximately 29% (4.4 X 10 SFU C2/ml) were
utilized by the EAC14 complexes. Despite the fact that the supernate
LTA
from the C2 incubated witii EAC.L4 liad slightly more residual C2 activity
LTA
(approximately 71% of the C2 activity still remained in the supernate
after incubation with EAC14 ), a difference of only 6% is within ex-
LTA
perimental variance of this assay. Therefore, it was concluded that LTA
had no apparent effect on C2 uptake by EAC14.
Inhibition of lysis of EA by LTA from other bacterial sources.
Additional evidence indicating that LTA might be primarily responsible
for the C inhibition phenomenon came from hemolytic assays utilizing
LTA from other sources.
I)r. R. Doyle (Dept, of Microbiology ami


TABLE ll
Percent Inhibition and PUA Titer of F.As
Treated with LTA Containing Extracts from Several Sources
Source of LTA3
1^
Percent Inhibition
PHAC
S. mutans BHT
40
1600
S. mutans AHT
35
1600
L. casei (ATCC 7469)
75
3200
B. subtilis (gta B290)
70
1 600
aThe LTA extracts from all sources were used at a concentration of
50 pg/ml in VBS.
^EAs were treated with the appropriate LTA-extracl and bemolvsis
was developed by incubation of the cells with several dilutions
of human C (37/60 minutes). Values represent inhibition of CH,._
units.
c
PHA titers are expressed as the reciprocal of the final dilution
of specific anti-LTA which caused hemagglutination.


also capable of stimulating osteoclast mediated bone resorbtion (17).
Even without profound activation of complement, the possession and
release of complement inhibitory substances might, confer a certain
degree of survival value on the organisms producing them. Thus in
the face of immunological challenge, the complement system nay he
blocked from reacting against the bacteria producing such factors.
It may be more than coincidence that gram positive organisms such as
Micrococcus lysodeikticus lacking LTA in their cell membranes are also
susceptable to lysis by the synergism of lysozyme and complement (151)-
All other gram positives containing intact LTA in their membranes are
notoriously resistant to complement lysis even in the presence of
lysozyme (151).
Three lines of evidence have been obtained which suggest that
the active inhibitory factor is 1 ipteichoic. acid (LTA). The inhibitor
co-purified with LTA when extracellular material from spent culture
was fractionated by gel-CiLtrat ion and was purified by adsorbtion to
phospholipid vesicles. Sheep erythrocytes which had been treated with
S. mutans BUT extracellular extract became resistant to lysis by com
plement and they also became coated with LTA as judged by PHA using
antibodies monospecific for purified LTA. The amount of LTA present
on the cells paralleled the degree of lytic, resistance that was
acquired by the treatment. Purified LTA and LTA-rich fractions from
other bacteria aLso caused sheep erythrocytes to hceme resistant to
complement mediated hemolysis. Again, P11A assays indicated that cells
which became resistant to lysis had LTA on their surfaces.
Experiments using crude extracellular I.l'A (LTAcx) provided evi
dence for the consumption of whole human complement activity.
When


Figure lr>. Carbohydrate analysis of J.TA containing preparations
by gas liquid chromatography. Abbreviations: (MAN)
Mannitol; (GLC) Glucose; (GAT.) Galactose. Mannitol
was incorporated as an internal, standard with all
samples.


Figure 2.
Dose response inhibition of whole human complement
after incubation with varying concentrations of
LTAcx. The non-treated control is abbreviated
as NIC.


BIOGRAPHICAL SKETCH
Louis (Loui) Silvestri was born in Peckville. PA on January II,
1952. He spent most of his years in Archbald, PA.
Loui attended a parochial grade school (St. Thomas of Aquinas),
a Jesuit preparatory high school (Scranton Preparatory School) and a
college heavily influenced by Augustinian Catholicism (Villanova
University).
Loui's higher education was continued at the University of Florida
(Gainesville, FL) where, under the tutelage of Dr. Edward Hoffmann, he
received his Ph.D. However, earning that degree became more of a
challenge than originally anticipated.
Loui is currently employed at the University of Alabama (Birmingham.
AL) as a post doctoral fellow under the direction of Dr. Robert Stroud.


moles CARB (GLUC)/ml
c
A BCD
E
\
CL
cn
a>
o
E
c
VOLUME (ml)


Figure 23. Effect of LTApcx on the ability of Cls to hydrolixe
TAMe. As the synthetic substrate TAMo is hydro!iced,
there is an increase in A.,,_ absorbing material. in
- / q /
this experiment, Cls and TAMe were incubated together
in the presence of LTApcx (100 pg/ml) at room temper-
ature_(24C). Symbols: () Cls and TAMe plus buffer;
(o) Cls and TAMe plus LTApcx.


Abstract of Dissertation Presented to the
Graduate Council of the University of Florida
in Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy
INHIBITION OF HUMAN COMPLEMENT BY EXTRACELLULAR
LIPOTEICHOIC ACID FROM STREPTOCOCCUS MUTANS BHT
By
Louis Joseph Silvestri
December, 1977
Chairman: Edward M. Hoffmann
Major Department: Microbiology and Cell Science
A number of biological and chemical similarities exist between
the lipopolysaccharides (EPS) of gram negative microorganisms and the
lipoteichoic acids (LTA) of gram positive organisms. The potent
affects of LPS on the complement system are. well documented; however,
the effect of LTA on this host defense system has not been adequately
studied. Furthermore, all studies thus far conducted have been limited
to the interaction of LTA with whole fluid phase complement. Ln this
investigation it was demonstrated that extracellular LTA from the
cariogenie microorganism Strep tocoecus mutans BHT was not only capable
of spontaneously binding to sheep erythrocyte target cells hut was
also capable of rendering them refractory to complement mediated Ivsis.
Purification of the LTA to homogeneity was achieved by a combination of
gel filtration and adsorhtion to phospholipid choline vesicles (arti
ficial membranes). By utilizing various cellular complement component
intermediate complexes and functionally purified complement components,
experiments were conducted to define the site and mechanism of inhibition


TABLE 4
Percent
Recovery
a
of LTA During Octyl Sepharose Purification
LTA Source
Reciprocal of
Initial Dilution
Initial
Concentration ,
of LTA (ug/ml)
Total volume
of Sample (ml)
Calculated
Total Weight
LTA in Sample
of
(mg)
Percent
Recovery
of LTA
LTAppx
400
0.500
9.0
1.80
100.0
Peak I
2
0.250
87 .0
0.04
2.2
Pooled column Effluent
from all areas not Loca
Under Peak I or 11
ted
?
0.000
273.4
0.00
0.0
Peak II (LTAosx) Before
Passage through LK20
100
0.500
31.6
1.58
87.8
Peak II ''LTAosx) After
Passage tnrough LH20
100
0.25
62.3
1.. 56
86.5
As determined by ?HAg
Determined by methods described in Table 3
Calculated by multiplying the corresponding values for the first three columns


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
Edward M. Hoffmann, Ich^aijhnan
Professor of Microbiology and Cell Science
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
L. William Clem
Professor of Immunology and Medical
Microbiology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
Briah" Gebhardt/
Associate Professor of Pathology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
Arnold S. Bleiweis
Professor of Microbiology and Cell Science


modulating the action of tills enzyme, properdin at least indirectly
plays a role in stabilizing the classical pathway sequence.
The recognition of foreign substances by a host usually leads to
the neutralization and eradication of these substances by immune lympho
cytes, phagocytic cells, specific antibodies, complement, or an amalga
mation of these factors. However, in instances where antigenic substances
interact directly with host tissue, the reactions of the host's immuno
logical defense system could sometimes result in a considerable amount
of autodestruction. LTA represents a class of antigens that are capable
of spontaneous cytophilic binding to mammalian tissue (101,102,103).
As a result, host tissue acquires a new "ani tgenic. face" and may now
react with natural or induced antibodies to the LTA. Furthermore, anti
bodies directed primarily at LTA determinants may cross react with
similar determinants of the host's tissue. Such a mechanism has been
proposed for the high incidence of rheumatic fever and glomerulonephritis
in patients recovering from post streptococcal infections (104,103).
Recently, acylated heteropolysaccharides (LTA) isolated from the cell
membranes of several Lactobacillus species were shown to replace pigeon
excreta antigens in complement consumption tests diagnostic, for pigeon
breeders disease (9,106). Thus, precedence mav already be established
for LTA's role in the manifestation of several clinical maladies. In
addition, the. chemical and biological similarities between LTA and LPS
(1,2) plus the abilitv of LTA to stimulate bone resorbtion (17) make
LTA a likely candidate for a role, in periodontal disease. On the other
hand, LTA lacks some of the biological activities associated with LPS
such as pvrogenicity in rabhits (2,107) and a mitogenic effect on B-rclls
(2). Since these activities have been shown to reside with the complex


Figure 6.
Passive hemagglutination (PHA) of FA treated with
varying concentrations of LTAex.


If)
Streptococcus mutans BUT was capable of inhibiting complement mediated
cytolysis of target sheep erythrocytes.
(2) To purify the extracellular LTA of S. mutans BUT to homogenity.
(3) To describe the nature of any ant i-c*.omp 1 omentary activity
that purified extracellular LTA may exhibit.
(4) To determine the site of action of any such inhibition.
(5) To determine the mechanism by which purified extracellular LTA
may enhibit anti-complementary activity.
(6) To determine if the LTA from other gram positive genera and
species can be shown to demonstrate anti-complementary activity.


TABLE 1
Partial Purification
of LTA by A5-M Gel
Filtration
Poo 1ed
Fraction
Test Tube
Numbers
Pooled
Probable
Content
Percent
Inhibition
of Lysis3
PILA ,
Ti ter 1
A
32-40
Void Volume Material
NDC
ND
B
41-46
LTA Plus Low Percent Carbohydrate
47.4
6400
C
47-60
LTA Plus High Percent Carbohydrate
39,1
6400
D
61-71
Carbohydrate
0.0
<100
E
72-84
TA, Carbohydrate and Nucleic Acid
0.0
<100
F
85-100
Nucleic Acid
0.0
<100
CX
All of Above
31.2
3200
EA were prepared with the LTA source at a concentration of 50 yg/nl. Hemolysis was developed by
incubation of the cells with several solutions of human C (37,'60 minutes). Values represent inhibi
tion of CI!_ units.
oO
PHA titers arc expressed as the reciprocal of the final dilution of anti-LTA which still resulted in
hemagglutination when incubated with the EA,,,.,.
Not determined.


TABLE OF CONTENTS
PAGE
ACKNOWLEDGMENTS i i
LIST OF TABLES v
LIST OF FIGURES vi
GLOSSARY OF ABBREVIATIONS viii
ABSTRACT ix
INTRODUCTION 1
MATERIALS AND METHODS 17
RESULTS 11
DISCUSSION IOS
LITERATURE CITED 120
BIOGRAPHICAL SKETCH 132
i v


PERCENT HEMOLYSIS


PERCENT INHIBITION OF LYSIS
O % PHA
v50 % PHA
I 00 % PHA
RECIPROCAL OF ANTI-LTA DILUTION
JO


A 280
TEST TUBE NUMBER
RSC


GLOSSARY OF ABBREVIATIONS
A:
Antibody
C:
Complement
Cl, C2
-C9:a Complement components. Horizontal bars
above the component designation denotes
a biologically active state.
CVF:
Cobra venom factor
E:
Erythrocyte
EDTA:
(Disodium) Ethylenediamine tetrnacetic acid
EGTA:
Ethy leneglyco1-b is (B Amino Ethyl Ether) N,N" tetra
acetic acid
LTAcx:
Crude extracellular lipoteichoic acid
LTApcx:
Extracellular LTA purified via phosphatidyl-choline
vesicle adsorbtion
LTAppx:
Partially purified extracellular LTA
LTAosx:
Extracellular LTA purified via Octyl Sepharose gel.
column adsorb Lon
IPS:
L i p o p o 1 y s a c e h a r i d e
PHA:
Passive hcmagglutination
PHAg:
Passive hemagglutination (modified method)
TA:
Teic.hoic acid
TAME:
p-Tosy1-1-arginine methylester
All comp 1 omen l nomom: 1 ;i tu rc follows the WHO recommetidaC intis
(Bull. Wld. Hlth. Org. 39:>39, 1963).
v i i i.


Figure 12.
Partial purification of LTA by A5M gel filtration
with LTA enriched starting material. Symbols: ()
A,-, absorbance: (o) Pi concentration in ;i-mo 1 os/'mL
as determined by the Lowry Pi assay: (+) Antigenic it
as determined by PHA using antisera directed against
LTA backbone.


suJ.fate, heparin, polyiosinic acid, chondroitin and many other polv-
anionic compounds (39,40) in addition to DNA, RNA (135,156), and
carrageenin (157). It was dismaying to find that although LTApcx
maintained anti-complementary activity with the appropriate cellular
intermediates, all fluid phase inhibition of Cl was abrogated (Figures
17 and 18). All subsequent experiments attempting to define Clq, Cls,
or Cls dysfunction were negative. The only experiments that gave sug
gestive results were the Cl transfer assavs. Even here, instead of the
anticipated inhibition of Cl transfer, over 20% enhancement of transfer
was observed (Table 10). Thus, in light of these data obtained with
purified LTA it was necessary to devise new molecular models to explain
the mechanism of lytic inhibition of certain complement intermediates
by highly purified LTA. Some possibilities are discussed below:
1). Attachment of LTA sterically blocks the affixation of Cl to the
Fc portion of the immunoglobulin. Thus, if Cl does not attach properly,
or is prevented from attaching at all, the complement cascade will never
be initiated.
2). Although Cls activity was not effected fluid phase, perhaps such
activity would be abrogated once the Cl molecule became associated with
the cell membrane. If so, EAC1 would no longer be capable of hydrofining
C4 or C2 again, the cascade would he-terminated.
3). LTA directly interacts with fluid phase C4 or ('2 thus preventing
them from combining with the appropriate sites on the membrane.
4). The attachment of LTA lends to increased fluidity of the mem
brane resulting in the displacement of looselv attached molecules. If
some of those less tenacious molecules include any of the early comple
ment components, the physical loss of these components would terminate
the lytic attack sequence.


PERCENT INHIBITION OF LYSIS
CELLULAR INTERMEDIATES TREATED
WITH LTAcx (125 /xg/ml)


m L
TABLE in
_ Comparison of Che Relative Numbers of Effective
Cl Molecules Capable of Transfer from EAC1 Treated with LTApcx
Sample
Experiment
Number
Effective Number of Cl
Molecules Transferred/Cell
EAClLTAa
1
175
eacTlta
2
124
eacTlta
3
153
EAcTDVBb
1
137
eacTdvb
2
115
faltacT C
1
185
ealtacT
2
1 78
ealtacT
3
21 5
eadvbcI
1
132
eadvbcT
2
182

EAC1 were generated and treated with LTApcx at 100 pg/ml in PVB.
After extensive washing, the Cl capable of transfer was titrated.
k Control EAC1 treated with DVB for 10 /1 minutes
p
F.A were prepared (100 ¡ig LTApcx/ml) and aftor_the cells were
extensivo Iy washed EA Cl were generated. The Cl capable of
transfer was then titrated.
Control EAC1 treated with DVB while in the FA slate. FACI pre
paration and Cl transfer exactly paralleled the T.TA treated cells.
d


Figure 5.
Inhibition of complement mediated lysis of F.A
treated with varying concentrations of LTAex.


13.
Purification of i.TA by Octyl Eoplmrose
hydrophobic ge! chromatography
14. Simultaneous removal of salt and propanol
from LTAosx by LH-20 gel chromatography 69
15. Carbohydrate analysis of ETA containing
preparations by gas liquid chromatography 7 6
16. Passive hemagglutination (PHA) titration
and inhibition of complement mediated lysis
of EA treated with varying concentrations
of LTApcx 81
17. Effect of ETApcx on the complement
mediated lysis of various cellular
complement component intermediates 83
18. Effect of LTAcx and LTApcx on function
ally purified human Cl. 86
19. Immunodiffusion and precipitation analysis
of various steps in the purification of
human Clq 88
20. Disc gel electrophoresis of purified
human Clq
21. DEAE elution profile of human CIs 93
22. Immunoelectrophoresis of human C.ls and
CIs 95
23. Effect of ETApcx on the ability of CIs
to hydrolize TAMe 99
24. Difference in complement mediated lytic
susceptabilitv of LTApcx treated EAC4 versus
EAC4 103


LTA ran be immunogenic (2). Of particular interest is the fact that the
attachment of streptococcal LTA to erythrocytes could he reversibly
transferred from the erythrocytes to other tissue cells (104,12b). The
possible significance of this "transferability" in relation to rheumatic
fever and glomerulonephritis and pigeon breeders disease has been pre
viously discussed (9, 104106). However, despite this precedence the
significance of the binding of LTA to oral epithelial cells in gingival
pockets has not yet been investigated. Not only does LTA mediate bone
resorbtion as previously indicated, but spontaneous hybrid micells of
LPS and LTA are known to occur,' thus compounding the possibility of in
situ immunological modulation. There is little doubt of the availability
of extracellular LTA in this environmentStreptococcus mutans BUT alone
has been reported to produce excess of 50 ng of LTA/ml in culture media
(20). Recently, Wicken and Knox have studied the excretion of extra
cellular LTA from this organism in a chemostnt under steady state loga
rithmic growth conditions. Results indicated that a generation time of
10-14 hours (estimated to reflect that actual in vivo growth rate of this
organism in the oral cavity) produced the maximal amount of extracellular
LTA (1). Considering its ubiquity and the cariogenic nature of Strepto
coccus mutans BUT (127-130), the secretion of copious amounts of biologi
cally active LTA into the oral cavity has the potential of considerable
influence on the host-parasite relationship.
The objectives of the project were then defined as follows:
(l) To establish if an LTA-centa ining extracellular extract of
Personal common ¡cat ion of A. .!
K i cken.


A Millipore 15 ml analytical filter holder (Millipore Corn.,
Bedford, MA) was loaded with a 3.0 p fluoropore membrane (Millipore
Corp.) and washed with several, volumes of the chloroform/methanol
solvent. The test tubes were all sequentially decanted into the
apparatus and the contents were allowed to filter bv gravity through
the membrane. Each test tube was washed with several volumes of
warmed chloroform and decanted into the filtering apparatus. Finally,
the barrel and filter were washed in situ with warm chloroform. The
filter was removed after air drying in situ and placed in 10.0 mi
of deionized water warmed to approximately 40C. All centrifuge tubes
and the barrel of the filtering apparatus were washed with warm
deionized water and all products were combined. The resulting
product was passed through a 25 mm Swinnex filter (Millipore dorp.)
loaded with a 5 u microporous membrane (Millipore Corp.) to remove
particulate debris. The membrane was washed i_n s_itu with several
volumes of warm deionized water. The filtrate was collected directly
into a lyophilization flask and was then shell, frozen and lyophi 1 ized.
The final product was stored in a dessic.ator at -20C.
14
C Phosphatidyl choline analysis In order to detect any
phospholipid contamination of the LTA throughout the previously
described PCV purification, radioactive PC was used to Label the phos
pholipids iu the vesicles. Approximately 2.3 pCi (3 X 10 DPM)
1 4
of C labeled phosphatidyl choline (Amersham Searle Corp., Arlington
Heights, TL) were added to 40 mg of phosphatidyl choline dipalmitovl
in a 30 ml Corex centrifuge tube. Phosphatidyl choline vesicles
were prepared from this and the non-labe Led contents of three
other tubes bv the methods previous!v described. Fifty microliter


cell wall material. 3). LTA or TA may contribute to the overall elec
trostatic charge of gram positive organisms. Although membrane
localized, the long polar tails of many LTA penetrate the thick pepti-
doglycan layer and become externalized (107). These, together with the
TA which are covalently linked to the cell, wall (]08) present a myriad
of antigenic faces to the external environment (ILl),l20). This antigenic
presentation is of serological import since these antigens are often
genus, species, group, or type specific (103,120). In addition, these
polar tails generate a net negative charge by exposing the phosphate
groups of the polyglycerol or polyribito.l backbone. This net negative
charge has been teleologically assigned the function of maintaining elec
trostatic repulsion and dispersion of the bacterial cell (121). Since
LTA has been shown to sequester certain cations such as magnesium (122),
an additional function as a site of divalent cationic convergence has
ilso been postulated. The association with magnesium ions appears to
be more than casual since protoplasts of Lac tobac111us easel placed in
a magnesium ion free or chelated medium rapidly lose their LTA from the
cell membrane.
Anti-LTA titers (of both the TgM and Igf. classes) have been regularly
reported in mice, rabbits, and man (2,121). Several clinical studies
have reported increases in nnti-LTA titerincluding antibodies of the
class IgAafter acute gram positive infections (I2A.123). Pigs, guinea
pigs, and rats exhibit a low level of natural immunity to LTA and recently,
there have boon reports of salivary IgA product ion as a result of gastric
intubation of monkeys with Streptococcus mut ans (>713 serotype C.. There
is no doubt that TA and LTA of all gram positive genera thus far inves
tigated contain antigenic moieties and that under certain circumstances


110
preincubated with various concentrations of LTA, whole human sera lost
complement activity .n a dose-dependent fashion. Individual component:
titrations revealed that not only C3, but the early components Cl, C'>,
and C2 were consumed to some degree. However, no C3 consumption was
observed if the preincubation was performed with Isolated C3 or in the
presence of EDTA. If EGTA-Mg were substituted as the chelating agent,
only a minimal restoration of C3 consuming activity was observed.
These results indicated that not only were calcium and magnesium ions
necessary for the anti-complementary activity, but there was a require
ment for some factor(s'> in whole sera as well. This "factor" is most
likely natural antibody directed at LTA or some component of the crude
extract. This resulted in the formation of a typical, antigen-antibody
complex with subsequent classical complement consumption.
Experiments using sheep erythrocytes in various stages of com
plement component fixation provided evidence that LTA was not only
capable of spontaneously adsorbing to the surface of these cells, but
also rendered many of the intermediates refractory to lysis. When
sheep red blood cells, EA, or F.AC1 were treated with LTA, all became
resistant to complement lysis. Lipoteichoic acid treated FACIA were
somewhat less resistant to lysis and all ceLl.utar complement inter
mediates beyond EAC14 were no longer protected.
Conversion of cells to hemolytic resistance by treatment with
LTAcx can aid in the interpretation of the C2 consumption data depicted
in Figure 4. As indicated, the degree of C2 consumption was dispropor
tionate compared to loss of Cl and C4 activity. However, the commer
cially available human C2 used in these sftul ios had a fairly low titer.
As a result, the dilutions made, after the preincubat ion step were not
sufficient to prevent substantial amounts of the LTA from binding to


loo
EA Despite the lower concentration, previous data with LTA treated
LTA
EA (Figure LG) indicated that even at this concentration, inhibition

should have been significant if indeed Clq were the site of inhibition.
8
Instead of this anticipated inhibition, 1.36 X 10 SFU/ml of Clq were re-
8
covered from the incubation mixture originally containing 1.50 X 10 SFU
Clq/ml. Due to assay variation this difference was considered insignificant.
Effect of LTApcx on Cl transfer. In another attempt to elucidate
the effect of LTA on the Cl molecule, the interference of the normal
ability of Cl to transfer from cell to cell under conditions of high
ionicity was investigated. Two different types of transfer tests were
performed. In type I, EA were treated with LTA and then Cl was added.
In type II, EAC1 were prepared and then LTA was added. Not so sur
prisingly there was no inhibition of Cl transfer as measured by hemo
lysis of EACA cells. However, there was an increase in the Cl trans
ferability of cells containing LTA. As can he seen in Table 10, this
phenomenon was repeatable and was observed in both types of experiments.
Differences in complement mediated lytic susceptabilitv of LTAcx
treated EACA versus EAC14. Buffer or LTAcx (250 pg/ml) was used to
treat EACA using procedures described in Materials and Methods. Various
limiting concentrations of human C were then added to aliquots of the
cells and lysis was developed as previously described. Alternately.
EAC1A were prepared using various limiting concentrations of human Cl.
Aliquots of cells were then treated with LTAcx (250 pg/ml) or with buf
fer. After the cells were washed extensively in buffer, lysis was
developed as previously described. As shown in Figure 24, EAC14 treated
with LTA are considerably more refractory to complement mediated lysis
than are EACA treated with cl.
LTA


Figure 11.
Partial purification of LTA hv A5-M gel filtration.
Symbols: (*) A,,^() absorbance (maximal absorbance
wavelength for nucleic acids): (*) A^^_ absorbance
(maximal absorbance wavelength for carbohydrates as
determined by the Phenol Sulfuric Acid assay); (A)
Pi concentration in n-moles as determined by the
Lowry Pi assay; (+) Antigenicity as determined by
Ouchterlony gel diffusion using an antisera
directed against LTA backbone.


myriad of microorganisms in their immediate environment. Obviously,
more research in this area is needed before such speculation can be
substantiated with fact.


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
L. 0. Ingram fj
Associate Professor of Microbiology and
Cell Science
This dissertation was submitted to the Graduate Faculty of the
Department of Microbiology and Cell Science in the College of Arts
and Sciences and to the Graduate Council, and was accepted as
partial fulfillment of the requirements for the degree of Doctor of
Philosophy.
December 1977
Dean, Graduate School


OPTICAL DENSITY (247nm
10 20 30 40 50 60 70 80 90 100 NO 120
MINUTES AT 24C


ACKNOWLEDGEMENTS
In all sincerity, no occasion or project thus far undertaken has had
a more humbling effect on my life than the completion of this Ph.D.disser
tation. It is unfortunate that only now in retrospect can I clearly see the
tremendous debt I owe for the help, patience, understanding, and knowledge
so generously contributed by my mentor, my friends and wife.
If I were to formally thank everyone who contributed in someway to the
successful completion of my Ph.D. dissertation, it is likely that my acknow
ledgements would read like the listings of a telephone hook. I do however
feel compelled to thank a few very special people.
First of all I would like to thank my mentor and friend, Dr. E.M.
Hoffmann. Ed possesses the rare ability to earn respect rather than having
to demand it. His tolerance for my idiosyncrasies was unlimited (almost).
He gave me direction yet left me with alternatives; he challenged my intellect
yet never made me feel ignorant; he provided the foundation on which 1 am
still building my scientific character. Most of all, lie was (and is) a friend
I would also like to thank the. members of our laboratory "family"
(Suzanne, Jean, Bert and Torn) for their help, patience and tolerance
during these difficult last clays. One cannot help but reflect upon the many
events that shape the complex web of friendships within the laboratory.
All of you will always he regarded as the closest, of triends.
1 wish to express mv gratitude to Ron Craig not only for his friend
ship, but also for the professional technical assistance that lie afforded.
I would also like to express mv appreciation to the employees of leach
ing Resources, for dedication above and beyond the call of duty. I would


% INHIBITION OF CH, UNITS


1 2
26. Mil 1 1 e.r-Eberhard, II. .1., U. R. Nilsson. A. P. Ha I niasso, M. .r. Pol lev,
and M. A. Calcott. 1966. A molecular concept of immune cytolvsis.
Arch. Pathol. 82:205.
27. Reid, K. B. M., D. M. Lowe, and R. R. Porter. 1972. Isolation and
characterization of Clq, a subcomponent of the first component
of complement from human and rabbit, sera. Riochem. .1. 130:749.
28. Calcott, M. A., and H. .J. Mill ler-Eborhard. 1972. Clq protein of
human complement. Biochemistry. 11:3443.
29. Mller-Eberhard, H. J. 1975. Complement. Ann. Rev. Biochem. 44:697.
30. Taranta, A., and E. C. Franklin. 1961. Complement fixation by anti
body fragments. Science. 134:1981.
31. Augener, W., H. M. Orey, N. R. Cooper, and H. J. Mller-Eberhard.
1971. The reaction of monomeric and aggregated immunoglobulins
with Cl. Immunochemistry. 8:1011.
32. Isliker, H. 1974. Interaction of Clq with IgG and fragments thereof.
Adv. Biosci. 12:2 7 0.
33. Valet, G., and N. R, Cooper. 1974. Isolation and characterization of
the proenzyme form of the Clr subunit of the first complement com
ponent:. J. Immunol. 112:1667.
34. Val.et, C. and N. R. Cooper. 1974. isolation and characterization
of the proenzyme form of the Cls subunit of the first complement'
component. J. Immunol. 112:339.
35. Sakai, K., and R. M. Stroud. 1973. Purification, molecular proper
ties, and activation of Cl proesterase, Cls. .1. Immunol. 110:1010.
36. Sakai, K., and R. M. Stroud. 19 74. The act ivat ion of Cls with
purified Clr. Immunochemistry. 11:191.
37. Becker, E. L. 1956. Concerning the mechanism of complement action.
IT. The nature of the 1st component of guinea pip, complement. J.
Immunol. 77:469.
38. Nagnlci, K. and R. M. Stroud. 1969. The relationship of the hemo
lytic activity of active Cls to its I'AMe esterase action: A new
method of purification and assay. .!. Immunol. 102:421.
39. I.oos, M. E. Volanakis, and R. M. Stroud. 1976. Mode of inter
act inn of different polyanions with the first (Cl, Cl), the second
(C2), and the fourth (C4) component of comp 1 oment11 I. Inhibition
of C4 and C2 binding stte(s) on Cls bv polyanions. Immunochemistry
L3: 789.
40. Rnepplo, E., H. H. Hill, and M. Lons. 1976. J-lode of interaction of
different pol.yanions with the first (Cl, CL), the second (C2),and
the fourth (C4) component of complement1. Effect on fluid phase
Cl and on Cl hound to EA or to EAC4. Tmmunoehem i strv. 13:251.


hydroxyprol ino, 27 hydroxy lysine and 13" glycine. This unusual col Ingyn-
like composition makes it unlike any plasma protein yet described (28,29).
When complement is activated by ant ihody-antigen complexes such as
exists on the surface of an antibody sensitized erythrocyte (EA), it
undergoes a self assembly process sequentially depositing the entire
fluid phase cascade onto the surface of the target. Specifically, Clq
recognizes a previously sequestered binding site located in the Fc frag
ment of IgG and IgM (30,31). The three polypeptide chains of Clq are
physically arranged in a manner perhaps analagous to a six headed mace
or bola with each "head" representing a binding site for an IgG molecule
(32). Thus each Clq molecule has six binding sites for IgG (and presum
ably the same number for IgM). Internal activation of Cl probably is the
result of a conformational change in Clq which in turn induces a change
in the proenzyme Clr (33). Once Clr is activated to Clr it is endowed
with enzymatic activity through which the proenzyme Cls is converted to
CL esterase (C is )(34,35 3f>) Cls is a serine esterase and is inhibited
by diisopropyIphosphofluoridate (DFP) (37). This esterase activity can
be used to hydrolyze the synthetic substrat es p-Tosy1 I argintne methv-
lester (TAMe) and N-nc.ety 1-1-tyros ine ethvlester (ATF.e) (38). Recently,
I.oos and Raepple have demonstrated that many polyanions were capable of
inhibiting the activity of Cl either In* in! orfering with Clq binding to
the antibody-antigen complex, or by preventing interaction of C4 and ('.2
with Cls (39,AO). Although binding of Cl usually leads to activation,
the two processes are not integral I t;C with modified trvptopiian (41)
and the human immunog1 obu 1 in subclass IgG4 (42)both bind Clq but do
not activate Cl.
After activation, Cls enzymatically cleaves C4 into a large (C4b)


TABLE 7
Summarized Chemical Composition of Various LTA Containing Sources
LTA Source
3
Carbohydrate'
Percent Composition of Dry
Protein
Amino Acid Bio-Rad
Weight
LTAb
c
Pi
, f
A220
A260
A280
Analysis
Protein Assay
LTA ppx
23-32
21-28
16-22
21-26
1.8-2.5
.555
.090
.082
Combined Fractions
from Octyl Sepharose
(except Peak II)
57-65
43-56
36-48
<5
0.9-1.3
. 392
. 105
.071
LTAos x
(Peak II, Octyl
Sepharose)
15-30
4-6
72-85
3.1-4.0
.368
.091
.074
Combined Super-
nates from PCV
washings
49-58d
26-34d
16-22d
<5
NA6
NA
NA
NA
LTA;) ex
< 5
-
<2.5
<95
5.8-6. o
. 160
.093
. Ob9
a
Percent carbohydrat
e was determined
by gas liqu
id
chromatography as
described
in Materials
and Met ho
CS .
Percent LTA was determined by PHAg.
Percent phosphate was determined by the Lowry Phosphate assay.
These values were corrected for weight differences due to contaminating phospholipid vesicles.
Because of the high percent phospholipid vesicle contamination in this sample, valid determinations for total
Pi and optical densities were not possible.
Ultra violet light absorbance determinants were made with the indicated materials at a concentration of
100 al/tnl In distilled water.


CaCi,; and MgCl? (DGVB), 1'DTA containing Veronal buffer (0.04 M
EDTA-GVB) and gelatin Veronal buffer with added C.a('l0 and MgCl,
(GVB) were prepared as previously described by Hoffmann (131).
Human complement (HuC). Fresh human blood samples were obtained
from the Gainesville Plasma Corp., Gainesville, FL. The blood was
allowed to clot at room temperature for about 60 minutes, and
the serum was separated by centrifugation at 500 X g at 0UC. The
serum was collected and stored at -70C.
Guinea pig complement (GPC). Fresh frozen guinea pig complement
was purchased from Pel Freeze Laboratories (Rogers, AR). The serum
was shipped in dry ice and it was stored at ~70cC after arrival
in the laboratory.
Complement: components. Purified guinea pig Cl and C2 were
prepared according to Nelson et al. (132) and Ruddv and Austin
(133,134). Functionally purified guinea pig C3, CP> and C9 and
human Cl, C5, C6 and C7 were purchased from Cordis Laboratories
(Miami, FL).
Erythrocytes (JE). Sheep blood was taken by venipuncture from
a single animal maintained at the animal research laboratory of the
J. Hillis Miller Health Center (Gainesville, Fh). One hundred
milliliter volumes of blood were rol lor ted in equal volumes of
sterile Alsevors solution (135) and the blood was stored at 4nC
for up to three weeks.
Antibody sensitized sheep erythr oey_tes (F.A) Rabbit anti -
sheep E stromata was obtained from Cordis Laboratories (Miami, FL).
Sensitization of washed sheep F was performed as recommended by
the supplier.


component intermediates were treated with LTAcx and analyzed for sus
ceptibility to complement mediated lysis. The LTAcx treated cells were
also tested for bound LTA using PHA with anti-LTA. Results indicated
that L, EA, and EACH4 were all refractory to complement mediated 1 vs is
and that LTA was detectable on the surfaces of the cells (Figures 7 and
8). However, EACL423567 which had been treated with LTAcx were not re
sistant to lysis despite the fact that LTA was detectable on the cells
(Figure 8). Thus, the inhibitor appeared to affect a complement com
ponent required for lysis os EAC14, but which was unnecessary for lysis
of EAC1423567.
In an attempt of focus on the site of inhibition, the ability of
LTAcx to affect the hemolytic susceptibility of EAC 142 was examined.
This intermediate possesses C3 convertase activity ((,42) which is in
volved in the generation of SAC1423 and SAC!4235. However, Cl is not
required for lysis of the intermediate once SAC142 have been formed (148).
Failure of LTAcx to inhibit this intermediate would indicate that C3
convertase was not the. step in the complement sequence affected hv the
LTAcx.
Sheep EAC142 were treated with LTAcx according to the protocol
that has been described. For this experiment, three different' amounts
of C2 were used to generate EACL42 from EAC 14. The results clearlv
indicated that there was no inhibition of the intermediate complex
EAC42 (Figure 9). resting by PHA with antibodies specific for LTA
confirmed the presence of LTA on the surfaces of the cells at the same
relative concentrations found when the other intermediate complexes
were tested.
Effect (if LTAcx on ant i-sheep erythrocyte antibodies.
Some


and small fragment (C4a) (43). The cleavage of 04 exposes a membrane
attachment site on the C4b molecule, and it will attach to the nntihodv-
antigen complex at a site juxtaposed to the Cl.-antibodv complex (44,43).
Cls then cleaves 02 into C2a and 02b (46) with C2a attaching to the C.4b
site and C2b being released into the fluid phase. Thus, the molecular
complex C4b2a is formed and is referred to as C3 convertase because it
is capable of splitting and activating C3 (47,48). C3 convertase is
also an esterase, and although C3 is its natural substrate, it also
hydrolyzes the ester bond of acety1-glycl- lysine methyl ester (49). The
catalytic site of C3 convertase is believed to reside in the C2a sub
unit and even after release from the C4b complex, cytolvtically inactive
C2a retains esterase activity, but is no longer capable of cleaving C.3
(49). The enzymatic half-life of C4b2a is quite ephemeralonly 10
minutes at 37. However, if the 02 is first oxidized by treatment with
iodine (applicable to human but not guinea pig C2). not only is the
binding of C2a to C4b enhanced, but the half life of the binolecular
complex is increased 20 fold (50). No doubt the transient association
of C2a with the (¡42 and (¡423 complex plays a vital ro 1 e in controlling
the complement reaction hv temporarily limiting the functional associa
tion of these complex enzymes.
Once C3 is cleaved into 03a and 03b, the small 03a fragment is
released into the fluid phase and 03b becomes associated with the 04h2a
complex and with other non-hemolvtio sites on the largel membrane (47).
The association of 03b with the 03 convert .use modulates its activity .so
that now ('3 becomes the natural substrate of this ( r l.nnl ecu 1 nr complex.
The 0423b complex is referred to as 05 convertase (31) and like (.42, is
a highly sneoinLLzed protease.
lust as 0.3 is the only known protein


7
susceptible target cells is not clearlv understood. One hypothesis., in
light of the newly discovered trihut yrina.se act ivi.tv of 07, is that the
lytic event is caused by an enzymatic attack on the membrane (63). How
ever, no enzymatic degradation products have ever been discovered in
either lysed cell membranes or in ruptured synthetic lipid hi 1 avers (64).
The two most favored models are tin? "doughnut" insertion hypothesis (65)
and the C8 insertion model (29). The former model purports that the
C5b-9 complex inserts inself into the membrane as a "prefabricated hole"
allowing the exchange of inLra and extracellular material via an internal
hydrophilic channel (65). However, the. model fails to explain hew the
hydrophilic complement components enter the hydrophobic expanses of the
membrane. In addition, although electron microscopy has revealed apparent
ultrastructure doughnut shaped "lesions" on the surface of cells Ivsed by
complement (66), freeze etching techniques have shown that the ultra
structure alterations are confined to the outer loaf lot of the membrane,
i.e. the lesion does not penetrate the membrane (67). The C8 insertion
model embraces most of the salient features of the doughnut mode 1, but
in addition postulates that the u and y chains of C3 are inserted into
the channel formed by the surface macromo 1 ocular complex. The and y
chains thus extend into the membrane hi Inver causing disruption of orderly
structure.
In addition to the restraints placed on tho complement cascade due
to the rapid decay of several of the intermediates, there are two
naturally occurring inhibitors of complement present in the sera of man
and probablv in all vertebrates. The first inhibitor is referred to ts
Cls inhibitor and, as the name implies, it directly abrogates the hemo
lytic and esternlytie activity of hi (68,69). The second inhibitor is


Complement componen!: intermediate compiexes. Sheep E in
various stages of complement fixation were used in this study.
EAC1, EAC14 and EAC142 were prepared by methods described by Borsos
and Rapp (136). EAC1423567 were prepared by the procedure described
by Hoffmann (137). Unless otherwise indicated, guinea pig Cl,
C8 and C9 were used in all instances, and the remaining C components
were from human serum.
Treatment of cells and cellular intermediates with LTAcx.
Unless otherwise indicated, cells were washed and suspended in VBS
q
at a concentration of 10 /ml. Equal volumes of these cells and
LTAcx were mixed and incubated at 37 for 20 minutes with continuous
shaking. The mixture was then placed in an ice bath for 10 min
utes. At tlie end of incubation DGVB was added to the mixture and
it was centrifuged at 500 g for five minutes. The supernote was
discarded and the cells were suspended and washed thrice with
DGVB (0 for 10 minutes at 500 g) to remove any unbound material.
g
The cells were then resuspended in DGVB at a concentration of 10 /ml.
A sample of the cells were tested for cell-bound I.TA using passive
hemagglutination with rabbit anti-l.TA. The remaining cells wore
used in experiments to detect acquired resistance to hemolysis.
Passive hemagglutination .(PHA) Passive hemaggl ut ¡.nation was
carried out using a microtitration system. fifty ill. of a VBS
dilution of anti-UTA we re added to the first row of wells of a round
bottom microtiter plate (Cook Engineering Co., Alexandria, VA)
and 25 ui (one dron from the calibrated pipetes suppl ied with the
system) of VBS were added to the other weils on the plate. The
anti-serum was serially diluted in situ and one drop of LTAcx


Figure 18.
Effect of LTAcx and LTApcx on functionally purified
human Cl. The upper graph represents a residual Cl
titration after incubation with LTAcx (5D0 pg/ml).
The lower graph represents the results from an analo
gous experiment using LTApcx (500 pg/ml) instead of_
LTApcx in the incubation mixture. Symbols: (*) Cl
incubated with buffer; (o) Cl incubated with the
appropriate L'L'A containing extract.


127
97. Gotze, 0., and H. J. Mi.il .1 er-Ehcrhard. 1974. The role of properdin
in the alternate pathway of complement activation. .1. Exp. Mod.
139:44.
98. Gotze, 0. 1975. Proteases of the properdin system. Tn Proteases
and Biological Control. E. Reich, D. B. Rifkin, and E. Shaw, ed.
Cold Spring Harbor Laboratory, 'Cold Spring Harbor, N. Y. p 255.
99. Iledicus, R. G. 0. Gotze, and H. J. Mill ler-Eherhard. 197b. .Alter
native pathway of complement recruitment of precursor properdin
by the labile C3/C5 convertase and the potentiation of the path
way. J. Exp. Med. 144:1076.
100.Vallota, E. H., .J. Forristal, R. E. Spitzer, N. C. Davis, and C. P.
West. 1970. Characteristics of a non-complement-dependent C3-reac-
tive complex formed from factors in nephritic and normal serum. J.
Exp. Med. 131:1306.
101. Gorzynski, E. A., E. Neter, and E. Cohen. 1960. Effect of lysozyme
on the release of erythrocyte modifying antigen from staphylococci
and Micrococcus lysodeikticus. J. Bacterio!. 80:207.
102. Chorpenning, F. W., and M. C. Dodd. 1966. Heterogenic antigens of
gram-positive bacteria. J. Bacterio!. 91:1440.
103. Hewett, M. J., K. W. Knox, and A. J. Wicken. 19 70. Studies o: the
group F antigen of 1 nctobarillus: Detection of antibodies \v
hemagglutination. J. Gen. Microbiol. 60:315.
104. Zabriski, J. B. 1967. Mimetic relationships between group A strep
tococci and mammalian tissues. Adv. Immunol. 7:147.
105. Click, A. L., R. A. Getnick, and R. M. Cole. 1971. Electron
microscopy of group A streptococci after phagocytosis by human
monocytes, infect. Immun. 4:772.
106. Berrens, L. and C. L. 11. Guikers. 1972. An immunochemical study
of pigeon-breeder's disease. Int. Arch. Allergy. 4_3: 347.
107. Knox. K. W., and A. J. Wicken. 1973. immunological properties
of teicboic acids. Bacterio!.. Rev. 37:215.
103. Baddilcy, J. 1970. Structure., biosynthesis, and function of
teichoic acids. Account. Chem. Res. 5:98.
109. Rictshel, I'. Tii. II. Got tort, O. huderitz, and 0. Westphal. |972.
Nature and linkage of the fatty acids present in the lipid-A
component of salmonella 1 i popo 1ysaccharide. Fur. .1. Biochen. 28:16b.
110. Wicken, A. .1., and K. W. Knox. 1970. Studies on the group F anti
gens of 1actobaci!1i: Isolation of a teichoic acid-lipid complex
from Kactobaci11 us ferment l. NTGG 6991. .1. Gen Microbiol. 60:293.


RECIPROCAL OF
ANTI-LTA DILUTION (x 10
CM
imlr
0 % PHA
-50 % PHA
100 % PHA
CELL INTERMEDIATES TREATED
WITH LTAcx (125fg/ ml)


Figure 4. Complement component titration of whole human .era
after treatment with LTAcx. The sera were incubated
with the LTAcx (500 ug/ml) then titrated for resi
dual activity of the components indicated as describe
in Materials and Methods.


Figure 22. Immunoelectrophoresis of human Cls and Cls.
Well A and D contained purified Cls; well B
contained purified Cls; well C contained
whole human sera. Trough 1 contained anti
Cls (Cls) ; troughs 2 and 3 contained a mix
ture of 7 5% anti whole human sera and 25%
anti Cls (Cls).


substrate for C42, Cr> is the only known substrate lair C423.
Once C5 is cleaved into C5a and C,5h, ('.5a is, released in the fluid
phase and C5b transiently acquires the ability to hind one molecule
each of C.6 and C7 (52,53). With this, a sol f-assemblv process is .ini
tiated and results, without any further enzymatic activity, in the form
ation of the stable C5b~9 complex (54). It should he noted that the
small by-product fragments C3a and C5a are endowed with marked phlogo-
genic activity (55,56,57). Some of these activities include release of
histamine from mast cells, contraction of smooth muscle tissue, directed
chemotaxis of polymorphonuclear leukocytes,and vasodilation both in con
junction and independent of histamine activity (58). Such potent pharma
cological activities obviously play a major role in the normal course
of the inflammatory response. i
Once the C5b67 complex is formed, it too can bind nonspec.ificallv
to areas on the membrane other than at the location of the C5 convertase
(52). The trimolecular association of 0567 provides the molecular arrange
ment for the adsorptive binding of one molecule of 08 which in turn pro
vides a binding region for up to six molecules of Oh (34). A Low grade
lesion of the target membrane occurs with only the addition of 08 to the
complex (59); but with the. binding of 09, a ten component mao romo I ecu 1 ar
complex is formed which greatly enhances the rate of target' cell ovto-
lysis (54). it should lv noted that the C5bb7 complex or even the Olhu/
complex can attach to non-sons i t i zed "innocent by-stander" cells and thus
promote' a terminal cytolytic event. This phenomenon has been termed
"reactive lysis" (60) and is controlled by the rapid decay of the unbound
comp lex (61,62).
Tlie precise mechanism hv which complement mediates cytelysis of


Figure 14.
Simultaneous removal of salt and propanol free LTAosx
by LH-20 gel chromatography. Symbols: (*) absor
bance; (o) Volurne/test tube: ( + ) Antigenicity as deter
mined by PHA; (Shaded Area) relative degree of precipi
tation of salt and other low molecular weight materials
as determined by AgNO test.


Figure .17. Kffec.t of f.TApcx on the complement mediated 1vsis of
various cellular complement component intermediates.


INHIBITION OF HUMAN COMPLEMENT BY EXTRACELLULAR
LIPOTEICHOIC ACID FROM STREPTOCOCCUS MUTANS 1U1T
By
LOUIS JOSEPH SILVESTRT
A DISSERTATION PRESENTED TO THE
GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DECREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
10 7 7


TABLE 9
Effect of LTApcx on the Ability of Cls to consume C4 and C2 Activity
Exp t.
Group
DGVB
Cls
C4
C2
LTApcx
(50 Mg/ml)
LTApcx
200 pg/ml)
Residuu L
C4 or C2
Activity
Consumption of
C4 or C2
Activity
Pi
Ml
Ml
Ml
Ml
Ml
SFU/mia
%
la
100
100
100
9
6.2 X 10
86
b
100
100
100
9
7.1 X 10
84
c
100
100
100
8.6 X 109
81
d
100
100
100
4.3 X 1010
4
e
200
100
4.5 X 10i0
b
NA
2a
100
100
100
9
6.5 X 10
93
b
100
100
1.00
9
9.1 X 10
90
c
100
100
100
O
o
1
X
r-4
r|
87
a
100
100
100
9.4 X 1010
0
j
200
100
9.2 X 101U
NA
Site forming unit.
b
Not applicable
o


and LTApcx were mixed and
volumes of Cls, functional Lv pure C4 or 02,
incubated at 37/5 minutes. The incubation mixture was then serially
diluted in DCVB, and the residual titers of 04 or C2 were determined
and compared against a buffer treated control. As shown in. Table 9. no
appreciable difference in Cls activity can be observed when even 200 pg/ml
of LTApcx were used. Also note that LTAprx preincubated with either C.4
(Expt. Group 2d) had no significant effect on residual activity.
Effect of LTApcx on the ability of Cls to hydrolyze TAlie. The
ability of Cls to hydrolyze the synthetic substrate TAMe is another index
of Cls activity. As can be seen in Figure 23, essentially no inhibition
of Cls activity was observed when Cls is preincubated with LTApcx and
TAMe.
Effect of LTApcx on the ability of Clq to bind to target cells.
Since Clq is the recognition unit of the classical pathway of complement,
any alternation in its ability to react with the antigen-antibody com
plexes on the surface of EA would have profound affects on the ability
of complement to lyse those cells. Therefore, equal volumes of puri
fied human Clq and LTApcx (10 pg/ml) were pro incuba ted at 30/1.5 minutes.
After preincubation, Clq was serially diluted in DCVB and (Mr and Cls
reagents were added. Hemolysis was then developed as described (Materi
als and Methods). Again there was no apparent inhibition (results not
shown) of activity. The major criticism of this experiment is that the
LTApcx concentration list'd to pre incubate with Clq is 10-fold less than
what was normally used in fluid phase inaetLvations. Ihe reason for the
use of this lower conoentration was to insure that the LTA would ho
sufficiently diluted at the time of EA addition. If significant amounts
of t.TA were present in the incubation mixture, EA( ^ would form, thus
generating a false positive inhibition duo to the refractory nature of


Lipid A of IPS 008,109) and since the unique sugars and hydroxyacy!
esters of Lipid A are absent in LTA, it is not surprising that associated
activities are absent as well. As a class, teichoic and lipoteichoic
acids are wail and membrane components of gram positive bacteria (107,108).
LTA is typically membrane associated and consists of a glycolipid cova
lently linked to a polyg1ycerolphosphate backbone which may carry carbo
hydrate and D-alanine substituents (2). Teichoic acids (TA), however,
are never associated with cell membranes; they lack the terminal givco-
lipid coupling, and they may have a backbone of either polyelvcerol-
phosphate or polyribitol phosphate (2). LTA may be converted function
ally to polyglycerol TA by spontaneous dencvlntion in an aqueous environ
ment, or mild alkaline, or acidic hydrolysis (L07). The molecular
weight of LTA (93) is probably between 3000-12000 hut because of its
tendency to form micelles in an aqueous environment, the apparent mole
cular weight as determined by gel. filtration is approximately four mil-
2
lion (110). Because LTA possess the glycolipid moiety, they are amphi-
pathic molecules exhibiting a propensity to spontaneously associate with
proteins and biological membranes (103). Mammalian red blood cells can be
"coated" by spontaneous adsorbtion with an LTA containing extract and the
cells can subsequently he agglutinated with an anti-LTA serum. Passive
hemagglutination (1HA) performed in this manner with sheep red blood
colls lias previously been reported bv manv invest igators who discovered
' Personal oQjnmun Lea t ions from R. Craig, Dept. of Ml'S, Cniv. of PI.; K.
Knox and A. J. Wickcn, Institute for Dental Research, Sydney, Australia;
and personal unpublished data.
>
Data supported bv personal experience (see Figures 11 and 12), and
personal communication from R. Craig.


released. Cl is not: attached to the membrane at all, hut rather is
combined with the Fc region of the hemolysin antibody. Therefore, this
model would predict that either Cl is released from the antigen-anti
body complex (very much akin to the predictions and shortcomings of
model one) or that the entire antibody-Cl complex is released from the
cell membrane (with or without the accompanying antigen). Such a
mechanism is somewhat exotic, but not totally improbable. Recent evi
dence suggests that the binding of serum albumin, immunoglobulins, or
complement can effect a release of phospholipids from liposomes (160.
161,162). Perhaps the attachment of LTA can likewise evoke such a
release of cell membrane constituents and in the process, release the
131
immune complexes as well. Experiments utilizing f labelled hemolvsin
antibody would demonstrate, whether the antihodv was maintained on the
131
cell surface or released into the medium. Likewise, I labe Lied Cl
could be used to determine if Cl were released.
Of ail the proposed models, number five most 1ikelv portrays the
actual mechanism of inhibition. This model assorts that the affixation
of LTA to the surface of the cell delays or prevents the rapid associa
tion of C4b (or C2a) with its respective site on (lie ceil membrane. As
previously discussed, once C4 is cleaved bv Cl, the cleavage results in
the formation of a short-lived binding site on the C4h fragment. A high
density of LTA on the surface of the cell might sequester C4h f inding
sites or perhaps change the electrostat to charge of the cell surface
sufficiently to effect the kinetics of the C4b attachment. The end
result in either case would bo the nonproduct ivo consuinpt ion of C4
molecules. This is consistent with the results from the residual C4
titration studies in which no alteration of C4 consumption was observed


igure 3.
Titration of C3 in whole human serum after treatment
with LTAcx. SvmhoJs: (*) Non-treated control; (<')
Serum treated with LTAcx at a concentration of 230
yg/ml. After incubation, sera were titrated for
residual C, 3 activity according to procedures
described in Materials and Methods.


TABLE 3
II. Results from Partial Purification of LTAa
Samp le
A 2 60
. b, ,
Protein (mg)
Amount of LTA inL
Sample (mg)
Weight of
Sample (mg)
Dialyzed, Non-Inoculated
Todd-Hewitt Broth
3.7xl05
6.5xl03
O
O
3.3x10
Supernate from Inoculated
but Ron-fractionated Broth
3.6xlOJ>
6.7xl03
9.1
ND
PTGC Retntate Fraction
of Supernate (LTAcx)
1.25x103
9.5xl02
11.0
2.3x10
Peak II from A5M After
Desalting (LTAppx)
1.7 6x10^
6.0
8.2
3.6x10
Unless otherwise indicated, all data are expressed in the units indicated and represent values extrapolated
back to the undilute sample times -total volume.
Values determined
by Bio Rad Protein Assay.
These values were calculated by determining the minimal concentration of purified LTA that can still be
detected by PHAg. Equating this value with the PHAg end point for all other LTA sources, the hypothetical
LTA concentration in the starting well can be calculated by serial twofold interpolations.


1.). phLogogenic activity mediated via complement reaction hv-products
2). increased opsonic susrept ib i 1 i ty of foreign substances
3). irreversible physinchemieal membrane damageand ultimately, evto-
lysisof susceptab]e target cells. Although the importance of comple
ment as a component of the host defense system has been suspected for
quite some time, only recently has its biomedical significance been
firmly established. Indeed, the participation of complement in host
resistance to infections and in several disease mechanisms is a topic
which has generated considerable research interest in recent years (21,22).
The classical pathway of complement contains eleven discrete glyco
proteins representing nine distinct components referred to sequentially
as Cl through C9. Cl is actually a multimolecular complex of three dis
tinct proteins (Clq, (Hr, and Cls) and the aggregate is held together hv
the divalent calcium ions (23). Removal of calcium ions hv chelating
agents such as ethylened iam inetetraaoot ic acid (IIDTA) results in the dis-
association of Cl into its subcomponents with concomitant loss of activity
(24). Activation of the classical pathway is characterized hv a depen
dence on IgG or igM antibodies complexed with antigens. The classical
pathway also specifically requires the components Cl, C.2, and C4 as well
as the divalent cations calcium and magnesium. Although the components
(13, and C5 through (19 are usually considered part of the classical system,
they are shared by the alternative pathway and thus are not considered as
unique components of the c lass lea 1 system per so.
The recognition and ¡nit ¡at ion funct Ion with respect to immuno
globulins resides with the Clq subcomponent (23,2b). Clq itself is a
rather peculiar protein consisting of three different polypeptide chains
(27). Chetni ea l l v, Clq contains .approximately 1OT carbohydrate, 32


TABLE 6
Percent:
Recover/
a
of
LTA from Various
Steps of PCV Purification
LTA Source
Reciprocal of
Initial Dilution
Initial
Concentration
of LTA (yg/ml)
Total Volume
of Sample (ml)
Calculated Total0
Weight of LTA
in Sample (mg)
Percent Recovery
of LTA
LTAppx
600
0.500
6.0
o
co
100.0
Decant
5
0.125
48.0
0.03
1.6
1st Wash
1
0.000
48.0
0.00
0.0
2nd Wash
1
0.000
O
co
' 0.00
0.0
3rd Wash
1
0.000
48.0
0.00
0.0
LTAncx
1000
1.000
1.5
1.50
83.3
As determined by ?HAg
D Determined by methods described in Table 3
u Calculated by multiplying the corresponding values of the first 3 columns


LTST OF TARI,ES
TABLE page
1. Partial Purification of LIA by A5-M
Gel Filtration 57
2. I. Results from Partial Purification
of LTA A2
3. II. Results from Partial Purification
of LTA A3
4. Percent Recovery of LTA During Octyl
Sepharose Purification 70
_ 14
5- Distribution of C-Phosphatidv1 Choline
During PCV Purification of LTA 72
6. Percent Recovery of LTA from Various
Steps of PCV Purification 73
7. Summarized Chemical Composition of Various
I.TA Containing Sources 74
8. Specific Activity Determinations of Purified
LTA 7 9
9. Effect of LTApex on the Ability of C.Ts to
Consume C4 and C2 Activitv 7
10. Comparison of the Relative Numbers of
Effective Cl Molecules Capable of Transfer
from EAC1 Treated with LTApex K>1
11. The inhibition of EA Lysis by I.ipoteiehoir
Acids f rom Several Bari erial Sources K'7
v


Figure 1. Titration of whole human complement after incubatLo
with crude extracellular lipoteichoic acid (LTAex).
Symbols: (o) Non-treatcd control: () Serum treated
with LTAex at 500 tig/ml .


A 220
TEST TUBE NUMBER
VOLUME / TEST TUBE


LITERATURE CITED
1. Wicken, A. J., and K. W. Knox. 1977. Hicrobiology 1977 Biological
properties of lipoteichoic acids. American Society for Micro
biology, Washington, D. C. p 360.
2. Wicken, A. J., and K. W. Knox. 1975. Lipoteichoic acidsA new
class of bacterial antigens. Science. 187:1161.
3. Gewurz, H., H. S. Shin, and S. E. Mergenhagen. 1963. Interactions
of the complement system with endotoxJc 1ipopolysaccharides:
Consumption of each of the six terminal complement components.
J. Exp. Med. 1_28:1049.
4. Gewurz, H., S. E. Mergenhagen, A. Nowotny, and .1. K. Phillips. 1963.
Interactions of the complement systems with native and chemically
modified endotoxins. J. Bacteriol. 95:397.
5. Marcus, R. L., II. S. Shin, and M. M. Mayer. 1971. An alternate
complement pathway: C3 cleaving activity not due to C4, 2a on
endotoxic lipopolysaccharide after treatment with guinea nig
serum. Relation to properdin. Proc. Natl. Acad. Sei. U. S. A.
68:1351.
6. Phillips, J. K., R. Snyderman, and S. E. Mergenhagen. 172. Acti
vation of complement by endotoxin: A role for globulin, Cl, C4,
and C2 in the consumption of terminal complement components by
endotoxin coated erythrocytes. J. Immunol,. 109: 334.
7. Mergenhagen, S. E., R. Snyderman, and .1. K. Phillips. 1973. Acti
vation of complement by endotoxin. ,1. Infect. Dis. 128:386.
3. El Imn, I., I. Green, and M. Frank. 1970. Genetic controlled total
deficiency of the fourth component of complement in the guinea
p i g.. Sei once. 1 20: 74 .
9. Hu is in1 '1 Veld, J. II. .1., and 1.. Barrens. 1976. Inactivation of
hcmolyt ic complement in human serum hv an acvlated po 1 ysaecharido
frem a gram-positive3 rod: Possible significance in pineon-breeder's
disease. Infect. Immunity. 13:1,619.
10. Gonco, R. ,J. P. A. Mashirno, (.. Prvgier, and S. A. Ellison. 1974.
Antibody mediated effects on the periodontium. .1. Periodontol Res.
4_5 (part II) : 330.
1L. Mergenhagen, S. E.,T. K. Tempe!, and R. Snvderman. ¡970. Immunolo
gic reactions and periodontal inflammation. .1. Dent. Res. 49:256.
1,20


referred Lo as C3b inactivator and cleaves both soluble and cell bound
C3b into two antigeniral1y distinct fragments, C3c and C3d (70). As a
result, 0423 loses C5 convertaso activity, and C3b activation of both
tIte alternative pathway and the immune adherence phenomenon is abolished
(71,72,73). This latter activity can be visualized by the clustering of
cells bearing C3b on their surface around other cells displaying C3b
receptors. Such receptors have been shown to be present on human ervthro-
cytes, polymorphonuclear leukocytes, platelets, macrophages, and on u
lymphocytes (74,75). The attachment of C3b not only plays a direct role
in the increased opsonization of target cells (76), but C3b binding to B
lymphocytes has been postulated to play a role in B-cell activation as
well (77).
The second pathway by which complement may be activated is referred
to as the alternative or properdin pathwav. Historically, the existence
of this pathway had been suggested as early as 1954. At that, time,
Pillemer and his associates reported the discovery of a new protein in
normal human sera (78). Properdin, as it was called, was capable of
reacting nnn-specifically with diverse naturally occurring po 1 ysaccharides
and 1ipopo Iysaccharides ultimately resulting in the activation of comple
ment. This process ostensibly occurred without the interaction of anti
body and was proposed as a major pathway bv which susceptible bacteria
and viruses were destroyed. However, this provocative hypothesis was
discarded as apocryphal and the described activities wore attributed to
tile presence of natural antibodies (79). The controversy remained un
resolved until recent years when rigorous immunorhemienl techniques wore
employed in the isolation, purification, and determination of function
of many of these components. i'lie unanticipated complexity of the properdin


assume that the values given for the final products are at least a
close indication of the total percent protein available in each product.
Although the values may seem high, it should he remembered that (1) the
total amount of protein available in the sample represents a lower limit
for the accuracy of the assay, and (2) the standard protein curve
(human albumin) used to convert optical density readings to pg of pro
tein may not accurately correlate the reactions of the limited number
of amino acid residues available in the final product. Attempts to
verify these values with the biuret reaction (153) and the I.owrv Pro
tein Assay (154) were unsuccessful. Biuret was too insensitive whereas
the Lowry proved to be unreliable due to its reaction with glycerol to
give a false positive reaction. Despite this shortcoming all other
factors indicate that l.TApcx represents the most highly purified LIA
from _S. mu tans BHT that any laboratory has yet achieved.
The results from the final experiments to determine the site and
mechanism of complement inhibition by LTA were equivocal. Like the
purification of LTA, establishing the site and mechanism of inhibition
proved to be considerably more challenging than anticipated. Prelimin
ary data utilizing LTAcx quite consistently suggested that Cl was the
site of action and interference with binding affinity (Clq dysfunction)
or with esterase activity (Cls dysfunction) was the mechanism. These
conclusions were based on the fact that !',ACl4irt,. hut not F.ACJ42 were
inhibited and also that the titer of fluid phase CL preincubuted with
LTAcx was significantly reduced. Considering the pnlvanion ic nature of
LTA conferred hv the polar po 1 yglycero!. phosphate backbone, it appeared
that LTA represented a model system for polvanionic interference of Cl
funct ion.
Such activity hits been ascribed to dexcran sulfate polyvinyl


ester (IMS) derivatives ns described in Material and Methods.
An inter
nal mannitol standard is included with all samples. The I/IAppx chroma
tograph represents the typical carbohydrate profile achieved with
partially purified LTA. The tracings for LTAosx and LTAppx contrast
the qualitative and quantitative differences in carbohydrate content.
i
The second two chromatograms ,deacylated cardiolipin (f. P^) and cardio
lipin, were included as a comparison of how a naked polyglycerol phos
phate backbone might be expected to react under the described conditions.
The base line instability of the G.^P^ looks remarkably similar to the
profile of the purified LTApcx, The procedure for purifying deacylated
cardiolipin requires passage through Sephadex columns. It is quite con
ceivable that the minute quantities of unidentified carbohydrates which
are indicated may be due to dextran contamination from the column.
However, it would be difficult to account for the same source of contam
ination for the LTApcx since gel chromatography was not used in the final
purification. On the other hand, the similarity of the indicated chroma
togram tracings may be more than mere coincidence and may reflect actual
reactions of the derivatizing agent with the polyglycerol phosphate
backbone. This latter hypothesis is supported by the fact that an
unidentified trailing "carbohydrate" peak of significant mass appears
in both the cardiolipin and G P0 chromatographs. The Rf value of this
peak is similar (but yet suspiciously disparate) to the retention tine
normally observed for 8-glucose. However, if indeed this peak docs
represent 8-glucose, one is hard pressed to rationalize why a corres
ponding -glucose peak does not occur as well. In either case, it is
Deacylated cardiolipin was prepared bv the method of Wilkinson (14D)
and was kindly provided bv R. Craig, University of Florida.


Figure Hi. Passive hemagglutination (PIIA) titration and inhibition
of complement mediated lysis of FA treated with varying
concentrations of TifApc.x.


LIST OF FIGURES
FIGURE
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
PAGE
Titration of whole human complement
after incubation with crude extra
cellular lipoteichoic acid (LTAcx) 33
Dose response inhibition of whole
human complement after incubation
with varying concentrations of LTAcx 35
Titration of C3 in whole human serum
after treatment with LTAcx 38
Complement component titration of
whole human sera after treatment
with LTAcx 4D
Inhibition of complement mediated
lysis of FA treated with varying
concentrations of LTAcx 4 3
Passive hemagglutination (FHA) of F.A
treated with varying concentrations
of LTAcx
Effects of LTAcx treatment on the
lysis of various complement component
intermediates 48
PUA of various LTAcx treated complement
component intermediates 30
Effect of LTAcx treatment on the ivsis
ol MAC 142
Effect of LTAcx on hemolytic antibody
t i t rat ion 3 3
Partial purification of l.TA hv A3-M
gel filtration 39
Partial purification of LTA by A*5M
gel filtration with 1TA enriched
starting material M
v 1


L 2
69. Pensky, .1. L. R. levy, and I. H. I.i'pnw. 1961. Partial 'unification
of a serum inhibitor of C1 esterase. .1. Biol. Chem. 236:1674.
70. Ruddy, S., and K. F. Austen. 1971. C3b inactivator of nan. .
Immunol. 107:742.
71. Miii ler-Eberhard, II. .1., and 0. Gcitze. 1972. Cl proactivator con-
vertase and its mode of action. J. F.xp. Med. 135:1003.
72. Alper, C. A., F. S. Rosen, and P. J. i.achmann. 1972. Inactivator
of the third component of complement as an inhibitor in the
properdin pathway. Proc. Nat. Acad. Sci. U. S. A. 69:2910.
73. Tamura, N., and R. A. Nelson, Jr. 1967. Three naturally occurring
Inhibitors of components of complement in guinea pie and rabbit
serum. J. Immunol. 99:582.
74. Henson, P. M. 1969. The adherence of leukocytes and platelets in
duced by fixed IgG antibody or com]') 1 ement. Tmmunologv. 16:107.
75. Lay, W. II., and V. Nussenzweig. 1968. Receptors for complement on
leukocytes. J. Exp. Med. 128:991.
76. Henson, P. M. 1972. Complement-dependent adherence of cells to
antigen and antibody. Mechanisms and consequences. Bi o 1 og i c a1
Activities of Complement. Karger and Basel, N. Y., N. Y. p 173.
77. Dukor, P., and K. U. Hartmann. 1973. Itvpothesis--Bound Cl as the
second signal for B-cel1 activation. Cell Immunol. 7:3*9.
78. Plllemer, L., L. Blum, T. H. Lepow, O. A. Ross, I'. W. 1 odd, and
A. C. Ward law. 1954. The properdin system and immunity: Demon
stration and isolation of a new serum protein, properdin, and
its role in immune phenomena. Science. JL20:279.
79. Nelson, R. A., Jr. 1958. An alternative mechanism for the properdin
system. J. Exp. Med. 108:515.
80. Pillemer, L., M. D. Schoenberg, L. Blum, and L. Wur z 1 55. Proper
din system and immunity. II. Interaction of the properdin svstem
with polysaccharides. Science. 122:543.
81. Fine, D. P. 1974. Activation of the classical and alternate path
ways of endotoxin. J. Immunol. 112:2.
82. Sehreiher, R. D. I!. C. Medians, C. Col/.e, and II. J. Miiller-Fherli.ini
1975. Properdin-and nephritic factor-dependent C3 convertases:
Requirement of native C3 for enzyme formation and the function of
bound C3G as properdin receptor. J. Exp. Med. I't2:76c.
83. Giitze, 0., and H. J. MM 11 or-Eherhard. |971. The (13-activator svstem:
An alternate pathway of complement activation. J. Exp. Med. 134:90


INHIBITION OF HUMAN COMPLEMENT BY EXTRACELLULAR
LIPOTEICHOIC ACID FROM STREPTOCOCCUS MUTANS 1U1T
By
LOUIS JOSEPH SILVESTRT
A DISSERTATION PRESENTED TO THE
GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DECREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
10 7 7

ACKNOWLEDGEMENTS
In all sincerity, no occasion or project thus far undertaken has had
a more humbling effect on my life than the completion of this Ph.D.disser
tation. It is unfortunate that only now in retrospect can I clearly see the
tremendous debt I owe for the help, patience, understanding, and knowledge
so generously contributed by my mentor, my friends and wife.
If I were to formally thank everyone who contributed in someway to the
successful completion of my Ph.D. dissertation, it is likely that my acknow
ledgements would read like the listings of a telephone hook. I do however
feel compelled to thank a few very special people.
First of all I would like to thank my mentor and friend, Dr. E.M.
Hoffmann. Ed possesses the rare ability to earn respect rather than having
to demand it. His tolerance for my idiosyncrasies was unlimited (almost).
He gave me direction yet left me with alternatives; he challenged my intellect
yet never made me feel ignorant; he provided the foundation on which 1 am
still building my scientific character. Most of all, lie was (and is) a friend
I would also like to thank the. members of our laboratory "family"
(Suzanne, Jean, Bert and Torn) for their help, patience and tolerance
during these difficult last clays. One cannot help but reflect upon the many
events that shape the complex web of friendships within the laboratory.
All of you will always he regarded as the closest, of triends.
1 wish to express mv gratitude to Ron Craig not only for his friend
ship, but also for the professional technical assistance that lie afforded.
I would also like to express mv appreciation to the employees of leach
ing Resources, for dedication above and beyond the call of duty. I would

especially like to acknowledge the professional artistic assistance of
Margie Summers, Margie Niblack and John Knaub.
I would like to thank Steve Hurst for being the.ro when T needed a
friend and for helping with some of the last minute photography.
The typist, Joanne Hall, deserves a particularly special mention.
If not for her personal concern and dedication the deadlines would never
have been met. She worked on this dissertation under conditions for whi
no degree of monetary reinbursement could possibly compensate. I thank
you Joanne and I sincerely hope you never have to go through that again!
Finally, and most importantly, I wish to thank my wife Lyn for her
infinite patience and encouragement. Her attitudes, her ideals, her
"being" is so much a part of me that it would be hopelessly futile to
list all the things for which X am indebted to her. She is a friend
and lover, a typist and an occasional laboratory technician. She is my
driving force in life and rightfully so, I dedicate this dissertation
to her.
" And leitk one loud ivotraieoriaieoi'iaierrraieo'i'ia he jumped at the
end 0^ the tablecloth, pulled it to the ground, mapped himself] up ti; it
three times, 'wiled to the other end of the loom, and aftci a ter'iiblc
struggle got lids head into dai/light again and said cheerfully
-- have 1 Hien?"
(tom "The IlmiSe at Pee/i Co'iml" bit A. A. M(fne)
i i t

TABLE OF CONTENTS
PAGE
ACKNOWLEDGMENTS i i
LIST OF TABLES v
LIST OF FIGURES vi
GLOSSARY OF ABBREVIATIONS viii
ABSTRACT ix
INTRODUCTION 1
MATERIALS AND METHODS 17
RESULTS 11
DISCUSSION IOS
LITERATURE CITED 120
BIOGRAPHICAL SKETCH 132
i v

LTST OF TARI,ES
TABLE page
1. Partial Purification of LIA by A5-M
Gel Filtration 57
2. I. Results from Partial Purification
of LTA A2
3. II. Results from Partial Purification
of LTA A3
4. Percent Recovery of LTA During Octyl
Sepharose Purification 70
_ 14
5- Distribution of C-Phosphatidv1 Choline
During PCV Purification of LTA 72
6. Percent Recovery of LTA from Various
Steps of PCV Purification 73
7. Summarized Chemical Composition of Various
I.TA Containing Sources 74
8. Specific Activity Determinations of Purified
LTA 7 9
9. Effect of LTApex on the Ability of C.Ts to
Consume C4 and C2 Activitv 7
10. Comparison of the Relative Numbers of
Effective Cl Molecules Capable of Transfer
from EAC1 Treated with LTApex K>1
11. The inhibition of EA Lysis by I.ipoteiehoir
Acids f rom Several Bari erial Sources K'7
v

LIST OF FIGURES
FIGURE
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
PAGE
Titration of whole human complement
after incubation with crude extra
cellular lipoteichoic acid (LTAcx) 33
Dose response inhibition of whole
human complement after incubation
with varying concentrations of LTAcx 35
Titration of C3 in whole human serum
after treatment with LTAcx 38
Complement component titration of
whole human sera after treatment
with LTAcx 4D
Inhibition of complement mediated
lysis of FA treated with varying
concentrations of LTAcx 4 3
Passive hemagglutination (FHA) of F.A
treated with varying concentrations
of LTAcx
Effects of LTAcx treatment on the
lysis of various complement component
intermediates 48
PUA of various LTAcx treated complement
component intermediates 30
Effect of LTAcx treatment on the ivsis
ol MAC 142
Effect of LTAcx on hemolytic antibody
t i t rat ion 3 3
Partial purification of l.TA hv A3-M
gel filtration 39
Partial purification of LTA by A*5M
gel filtration with 1TA enriched
starting material M
v 1

13.
Purification of i.TA by Octyl Eoplmrose
hydrophobic ge! chromatography
14. Simultaneous removal of salt and propanol
from LTAosx by LH-20 gel chromatography 69
15. Carbohydrate analysis of ETA containing
preparations by gas liquid chromatography 7 6
16. Passive hemagglutination (PHA) titration
and inhibition of complement mediated lysis
of EA treated with varying concentrations
of LTApcx 81
17. Effect of ETApcx on the complement
mediated lysis of various cellular
complement component intermediates 83
18. Effect of LTAcx and LTApcx on function
ally purified human Cl. 86
19. Immunodiffusion and precipitation analysis
of various steps in the purification of
human Clq 88
20. Disc gel electrophoresis of purified
human Clq
21. DEAE elution profile of human CIs 93
22. Immunoelectrophoresis of human C.ls and
CIs 95
23. Effect of ETApcx on the ability of CIs
to hydrolize TAMe 99
24. Difference in complement mediated lytic
susceptabilitv of LTApcx treated EAC4 versus
EAC4 103

GLOSSARY OF ABBREVIATIONS
A:
Antibody
C:
Complement
Cl, C2
-C9:a Complement components. Horizontal bars
above the component designation denotes
a biologically active state.
CVF:
Cobra venom factor
E:
Erythrocyte
EDTA:
(Disodium) Ethylenediamine tetrnacetic acid
EGTA:
Ethy leneglyco1-b is (B Amino Ethyl Ether) N,N" tetra
acetic acid
LTAcx:
Crude extracellular lipoteichoic acid
LTApcx:
Extracellular LTA purified via phosphatidyl-choline
vesicle adsorbtion
LTAppx:
Partially purified extracellular LTA
LTAosx:
Extracellular LTA purified via Octyl Sepharose gel.
column adsorb Lon
IPS:
L i p o p o 1 y s a c e h a r i d e
PHA:
Passive hcmagglutination
PHAg:
Passive hemagglutination (modified method)
TA:
Teic.hoic acid
TAME:
p-Tosy1-1-arginine methylester
All comp 1 omen l nomom: 1 ;i tu rc follows the WHO recommetidaC intis
(Bull. Wld. Hlth. Org. 39:>39, 1963).
v i i i.

Abstract of Dissertation Presented to the
Graduate Council of the University of Florida
in Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy
INHIBITION OF HUMAN COMPLEMENT BY EXTRACELLULAR
LIPOTEICHOIC ACID FROM STREPTOCOCCUS MUTANS BHT
By
Louis Joseph Silvestri
December, 1977
Chairman: Edward M. Hoffmann
Major Department: Microbiology and Cell Science
A number of biological and chemical similarities exist between
the lipopolysaccharides (EPS) of gram negative microorganisms and the
lipoteichoic acids (LTA) of gram positive organisms. The potent
affects of LPS on the complement system are. well documented; however,
the effect of LTA on this host defense system has not been adequately
studied. Furthermore, all studies thus far conducted have been limited
to the interaction of LTA with whole fluid phase complement. Ln this
investigation it was demonstrated that extracellular LTA from the
cariogenie microorganism Strep tocoecus mutans BHT was not only capable
of spontaneously binding to sheep erythrocyte target cells hut was
also capable of rendering them refractory to complement mediated Ivsis.
Purification of the LTA to homogeneity was achieved by a combination of
gel filtration and adsorhtion to phospholipid choline vesicles (arti
ficial membranes). By utilizing various cellular complement component
intermediate complexes and functionally purified complement components,
experiments were conducted to define the site and mechanism of inhibition

by LTA. The site of inhibition was determined to occur between the
formation of the SAC1 and SAcl42 complex. Be cause Cl. is no longer
necessary after formation of the C'l convertase (SAC42), lack of inhi
bition after this step implies a direct effect on Cl activity. Although
experimental data derived from utilizing Cl, Clq, Cls, and Cls were
suggestive, data did not. unequivocally establish this as the precise
mechanism of inhibition. No evidence for fluid phase consumption of
hemolysin Ab, Cl, C4, or C2 by LTA could be demonstrated. Evidence for
the inhibitory activity of LTA from several unrelated genera is pre
sented and the possible role of LTA in periodontal disease is discussed.
x

INTRODUCTION
As reviewed hv Wicken and Knox (1,2), a number o!" chemical and
biological similarities exist between the. 1 ipopo Lysncchar ides (bPS) of
gram negative bacteria and the lipoteichoic ac.ids (TLA) of gram positive
organisms. Because of these similitudes our laboratory began to inves
tigate whether LTA possessed anttcomplemenlary activity analogous to
that associated with the LPS endotoxin (3-8). Although there have been
concentrated efforts to define the site and mechanism of LPS inhibition of
complement, very few investigators have reported data on the possible
effects of LTA on the complement system (9). This is somewhat surprising
since the interaction of LTA, LPS, and complement almost certainly play a
significant role in the etiology of periodontal diseases. Bacterial pro
ducts and serum components in the gingival crevices of the oral cavitv
have been shown to activate complement by both the classical (10,11) and
the alternative pathways (12,13). in fact recent evidence suggests that
bone loss (a major clinical manifestation of acute periodontal disease)
may occur via osteoclast activation due to the interaction of como lenient
and prostaglandin E (14). Prostaglandins are naturally occurring cyclized
derivatives of unsaturated long chain fatty acids (15) and their concen
trations are dramatically elevated in inflamed gingival tissues (16). It
is of interest to note, that both LTA and LPS are also capable of initiating
osteoclast mediated bone resorbt ion (17) and this activity proceeds with
out the contribution of complement or prostaglandins. The potential for
svnergism cannot be overlooked, and indeed LPS endotoxins have long boon
implicated as participants in the development of periodontal lesions (4-8).
Analogous LTA activity could he of significant clinical import especially
1 '

in light of the fact that gram positive bacteria represent the major
cellular constituent of dental plaque at fhe early stages of plaque
formation (18). Most of the gram positive organisms found in dental
plaque have been isolated, cultured, and identified. The production of
copious amounts of extracellular LTA by several of these organisms has
been well established (19,20). In fact, growing under conditions esti
mated to reflect the growth rate in the oral cavity, Wicken and Knox have
shown that the cariogenic bacterium Streptococcus mutans BUT produces
some eleven fold greater amount of extracellular LTA in the culture fluid
than that contained within the cells themselves (1,2). Therefore, if an
effect on complement by LTA can be demonstrated in vitro, an in vivo
model can be readily envisioned. Preliminary experimentation with a
crude LTA containing extract from S. mutans BUT did indeed indicate that
complement activity was consumed. However, consumption or alteration
of complement activity can he due to a number of specific or non-specific
factors. Because of the complexity of this system, a thorough under
standing of the possible interactions is necessary before anv model
attempting to define a site and mechanism of inhibition can he elucidated.
The complement system of vertebrates is comprised of at least-
eighteen discrete plasma proteins capable of interacting in a specific
and sequential fashion. There are two pathways by which this biochem
ical cascade may he initiated and they arc referred to as t lie classical
and the alternative pathways of complement activation. However, regard
less of how the activation scheme is initiated, the biological consequen
ces of activation are the same for both pathways:
1
Silvestri et al. 1.97b. Abst. Ann. Meeting, ASM, p77.

1.). phLogogenic activity mediated via complement reaction hv-products
2). increased opsonic susrept ib i 1 i ty of foreign substances
3). irreversible physinchemieal membrane damageand ultimately, evto-
lysisof susceptab]e target cells. Although the importance of comple
ment as a component of the host defense system has been suspected for
quite some time, only recently has its biomedical significance been
firmly established. Indeed, the participation of complement in host
resistance to infections and in several disease mechanisms is a topic
which has generated considerable research interest in recent years (21,22).
The classical pathway of complement contains eleven discrete glyco
proteins representing nine distinct components referred to sequentially
as Cl through C9. Cl is actually a multimolecular complex of three dis
tinct proteins (Clq, (Hr, and Cls) and the aggregate is held together hv
the divalent calcium ions (23). Removal of calcium ions hv chelating
agents such as ethylened iam inetetraaoot ic acid (IIDTA) results in the dis-
association of Cl into its subcomponents with concomitant loss of activity
(24). Activation of the classical pathway is characterized hv a depen
dence on IgG or igM antibodies complexed with antigens. The classical
pathway also specifically requires the components Cl, C.2, and C4 as well
as the divalent cations calcium and magnesium. Although the components
(13, and C5 through (19 are usually considered part of the classical system,
they are shared by the alternative pathway and thus are not considered as
unique components of the c lass lea 1 system per so.
The recognition and ¡nit ¡at ion funct Ion with respect to immuno
globulins resides with the Clq subcomponent (23,2b). Clq itself is a
rather peculiar protein consisting of three different polypeptide chains
(27). Chetni ea l l v, Clq contains .approximately 1OT carbohydrate, 32

hydroxyprol ino, 27 hydroxy lysine and 13" glycine. This unusual col Ingyn-
like composition makes it unlike any plasma protein yet described (28,29).
When complement is activated by ant ihody-antigen complexes such as
exists on the surface of an antibody sensitized erythrocyte (EA), it
undergoes a self assembly process sequentially depositing the entire
fluid phase cascade onto the surface of the target. Specifically, Clq
recognizes a previously sequestered binding site located in the Fc frag
ment of IgG and IgM (30,31). The three polypeptide chains of Clq are
physically arranged in a manner perhaps analagous to a six headed mace
or bola with each "head" representing a binding site for an IgG molecule
(32). Thus each Clq molecule has six binding sites for IgG (and presum
ably the same number for IgM). Internal activation of Cl probably is the
result of a conformational change in Clq which in turn induces a change
in the proenzyme Clr (33). Once Clr is activated to Clr it is endowed
with enzymatic activity through which the proenzyme Cls is converted to
CL esterase (C is )(34,35 3f>) Cls is a serine esterase and is inhibited
by diisopropyIphosphofluoridate (DFP) (37). This esterase activity can
be used to hydrolyze the synthetic substrat es p-Tosy1 I argintne methv-
lester (TAMe) and N-nc.ety 1-1-tyros ine ethvlester (ATF.e) (38). Recently,
I.oos and Raepple have demonstrated that many polyanions were capable of
inhibiting the activity of Cl either In* in! orfering with Clq binding to
the antibody-antigen complex, or by preventing interaction of C4 and ('.2
with Cls (39,AO). Although binding of Cl usually leads to activation,
the two processes are not integral I t;C with modified trvptopiian (41)
and the human immunog1 obu 1 in subclass IgG4 (42)both bind Clq but do
not activate Cl.
After activation, Cls enzymatically cleaves C4 into a large (C4b)

and small fragment (C4a) (43). The cleavage of 04 exposes a membrane
attachment site on the C4b molecule, and it will attach to the nntihodv-
antigen complex at a site juxtaposed to the Cl.-antibodv complex (44,43).
Cls then cleaves 02 into C2a and 02b (46) with C2a attaching to the C.4b
site and C2b being released into the fluid phase. Thus, the molecular
complex C4b2a is formed and is referred to as C3 convertase because it
is capable of splitting and activating C3 (47,48). C3 convertase is
also an esterase, and although C3 is its natural substrate, it also
hydrolyzes the ester bond of acety1-glycl- lysine methyl ester (49). The
catalytic site of C3 convertase is believed to reside in the C2a sub
unit and even after release from the C4b complex, cytolvtically inactive
C2a retains esterase activity, but is no longer capable of cleaving C.3
(49). The enzymatic half-life of C4b2a is quite ephemeralonly 10
minutes at 37. However, if the 02 is first oxidized by treatment with
iodine (applicable to human but not guinea pig C2). not only is the
binding of C2a to C4b enhanced, but the half life of the binolecular
complex is increased 20 fold (50). No doubt the transient association
of C2a with the (¡42 and (¡423 complex plays a vital ro 1 e in controlling
the complement reaction hv temporarily limiting the functional associa
tion of these complex enzymes.
Once C3 is cleaved into 03a and 03b, the small 03a fragment is
released into the fluid phase and 03b becomes associated with the 04h2a
complex and with other non-hemolvtio sites on the largel membrane (47).
The association of 03b with the 03 convert .use modulates its activity .so
that now ('3 becomes the natural substrate of this ( r l.nnl ecu 1 nr complex.
The 0423b complex is referred to as 05 convertase (31) and like (.42, is
a highly sneoinLLzed protease.
lust as 0.3 is the only known protein

substrate for C42, Cr> is the only known substrate lair C423.
Once C5 is cleaved into C5a and C,5h, ('.5a is, released in the fluid
phase and C5b transiently acquires the ability to hind one molecule
each of C.6 and C7 (52,53). With this, a sol f-assemblv process is .ini
tiated and results, without any further enzymatic activity, in the form
ation of the stable C5b~9 complex (54). It should he noted that the
small by-product fragments C3a and C5a are endowed with marked phlogo-
genic activity (55,56,57). Some of these activities include release of
histamine from mast cells, contraction of smooth muscle tissue, directed
chemotaxis of polymorphonuclear leukocytes,and vasodilation both in con
junction and independent of histamine activity (58). Such potent pharma
cological activities obviously play a major role in the normal course
of the inflammatory response. i
Once the C5b67 complex is formed, it too can bind nonspec.ificallv
to areas on the membrane other than at the location of the C5 convertase
(52). The trimolecular association of 0567 provides the molecular arrange
ment for the adsorptive binding of one molecule of 08 which in turn pro
vides a binding region for up to six molecules of Oh (34). A Low grade
lesion of the target membrane occurs with only the addition of 08 to the
complex (59); but with the. binding of 09, a ten component mao romo I ecu 1 ar
complex is formed which greatly enhances the rate of target' cell ovto-
lysis (54). it should lv noted that the C5bb7 complex or even the Olhu/
complex can attach to non-sons i t i zed "innocent by-stander" cells and thus
promote' a terminal cytolytic event. This phenomenon has been termed
"reactive lysis" (60) and is controlled by the rapid decay of the unbound
comp lex (61,62).
Tlie precise mechanism hv which complement mediates cytelysis of

7
susceptible target cells is not clearlv understood. One hypothesis., in
light of the newly discovered trihut yrina.se act ivi.tv of 07, is that the
lytic event is caused by an enzymatic attack on the membrane (63). How
ever, no enzymatic degradation products have ever been discovered in
either lysed cell membranes or in ruptured synthetic lipid hi 1 avers (64).
The two most favored models are tin? "doughnut" insertion hypothesis (65)
and the C8 insertion model (29). The former model purports that the
C5b-9 complex inserts inself into the membrane as a "prefabricated hole"
allowing the exchange of inLra and extracellular material via an internal
hydrophilic channel (65). However, the. model fails to explain hew the
hydrophilic complement components enter the hydrophobic expanses of the
membrane. In addition, although electron microscopy has revealed apparent
ultrastructure doughnut shaped "lesions" on the surface of cells Ivsed by
complement (66), freeze etching techniques have shown that the ultra
structure alterations are confined to the outer loaf lot of the membrane,
i.e. the lesion does not penetrate the membrane (67). The C8 insertion
model embraces most of the salient features of the doughnut mode 1, but
in addition postulates that the u and y chains of C3 are inserted into
the channel formed by the surface macromo 1 ocular complex. The and y
chains thus extend into the membrane hi Inver causing disruption of orderly
structure.
In addition to the restraints placed on tho complement cascade due
to the rapid decay of several of the intermediates, there are two
naturally occurring inhibitors of complement present in the sera of man
and probablv in all vertebrates. The first inhibitor is referred to ts
Cls inhibitor and, as the name implies, it directly abrogates the hemo
lytic and esternlytie activity of hi (68,69). The second inhibitor is

referred Lo as C3b inactivator and cleaves both soluble and cell bound
C3b into two antigeniral1y distinct fragments, C3c and C3d (70). As a
result, 0423 loses C5 convertaso activity, and C3b activation of both
tIte alternative pathway and the immune adherence phenomenon is abolished
(71,72,73). This latter activity can be visualized by the clustering of
cells bearing C3b on their surface around other cells displaying C3b
receptors. Such receptors have been shown to be present on human ervthro-
cytes, polymorphonuclear leukocytes, platelets, macrophages, and on u
lymphocytes (74,75). The attachment of C3b not only plays a direct role
in the increased opsonization of target cells (76), but C3b binding to B
lymphocytes has been postulated to play a role in B-cell activation as
well (77).
The second pathway by which complement may be activated is referred
to as the alternative or properdin pathwav. Historically, the existence
of this pathway had been suggested as early as 1954. At that, time,
Pillemer and his associates reported the discovery of a new protein in
normal human sera (78). Properdin, as it was called, was capable of
reacting nnn-specifically with diverse naturally occurring po 1 ysaccharides
and 1ipopo Iysaccharides ultimately resulting in the activation of comple
ment. This process ostensibly occurred without the interaction of anti
body and was proposed as a major pathway bv which susceptible bacteria
and viruses were destroyed. However, this provocative hypothesis was
discarded as apocryphal and the described activities wore attributed to
tile presence of natural antibodies (79). The controversy remained un
resolved until recent years when rigorous immunorhemienl techniques wore
employed in the isolation, purification, and determination of function
of many of these components. i'lie unanticipated complexity of the properdin

system has spawned a multiplicity of models attempting to elucidate its
precise mode of initiation and function. Clearly, a plethrn of diverse
stimuli are capable of activating this pathway, and this fact alone im
poses a formidable constraint on any molecular model. Some of the more
common naturally occurring activators of the alternative pathway Include
bacterial and fungal cell wall constituents such as Iipopolysaccharide,
zymosan, and inulin (a poly fructose) (71,80-83). In addition, aggregates
of some immunoglobulin classes (84,85), some types of animal cell, mem
brane constituents (86,87), and antibody-coated budding virus infected
cells (88,89) also stimulate this pathway. The alternative pathway can
even be activated by substances of relatively defined chemical nature
such as benzyl-B-D-fruetopyranoside (90), polyglucose with repetitions
a 1-3 and branched a 1-6 linkages (91), d in itrophonylated albumin (92),
and many polyanionic substances. Cobra venom factor (a non-1ipolyt ic,
non-hemolytic glycoprotein isolated from the venom of the cobra Naga
naja) is also a potent activator of complement cytolytic potential, but
it appears to act as a C3b analog and is thus unique in its mode of
alternative pathway activation (93,94,95). Potentiation of this system
requires devalent magnesium ions and the interaction of at least five
novel serum proteins. By convention, the names of these proteins are IF
(or initiating factor), P or P (properdin). Factor B (C3 pnmit i vator) ,
Factor B (C3 activator), and Factor I) or 1) (C3 pronet i vator convurtaso).
To date, all of the above components have been isolated, purified, and
characterized ns to molecular weight, electrophoretic mobility, and sedi
mentation coefficients (83,96-98). CJb (of the classical pathway) plays
an intregal role in the alternative pathway (71,96,99), and thus it in
essence forms the junction point of the two systems. Because all terminal

components ((3, C5-9) are .shared, the biological consequences of acti
vation enc.ompass all the processes previously described (immune adherence
opsonic activity, anaphyIntoxin production, membrane attack complexes,
etc,.).
There are similarities between some of the more salient features
of the classical pathway compared with those of the alternative pathway.
Analogous to Clq, IF seems to function as the recognition unit for the
properdin pathway, but its relationship to another factor (referred to
as a C3 nephritic factor from the sera of patients with membranoprolifern
tive glomerulonephritis (1.00) and its mode of activation is poorly under
stood (96). Factor D is capable of enzymatically cleaving Factor B into
Ba and Bb (29,94). In the presence of C3h, a himolecular complex C3bBb
is formed (29) which is endowed with C3 splitting .activity similar to
the C3 convertase (C4h2a) of the classical pathway. Furthermore, just
as C4b anchors the classical convertase. to the membrane allowing C2a to
exert its enzymatic activity, so toe cytophilic C3h anchors the C3hBb
complex to the membrane allowing the enzymatic activity of Factor Bb to
be. expressed (83). Both complexes merelv gain additional C3b to modulate
05 cleaving activity (99). Thus, the presence of Cl nol oniv prevents an
"abort" due to rapid decay of either convertase, but because C3b is
utilized as part of the alternative pathway convertase, it participates
in a type of ampl ificat ion Loop. In other words, the more C3b that is
formed from either pathway, the more C3 leaving potential is endowed
upon the properdin C3 convertase. Froperdi.n (P) seems to stabilize the
fragile C 3 kill) complex but its possible rol' in stabilizing the classical
03 convertase has not been invest igated (Qr|). Noteworthy, however, is
the potent effect properdin exerts on tin* C3b inhibitor (99). By

modulating the action of tills enzyme, properdin at least indirectly
plays a role in stabilizing the classical pathway sequence.
The recognition of foreign substances by a host usually leads to
the neutralization and eradication of these substances by immune lympho
cytes, phagocytic cells, specific antibodies, complement, or an amalga
mation of these factors. However, in instances where antigenic substances
interact directly with host tissue, the reactions of the host's immuno
logical defense system could sometimes result in a considerable amount
of autodestruction. LTA represents a class of antigens that are capable
of spontaneous cytophilic binding to mammalian tissue (101,102,103).
As a result, host tissue acquires a new "ani tgenic. face" and may now
react with natural or induced antibodies to the LTA. Furthermore, anti
bodies directed primarily at LTA determinants may cross react with
similar determinants of the host's tissue. Such a mechanism has been
proposed for the high incidence of rheumatic fever and glomerulonephritis
in patients recovering from post streptococcal infections (104,103).
Recently, acylated heteropolysaccharides (LTA) isolated from the cell
membranes of several Lactobacillus species were shown to replace pigeon
excreta antigens in complement consumption tests diagnostic, for pigeon
breeders disease (9,106). Thus, precedence mav already be established
for LTA's role in the manifestation of several clinical maladies. In
addition, the. chemical and biological similarities between LTA and LPS
(1,2) plus the abilitv of LTA to stimulate bone resorbtion (17) make
LTA a likely candidate for a role, in periodontal disease. On the other
hand, LTA lacks some of the biological activities associated with LPS
such as pvrogenicity in rabhits (2,107) and a mitogenic effect on B-rclls
(2). Since these activities have been shown to reside with the complex

Lipid A of IPS 008,109) and since the unique sugars and hydroxyacy!
esters of Lipid A are absent in LTA, it is not surprising that associated
activities are absent as well. As a class, teichoic and lipoteichoic
acids are wail and membrane components of gram positive bacteria (107,108).
LTA is typically membrane associated and consists of a glycolipid cova
lently linked to a polyg1ycerolphosphate backbone which may carry carbo
hydrate and D-alanine substituents (2). Teichoic acids (TA), however,
are never associated with cell membranes; they lack the terminal givco-
lipid coupling, and they may have a backbone of either polyelvcerol-
phosphate or polyribitol phosphate (2). LTA may be converted function
ally to polyglycerol TA by spontaneous dencvlntion in an aqueous environ
ment, or mild alkaline, or acidic hydrolysis (L07). The molecular
weight of LTA (93) is probably between 3000-12000 hut because of its
tendency to form micelles in an aqueous environment, the apparent mole
cular weight as determined by gel. filtration is approximately four mil-
2
lion (110). Because LTA possess the glycolipid moiety, they are amphi-
pathic molecules exhibiting a propensity to spontaneously associate with
proteins and biological membranes (103). Mammalian red blood cells can be
"coated" by spontaneous adsorbtion with an LTA containing extract and the
cells can subsequently he agglutinated with an anti-LTA serum. Passive
hemagglutination (1HA) performed in this manner with sheep red blood
colls lias previously been reported bv manv invest igators who discovered
' Personal oQjnmun Lea t ions from R. Craig, Dept. of Ml'S, Cniv. of PI.; K.
Knox and A. J. Wickcn, Institute for Dental Research, Sydney, Australia;
and personal unpublished data.
>
Data supported bv personal experience (see Figures 11 and 12), and
personal communication from R. Craig.

1.3
erythrocyte-sensitizing antigens in cell free saline washings or spent
culture fluid from several gran positive organisms (101.102). These so
called "Rantz antigens" were recently shown to possess properties asso
ciated with LTA (111). Because only acylated LTA will hind to erythro
cytes, PHA provides a means of quantitating the amount of LTA in a
preparation without having to contend with deacylated TA contamination.
The biological role of TA and LTA to the microorganism has been a
subject of considerable disputation by several investigators in recent
years. Thus far, at least three roles have been tentatively assigned:
1). TA and LTA seem to function as "carrier" molecules for membrane
and cell wall components, i.e. amphipathic LTA may be used by the cell
to transport needed hydrophobic molecules through hydrophilic zones
which would otherwise pose an almost impenetrable harrier. Fielder and
Glaser have established that intracellular LTA servos as a lipid carrier
for the biosynthesis of cell wall ribitol teichoic. acid in Staphylococcus
aureus (112,113). Chaterjee and Wong (114) have demons!'rated that LTA
may serve as the acceptor in which nascent peptidoglyran polymers are
synthesized. 2). LTA seems to be involved in ceLl wal.1 division and
regulation. Holtje and Tomasz have reported that LTA exhibits an inhi
bitory effect on the function of nutolytic enzymes during the division
cycle of pneumococcus (115). It is interesting to note that similar
functions have been described by Cleveland, et al. working with a strain
of Streptococcus faecal is (11 A.117,118). In these systems, LTA is
deacylated and released into the environment as TA. Once the concentra
tion of LTA is sufficiently lowered, or the concentration of nutolytic
enzymes is sufficiently elevated, cell wall autolysis begins at the divi
sion zone.
This nutolytic activity thou allows for insertion of additional

cell wall material. 3). LTA or TA may contribute to the overall elec
trostatic charge of gram positive organisms. Although membrane
localized, the long polar tails of many LTA penetrate the thick pepti-
doglycan layer and become externalized (107). These, together with the
TA which are covalently linked to the cell, wall (]08) present a myriad
of antigenic faces to the external environment (ILl),l20). This antigenic
presentation is of serological import since these antigens are often
genus, species, group, or type specific (103,120). In addition, these
polar tails generate a net negative charge by exposing the phosphate
groups of the polyglycerol or polyribito.l backbone. This net negative
charge has been teleologically assigned the function of maintaining elec
trostatic repulsion and dispersion of the bacterial cell (121). Since
LTA has been shown to sequester certain cations such as magnesium (122),
an additional function as a site of divalent cationic convergence has
ilso been postulated. The association with magnesium ions appears to
be more than casual since protoplasts of Lac tobac111us easel placed in
a magnesium ion free or chelated medium rapidly lose their LTA from the
cell membrane.
Anti-LTA titers (of both the TgM and Igf. classes) have been regularly
reported in mice, rabbits, and man (2,121). Several clinical studies
have reported increases in nnti-LTA titerincluding antibodies of the
class IgAafter acute gram positive infections (I2A.123). Pigs, guinea
pigs, and rats exhibit a low level of natural immunity to LTA and recently,
there have boon reports of salivary IgA product ion as a result of gastric
intubation of monkeys with Streptococcus mut ans (>713 serotype C.. There
is no doubt that TA and LTA of all gram positive genera thus far inves
tigated contain antigenic moieties and that under certain circumstances

LTA ran be immunogenic (2). Of particular interest is the fact that the
attachment of streptococcal LTA to erythrocytes could he reversibly
transferred from the erythrocytes to other tissue cells (104,12b). The
possible significance of this "transferability" in relation to rheumatic
fever and glomerulonephritis and pigeon breeders disease has been pre
viously discussed (9, 104106). However, despite this precedence the
significance of the binding of LTA to oral epithelial cells in gingival
pockets has not yet been investigated. Not only does LTA mediate bone
resorbtion as previously indicated, but spontaneous hybrid micells of
LPS and LTA are known to occur,' thus compounding the possibility of in
situ immunological modulation. There is little doubt of the availability
of extracellular LTA in this environmentStreptococcus mutans BUT alone
has been reported to produce excess of 50 ng of LTA/ml in culture media
(20). Recently, Wicken and Knox have studied the excretion of extra
cellular LTA from this organism in a chemostnt under steady state loga
rithmic growth conditions. Results indicated that a generation time of
10-14 hours (estimated to reflect that actual in vivo growth rate of this
organism in the oral cavity) produced the maximal amount of extracellular
LTA (1). Considering its ubiquity and the cariogenic nature of Strepto
coccus mutans BUT (127-130), the secretion of copious amounts of biologi
cally active LTA into the oral cavity has the potential of considerable
influence on the host-parasite relationship.
The objectives of the project were then defined as follows:
(l) To establish if an LTA-centa ining extracellular extract of
Personal common ¡cat ion of A. .!
K i cken.

If)
Streptococcus mutans BUT was capable of inhibiting complement mediated
cytolysis of target sheep erythrocytes.
(2) To purify the extracellular LTA of S. mutans BUT to homogenity.
(3) To describe the nature of any ant i-c*.omp 1 omentary activity
that purified extracellular LTA may exhibit.
(4) To determine the site of action of any such inhibition.
(5) To determine the mechanism by which purified extracellular LTA
may enhibit anti-complementary activity.
(6) To determine if the LTA from other gram positive genera and
species can be shown to demonstrate anti-complementary activity.

MATERIALS AND METHODS
Crude extracellular LTA (LTAcx) The initial studies were
carried out utilizing LTAcx prepared in Australia by the method
of Wicken and Knox (110). Streptococcus mutans RUT was grown
to late stationary phase in a New Brunswick Microfirm fermentar at
37C, under anerobic conditions (95 N and 5/- C.O^) in a complex
medium.
Later experiments utilized LTAcx prepared at Gainesville,
Florida. The original method was modified as follows. A Pell icon
2
Cassette system (Millipore Corp., Bedford, MA) equipped with 1.0 ft
of PTGC filter material was used to dialyze Todd-Howitt broth (Pifo.o
Laboratories, Detroit, Ml). A 100 mi culture of early log phase
_S. mutans BHT was inoculated into 10 liters of dialyzed medium
and incubated at 37 for 24 hours. The cells were harvested using
a Delaval Gyrotester (Poughkeepsie, NY). The supernote was passed
2
through the Pellicon Cassette system (loaded with 1.0 ft of 0.45 u
microporous membrane) to remove remaining cells and debris. The cell-
free spent fluid was then fractionated and eoncontrated by passage
through 5.0 ft PTGC membrane (nominal molecular weight' exclusion
limit of 10,000). The filter retentte was washed in s_itu with several
liters of water, collected and 1vophi 11 zed. The freeze-dried retentte,
designated as LTAcx. was stored in a dessicator at -20C.
So lut i ons for complement assays. Tsotonic Veronal buffered
sodium chloride (VRS), dextrose gelatin Veronal buffer with added

CaCi,; and MgCl? (DGVB), 1'DTA containing Veronal buffer (0.04 M
EDTA-GVB) and gelatin Veronal buffer with added C.a('l0 and MgCl,
(GVB) were prepared as previously described by Hoffmann (131).
Human complement (HuC). Fresh human blood samples were obtained
from the Gainesville Plasma Corp., Gainesville, FL. The blood was
allowed to clot at room temperature for about 60 minutes, and
the serum was separated by centrifugation at 500 X g at 0UC. The
serum was collected and stored at -70C.
Guinea pig complement (GPC). Fresh frozen guinea pig complement
was purchased from Pel Freeze Laboratories (Rogers, AR). The serum
was shipped in dry ice and it was stored at ~70cC after arrival
in the laboratory.
Complement: components. Purified guinea pig Cl and C2 were
prepared according to Nelson et al. (132) and Ruddv and Austin
(133,134). Functionally purified guinea pig C3, CP> and C9 and
human Cl, C5, C6 and C7 were purchased from Cordis Laboratories
(Miami, FL).
Erythrocytes (JE). Sheep blood was taken by venipuncture from
a single animal maintained at the animal research laboratory of the
J. Hillis Miller Health Center (Gainesville, Fh). One hundred
milliliter volumes of blood were rol lor ted in equal volumes of
sterile Alsevors solution (135) and the blood was stored at 4nC
for up to three weeks.
Antibody sensitized sheep erythr oey_tes (F.A) Rabbit anti -
sheep E stromata was obtained from Cordis Laboratories (Miami, FL).
Sensitization of washed sheep F was performed as recommended by
the supplier.

Complement componen!: intermediate compiexes. Sheep E in
various stages of complement fixation were used in this study.
EAC1, EAC14 and EAC142 were prepared by methods described by Borsos
and Rapp (136). EAC1423567 were prepared by the procedure described
by Hoffmann (137). Unless otherwise indicated, guinea pig Cl,
C8 and C9 were used in all instances, and the remaining C components
were from human serum.
Treatment of cells and cellular intermediates with LTAcx.
Unless otherwise indicated, cells were washed and suspended in VBS
q
at a concentration of 10 /ml. Equal volumes of these cells and
LTAcx were mixed and incubated at 37 for 20 minutes with continuous
shaking. The mixture was then placed in an ice bath for 10 min
utes. At tlie end of incubation DGVB was added to the mixture and
it was centrifuged at 500 g for five minutes. The supernote was
discarded and the cells were suspended and washed thrice with
DGVB (0 for 10 minutes at 500 g) to remove any unbound material.
g
The cells were then resuspended in DGVB at a concentration of 10 /ml.
A sample of the cells were tested for cell-bound I.TA using passive
hemagglutination with rabbit anti-l.TA. The remaining cells wore
used in experiments to detect acquired resistance to hemolysis.
Passive hemagglutination .(PHA) Passive hemaggl ut ¡.nation was
carried out using a microtitration system. fifty ill. of a VBS
dilution of anti-UTA we re added to the first row of wells of a round
bottom microtiter plate (Cook Engineering Co., Alexandria, VA)
and 25 ui (one dron from the calibrated pipetes suppl ied with the
system) of VBS were added to the other weils on the plate. The
anti-serum was serially diluted in situ and one drop of LTAcx

treated cells was added to each well. Controls for spontaneous
or nonspecific agglutination consisted of wells that contained anti
serum and sheep E which had never been exposed to LTAcx. Treated
sheep E plus VBS constituted another control. The microtiter plate
was incubated at 37C on a Cordis Micromixer (Cordis Laboratories,
Miami, FL) for 15 minutes. The plates were removed from the mixer
and the cells were allowed to settle for two hours at 37C, followed
by three hours at room temperature.
Modified passive hemagglutination (PHAg). A modification of
the above technique was used to semi-quantitate the amounts of LTA
present in various preparations. The same apparati were used, hut
instead of antibody, LTA-containing extracts were added to the
bottom wells and serially diluted in situ as described. After
each LTA source was diluted, one drop of sheep erythrocvtes (10 /ml
in VBS) was added to each well and the plate was then incubated
at 37C for 20 minutes and at 0C for 10 minutes. The cells were
kept in suspension by vibrating the plate on a Cordis Micromixer
during both incubation periods. One drop of OVB was then added to
each well and the plate was centrifuged at 200 g for 5 minutes.
The entire plate was then abruptly inverted over absorbent paper
towels and allowed to drain for approximately one minute. One
drop of OVB was again added lo each well and the plate was vibrated
at 0C for 5 minutes to resuspend the pellet. An additional drop
of OVB was added per well and the plate was again centrifuged at
200 g for 5 minutes. This washing procedure was repeated three
times and the cells were then finally resuspended in one drop
of OVB. One drop of anti-ETA (diluted 1:1000 in VBS) was then

added to each well and the plate was Incubated at 37C for 15
minutes on a Cordis Micromixer. The plate was removed from the
mixer and the cells were allowed to settle for two hours at 37C,
followed by three hours at room temperature.
Inhibition of complement mediated lysis. E A coated with
in DGVB and 0.4 ml of DGVB diluted HuC. The HuC was diluted so
that a maximum of 80 percent lysis was produced in EA which had
not been treated with LTAcx. The mixture was incubated at 37C
with continuous shaking for 60 minutes. One milliliter of ice
cold EDTA-GVB was added, the mixture was centrifuged for 5 minutes
at 500 g at 0C and the superna tent fluid was recovered. The
optical density of the supe run tent fluid was determined at a wave
length of 414 nm. Inhibition of hemolysis was calculated for each
concentration of LTAcx used by comparing the extent of lysis in
each assay with a control reaction mixture which contained EA
that had not been treated with LTAcx.
Ef fee t of LTAcx on t_he_ t it or of antibodies spo c_i f ic for _shee_p
erythrocyte stromata. Because LTA associate with some proteins (138)
it was necessary to perform a hemolytic antibody titration to
determine if the ability of the immunoglobulins to fix complement
at the cell surface was being affected by LTAcx treatment. The
possibility of similar ,mt igens in LTAcx and sheep erythrocyte
stromata was also considered. Equal volumes of LTAcx (500 ug/ml
in VBS) and rabbit anti-sheep E stromata were incubated together
at 37C for 20 minutes. A control consisted of incubating an

equal volume mixture of VBS and anti-sheep erythroovte stromata
for the same time at the same temperature. The ant ihodies were,
then titrated using limiting amounts of complement (1.35).
Cl fixation and transfer. The number of Cl molecules hound
to an antigen-antibody complex can be measured by the Cl fixation
and transfer test described by Borsos and Rapp (139). In a mod
ification of this procedure, an attempt was made to quantitate
the number of Cl molecules fixed to EA which had previously been
treated with LTApcx. Buffer controls and EAt_a were prenared
LEA
as previously described, and after washing were resuspended at
g
10 cells/ml in DGVB. Equal volumes of EA^^^ and EA were in
cubated with Cl at 30C for 15 minutes. The cell mixtures were
washed twice with DGVB, and resuspended in GVB at a cell concen
tration of 1 X 10^/ml, 5 X 10^/ml, and 1 X lO'Vml. One volume
of each cell concentration was added to one volume of EAC4 (at
8 ~ 1
1 X 10 cells/ml) to permit transfer of Cl from EA Cl to EAC4.
The cells were incubated at 30C for 15 minutes, and then C2 and
C-EDTA were added In relative excess as described previously.
A variation of the Cl transfer assay was performed by treating
preformed EAC1 with ETA or buffer control .as described. The
resulting EACl^ were resuspended to 1 X 10* cells/ml in GVB and
the amount of Cl capable of transfer was measured as described
above.
Gel filtration. I.TAcx was fractionated on a 2.5 cm X 100.0 cm
column of Bio-Gel A-5M, 200-400 mesh (Biorad Laboratories, Richmond,
'in this; instance, "x" represents ETA or the appropriate buffer
treated control.

CA) using a modification of the method described by Wlcken and
Knox (110). The column was equilibrated and eluted using 0.01 M
Tris carbonate (Sigma Chemical Co., St. Louis, MO), pH 6.8.
Hydrophobic Affinity Column chromatography.' Because of the
hydrophobic nature of the fatty acid moieties of lipoteichoic acid,
adsorbtion to a stationary phase of a chromatographic coLumn was
used in an attempt to further purify the LTA. LTAppx in buffer A
(0.01 M Tris carbonate pH 6.8, 1.0 M Nad was loaded on a 25.0 X 2.25 cm
column packed with Octyl Sepharosc (Pharmacia Fine Chemicals,
Piscatawav, NJ) and equilibrated in the same buffer. After eluting
with 150 ml of starting buffer A, the reservoir was then changed
to buffer B (0.01 M Tris carbonate pH 6.8) and another 100 ml were
eluted. Buffer C consisted of 250 ml of a gradient ranging from
10-70 % propanol (by volume) in 0.01 M Tris carbonate, pH 6.8.
Octyl Sepharose is a derivative of the cross linked agarose
Sepharose CL-4B. The terminal, n-ortyl groups of this agarose gel
confer a hydrophobicity to the matrix. By exploiting this property
it was hoped that polar or neutral non-interacting components
would be removed by elution with solutions of high ionic strength.
The lipoteichoic acid would then he eluted from the matrix with
an organic solvent such as propanol. (It is imperative that all
tubing, connections and gaskets used throughout the column lie
constructed of a material that is resistant to organic solvents).
^This method represents a modification of a procedure described
by A..I. Wicken and K. Knox (Sydney, Australia) via personal commun-
ica tion.

Removal of salt and prop an oJ_ from LTA contain in g e :< tract s .
Removal of salts and/or propanol from various preparations was rapidly
and quantitatively accomplished by gel filtration utilizing LH20
(Pharmacia Fine Chemicals, Piscatawav, NJ) as the solid phase support
matrix. The most commonly employed column was 50.0 cm X 2.5 cm but
a larger 65.0 cm X 3.0 cm column was sometimes utilized. The column
was packed and equilibrated with deionized water. Sample preparations
usually involved rotary flash-evaporation (Buchler Instruments. Fort
Lee, NJ) in order to reduce the volume of sample to 15-20 ml. Elution
of product was carried out at a pressure head of approximately 50 cm
water and approximately 4.0 ml effluent were collected per tube.
Phosphatidyl choline vesicle (PCV) purification of_LTA
(a) Preparation of PCV. Although reported as the method of choice by
other investigators,^ in our hands Octyl Sepharose purification
of LTA from Streptococcus mutans BUT resulted in a product still
highly contaminated with polysaccharides. in an attempt to achieve
homogeneous purification of LTA, a modification of the above mentioned
hydrophobic adsorbtion principle was employed. In this procedure,
artificial membrane vesicles were prepared with DL^phosphatidyl,
choline dipalmitoyl (PC) (Sigma Chemical Co.) as the sole constituent
via a modified method of Hill (1.40). In brief, 40.0 me, of PC was
placed in each of several 10 mL high speed glass Corex centrifuge
tubes (Corning Glass Works, Corning, NY) and dissolved with one ml
chloroform. The solvent was gently evaporated in a 50C water bath
while rotating the tubes so as to coat the bottom 5 or 6 cm of the
tube with PC. Once dry, the lubes were placed in a 1yophi1 i cat ton
flask and any residual solvent was removed in vacuo. One milliliter
'wichen, A.J.-, and Knox, K.Personal communication.

of 0.01 M Tris carbonate pH 6.8 was then added to each tube and they
were placed in a 50C water bath. Once warmed, the tidies were
vigorously vortexed (Vortex Genie Mixer, Scientific Industries Inc.,
Bohemia, NY) and the cycle of warming and vortexing was continued
until a milky emulsion was formed. Fifteen milliliters of 0.01 M
Tris carbonate were then added to each tube and the tubes were centri
fuged at 27,000 g for 30 minutes. The supernatent fluids were then
decanted, the pellets were resuspended in 1.0 mi Tris carbonate
buffer and warmed to 50C in a water bath. The tubes were gently
swirled (but not aggitated) to dissolve and resuspend the pellet .
The resulting phosphatidyl choline vesicles (PCV), devoid of very small
vesicles, were then used to adsorb LTA from LTAppx.
(b) Preparation of PCV-LTA. Two milliliters of LTAppx at a concen
tration of 1.5 mg/ml in 0.01 M Iris carbonate, pH 6.8 were added to
each centrifuge tube containing 1.0 ml of PCV. The tubes were covered
with parafilm (American Can Co. Neehaw, I/S) and incubated for
90 minutes in a 37 shaker water bath. Thirteen milliliters of
0.01 M Tris carbonate were then added to each test tube and tbev were
centrifuged at 27,000 g for 45 minutes. The superoates were discarded
and the pellets were gently resuspended in 1.0 ml of Tris carbonate
buffer at 50C as previously described.
Fifteen milliliters of buffer were then added to end) pellet,
the tubes were gentlv swirled and then centrifuged as described.
The pellets were washed three times in this manner. The final pellet
was drained and then dissolved in 5.0 mi of chloroform/methanol.
(3+1 v/v). The tubes were then covered with aluminum foil and
allowed to sit at room temperature for 60 minutes.

A Millipore 15 ml analytical filter holder (Millipore Corn.,
Bedford, MA) was loaded with a 3.0 p fluoropore membrane (Millipore
Corp.) and washed with several, volumes of the chloroform/methanol
solvent. The test tubes were all sequentially decanted into the
apparatus and the contents were allowed to filter bv gravity through
the membrane. Each test tube was washed with several volumes of
warmed chloroform and decanted into the filtering apparatus. Finally,
the barrel and filter were washed in situ with warm chloroform. The
filter was removed after air drying in situ and placed in 10.0 mi
of deionized water warmed to approximately 40C. All centrifuge tubes
and the barrel of the filtering apparatus were washed with warm
deionized water and all products were combined. The resulting
product was passed through a 25 mm Swinnex filter (Millipore dorp.)
loaded with a 5 u microporous membrane (Millipore Corp.) to remove
particulate debris. The membrane was washed i_n s_itu with several
volumes of warm deionized water. The filtrate was collected directly
into a lyophilization flask and was then shell, frozen and lyophi 1 ized.
The final product was stored in a dessic.ator at -20C.
14
C Phosphatidyl choline analysis In order to detect any
phospholipid contamination of the LTA throughout the previously
described PCV purification, radioactive PC was used to Label the phos
pholipids iu the vesicles. Approximately 2.3 pCi (3 X 10 DPM)
1 4
of C labeled phosphatidyl choline (Amersham Searle Corp., Arlington
Heights, TL) were added to 40 mg of phosphatidyl choline dipalmitovl
in a 30 ml Corex centrifuge tube. Phosphatidyl choline vesicles
were prepared from this and the non-labe Led contents of three
other tubes bv the methods previous!v described. Fifty microliter

27
samples from the C containing test tube were taken at each step
of the purification and placed in empty glass scintillation rials.
The samples were heated to 50C in a drying oven to remove the
solvent from the sample. Once dry, 50 ul of chloroform were used to
redissolve all samples and then 5.0 ml scintiJlation fluid containing
toluene (scintillation grade, Mal 1 inckrod t, St. Louis, MO), 0.4/' PPO
(2,5 diphenyloxazole), and 0.01% POPOP (1,4-di (2- (5-phenyloxazolv 1)-
benzene) were added to each vial. The degree of ^C-PCV contam
ination of the final product was determined by placing the entire
LTA-containing-fluoropore filter .in a scintillation vial with 5.0 ml
scintillation fluid. The possible influence of quenching by the
fluoropore filter was investigated by adding equal aliquots of
14
C-PC to two scintillation vials one of which contained a fluoropore
filter in addition to scintillation fluid. No appreciable difference
in CPM was observed. Disintegrations per minute (DPM) values were
calculated from a standard quench curve constructed for use with chloro
form. Standard ratios were determined for each sample and percent
efficiencies were extrapolated from the standard quench curve. This
volume was then used to correct counts per minute (CPM) to DPM. Unless
otherwise indicated, the samples were counted for 10 minutes in a Beckman
LS-.133 liquid scintillation counter (Beckman Instruments, Fullerton, CA).
Col orine trie assays. Phosphorous was determined hv the method
of Lowry et al. (141) with absorbancies measured at 820 nm. Total
carbohydrate was measured by the phenol sulfuric acid assay as des
cribed by Dubois ct al. (142). Total protein was performed on samples
using the Bio-Rad Protein Assav (Bio-Rad Laboratories, Rockville Center,
NY). Samples and the standard curve were prepared following the
manufacturers recommrndations.

Carbohydrate analysis was performed
Gas Liquid Chromatography.
after treatment of the samples with 1.0 N F^SO, in sealed ampules
for 8 hours at 105aC. Upon cooling, the seal was broken and exactly
0.2 ml of mannitol (either at 5.0 mg/ml or 1.0 mg/ml depending on
carbohydrate concentration of the sample) was added as an internal
standard. The contents of each vial were quantitatively transferred
to 15 ml centrifuge tubes (Corning Glass Works) containing 0.5 g
BaCO^. Each centrifuge tube was heated in a boiling water bath
and alternately vortexed until the pH approached neutrality as
indicated by full-range pH paper (Micro Essential Laboratory,
Brooklyn, NY). All tubes were centriufged at 500 g for 5 minutes
and the supernates were removed and collected in appropriately labeled
13 mm screw cap tubes fitted with teflon lined lids. The centrifuge
tubes containing BaCO^ were washed once with one ml of deionized
water and the supernates were appropriately pooled.
After lyophilization, the hydrolyzed carbohydrates were con
verted to trimethylsilyl ester (IMS) derivatives by the addition
of 0.2 or 1.0 ml (depending upon carbohydrate concentration) of
TRI SIL Z (Pierce Chemical Go.). Samples were warmed to approx
imately 60C in a water hath for 15-30 minutes before use and
assayed using a Packard 800 series gas chromatograph equipped with a
flame ionization detector. The gas chromatographic column (153 cm X
4 cm) was packed wi th SE-40 ULTRAP1IASK 33 on Chromosorb W (IIP) 80/L00
mesh matrix (Pierce Chemical Co., Rockford, TL). Column and detector
temperatures were set at lf)0C and 1F5"C respectively. The N,, carrier
gas was set at approximately 30 co/minute.

Amino acid analysis. Amino acids and amino sugars were measured
on a JEOL model JLC-6AH automated amino acid analyser (JEQL, Inc..
Cranford, NJ). Sample hydrolysates were prepared as described bv
Grabar and Burt in (143).
Clg, Cls, and Cls purification. Highly purified human Clq
was prepared from whole human sera by the method of Yonemasu and Stroud
(144). Highly purified human Cls and Cls were prepared by a minor modi
fication of the method described by Sakai and Stroud (35). For the final
resolution step, Bio-Rad Cellex-D DEAE with binding capacity of 1.07 meq/g
(Cellex-D, Bio-Rad Laboratories, Rockville Center, NY) was substituted for
fibrous DEAE cellulose Whatman DE-23. The DEAE was washed and prepared
according to the manufacturer's specifications. Final elution of the pro
duct was accomplished with the use of the same eluting buffer as described,
but instead of a stepwise elution of the column, an ionic gradient from
0.2 0.4 RSC (relative sodium chloride concentration) was utilized.
Disc acrylamide gel electrophoresis o f_C Lc^, Cls and Cls. This was
carried out essentially as described by Yonemasu and Stroud (1.44) but with
out the use of sodium dodecyl sulfate (SDS).
Cls Inhibition assays. The ability of Cls to consume C2 activity was
assayed by a modification of the method described by Sakai and Stroud (3.5).
Briefly, 0.1 ml of Cls (approximately 8.0 X 10^ site forming units, SFU/ml)
plus 0.1 ml LTApox (100 t|g/m! in DVB) were incubated at 30 for 15 minutes.
One tenth milliliter of C2 was then added at a concent ration of approxi
mately 9.0 X IQ7 effective mo I ecu 1es/m1 and incubated at 37^0 for 30 min
utes. At the end of the incubation, 9.7 ml cold DCVR were added to the
mixture resulting in a 1:1.00 dilution of the 0,2. The C2 was then serially
diluted and 0.1 ml aliquots from each dilution were added to 0.I mi ot

EAC14 (10 cells/ml in DGVB). The mixture was incubated at 30'JC for JO
minutes and cooled to 0C in an ice bath for 2.0 minutes. Three tenths
of a milliliter of C-EDTA (1:37.5 in 0.04 M EDTA-GVB") were then added to
each test tube and the mixtures were incubated at 37C for AO minutes.
At the end of the incubation period, 1.0 ml of cold EDTA-GVB was added,
the tubes were centrifuged, and the supernates read for release of oxv-
hemaglobin at a wave length of 414 nra. External controls consisted of C2
with no Cls nor LTApcx, C2 with Cls but not LTApcx, and C2 with LTApcx
but no Cls. The usual internal controls (spontaneous lysis, color cor
rection, no C2, and total lysis) were included at all times. Results were
expressed as percent inhibition of C2 consuming ability compared with a
control containing only Cls and C2.
The ability of Cls to hydrolize the synthetic substrate p-Tosy1-1-
arginine methylester (TAMe) was determined as described hv Nagaki and
Stroud (38). Inhibition assays were performed by incubating equal volumes
of Cls (approximately 8.0 X 1.0^ SFU/ml) and LTApcx (approximately 100 g/m
at 37 for 10 minutes. Residual Cls activity was then determined as des
cribed (38-40).
Clq inhibition assays. The effect of LTApcx on the ability of puri
fied Clq to bind to antibody sensitized sheep erythrocytes was determined
by methods described by Loos et al. (39) and Raepple et al. (40). Equal
8
volumes of Clq (approximately L.3 X 10 SFU/ml) and LTApcx (10 hg/ml)
were incubated at 37 for 10 minutes. Residual Clq activity was then
determined as indicated above.

RESULTS
Inhibition of whole human complement by erudo extracelLuIar
lipoteichoic acid (LTAcx) To determine whether LTAcx had any effect on
whole human complement, equal, volumes of LTAcx and whole human complement
were preincubated at 37/30 minutes. After pre tncuabt ion, tin.' complement
source was serially diluted in DGVR and the residual hemolytic activity
was titrated. As shown in Figure 1, approximately 50% of the whole com
plement hemolytic activity (measured in CH,.^ units) was consumed. Further
more, as seen in Figure 2, this consumption was dependent on the concen
tration of the LTAcx used.
Titration of complemcnt components in whole human sera after treat
ment with LTAcx. One mechanism for fluid phase consumption of whole com
plement could have been the interaction of natural antibodies in the human
sera with LTA or some other antigenic substance in the crude extract. The
result would be the fixation of Cl and subsequent .activation of C4 and C2
via classical pathway. Another explanation for decreased hemolytic activ
ity could have been the activation of the alternative pathway in a manner
analogous to LPS. To differentiate between these two modes of activation,
individual component titrations were performed on human sera incubated
with LTAcx. Tn addition, C3 titrations were carried out in the presence
of ethy Lenoglvcol-bis (b Amino Ethyl Ether) N,N totrnaectic acid (L(.IA)
and Mg ions. This chelating agent preferentially hinds Ca ions (1 4 5,1 4M .
and by reinforcing the F.OTA buffer with Mg ions one can effectively deplete
the available Ca ions yet maintain relatively high levels of Mg Ions.
Thus, the Ca ion dependent classical pathway is blocked, hut the
alternative pathway can function relatively unimpaired (145,147).

Figure 1. Titration of whole human complement after incubatLo
with crude extracellular lipoteichoic acid (LTAex).
Symbols: (o) Non-treatcd control: () Serum treated
with LTAex at 500 tig/ml .

DECIMAL EXPRESSION OF CHU DILUTION! MO'
ro

Figure 2.
Dose response inhibition of whole human complement
after incubation with varying concentrations of
LTAcx. The non-treated control is abbreviated
as NIC.

RESIDUAL CHc UNITS/ml
600
500
CONCENTRATION OF LTAcx(/g/ml) USED IN
PREINCUBATION WITH WHOLE HUMAN
COMPLEMENT(Chu)

i 6
A typical component titration in serum treated with I.TAox is
depicted in Figure 3. In this example, the LTAcx treated serum was
serially diluted in DGVB. Next F.AC142, C5, 6, /, and 08-9 were added
sequentially to the dilutions. Since all components were added in
excess, C3 became the limiting factor in contributing to the hemolysis
of the target cells. Percent lysis in each test tube was mathematically
converted to Z (the average number of SAC1423 sites per cell) and this
was plotted against the reciprocal of the serum dilution. Percent inhi
bition of site forming units (SFU) was then calculated from 7.-1 values
or percent inhibition of CHr units was determined from values asso-
.30
dated with Z= 0.69. Figure 4 represents a composite of multiple com
ponent. titrations from whole human sera treated with I.TAcx. As can be
seen in this figure, Cl and C4 activities were consumed to some degree,
however, more than 50% inhibition of C2 activity was observed. As
indicated, C.3 activity was al.so consumed during preincubation of com
plement with LTAcs, but incubation with purified C3^~ produced no inhi
bition of C3 hemolytic potential. No C3 consumption occurred if the
incubation was performed in the presence of the chelator ethvlenodiamine
tetra acetic acid (KD'i'A) and less than 7 if incubated in the presence
of EOTA-Mg ions. The above results indicated the necessity for divalent
cations as cofactors mediating the consumption of C3 in the presence of
LTAcx. in addition, there? appeared to be a requirement for other serum
factors (possibly natural, AB and/or components of the alternative path
way' since purified (13 activity remained unaffected hv incubation with
LTAcx.

igure 3.
Titration of C3 in whole human serum after treatment
with LTAcx. SvmhoJs: (*) Non-treated control; (<')
Serum treated with LTAcx at a concentration of 230
yg/ml. After incubation, sera were titrated for
residual C, 3 activity according to procedures
described in Materials and Methods.

Z(AVERAGE NUMBER OF SAC 1423 SITES/ml)
RECIPROCAL OF (HUMAN SERA) C3 DILUTION

Figure 4. Complement component titration of whole human .era
after treatment with LTAcx. The sera were incubated
with the LTAcx (500 ug/ml) then titrated for resi
dual activity of the components indicated as describe
in Materials and Methods.

% INHIBITION OF CH, UNITS

Inhibition of complement Lysis of LTAcx treated EA. During an
experiment in which EA treated with LTAcx were tested for reactive
lysis, it was discovered that the LTAcx treated cells exhibited Less
hemolysis than even the buffer treated controls. This serendipitous
observation led to the discovery that LTA treated EA were refractory
to complement mediated lysis. To confirm these results, various concen
trations of LTAcx were used to treat EA. After the treated cells were
extensively washed they were tested for their susceptibility to lysis
by complement. The same cells were also tested for the presence of
cell-bound LTA using the passive hemagglutination technique (PHA) with
anti-LTA. The results shown in Figures 5 and 6 indicated that both
the extent of inhibition of hemolysis and PHA titers were LTAc.x dose
dependent. There was a decline in both activities only after the LTAcx
had been diluted to a concentration of 62.5 pg/ml The decrease in
titer below this concentration indicated that the test cells wore no
longer saturated with LTA. There was a concomitant drop in inhibition
of lysis at 62.5 pg/ml. EA which were treated with uninoculated cul
ture medium (dialyzed Todd-IIewitt broth) were unaffected when comple-
emnt was added.
Effect of LTAcx on lysis of sheen E and sheep E cellular
intermediates The treatment of EA with LTAcx caused the cells to
become relatively resistant to complement mediated lysis. This could
have been due to an effect on the ant¡body molecules, an effect on one
or more of the complement components, or an alteration of the cell
membrane .
To further investigate the nature of the complement inhibition
associated with LTAcx, sheep E. sheep EA, and various sheep E complement

Figure 5.
Inhibition of complement mediated lysis of F.A
treated with varying concentrations of LTAex.

PERCENT INHIBITION OF LYSIS
80-1
70-
3
.25
LTAcx (/xg/ml) USED IN EAltacx PREPARATION

Figure 6.
Passive hemagglutination (PHA) of FA treated with
varying concentrations of LTAex.

RECIPROCAL OF ANTI-LTA DILUTION
0 % PH A
~50 % PH A
IOO % PH A
LTAcx (|jg/ml) USED IN EAltac* PREPARATION

component intermediates were treated with LTAcx and analyzed for sus
ceptibility to complement mediated lysis. The LTAcx treated cells were
also tested for bound LTA using PHA with anti-LTA. Results indicated
that L, EA, and EACH4 were all refractory to complement mediated 1 vs is
and that LTA was detectable on the surfaces of the cells (Figures 7 and
8). However, EACL423567 which had been treated with LTAcx were not re
sistant to lysis despite the fact that LTA was detectable on the cells
(Figure 8). Thus, the inhibitor appeared to affect a complement com
ponent required for lysis os EAC14, but which was unnecessary for lysis
of EAC1423567.
In an attempt of focus on the site of inhibition, the ability of
LTAcx to affect the hemolytic susceptibility of EAC 142 was examined.
This intermediate possesses C3 convertase activity ((,42) which is in
volved in the generation of SAC1423 and SAC!4235. However, Cl is not
required for lysis of the intermediate once SAC142 have been formed (148).
Failure of LTAcx to inhibit this intermediate would indicate that C3
convertase was not the. step in the complement sequence affected hv the
LTAcx.
Sheep EAC142 were treated with LTAcx according to the protocol
that has been described. For this experiment, three different' amounts
of C2 were used to generate EACL42 from EAC 14. The results clearlv
indicated that there was no inhibition of the intermediate complex
EAC42 (Figure 9). resting by PHA with antibodies specific for LTA
confirmed the presence of LTA on the surfaces of the cells at the same
relative concentrations found when the other intermediate complexes
were tested.
Effect (if LTAcx on ant i-sheep erythrocyte antibodies.
Some

Figure 7.
Effect of LTAcx treatment on the lysis of various
complement component, intermediates. Each cellular
intermidiate was prepared and then treated with
LTAcx (125 ug/ml). Lysis was developed using
procedures described in Materials and Methods.
Percent inhibition was calculated by comparison
against buffer treated controls.

PERCENT INHIBITION OF LYSIS
CELLULAR INTERMEDIATES TREATED
WITH LTAcx (125 /xg/ml)

Figure 8.
PHA of various LTAcx treated complement component
intermediates.

RECIPROCAL OF
ANTI-LTA DILUTION (x 10
CM
imlr
0 % PHA
-50 % PHA
100 % PHA
CELL INTERMEDIATES TREATED
WITH LTAcx (125fg/ ml)

Figure 9.
Effect of LTAcx treatment on the lysis of EAC142.
Various limiting concentrations of C2 were used to
prepare EAC142 cellular intermediates. The cells
were then treated with LTAcx (250 yg/ml) and lysis
was developed using procedures described in Materials
and Methods. Symbols: (o) EAC142 incubated with
LTAcx; () EAC142 incubated with buffer.

RECIPROCAL OF HUMAN C2 DILUTION
Ln
NJ

substances in LTAcx might be capable of interacting with the anti
bodies used to sensitize sheep E. This interaction could then lead to
an impairment of Cl activation and result in reduced lysis. Such a
mechanism might be the reason why E and EA become resistant to lysis
after treatment with LTAcx. Therefore, antibodies to sheep erythro
cyte stromata were incubated with LTAcx. The mixture was then diluted
to the point where the LTAcx-related inhibition could not be detected
and the antibodies in the mixture were titrated (135). It was found
that antibodies that had. been preincubated with LTAcx had the same
titer as antibodies that were incubated for the same time and temperature
with VBS (Figure 10).
Partial purification of LTA. Partial purification of LTA and
the complement inhibitor was accomplished by gel filtration of the LTAcx
through an A-5M Biogel column. The results of a typical experiment arc
shown in Figure 11. Areas of antigenicity were resolved hv immunodif
fusion in an agarose gel utilizing an anti-serum specific for the LTA
backbone. Fractions were pooled as indicated (A-F), and each pool was
dialyzed against water and subsequently lvophilized. Note that pools B,
C, and E contained high levels of phosphorus and that the zones of anti
genicity were also located in these areas. Utilizing extracellular
extracts from S. rnutans and other microorganisms, similar fractionation
profiles under comparable conditions were obtained by Ktoken and Knox
(lit)) and Blewieis and (iraig. Analysis bv these workers revealed that
the second phosphorous containing peak (peak II) contained LTA whereas
the trailing phosphorus peak contained deucvlatcd LTA and wall teichoic
Personal communication.

Figure 10.
Effect: of LTAcx on hemolytic antibody titration.
Antibodies to sheep red blood coll stromata (Ah) were
incubated with LTAcx (SOD ug/ml) and residual hemolys
activity was titrated by procedures described in
Materials and Methods. Lysis of cells was developed
with whole guinea pig complement. Symbols: (o) Ab
incubated with LTAcx; () Ah incubated with buffer.

PERCENT HEMOLYSIS

r-H
acids. Because peak IT represented partially purified extracellular
lipoteichoic acid, the recovered material was designated LTAppx.
A sample of each pool was rehydrated to 50 pg/ml and reacted with
EA, according to standard procedures (Materials and Methods). Each EA
preparation was analyzed using the complement inhibition assay and
tested for bound LTA by PHA. Only the pools containing LTA (as demon
strated by PHA) caused inhibition of complement mediated lysis (Table 1).
Despite the excellent separation of LTA from most of the material
that absorbed light, at a wave length of 260 nm, and presumably from
all deacvlated LTA or TA, two persistent problems arose with this puri
fication procedure:
1). Polysaccharide contamination accounted for a major portion of
the mass recovered in peak LI, and
2). The total mass of LTAppx under peak II was almost immeasurable
smal1.
In an attempt to at least increase the yield of peak ¡I material,
a Millipore Cassette system was employed to both concentrate and frac
tionate the spent culture superante (Materials and Methods). This method
of LTA enrichment proved highly successful as evidenced by the. results
in Figure 12. Even after values are corrected for the greater mass of
crude extract applied on the latter column the mass yield of Ll'Anpx was
some fifteen fold greater than that obtained with previously employed
procedures (Figure 11).
An analysts of results tracing the partial purification of LTA
is summarized in Tables 2 and 3. It should he noted that the total
amount of Pi, mass, protein, and absorbing material decreased
several thousand fold in the purification process, whereas the total

TABLE 1
Partial Purification
of LTA by A5-M Gel
Filtration
Poo 1ed
Fraction
Test Tube
Numbers
Pooled
Probable
Content
Percent
Inhibition
of Lysis3
PILA ,
Ti ter 1
A
32-40
Void Volume Material
NDC
ND
B
41-46
LTA Plus Low Percent Carbohydrate
47.4
6400
C
47-60
LTA Plus High Percent Carbohydrate
39,1
6400
D
61-71
Carbohydrate
0.0
<100
E
72-84
TA, Carbohydrate and Nucleic Acid
0.0
<100
F
85-100
Nucleic Acid
0.0
<100
CX
All of Above
31.2
3200
EA were prepared with the LTA source at a concentration of 50 yg/nl. Hemolysis was developed by
incubation of the cells with several solutions of human C (37,'60 minutes). Values represent inhibi
tion of CI!_ units.
oO
PHA titers arc expressed as the reciprocal of the final dilution of anti-LTA which still resulted in
hemagglutination when incubated with the EA,,,.,.
Not determined.

Figure 11.
Partial purification of LTA hv A5-M gel filtration.
Symbols: (*) A,,^() absorbance (maximal absorbance
wavelength for nucleic acids): (*) A^^_ absorbance
(maximal absorbance wavelength for carbohydrates as
determined by the Phenol Sulfuric Acid assay); (A)
Pi concentration in n-moles as determined by the
Lowry Pi assay; (+) Antigenicity as determined by
Ouchterlony gel diffusion using an antisera
directed against LTA backbone.

A 260
FRACTION NUMBER (5.0 ml)
i (n -moles)

Figure 12.
Partial purification of LTA by A5M gel filtration
with LTA enriched starting material. Symbols: ()
A,-, absorbance: (o) Pi concentration in ;i-mo 1 os/'mL
as determined by the Lowry Pi assay: (+) Antigenic it
as determined by PHA using antisera directed against
LTA backbone.

A 260
£
CO
O)
o
E
TEST TUBE NUMBER

TABLE 2
I. Results from Partial Purification of LTA
Sample
Pi (p-moles)^
b
Percent Lytic
Inhibition by
LTA Treated EA
PHA T
Dialyzed, Non-Inoculated
Todd-Hewitt Broth
1.1x105
0
0
Supernate from Inoculated
but Lion-Fractionated Broth
l.OxlO5
ND
ND
PTGC Retntate Fraction
of Supernate (LTAcx)
6.9xl02
AS
3200
Peak II from A5M After
Desalting (LTAppx)
6.OxlO1
55
3200
Data are expressed in the units indicated and represent values extrapolated back to the
undilute sample times total volume.
EA were prepared with the LTA source at a concentration of 250pg/ml. Hemolysis was
developed as described in TabLe 1.
PILA titers were determined by methods described in Table 1.

Figure 9. Effect of LTAcx treatment on the lysis of E AC 142.
Various limiting concentrations of C2 were used to
prepare EAC142 cellular intermediates. ihe cells
were then treated with LTAcx (250 vig/ml) and lysis
was developed using procedures described in Material
and Methods. Symbols: (o) KAC 1.42 incubated with
LTAcx; () F.AC142 incubated with buffer.

TABLE 3
II. Results from Partial Purification of LTAa
Samp le
A 2 60
. b, ,
Protein (mg)
Amount of LTA inL
Sample (mg)
Weight of
Sample (mg)
Dialyzed, Non-Inoculated
Todd-Hewitt Broth
3.7xl05
6.5xl03
O
O
3.3x10
Supernate from Inoculated
but Ron-fractionated Broth
3.6xlOJ>
6.7xl03
9.1
ND
PTGC Retntate Fraction
of Supernate (LTAcx)
1.25x103
9.5xl02
11.0
2.3x10
Peak II from A5M After
Desalting (LTAppx)
1.7 6x10^
6.0
8.2
3.6x10
Unless otherwise indicated, all data are expressed in the units indicated and represent values extrapolated
back to the undilute sample times -total volume.
Values determined
by Bio Rad Protein Assay.
These values were calculated by determining the minimal concentration of purified LTA that can still be
detected by PHAg. Equating this value with the PHAg end point for all other LTA sources, the hypothetical
LTA concentration in the starting well can be calculated by serial twofold interpolations.

amount of LTA in the sample, PHA titer, and percent lytic, inhibition of
EA remained relatively unchanged or increased in value.
Pur i f teat ion of LTA by hyd r o phobic, i n t e r ac. tion gel chroma tography .
A 25.0 X 2.25 cm column packed with Octyl Sepharose and equilibrated in
buffer A was prepared as described in Materials and Methods. Approxi
mately 6.0 mg of LTAppx dissolved in 10.0 ml of buffer A were applied
to the column. As can be seen in Figure 13, a small, amount of phosphate
containing material passed unimpeded through the column. A slightly
greater mass of polysaccharide was also excluded without binding. No
additional material eluted from the column with buffer B. Point C on
the graph marks the location where a 10-70 propanol gradient was begun.
Point D represents the point where a significant volume decrease per
test tube was observed. Since fractions were collected on a "drops per
tube" basis, the presence of propanol in the effluent causes a change in
surface tension of the drop resulting in a decreased volume per drop.
The ultimate result is a decrease in the volume per tube. This, test
tuhe volume provided a convenient means of monitoring the progress of
the propanol gradient.
It should be noLed that despite the use of a gradient (the original
procedure called for a single step-wise elution with 50 propanol)
significant amounts of carbohydrate eluted witli the I/LA. As indicated
on the the graph, all areas containing phosphates also contained LTA as
detected using PHA. The fact that a small amount of LTA passed unbound
through the column suggests that either the columns b i nding capacity was
exceeded, or perhaps the LTA was onlv partially aevlatcd and not capable
of tenacious hydrophobic binding.

Figure 13. Purification of LTA by Octyl Sephnroso hydrophobic gel
chromatography. Symbols: ( concentration of carbohydrate (n-moles/ml) as determined
by the Phenol-Sulfurie Acid assay. Concentrations were
determined using glucose as a standard carbohydrate.
(o) Concentration of Pi. (n-moles/ml) as determined by
the Lowry Pi assay; (+) zones of antigenicity as
determined by PHA using antisera directed against LTA
backbone; (A) elution with buffer A (1.0M NaCl, 0.01
M Tris-carbonate pH 6.8); (R) elution with buffer B
(0.01 M Tris-carbonate, pH 6.8); (C) elution with a
10-70 gradient of a propane 1-buffer B mixture; (D)
elution volume at which significant reductions of vol
ume/tube were observed, indicating elution of propanol.

moles CARB (GLUC)/ml
c
A BCD
E
\
CL
cn
a>
o
E
c
VOLUME (ml)

67
All test tubes containing greater than 25.0 n-moles Pi/ml were
pooled. The entire peak (approximately 22 ml) was loaded on to a
65.0 cm X 3.0 cm column packed with LH-20 equilibrated with deionized
water. Four and two tenths milliliter of effluent were collected per
test tube a a flow rate of approximately 30.0 ml/hour. The results
of this procedure, which simultaneously removed salt and propanol, are
shown in Figure 14. The column effluent was monitored at a x-zave length
of 220 nm and was also screened for LTA by PHA (-H-H-) using a single
dilution sample. In addition column fractions were tested for the pre
sence of chloride ions by placing one drop of a saturated AgNO^ solution
on a coverslip containing one drop from each test tube. Any resulting
precipitation was evaluated on a +1 to +5 basis and plotted accordingly.
It was empirically determined that not only Cl reacted with the AgNO^
resulting in insoluble AgCl, but the NaN^ and tris carbonate in the
buffers reacted as well. The presence of propanol was monitored indi
rectly by changes in test tube volume. Since LTA. azide, and tris
carbonate all absorb at a wave length of 220 nm tin? combination of ultra
violet light screening, the AgNO^ precipitation test, and visual inspec
tion of volume changes per test tube proved to be invaluable for rapidly
discerning the location and separation of LTA from contaminating salts
and solvents. The entire contents of peak I were pooled, frozen, and
lyophilixed. The final product was referred to as LTAosx (extracellu
lar lipoLeiehoic acid purified by Octyl Sepharose hydrophobic affinity
gel chromatography). The typical mass yield from such a procedure
was about 60-70. Percent recovery of LTA at various points in the
procedure is summarized in Table 4.

Figure 14.
Simultaneous removal of salt and propanol free LTAosx
by LH-20 gel chromatography. Symbols: (*) absor
bance; (o) Volurne/test tube: ( + ) Antigenicity as deter
mined by PHA; (Shaded Area) relative degree of precipi
tation of salt and other low molecular weight materials
as determined by AgNO test.

A 220
TEST TUBE NUMBER
VOLUME / TEST TUBE

TABLE 4
Percent
Recovery
a
of LTA During Octyl Sepharose Purification
LTA Source
Reciprocal of
Initial Dilution
Initial
Concentration ,
of LTA (ug/ml)
Total volume
of Sample (ml)
Calculated
Total Weight
LTA in Sample
of
(mg)
Percent
Recovery
of LTA
LTAppx
400
0.500
9.0
1.80
100.0
Peak I
2
0.250
87 .0
0.04
2.2
Pooled column Effluent
from all areas not Loca
Under Peak I or 11
ted
?
0.000
273.4
0.00
0.0
Peak II (LTAosx) Before
Passage through LK20
100
0.500
31.6
1.58
87.8
Peak II ''LTAosx) After
Passage tnrough LH20
100
0.25
62.3
1.. 56
86.5
As determined by ?HAg
Determined by methods described in Table 3
Calculated by multiplying the corresponding values for the first three columns

1 4
Phosphatidyl choline vesicle (PVC) purification of LTA using C
h 14
labelled phosphatidyl, choline. Approximately 5 X 10 DPM of C
labelled phosphatidyl choline were added to 40 mg of phosphatidyl
choline dipalmitovl. Phosphatidyl choline vesicles (PVC) were pre
pared as described in Materials and Methods. Three test tubes contain
ing identical volumes and concentrations of non-labelled PCV were pre
pared simultaneously and 2.0 ml of LTAppx (1.5 mg/ml) were added to each
test tube. Fifty microliter samples from the ^4C containing test tube
were removed at various steps during the purification process and an
alyzed as described (Materials and Methods). A standard chloroform
quench curve was constructed and all reported counts represent corrected
1 4
DPM values. Table 5 depicts the distribution of C counts at various
steps in the purification procedure. Utilizing this procedure as des
cribed, essentially no contaminating phospholipid could be detected in
the final product. The typical mass yield of product via PVC purifica
tion was about 10-15%. Percent recovery of LTA at various steps in the
procedure is summarized in Table 6.
Comparison and summary of LTApcx versus LTAosx. As indicated in
Table 7, both methods of LTA purification removed the majority of pro
tein as compared to the total amount ahailabie in the LTAppx. Both
methods ostensibly recovered > 80% of the original LTA. However, the
major difference between the two products is reflected in the percent
total, mass recovery and the concomitant increase in percent carbohydrate
in the final LTAosx product. This latter difference can he most readily
discerned by observing the composite gas chromatograph tracings in
Figure 15. The carbohydrate standard (CHO-STD) depicts the typical
chromatograph of glucose and galactose after preparing trimethvlsiIv1

14
Distribution of C-
Phosphatidyl Choline
During PCV Purification of LTA
Sample Source
Reciprocal of
Dilution Factor
DPM Aliquot'5
(x io'z)
Total DPM
in Sample
(X 10 )
Percent of
Total DPM
Calculated
Corresponding
Weight of PCV (mg)
14
C-PCV Suspended in Starting
Suf f er
60
830.00
4980.00
100.00
40.00
Supernate from Preliminary
Vesicle Washing (#1)
320
1.91
61.12
1.23
0.49
Supernate from Preliminary
Vesicle Washing (r2)
320
0.29
9.28
0.19
0.07
Decanting After Reaction of
PCV with LTAppx
320
2.95
94.40
1.90
0.76
1st Washing Supernate
320
2.82
90.24
1.81
0.72
2nd Washing Supernate
320
1.61
51.52
1.03
0.41
3rd Washing Supernate
320
3.44
110.08
2.21
0.88
Chloroform/Methanol Filtrate
72
590.00
4248.00
85.30
34 1 2
1st Chloroform/Methanol Washing
100
35.00
350.00
7.03
2.81
1st Chloroform Only Washing
' 60
1.09
6.54
0.13
0.05
2nd Chloroform Only Washing
60
0.58
3.45
0.07
0.03
Final Product (LTApcx)
1C
1.96
0.20
0.00
0.00
a Dilution factor was calculated
for analysis.
by dividing
t he
totaL volume of
the sample by the
volume of the
aliquot removed
^ DPM values were calculated from
CPMs and a
standard quench curve as described in
Materials and
Methods.
O contamination of che final product was determined by i icing the entire [,TA-conta in ing floroporo filter in
a scintillation vial and analyzing as described in Materi u _> and Methods.

TABLE 6
Percent:
Recover/
a
of
LTA from Various
Steps of PCV Purification
LTA Source
Reciprocal of
Initial Dilution
Initial
Concentration
of LTA (yg/ml)
Total Volume
of Sample (ml)
Calculated Total0
Weight of LTA
in Sample (mg)
Percent Recovery
of LTA
LTAppx
600
0.500
6.0
o
co
100.0
Decant
5
0.125
48.0
0.03
1.6
1st Wash
1
0.000
48.0
0.00
0.0
2nd Wash
1
0.000
O
co
' 0.00
0.0
3rd Wash
1
0.000
48.0
0.00
0.0
LTAncx
1000
1.000
1.5
1.50
83.3
As determined by ?HAg
D Determined by methods described in Table 3
u Calculated by multiplying the corresponding values of the first 3 columns

TABLE 7
Summarized Chemical Composition of Various LTA Containing Sources
LTA Source
3
Carbohydrate'
Percent Composition of Dry
Protein
Amino Acid Bio-Rad
Weight
LTAb
c
Pi
, f
A220
A260
A280
Analysis
Protein Assay
LTA ppx
23-32
21-28
16-22
21-26
1.8-2.5
.555
.090
.082
Combined Fractions
from Octyl Sepharose
(except Peak II)
57-65
43-56
36-48
<5
0.9-1.3
. 392
. 105
.071
LTAos x
(Peak II, Octyl
Sepharose)
15-30
4-6
72-85
3.1-4.0
.368
.091
.074
Combined Super-
nates from PCV
washings
49-58d
26-34d
16-22d
<5
NA6
NA
NA
NA
LTA;) ex
< 5
-
<2.5
<95
5.8-6. o
. 160
.093
. Ob9
a
Percent carbohydrat
e was determined
by gas liqu
id
chromatography as
described
in Materials
and Met ho
CS .
Percent LTA was determined by PHAg.
Percent phosphate was determined by the Lowry Phosphate assay.
These values were corrected for weight differences due to contaminating phospholipid vesicles.
Because of the high percent phospholipid vesicle contamination in this sample, valid determinations for total
Pi and optical densities were not possible.
Ultra violet light absorbance determinants were made with the indicated materials at a concentration of
100 al/tnl In distilled water.

Figure lr>. Carbohydrate analysis of J.TA containing preparations
by gas liquid chromatography. Abbreviations: (MAN)
Mannitol; (GLC) Glucose; (GAT.) Galactose. Mannitol
was incorporated as an internal, standard with all
samples.


ester (IMS) derivatives ns described in Material and Methods.
An inter
nal mannitol standard is included with all samples. The I/IAppx chroma
tograph represents the typical carbohydrate profile achieved with
partially purified LTA. The tracings for LTAosx and LTAppx contrast
the qualitative and quantitative differences in carbohydrate content.
i
The second two chromatograms ,deacylated cardiolipin (f. P^) and cardio
lipin, were included as a comparison of how a naked polyglycerol phos
phate backbone might be expected to react under the described conditions.
The base line instability of the G.^P^ looks remarkably similar to the
profile of the purified LTApcx, The procedure for purifying deacylated
cardiolipin requires passage through Sephadex columns. It is quite con
ceivable that the minute quantities of unidentified carbohydrates which
are indicated may be due to dextran contamination from the column.
However, it would be difficult to account for the same source of contam
ination for the LTApcx since gel chromatography was not used in the final
purification. On the other hand, the similarity of the indicated chroma
togram tracings may be more than mere coincidence and may reflect actual
reactions of the derivatizing agent with the polyglycerol phosphate
backbone. This latter hypothesis is supported by the fact that an
unidentified trailing "carbohydrate" peak of significant mass appears
in both the cardiolipin and G P0 chromatographs. The Rf value of this
peak is similar (but yet suspiciously disparate) to the retention tine
normally observed for 8-glucose. However, if indeed this peak docs
represent 8-glucose, one is hard pressed to rationalize why a corres
ponding -glucose peak does not occur as well. In either case, it is
Deacylated cardiolipin was prepared bv the method of Wilkinson (14D)
and was kindly provided bv R. Craig, University of Florida.

apparent: that the LTAosx stL.ll. contains stgni f leant amounts of carbo
hydrate contamination in contrast to the lower yield, hut highly puri
fied LTApcx.
A summary of specific activities relative to P11A activity is pre
sented in Table 3.
Inhibition of comp Iemeat mediated lysis of JL TApcx trented EA.
Once a highly purified preparation of LTA was obtained, it was necessary
to confirm the results that had been previously established with LTAcx.
As can be seen in Figure 16, not only were LTApcx treated EA refractory
to complement mediated lysis, but in addition the general profile was
remarkably similar to LTAcx treated EA. As indicated, LTApcx was used
in concentrations five-fold to ten-fold less than those, used with LTAcx
to achieve comparable degrees of inhibition. PHA titers typically Indi
cated LTA saturation of the cells.
Effect of LTApcx on the lysis of _var i nus j:_el^lulnr_ complemen t
component intermediates. Sheep E, EA and various complement Inter
mediates were treated with LTApcx (1.00 pe/ml in DVB) and washed exten
sively in DGVB. Percent lysis and inhibition of CH units were deter
mined as described in Materials and Methods. The results of these
experiments are summarized in Figure 17. As with LTAcx treated cells,
E, EA, and EACl were all refractory to lysis by complement. Likewise,
EAC142 and EACJ-7 intermediates wore totallv unaffected by the presence
of LTApcx on their cell surfaces. The onlv observable difference in the
activity of LTApcx on cellular intermediates versus LTAcx was that KALI'*
cells were inhibited to a lesser degree with LTApcx than LTAcx.
Effect of LTAcx and LTApcx on fluid pitase C 1 As previously
discussed, CL is not required for lysis once SAC¡42
are formed but it is

TABLE 3
Comparing Several Different Methods of Measurement
LTA
Source
PHAg
Protein
X 10~3
Carbohydrate
X 10~3
A260
X 10-3
Pi
X 10~3
Mass
X 103
LTAcx
400
(3.4)
H. D.b
(0.3)
(4.6)
(1.4)
LTAppv
2,400
(96)
(35.7)
(26,667.0)
(1,116.0)
(1,333.3)
LIAosx
3,000
(1,600)
(347.8)
(87,912.0)
(2,222.2)
(5,128.2)
LTApcx
32,000
(12,300.0)c
(6.400)c
(344,086.0)
(5,203.2)
(21,333.3)
1 Protein,
( tiXD TdSS
carbohydrate, phosphorus (Pi)
ed on the reciprocal of PHAg t
and mass calculations were made
iter) per mg.
on the basis
of activity
Not determined.
C These values are estimations since the amounts of material present were below the Limits of
detection for the methods.

Figure Hi. Passive hemagglutination (PIIA) titration and inhibition
of complement mediated lysis of FA treated with varying
concentrations of TifApc.x.

PERCENT INHIBITION OF LYSIS
O % PHA
v50 % PHA
I 00 % PHA
RECIPROCAL OF ANTI-LTA DILUTION
JO

Figure .17. Kffec.t of f.TApcx on the complement mediated 1vsis of
various cellular complement component intermediates.

70
60
50
40
30
20
! 0
CELLULAR INTERMEDIATES TREATED
WITH LTApcx (100 g/ml)
CO

84
essential until that point is reached (143). In addition, many poly-
anionic substances are known to directly affect Cl by interfering with
Clq binding or Cl esterase (Cls) activity (19,40). Because LTA is
polyanionic. due to the polyglycerol phosphate backbone and because
cellular intermediates beyond the EAC142 step were no longer inhibited,
it seemed reasonable to hypothesize that LTA was behaving like a poiv-
anion and directly affecting Cl.
To test this hypothesis, LTAcx and LTApcx were preincubated with
functionally purified human Cl. at 30/13 minutes. Residual Cl activity
was titrated as described by Rapp and Borsos (139) and activity was com
pared against buffer treated controls. The results shown in Figure 1.8
indicate that although LTAcx consumed Cl activity, purified LTApcx did
not.
Purification of human Clq, Cls and Cls. In an attempt to further
eludicate a possible site and mechanism of C inhibition, human Cl sub
components were purified by the methods and modifications previously
described (Materials and Methods). Although homogeneity beyond func
tional purity was not essential, the methods employed yielded highly
purified products. Figure 19 demonstrates Clq homogeneity bv immuno
diffusion against several monospecific antisera. Precipitation bands
of identity were observed in adjacent wells containing the whole human
serum starting material, the purified Clq final product, and a highly
enriched Clq prior to final precipitation (Figure L9, plate 5, well
numbers A, G, and E). As can be observed in Figure 20, disc gel electro
phoresis of the final product revealed a single dark staining band which
barely migrated into the separation gel. These results are eonsistant
with the observations of other investigators (L44).

Figure 18.
Effect of LTAcx and LTApcx on functionally purified
human Cl. The upper graph represents a residual Cl
titration after incubation with LTAcx (5D0 pg/ml).
The lower graph represents the results from an analo
gous experiment using LTApcx (500 pg/ml) instead of_
LTApcx in the incubation mixture. Symbols: (*) Cl
incubated with buffer; (o) Cl incubated with the
appropriate L'L'A containing extract.

AVERAGE NUMBER OF
16,000 8,000
4,000
2,000

Figure 19. Immunodiffusion and precipitation analysis of various
steps in the purification of human Clq. Purification
was achieved by repeated fractional precipitations of
whole human sera in buffers varying in ionic, strength,
pH, and concentrations EGTA or EDTA (144) .
Well designations:
(A) Whole human sera (starting material);
(B) Supernate from first precipitation;
(C) Supernate from second precipitation;
(D) Supernate from third precipitation;
(E) Material from resuspended pellet prior to
final precipitation.
(F) Final product (purified Clq).
Plate designation: (1) Center well contains nnti-lgC;
(2) Center well, contains Anti-IgA; (3) Center well
contains anti-whole human sera: (4) Center well
contains anti-IgM: (5) Center well contains anti-Clq.


Ht
. .
l&iSwSyt'S; *w8
ttHMi
^mMmsIwIIK
'' l
tt. S

Figure 20.
Disc gel electrophoresis of purified human Clq.
Cathode was at the top.


The procedures for Cls and Cls purification were modified only in
that an ionic gradient was used in the final, purification step of both
reagents rather than the stepwise elution utilized by Sakai and Stroud
(35). The rationalization for this modification was that a difference
in binding capacities of the DEAE matrix could have deleteriously
effected the elution characteristics of the Cls (Cls) at a fixed ionic
strength. The elution profile of Cls is shown in Figure 21. It should
be noted that two peaks of material which absorbed light at a wave length
of 280 nm were resolved during the gradient elution. Both peak II and
and peak III reacted with monospecific antisera to Cls, however, only
peak III contained Cls activity. Peak II presumably represents an in
active form of either Cls or Cls. No such ext raucous peak was resolved
during DEAE chromatography of cls.
Iminunoelectrophoretic analysis of purified human Cls and Cls on
1% Noble Agar is depicted in Figure 22. Results indicate a difference
in electrophoretic mobility of Cls and Cls which is consistent with the
observations of previous investigators (35). Also, there was a "gull
wing" pattern displayed bv Cls apparently representing microhetero-
geneity of the activated proesterase. This too has been observed In
previous investigators (35) .
Effect of LTApe.x on the ability _o_f Cls to consume C4 and C2 activity.
As previously discussed, activated Cl esterase (Cls) is capable of
cleaving C't into C4a and C4b (43) as well as cleaving C2 into C2n and
C2h (45). In either case, the active fragments rapidly decay and it not
quickly attached to membrane sites, lose their ability to do so. The
ephemeral nature of these active fragments can he used as sensitive in
dices of Cls activity. As described in Materials and Methods, equal

Figure 21. DEAE elution profile of human Cls. Peak I contains
Cls activating proteins (functionally pure Clr); Peak
II contains nonfunctional Cls; Peak III contains
functional, non-activated Cls. Symbols: () Absor
bance at (maximal absorbance for most proteins;
(o) Relative salt concentration (RSC) as measured
by electroconductivity. Arrows indicate the addition
of high ionic strength Sodium Chloride buffer.

A 280
TEST TUBE NUMBER
RSC

Figure 22. Immunoelectrophoresis of human Cls and Cls.
Well A and D contained purified Cls; well B
contained purified Cls; well C contained
whole human sera. Trough 1 contained anti
Cls (Cls) ; troughs 2 and 3 contained a mix
ture of 7 5% anti whole human sera and 25%
anti Cls (Cls).


and LTApcx were mixed and
volumes of Cls, functional Lv pure C4 or 02,
incubated at 37/5 minutes. The incubation mixture was then serially
diluted in DCVB, and the residual titers of 04 or C2 were determined
and compared against a buffer treated control. As shown in. Table 9. no
appreciable difference in Cls activity can be observed when even 200 pg/ml
of LTApcx were used. Also note that LTAprx preincubated with either C.4
(Expt. Group 2d) had no significant effect on residual activity.
Effect of LTApcx on the ability of Cls to hydrolyze TAlie. The
ability of Cls to hydrolyze the synthetic substrate TAMe is another index
of Cls activity. As can be seen in Figure 23, essentially no inhibition
of Cls activity was observed when Cls is preincubated with LTApcx and
TAMe.
Effect of LTApcx on the ability of Clq to bind to target cells.
Since Clq is the recognition unit of the classical pathway of complement,
any alternation in its ability to react with the antigen-antibody com
plexes on the surface of EA would have profound affects on the ability
of complement to lyse those cells. Therefore, equal volumes of puri
fied human Clq and LTApcx (10 pg/ml) were pro incuba ted at 30/1.5 minutes.
After preincubation, Clq was serially diluted in DCVB and (Mr and Cls
reagents were added. Hemolysis was then developed as described (Materi
als and Methods). Again there was no apparent inhibition (results not
shown) of activity. The major criticism of this experiment is that the
LTApcx concentration list'd to pre incubate with Clq is 10-fold less than
what was normally used in fluid phase inaetLvations. Ihe reason for the
use of this lower conoentration was to insure that the LTA would ho
sufficiently diluted at the time of EA addition. If significant amounts
of t.TA were present in the incubation mixture, EA( ^ would form, thus
generating a false positive inhibition duo to the refractory nature of

TABLE 9
Effect of LTApcx on the Ability of Cls to consume C4 and C2 Activity
Exp t.
Group
DGVB
Cls
C4
C2
LTApcx
(50 Mg/ml)
LTApcx
200 pg/ml)
Residuu L
C4 or C2
Activity
Consumption of
C4 or C2
Activity
Pi
Ml
Ml
Ml
Ml
Ml
SFU/mia
%
la
100
100
100
9
6.2 X 10
86
b
100
100
100
9
7.1 X 10
84
c
100
100
100
8.6 X 109
81
d
100
100
100
4.3 X 1010
4
e
200
100
4.5 X 10i0
b
NA
2a
100
100
100
9
6.5 X 10
93
b
100
100
1.00
9
9.1 X 10
90
c
100
100
100
O
o
1
X
r-4
r|
87
a
100
100
100
9.4 X 1010
0
j
200
100
9.2 X 101U
NA
Site forming unit.
b
Not applicable
o

Figure 23. Effect of LTApcx on the ability of Cls to hydrolixe
TAMe. As the synthetic substrate TAMo is hydro!iced,
there is an increase in A.,,_ absorbing material. in
- / q /
this experiment, Cls and TAMe were incubated together
in the presence of LTApcx (100 pg/ml) at room temper-
ature_(24C). Symbols: () Cls and TAMe plus buffer;
(o) Cls and TAMe plus LTApcx.

OPTICAL DENSITY (247nm
10 20 30 40 50 60 70 80 90 100 NO 120
MINUTES AT 24C

loo
EA Despite the lower concentration, previous data with LTA treated
LTA
EA (Figure LG) indicated that even at this concentration, inhibition

should have been significant if indeed Clq were the site of inhibition.
8
Instead of this anticipated inhibition, 1.36 X 10 SFU/ml of Clq were re-
8
covered from the incubation mixture originally containing 1.50 X 10 SFU
Clq/ml. Due to assay variation this difference was considered insignificant.
Effect of LTApcx on Cl transfer. In another attempt to elucidate
the effect of LTA on the Cl molecule, the interference of the normal
ability of Cl to transfer from cell to cell under conditions of high
ionicity was investigated. Two different types of transfer tests were
performed. In type I, EA were treated with LTA and then Cl was added.
In type II, EAC1 were prepared and then LTA was added. Not so sur
prisingly there was no inhibition of Cl transfer as measured by hemo
lysis of EACA cells. However, there was an increase in the Cl trans
ferability of cells containing LTA. As can he seen in Table 10, this
phenomenon was repeatable and was observed in both types of experiments.
Differences in complement mediated lytic susceptabilitv of LTAcx
treated EACA versus EAC14. Buffer or LTAcx (250 pg/ml) was used to
treat EACA using procedures described in Materials and Methods. Various
limiting concentrations of human C were then added to aliquots of the
cells and lysis was developed as previously described. Alternately.
EAC1A were prepared using various limiting concentrations of human Cl.
Aliquots of cells were then treated with LTAcx (250 pg/ml) or with buf
fer. After the cells were washed extensively in buffer, lysis was
developed as previously described. As shown in Figure 24, EAC14 treated
with LTA are considerably more refractory to complement mediated lysis
than are EACA treated with cl.
LTA

m L
TABLE in
_ Comparison of Che Relative Numbers of Effective
Cl Molecules Capable of Transfer from EAC1 Treated with LTApcx
Sample
Experiment
Number
Effective Number of Cl
Molecules Transferred/Cell
EAClLTAa
1
175
eacTlta
2
124
eacTlta
3
153
EAcTDVBb
1
137
eacTdvb
2
115
faltacT C
1
185
ealtacT
2
1 78
ealtacT
3
21 5
eadvbcI
1
132
eadvbcT
2
182

EAC1 were generated and treated with LTApcx at 100 pg/ml in PVB.
After extensive washing, the Cl capable of transfer was titrated.
k Control EAC1 treated with DVB for 10 /1 minutes
p
F.A were prepared (100 ¡ig LTApcx/ml) and aftor_the cells were
extensivo Iy washed EA Cl were generated. The Cl capable of
transfer was then titrated.
Control EAC1 treated with DVB while in the FA slate. FACI pre
paration and Cl transfer exactly paralleled the T.TA treated cells.
d

Figure 24.
Differences in complement mediated lytic susceptibility
of LTAcx treated EAC4 versus EAC14. Upper graph: EAC4
were treated with buffer or with LTAcx (23(.) ug/ml).
Various limiting dilutions of human Cl worn then added
to aliquots of the cells and lysis was developed accordin
to procedures described in Materials and Methods. Lower
graph: EAC14 were prepared with various limiting dilu
tions of Cl. Aliquots of cells were then treated with
LTAcx (250 pg/ml) or with buffer. After extensive
washing, lysis was developed according to procedures
described in Materials and Methods. Symbols: ()
Buffer treated cells; (o') LTAcx treated cells.

AVERAGE NUMBER OF SACI4/CELL
103
RECIPROCAL OF HUMAN C I DILUTION

LO 4
In addition to the above, mentioned experiments, several other
assays to elucidate the mechanism of inhibition were attempted. Unfor
tunately none of these experiments led to results that were consistent
with any models attempting to explain how some complement cellular
intermediates became refractory to complement lysis when pretreated
with LTA. These experiments and their summarized data are presented
below:
Cl uptake by EA EA were prepared with LTApcx at a con-
LTA LTA
centration of 100 pg/ml using procedures described in the Materials
and Methods. Buffer treated EA were also prepared at the same time.
10
Human Cl (approximately 1.0 X 10 SFU/ml) was reacted with aliquots
from each cell preparation and incubated for 1.5 minutes at 30C. The
cells were pelleted by centrifugation and the supernates analyzed for
9
residual Cl activity. Approximately 6.5 X 10 SFU Cl/ml remained in the
9
supernate of the buffer treated controls whereas approximately 6.8 X 10
SFU Cl/ml were titrated in the supernate of the EA treated cells.
LTA
Because values fluctuated by 5-8 / from one experiment to the next, this
slight degree of enhancement was not considered significant.
HU HU
Residual C4 titration after preincubat.ion of C4 with EAC1
~~ 9 LTA
Human C4 (approximately 4.0 X 10 SFU/ml) was added in equal volumes to
EACl which had been preincubated with either LTApcx (100 iig/ml) or with
buffer. The mixture was incubated at 30 for 15 minutes and residual C-'t
activity was titrated as described in the Materials and Methods. EA
were incubated with the C4 reagent as a negative control. Results in
dicated that there was approximately a 30% decrease in residual C.4

in
titer of the supernates previously incubated with EACl versus the negative
control which consisted of C4 incubated with EA. However, both EACl
LIA
and EACl consumed identical amounts of C4 (residual supertate C4
buffer 9 9
activity was 2.71 X 10 SFU/ml and 2.79 X 10 SFU/ml respectively). There
fore, it was concluded that LTA had no apparent effect on C4 uptake by
Residual C2 titration after preincubation with EAC14 Guinea
TO LTA
pig C2 (approximately 1.5 X 10 SFU/ml) was added in equal volumes to
EAC14 which had been preincubated with either LTApcx (100 pg/ml) or
with buffer. The mixture was incubated at 30 for 12 minutes and resi
dual C2 activity was titrated as described in Materials and Methods. EA
were incubated with the C2 as a negative control. Results indicated that:
9
approximately 35% (5.3 X 10 SFU C2/mi) of the available C2 was utilized
9
by the EAC14 complexes and approximately 29% (4.4 X 10 SFU C2/ml) were
utilized by the EAC14 complexes. Despite the fact that the supernate
LTA
from the C2 incubated witii EAC.L4 liad slightly more residual C2 activity
LTA
(approximately 71% of the C2 activity still remained in the supernate
after incubation with EAC14 ), a difference of only 6% is within ex-
LTA
perimental variance of this assay. Therefore, it was concluded that LTA
had no apparent effect on C2 uptake by EAC14.
Inhibition of lysis of EA by LTA from other bacterial sources.
Additional evidence indicating that LTA might be primarily responsible
for the C inhibition phenomenon came from hemolytic assays utilizing
LTA from other sources.
I)r. R. Doyle (Dept, of Microbiology ami

Immunology, Univ. of Louisville) provided samples of LTA purified from
Bacillus subtil is strain gta B290. Purified (,TA from Lactobacillus
casei ATCC 7469 was obtained From the Institute of Dental Research,
Sydney, Australia, and Dr. A. S. Rleiweis (Dept, of Microbiology and
Cell Science, Univ. of Florida) provided a sample of partially purified
LTA from Streptococcus tiutans strain AHT. Each preparation was mixed
with £A; the cells were thoroughly washed and analyzed using the pre
viously described techniques of PHA and susceptibility to whole comple
ment lysis. As depicted in Table 11, all preparations contained material
that reacted with anti-LTA by PHA and all such cellsespecially those
prepared with the purified L. casei--were more resistant to the hemo
lytic action of complement than were untreated controls.

TABLE ll
Percent Inhibition and PUA Titer of F.As
Treated with LTA Containing Extracts from Several Sources
Source of LTA3
1^
Percent Inhibition
PHAC
S. mutans BHT
40
1600
S. mutans AHT
35
1600
L. casei (ATCC 7469)
75
3200
B. subtilis (gta B290)
70
1 600
aThe LTA extracts from all sources were used at a concentration of
50 pg/ml in VBS.
^EAs were treated with the appropriate LTA-extracl and bemolvsis
was developed by incubation of the cells with several dilutions
of human C (37/60 minutes). Values represent inhibition of CH,._
units.
c
PHA titers are expressed as the reciprocal of the final dilution
of specific anti-LTA which caused hemagglutination.

DISCUSSION
Evidence has been provided for the inhibition of complement
mediated lysis of target cells by an extracellular material obtained
from Streptococcus mutans BHT. This material has been identified a;
lipoteiehoic acid (LTA) and is a plasma membrane constituent of most
gram positive bacteria (107,108).' Various gram positive bacteria
isolated from the oral cavity differ in the amount of LTA they excrete
into the external environment. S. mutans BHT is an example of a carin
genie streptococcus that not only produces copious amounts of LTA (1,20),
but its ubiquitous nature provides for a constant inundation of LTA
and other metabolites into the gingiva] crevices of the oral cavity.
The presence of a complement reactive component in the microenviron
ment of the gingival crevices could result in any number of biological
effects. Direct activation of the complement system (cither classical
or alternative) may result in the destruction of nearby "innocent by
stander" cells. This is particularly true if the activator is evto-
philie and thus capable of "sensitizing" nearby host cells. Activation
of complement in the gingival crevices can also result in osteoclast-
mediated bone resorbtion (14). This phenomenon is further complicated
by the fact LTA and LIS (and ostensibly hvdrid micolls of the two) are
1 Some bacteria are known to lack LTA in the i r membranes but in these
cases "LTA-like" molecules are inserted instead. Examples are the
lipomannan of Hicrococcus lysodeikti cus (150) and the F-antigen of
D iploeoeens pneumoniae (115).
108

also capable of stimulating osteoclast mediated bone resorbtion (17).
Even without profound activation of complement, the possession and
release of complement inhibitory substances might, confer a certain
degree of survival value on the organisms producing them. Thus in
the face of immunological challenge, the complement system nay he
blocked from reacting against the bacteria producing such factors.
It may be more than coincidence that gram positive organisms such as
Micrococcus lysodeikticus lacking LTA in their cell membranes are also
susceptable to lysis by the synergism of lysozyme and complement (151)-
All other gram positives containing intact LTA in their membranes are
notoriously resistant to complement lysis even in the presence of
lysozyme (151).
Three lines of evidence have been obtained which suggest that
the active inhibitory factor is 1 ipteichoic. acid (LTA). The inhibitor
co-purified with LTA when extracellular material from spent culture
was fractionated by gel-CiLtrat ion and was purified by adsorbtion to
phospholipid vesicles. Sheep erythrocytes which had been treated with
S. mutans BUT extracellular extract became resistant to lysis by com
plement and they also became coated with LTA as judged by PHA using
antibodies monospecific for purified LTA. The amount of LTA present
on the cells paralleled the degree of lytic, resistance that was
acquired by the treatment. Purified LTA and LTA-rich fractions from
other bacteria aLso caused sheep erythrocytes to hceme resistant to
complement mediated hemolysis. Again, P11A assays indicated that cells
which became resistant to lysis had LTA on their surfaces.
Experiments using crude extracellular I.l'A (LTAcx) provided evi
dence for the consumption of whole human complement activity.
When

110
preincubated with various concentrations of LTA, whole human sera lost
complement activity .n a dose-dependent fashion. Individual component:
titrations revealed that not only C3, but the early components Cl, C'>,
and C2 were consumed to some degree. However, no C3 consumption was
observed if the preincubation was performed with Isolated C3 or in the
presence of EDTA. If EGTA-Mg were substituted as the chelating agent,
only a minimal restoration of C3 consuming activity was observed.
These results indicated that not only were calcium and magnesium ions
necessary for the anti-complementary activity, but there was a require
ment for some factor(s'> in whole sera as well. This "factor" is most
likely natural antibody directed at LTA or some component of the crude
extract. This resulted in the formation of a typical, antigen-antibody
complex with subsequent classical complement consumption.
Experiments using sheep erythrocytes in various stages of com
plement component fixation provided evidence that LTA was not only
capable of spontaneously adsorbing to the surface of these cells, but
also rendered many of the intermediates refractory to lysis. When
sheep red blood cells, EA, or F.AC1 were treated with LTA, all became
resistant to complement lysis. Lipoteichoic acid treated FACIA were
somewhat less resistant to lysis and all ceLl.utar complement inter
mediates beyond EAC14 were no longer protected.
Conversion of cells to hemolytic resistance by treatment with
LTAcx can aid in the interpretation of the C2 consumption data depicted
in Figure 4. As indicated, the degree of C2 consumption was dispropor
tionate compared to loss of Cl and C4 activity. However, the commer
cially available human C2 used in these sftul ios had a fairly low titer.
As a result, the dilutions made, after the preincubat ion step were not
sufficient to prevent substantial amounts of the LTA from binding to

Che cells and being expressed In Che C2 deration. What anpenred to
be consumption of C2 activity was actual Iv the inability or the comple
ment system to lyse resistant cells. Because of the greater extent of
dilution, the same phenomenon did not influence Cl, C4 and C.3 titrations.
Because the EAC142 and EAC.l423.i67 intermediates were not effected
by LTA, some component no longer necessary for their stability was a
likely site of inhibition. C4 was probably not the site of attack
since this component is a necessary part of the C'3 convertase (152),
and EAC142 were not inhibited. Only C.1 is expendable after the EAC142
complex is formed and thus Cl seemed to be the most likely candidate
for the site of inhibition.
The. first consideration was the possibility that LTA was causing
inhibition of complement mediated lysis by blocking fixation of Cl to
antibodies specific for sheep erythrocytes or by blocking the site of
antibody attachment. The fact that the inhibitor functioned equai.lv
well when it was presented either before or after the addition of speci
fic antibodies to the cells indicated that blockage of antigenic sites
was not the mechanism of inhibition. This experiment did not rule out
the possibility that the inhibitory subsLanec could react with the Cl
fixation sites on immunoglobulin molecules. However, Figure 10 shows
that prelncuhation of LTAcx with anti-sheep L, hemolysins did not
decrease the hemolytic antibody titer of the serum. if LTA were capable
of binding or inactivating immunoglobulin molecules (either specifically
or non-spec if icaily) then the titer of the antiserum should have, been
reduced as a result of treatment with the bacterial extract.
There was some speculation that LTA might inhibit complement medi
ated lysis by inducing some alteration in the structure of the target

ceil membrane. However, one Would expect all of the complement com
ponent intermediate cellular complexes to lie equally affected bv I.iA,
when in actuality this was not the case. Lt is possible that some of
the complement components could block the attachment of the Inhibitor
to cell membranes so that the material would have no opportunity to
cause membrane alteration. This is an unlikely possibility because
even EAC1423567 had LTA on their surfaces.
The highly purified LTA necessary for the final site of action
and mechanism studies proved to be considerably more difficult to ob
tain than previously anticipated. As suggested by the results in
Figures 13 and 15, and Tables 4 and 7, the Octyl Sophorose method of
LTA purification did not sufficiently resolve the I.TA from tenacious
polysaccharide contamination. This method yielded almost quantitative
recovery of LTA (as determined by PHAg) and also a significant portion
of the total mass which was applied to the column. However, considering
the contaminated nature of the final product even when an elution gra
dient waS utilized, it was determined that a significant percentage of
the mass was probably polysaccharide. Indeed, gas Liquid chromatography
of extracellular LTA purified by Octyl Scpharose (L'l'Aosx) indicated
that as much as 30% of the final weight was carbohydrate, presumably
existing as polysaccharide (Figure 15, Table 7).
Tn contrast, the somewhat more elaborate method ol purifying LTA
by adsorbtion to phosphatidy l choline vesicLes (PCV) yielded a product
that was virtually devoid of all nucleic acid, protein, and carbohy
drate contamination (Figure 15. Tables 5,6, and 7). table 5 indicates
1 4
that although approximately 7% of the radioactive C used to label the
PCV was lost during washing procedures (and ostensibly, a percentage

of bound LTA as well), over 92% of the label could bo accounted for in
the chloroform/methanol filtrate and the first filter washing. Only
0.004% of the label was present in the final product therefore elimin
ating phosphatidyl choline as a source of contamination. Figure 15 and
Table 7 indicate that less than 5% polysaccharide contamination can be.
detected in the final product by gas liquid chromatography. Considering
the unusual profiles obtained from the gas liquid chromatography of
both cardiolipin and deacylated eardiolipin (Figure 15), it is likely
that the percentage of contaminating polysaccharide in the final LTApcx
preparation is even Less than 5%. As can he seen in Table 6, approxi
mately 85% of the LTA in the original partially purified extract can be
accounted for in the final product and washings. However, it should be
cautioned that the method used for these determinations (PHAg) is semi-
quantitative at best and is only considered accurate to within one two
fold dilution.
Although the percent protein of all partially purified samples
was determined by amino acid analysis, unfortunateLy the tremendous
quantity of purified material required in analysis for < 5% sensitivity
in analysis, exceeded the total amount of purified material available.
In fact after allocating fixed quantities of purified product for the
various other quantitative and complement assays, the required 5-6 mg
of purified LTA needed for amino acid analysis far exceeded the poten
tial amount available from the LTAppx. For this reason, the Bio-Rad
Protein Assay was used to estimate, the total amount protein in each
sample. As can he seen in Table 7, there was a relatively close cor
relation between values determined by amino acid analysis and those
determined using the Bio-Rad Assay. It is therefore reasonable to

assume that the values given for the final products are at least a
close indication of the total percent protein available in each product.
Although the values may seem high, it should he remembered that (1) the
total amount of protein available in the sample represents a lower limit
for the accuracy of the assay, and (2) the standard protein curve
(human albumin) used to convert optical density readings to pg of pro
tein may not accurately correlate the reactions of the limited number
of amino acid residues available in the final product. Attempts to
verify these values with the biuret reaction (153) and the I.owrv Pro
tein Assay (154) were unsuccessful. Biuret was too insensitive whereas
the Lowry proved to be unreliable due to its reaction with glycerol to
give a false positive reaction. Despite this shortcoming all other
factors indicate that l.TApcx represents the most highly purified LIA
from _S. mu tans BHT that any laboratory has yet achieved.
The results from the final experiments to determine the site and
mechanism of complement inhibition by LTA were equivocal. Like the
purification of LTA, establishing the site and mechanism of inhibition
proved to be considerably more challenging than anticipated. Prelimin
ary data utilizing LTAcx quite consistently suggested that Cl was the
site of action and interference with binding affinity (Clq dysfunction)
or with esterase activity (Cls dysfunction) was the mechanism. These
conclusions were based on the fact that !',ACl4irt,. hut not F.ACJ42 were
inhibited and also that the titer of fluid phase CL preincubuted with
LTAcx was significantly reduced. Considering the pnlvanion ic nature of
LTA conferred hv the polar po 1 yglycero!. phosphate backbone, it appeared
that LTA represented a model system for polvanionic interference of Cl
funct ion.
Such activity hits been ascribed to dexcran sulfate polyvinyl

suJ.fate, heparin, polyiosinic acid, chondroitin and many other polv-
anionic compounds (39,40) in addition to DNA, RNA (135,156), and
carrageenin (157). It was dismaying to find that although LTApcx
maintained anti-complementary activity with the appropriate cellular
intermediates, all fluid phase inhibition of Cl was abrogated (Figures
17 and 18). All subsequent experiments attempting to define Clq, Cls,
or Cls dysfunction were negative. The only experiments that gave sug
gestive results were the Cl transfer assavs. Even here, instead of the
anticipated inhibition of Cl transfer, over 20% enhancement of transfer
was observed (Table 10). Thus, in light of these data obtained with
purified LTA it was necessary to devise new molecular models to explain
the mechanism of lytic inhibition of certain complement intermediates
by highly purified LTA. Some possibilities are discussed below:
1). Attachment of LTA sterically blocks the affixation of Cl to the
Fc portion of the immunoglobulin. Thus, if Cl does not attach properly,
or is prevented from attaching at all, the complement cascade will never
be initiated.
2). Although Cls activity was not effected fluid phase, perhaps such
activity would be abrogated once the Cl molecule became associated with
the cell membrane. If so, EAC1 would no longer be capable of hydrofining
C4 or C2 again, the cascade would he-terminated.
3). LTA directly interacts with fluid phase C4 or ('2 thus preventing
them from combining with the appropriate sites on the membrane.
4). The attachment of LTA lends to increased fluidity of the mem
brane resulting in the displacement of looselv attached molecules. If
some of those less tenacious molecules include any of the early comple
ment components, the physical loss of these components would terminate
the lytic attack sequence.

1 h
5). The attachment of LTA to the cell membrane prevents the subse
quent attachment of the C4 or C2 active fragments (i.e. C4b or C2n
respectively). Thus, the activities of all complement components
would remain intact and no observable dysfunction should be observed.
However, if C4b or C2a were in the least impeded in their attachment
to the cell membrane, these active fragments would rapidly decay and
lose their ability to do so.
If the first model accurately portrayed the mechanism of inhibi
tion, one would predict a decrease in Cl uptake by EA This predic
tion was not corroborated by experimental results. In addition, this
model would not explain the high degree of inhibition of cells in the
EAC1 state where Cl is already attached.
If the second model were true, one would predict a decreased
consumption of fluid phase C4 or C2 after pre incubation with EA01 ...
L I A
Again, such was not the case. Neither residual C-'t activity when in
cubated with EAC1 nor residual C2 activity when incubated with
1-1 X
EACl4[TA was appreciably different from their buffer treated controls.
Model three would predict a decrease in fluid phase activity of
C4 or C2 when preincubated with LTA. As demonstrated in Table 8, no
such decrease in activity was observed.
Model, four maintains that the attachment of LTA would somehow
alter the membrane such that loosely attached components would he
released more readily. The first problem with this model is that the
attachment of the enrlv complement components to the membrane is quite
tenacious. In fact, some evidence suggests that membrane attachment
of cytophillis C4b is accompanied bv the formation of covalent bonds
(158,1V)). Once attached, it seems unlikely that (14b would be readily

released. Cl is not: attached to the membrane at all, hut rather is
combined with the Fc region of the hemolysin antibody. Therefore, this
model would predict that either Cl is released from the antigen-anti
body complex (very much akin to the predictions and shortcomings of
model one) or that the entire antibody-Cl complex is released from the
cell membrane (with or without the accompanying antigen). Such a
mechanism is somewhat exotic, but not totally improbable. Recent evi
dence suggests that the binding of serum albumin, immunoglobulins, or
complement can effect a release of phospholipids from liposomes (160.
161,162). Perhaps the attachment of LTA can likewise evoke such a
release of cell membrane constituents and in the process, release the
131
immune complexes as well. Experiments utilizing f labelled hemolvsin
antibody would demonstrate, whether the antihodv was maintained on the
131
cell surface or released into the medium. Likewise, I labe Lied Cl
could be used to determine if Cl were released.
Of ail the proposed models, number five most 1ikelv portrays the
actual mechanism of inhibition. This model assorts that the affixation
of LTA to the surface of the cell delays or prevents the rapid associa
tion of C4b (or C2a) with its respective site on (lie ceil membrane. As
previously discussed, once C4 is cleaved bv Cl, the cleavage results in
the formation of a short-lived binding site on the C4h fragment. A high
density of LTA on the surface of the cell might sequester C4h f inding
sites or perhaps change the electrostat to charge of the cell surface
sufficiently to effect the kinetics of the C4b attachment. The end
result in either case would bo the nonproduct ivo consuinpt ion of C4
molecules. This is consistent with the results from the residual C4
titration studies in which no alteration of C4 consumption was observed

1 18
when C4 was incubated with EAC1,_,,. This model is also consistant with
LTA
the fact no dysfunction of Cl, Clq, Cls, Cls, C4, or C2 could he demon
strated when incubated fluid phase with LTApcx.
This model would also predict that once C4b were attached to the
membrane, subsequent addition of LTA should have significantly less
impact on cascade disruption. As shown in Figure 17, this prediction
coincides well with the facts. Percent inhibition of lysis drops from
more than 65% in the case of EA treated with LTApcx (100 pg/nl) to less
than 20% in the case of EAC14 treated with the same concentration of
LTApcx. Furthermore, EAC142 are no longer inhibited as one would
LIA
expect if the C4b and C2a binding sites were already secured.
Although all data thus far presented are consistant with this
131
model, final proof would necessitate the 1 labelling of purified C4
and C2. Once labelled, one could determine if an excess of decayed CAn
and C2a fragments were released into the media after preincubation with
EACI.t, r EAC1.4 respectively.
L l A LTA
It is hoped that future research in this area may prove enlightening
not only in expanding upon the mechanism of inhibition but also upon
the specific role this extracellular metabolite, plays in the inflam
matory response of periodontal lesions.
It is apparent that the anti-complementarv activity of LTA is not
restricted to a single species or geneus (Table I!) and it may verv
well he that LTA plays ) significant role in protecting gram positive
organisms from immunologic destruction. If so, LTA could ho considered
a type of "virulence" factor and those organisms t li.it produce copious
amounts of extracellular LTA (such as S. mutans BUT) would not only
contribute to their own protection, but also to the protection of the

myriad of microorganisms in their immediate environment. Obviously,
more research in this area is needed before such speculation can be
substantiated with fact.

LITERATURE CITED
1. Wicken, A. J., and K. W. Knox. 1977. Hicrobiology 1977 Biological
properties of lipoteichoic acids. American Society for Micro
biology, Washington, D. C. p 360.
2. Wicken, A. J., and K. W. Knox. 1975. Lipoteichoic acidsA new
class of bacterial antigens. Science. 187:1161.
3. Gewurz, H., H. S. Shin, and S. E. Mergenhagen. 1963. Interactions
of the complement system with endotoxJc 1ipopolysaccharides:
Consumption of each of the six terminal complement components.
J. Exp. Med. 1_28:1049.
4. Gewurz, H., S. E. Mergenhagen, A. Nowotny, and .1. K. Phillips. 1963.
Interactions of the complement systems with native and chemically
modified endotoxins. J. Bacteriol. 95:397.
5. Marcus, R. L., II. S. Shin, and M. M. Mayer. 1971. An alternate
complement pathway: C3 cleaving activity not due to C4, 2a on
endotoxic lipopolysaccharide after treatment with guinea nig
serum. Relation to properdin. Proc. Natl. Acad. Sei. U. S. A.
68:1351.
6. Phillips, J. K., R. Snyderman, and S. E. Mergenhagen. 172. Acti
vation of complement by endotoxin: A role for globulin, Cl, C4,
and C2 in the consumption of terminal complement components by
endotoxin coated erythrocytes. J. Immunol,. 109: 334.
7. Mergenhagen, S. E., R. Snyderman, and .1. K. Phillips. 1973. Acti
vation of complement by endotoxin. ,1. Infect. Dis. 128:386.
3. El Imn, I., I. Green, and M. Frank. 1970. Genetic controlled total
deficiency of the fourth component of complement in the guinea
p i g.. Sei once. 1 20: 74 .
9. Hu is in1 '1 Veld, J. II. .1., and 1.. Barrens. 1976. Inactivation of
hcmolyt ic complement in human serum hv an acvlated po 1 ysaecharido
frem a gram-positive3 rod: Possible significance in pineon-breeder's
disease. Infect. Immunity. 13:1,619.
10. Gonco, R. ,J. P. A. Mashirno, (.. Prvgier, and S. A. Ellison. 1974.
Antibody mediated effects on the periodontium. .1. Periodontol Res.
4_5 (part II) : 330.
1L. Mergenhagen, S. E.,T. K. Tempe!, and R. Snvderman. ¡970. Immunolo
gic reactions and periodontal inflammation. .1. Dent. Res. 49:256.
1,20

12.
121
Attstrom, R. A., R. Laurel 1, !!. Larsson, and A. Sjoholm. 1975.
Complement factors in gingival crevice material from healthy and
inflamed gingiva in humans. J. Periodontal Res. 10:19.
13. Allison, A. C., and II. U. Schorlemmor. 1970. Activation of comple
ment by the alternative pathway as a factor in the pathogenesis
of periodontal disease. Lancet. 2:1001.
14. Raisz, L. G., A. L. Sandburg, J. M. Goodson, II. A. Simmons, and S.
Mergenhagen. 1974. Complement-dependent stimulation of prosta
glandin synthesis and bone resorption. Science. 185:789.
15. Dietrich, J. W., and L. G. Raisz. 1975. Prostaglandin in calcium
and bone metabolism. Clinical Orthopaedics and Related Research.
111:228.
16. Goodson, J. M. F. Dewhirst, and A. Brunotti. Prostaglandin levels
in human gingival tissue. J. Dent. Res. 5_2 (special issue) :182.
17. Hausmann, E., 0. Luderitz, K. Knox, and N. Weinfeld. 1975. Structural
requirements for hone resorption hv endotoxin and lipoteiehoic acid.
J. Dent. Res. B54 (special issue):94.
18. Carlsson, J. 1967. Presence of various types of non-hemolvtic strep
tococci in dental plaque and in other sites of the oral cavity in
man. Odontol. Revy. 18:55.
19. Joseph, R. and G. D. Shocktnan. 1975. Synthesis and excretion of
glycerol teichnie acid during growth of two streptococcal species.
Infect. Immun. 12:333.
20. Markham, J. L., K. W. Knox, A. J. Uicken, and M. J. lleve tt. 197".
Formation of extracellular lipoteiehoic acid by oral streptococci
and 1actobacilli. Infect, immun. 12:378.
21. Mll er-Eherhard, H. J. 1975. Textbook of_ I miminop.tt ho l.ogv ed P. A.
Micsehea, H. J. Mil 1 ler-Eberhard. Grue and Stratton, N. Y., N. Y.
2nd ed.
22. Austen, K. F. 1 974. (.hem is try and biologic activity of the comple
ment system. Trnnsp l.ant. Proc. 6:1.
23. Naff, (?. B. J. Pen sky, and I. 11. Lepov. 1964. The mae romo 1 ecu lar
nature of the 1st component of human J. Exp. ilc?d. !1_9: 593.
24. Lepow, [. H. G. B. Na f T, E. W. Todd, .1. Ienskv, and C. V. llinz, Jr.
Chromatographic resolution of the first component or human comple
ment into 3 activities. J. Exp. Med. H7:983.
25. Mii 1 1 er-KUerha rd, II. J. 1 *72 The molecular basis of
activities of G. Harvov Lec.t. 66:75.
the biological

1 2
26. Mil 1 1 e.r-Eberhard, II. .1., U. R. Nilsson. A. P. Ha I niasso, M. .r. Pol lev,
and M. A. Calcott. 1966. A molecular concept of immune cytolvsis.
Arch. Pathol. 82:205.
27. Reid, K. B. M., D. M. Lowe, and R. R. Porter. 1972. Isolation and
characterization of Clq, a subcomponent of the first component
of complement from human and rabbit, sera. Riochem. .1. 130:749.
28. Calcott, M. A., and H. .J. Mill ler-Eborhard. 1972. Clq protein of
human complement. Biochemistry. 11:3443.
29. Mller-Eberhard, H. J. 1975. Complement. Ann. Rev. Biochem. 44:697.
30. Taranta, A., and E. C. Franklin. 1961. Complement fixation by anti
body fragments. Science. 134:1981.
31. Augener, W., H. M. Orey, N. R. Cooper, and H. J. Mller-Eberhard.
1971. The reaction of monomeric and aggregated immunoglobulins
with Cl. Immunochemistry. 8:1011.
32. Isliker, H. 1974. Interaction of Clq with IgG and fragments thereof.
Adv. Biosci. 12:2 7 0.
33. Valet, G., and N. R, Cooper. 1974. Isolation and characterization of
the proenzyme form of the Clr subunit of the first complement com
ponent:. J. Immunol. 112:1667.
34. Val.et, C. and N. R. Cooper. 1974. isolation and characterization
of the proenzyme form of the Cls subunit of the first complement'
component. J. Immunol. 112:339.
35. Sakai, K., and R. M. Stroud. 1973. Purification, molecular proper
ties, and activation of Cl proesterase, Cls. .1. Immunol. 110:1010.
36. Sakai, K., and R. M. Stroud. 19 74. The act ivat ion of Cls with
purified Clr. Immunochemistry. 11:191.
37. Becker, E. L. 1956. Concerning the mechanism of complement action.
IT. The nature of the 1st component of guinea pip, complement. J.
Immunol. 77:469.
38. Nagnlci, K. and R. M. Stroud. 1969. The relationship of the hemo
lytic activity of active Cls to its I'AMe esterase action: A new
method of purification and assay. .!. Immunol. 102:421.
39. I.oos, M. E. Volanakis, and R. M. Stroud. 1976. Mode of inter
act inn of different polyanions with the first (Cl, Cl), the second
(C2), and the fourth (C4) component of comp 1 oment11 I. Inhibition
of C4 and C2 binding stte(s) on Cls bv polyanions. Immunochemistry
L3: 789.
40. Rnepplo, E., H. H. Hill, and M. Lons. 1976. J-lode of interaction of
different pol.yanions with the first (Cl, CL), the second (C2),and
the fourth (C4) component of complement1. Effect on fluid phase
Cl and on Cl hound to EA or to EAC4. Tmmunoehem i strv. 13:251.

121
41. Allan, R., and H. IslLker. 1974. Studies on the complement-binding
site of rabbit immunoglobulin GModification of tryptophan resi
dues and their role in anticomplementary activity of rabbit IgG.
Immunochemistry. 11:175.
42. Wilder, R. L., G. Green, and V. N. Sehumaker. Bivalent hapten-anti
body interactions. Immunochemistry. 12:54.
43. Patrick, R. A., S. B. Taubman, and I. H. Lepow. 1970. Cleavage of
the fourth component of human complement (C4) by activated Cls.
Immunochemistry. 7:217.
44. Schreiber, R. D., and H. J. M 1 ler-Eberhard. 1974. Fourth component
of human complement: Description of a three polypeptide chain
structure. J. Exp. Med. 140:1324.
45. Mller-Eberhard, H. .1., and I. H. Lepow. 1965. Cl esterase effect on
activity and physicochemical properties of the fourth component of
complement. J. Exp. Med. 121:819.
46. Policy, M. J., and H. J. MU 1. ler-Eberhard. 1968. The second component
of human complement: Its isolation, fragmentation by C'l esterase,
and incorporation into C'3 convertase. .J. Exp. Med. 128:533.
47. Mller-Eberhard, H. J., A. P. Dalmasso, and M. A. Cnlrott. 1966.
The reaction mechanism of Blc-globulin (C'3) in immune hemolysis.
J. Exp. Med. 1_2J3:33.
48. Shin, H. S., and 11. M. Mayer. 1968. The third component of the guinea
pig complement system. II. Kinetic study of the reaction of EAC'4,2
with guinea pig C'3. Enzymatic nature of fixation, and hemolytic
titration of C'3. Biochemistry. 7:2997.
49. Cooper, N. R. 1971. Enzymes of the complement system. Prog. Immunol.
JL: 5 6 7.
50. Policy, M. J., and I!. J. MU 11er-Ehorhard. 1967. Enhancement of the
hemolytic activity of the second component of human complement
by oxidation. J. Exp. Med. 126:1013.
51. Gold lust, M. B. H. S. Shin, C. 11. Hammer, and M. M. Mayer. 1974.
Studies of complement complex C5b,6 eluted from EAC-6: Reaction
of C5b,6 with EAC4b,3b and evidence on the role of C2a and C3b
in the activation of C5. J. Immunol. II3:998.
52. Arroyave, C. M. and li. J. MU 1 1 cr-F.be rhard. 1973. Interact ions
between human C5, C6, and C7 and their fimet ional significance
in complement dependent cytolysis. J. Immunol, ill:536.
53. Lachmann, P. J., and R. A. Thompson. 1970. Reactive lysis, (ho
complement mediated lysis of unsensitized colls. II. The charac
terization of activated reactor as C56 and the participation of
C8 and C.9. Exp. Med. 1 31 : 643.

12
54. Kolb, W. P. .1. A. Haxby, C. M. Arrovave, and 1!. .1. Mii I ] cr-Fberlmrd .
1972. Molecular ann Lysis of the monbrant attack mechanism of Cl .
J. Exp. Med. 1_35:549.
55. Jensen, J. A. 1967. Anuphylatoxin in its relation to the complement
system. Science. 1_55:1122 .
56. Dias Da Silva, W., J. W. Eisele, and !. H. Lepow. 1967. Complement
as a mediator of inflammation. J. Exp. Med. 126:1027.
57. Cochrane, C. G., and ii. J.. MU 1 ler-Eberhard. 1968. The derivation
of two distinct anaphylatoxin activities from the third and fifth
components of human complement. J. Exp. Med. 127:371.
58. Mahler, F., M. Intaglietta, T. E. Hugli, and A. R. Johnson. 1975.
Influences of C3a anaphylatoxin compared to other vasoactive
agents on the microcirculation of rabbit omentum. Microvasc. Res.
9: 345.
59. Stolfi, R. L. 1968. Immune lytic transformation: A state of irrever
sible damage generated as a result of the reaction of the eighth
component in the guinea pig complement system. J. Immunol. 100:46.
60. Thompson, R. A., and P. J. Lachmann. 1970. Reactive lysis: The com
plement-mediated lysis of unsensitized cells. .1. Exp. Med. 131:629
61. Kolb, W. P., and H. J. Mill ler-Eberha rd. 1973. The membrane attack
mechanism of complement verification of a stable C5-9 complex
in free solution. J. Exp. Med. 138:438.
62. Koethe, S. M., K. F. Austen, and I. Gigli. 1973. blocking of the
hemolytic expression of the classical C' sequence by products of
C1 activation via the alternate pathway. J. Immunol. 110:390.
63. Delage, J. M., G. Lenner-Netsch, and J. Simard. 1973. The tribu-
tyrinase activity of C7. Immunology. 24:671.
64.Inoue, K., and S. C. Kinsky. L970. Fate of phospholipids in lipo
somal model membranes damaged by antibody and complement.
Biochemistry. 9:4767.
65.
Mayer, M. M.
Nat. Acad.
1972. Mechanism of cytolvsis bv complement. Proc.
Sci. U. S. A. 69:2954.
66.
Humphrey, J.
membranes
!!. and R. R. Dotirmashkin.
caused bv complement. Ativan.
1969. The lesions in cell
10)1111111'' 1 11:75.
67.
lies, G. 11. ,
lesions in
P. Socman, 0. Naylor, and B
immune lysis: Surface rings
. Cinader. 1973. Mombram
, globule aggregates, am
transient openings. .1. Ge! I Biol. 56:528.
08. Gigli, 1., S. Ruddy, and K. F. Austen. 1968. The stoicbi ornotric
measurement of the serum inhibitor of the first component by the
inhibition of immune hemolysis. .1. Immunol. 100:1154.

L 2
69. Pensky, .1. L. R. levy, and I. H. I.i'pnw. 1961. Partial 'unification
of a serum inhibitor of C1 esterase. .1. Biol. Chem. 236:1674.
70. Ruddy, S., and K. F. Austen. 1971. C3b inactivator of nan. .
Immunol. 107:742.
71. Miii ler-Eberhard, II. .1., and 0. Gcitze. 1972. Cl proactivator con-
vertase and its mode of action. J. F.xp. Med. 135:1003.
72. Alper, C. A., F. S. Rosen, and P. J. i.achmann. 1972. Inactivator
of the third component of complement as an inhibitor in the
properdin pathway. Proc. Nat. Acad. Sci. U. S. A. 69:2910.
73. Tamura, N., and R. A. Nelson, Jr. 1967. Three naturally occurring
Inhibitors of components of complement in guinea pie and rabbit
serum. J. Immunol. 99:582.
74. Henson, P. M. 1969. The adherence of leukocytes and platelets in
duced by fixed IgG antibody or com]') 1 ement. Tmmunologv. 16:107.
75. Lay, W. II., and V. Nussenzweig. 1968. Receptors for complement on
leukocytes. J. Exp. Med. 128:991.
76. Henson, P. M. 1972. Complement-dependent adherence of cells to
antigen and antibody. Mechanisms and consequences. Bi o 1 og i c a1
Activities of Complement. Karger and Basel, N. Y., N. Y. p 173.
77. Dukor, P., and K. U. Hartmann. 1973. Itvpothesis--Bound Cl as the
second signal for B-cel1 activation. Cell Immunol. 7:3*9.
78. Plllemer, L., L. Blum, T. H. Lepow, O. A. Ross, I'. W. 1 odd, and
A. C. Ward law. 1954. The properdin system and immunity: Demon
stration and isolation of a new serum protein, properdin, and
its role in immune phenomena. Science. JL20:279.
79. Nelson, R. A., Jr. 1958. An alternative mechanism for the properdin
system. J. Exp. Med. 108:515.
80. Pillemer, L., M. D. Schoenberg, L. Blum, and L. Wur z 1 55. Proper
din system and immunity. II. Interaction of the properdin svstem
with polysaccharides. Science. 122:543.
81. Fine, D. P. 1974. Activation of the classical and alternate path
ways of endotoxin. J. Immunol. 112:2.
82. Sehreiher, R. D. I!. C. Medians, C. Col/.e, and II. J. Miiller-Fherli.ini
1975. Properdin-and nephritic factor-dependent C3 convertases:
Requirement of native C3 for enzyme formation and the function of
bound C3G as properdin receptor. J. Exp. Med. I't2:76c.
83. Giitze, 0., and H. J. MM 11 or-Eherhard. |971. The (13-activator svstem:
An alternate pathway of complement activation. J. Exp. Med. 134:90

1 2 6
84. Sandberg, A. L. A. G. Osier, H. Shin, and B. Oliveira. L970. The.
biologic activities of guinea pig antibodies, il. Modes of com
plement interaction with yl and y2 immunoglobulins. J. Immunol.
104:329.
85. Sandberg, A. L. 0. Gdtze, H. J. Miiller-F.berhard, and A. G. Osier.
1971. Complement utilization by guinea pig y 1 and y2 immunoglo
bulins through the C3 activator system. J. Immunol. 107:920.
86. Platts-Milis, T. A. E. and K. Tshizaka. 1974. Activation of the
alternate pathway of human complement by rabbit cells. J. Immunol.
113:348.
87. Poskitt, T. R. II. P. Fortwengler, ,Ir. and B. J. Funskis. 1973.
Activation of the alternate complement pathway by autologous red
stroma. J. Exp. Med. 138:715.
88. Joseph, B. S., N. R. Cooper, and M. B. A. Oldstone. 1975. Immunologic
injury of cultured cells infected with measles virus. I. Role of
TgG antibody and the alternative complement pathway, d. Exp. Med.
141:761.
89. Perrin, L. H., B. S. Joseph, N. R. Cooper, and M. B. A. Oldstone.
1976. Mechanism of injury of virus-infected cells by antiviral
antibody and complement: Participation of IgO, F(as'), and the
alternative complement pathway, d. Exp. Med. 14 3: 1 0271
90. Leon, M. A. 196], Inhibition in the properdin-dextran system. In
Immunochem i ca 1 Approaches to Problems in Mie rob i o .logy. M.
He ideiberger, O. d. Pleseia, and R. A. Day, ed. Rutgers-Un iver-
sity Press, New Brunswick, N. J. p 304.
91. Itiai, S., S. Ehisu, K. Kato, and S. Kotani. 1976. Activation of
complement through the alternate pathway hv microbial glucans.
J. Immunol. 116:1737.
92. Konig, W., D. B i t Ler-Siiennann, M. Piorich, M. I.imhert, i!. I'.
Sclior 1 emmer and II. lladding. 1974. DND-autigens activate the
alternate patiiwav of the complement system, d. Immunol. l_13:501.
93. Alper, C. A., and D. Bnlnvitch. 1976. Cobra venom factor: Evidence
for its being altered cobra C3. Science. 191:1275.
94. Hunsicker, 1.. f.. S. Ruddy, and K. F. Austen. 197!. Alternate C'
pathway: Factors Involved in CVF act tvaliou of (''!*". d. Immunol.
1 Ml: 128.
95. Cooper, N. R. 1973. Formation and Function of a complex of the C3
proactivator with a protein from cobra venom, d. Exp. Med. 137:451.
96. Sehreiher, R. D. 0. Gotxe, and H. d. Mill ier-Eberltard. 1976. Alter
native pathway of comp 1ement: Demonstrat ion and characterization
of initiating factor and its properd in-independent function, d.
Exp. Med. 144:1062.

127
97. Gotze, 0., and H. J. Mi.il .1 er-Ehcrhard. 1974. The role of properdin
in the alternate pathway of complement activation. .1. Exp. Mod.
139:44.
98. Gotze, 0. 1975. Proteases of the properdin system. Tn Proteases
and Biological Control. E. Reich, D. B. Rifkin, and E. Shaw, ed.
Cold Spring Harbor Laboratory, 'Cold Spring Harbor, N. Y. p 255.
99. Iledicus, R. G. 0. Gotze, and H. J. Mill ler-Eherhard. 197b. .Alter
native pathway of complement recruitment of precursor properdin
by the labile C3/C5 convertase and the potentiation of the path
way. J. Exp. Med. 144:1076.
100.Vallota, E. H., .J. Forristal, R. E. Spitzer, N. C. Davis, and C. P.
West. 1970. Characteristics of a non-complement-dependent C3-reac-
tive complex formed from factors in nephritic and normal serum. J.
Exp. Med. 131:1306.
101. Gorzynski, E. A., E. Neter, and E. Cohen. 1960. Effect of lysozyme
on the release of erythrocyte modifying antigen from staphylococci
and Micrococcus lysodeikticus. J. Bacterio!. 80:207.
102. Chorpenning, F. W., and M. C. Dodd. 1966. Heterogenic antigens of
gram-positive bacteria. J. Bacterio!. 91:1440.
103. Hewett, M. J., K. W. Knox, and A. J. Wicken. 19 70. Studies o: the
group F antigen of 1 nctobarillus: Detection of antibodies \v
hemagglutination. J. Gen. Microbiol. 60:315.
104. Zabriski, J. B. 1967. Mimetic relationships between group A strep
tococci and mammalian tissues. Adv. Immunol. 7:147.
105. Click, A. L., R. A. Getnick, and R. M. Cole. 1971. Electron
microscopy of group A streptococci after phagocytosis by human
monocytes, infect. Immun. 4:772.
106. Berrens, L. and C. L. 11. Guikers. 1972. An immunochemical study
of pigeon-breeder's disease. Int. Arch. Allergy. 4_3: 347.
107. Knox. K. W., and A. J. Wicken. 1973. immunological properties
of teicboic acids. Bacterio!.. Rev. 37:215.
103. Baddilcy, J. 1970. Structure., biosynthesis, and function of
teichoic acids. Account. Chem. Res. 5:98.
109. Rictshel, I'. Tii. II. Got tort, O. huderitz, and 0. Westphal. |972.
Nature and linkage of the fatty acids present in the lipid-A
component of salmonella 1 i popo 1ysaccharide. Fur. .1. Biochen. 28:16b.
110. Wicken, A. .1., and K. W. Knox. 1970. Studies on the group F anti
gens of 1actobaci!1i: Isolation of a teichoic acid-lipid complex
from Kactobaci11 us ferment l. NTGG 6991. .1. Gen Microbiol. 60:293.

128
111. Rant 7,, L. A., E. R. Randall, and A. Z. Zurkertnnn. 1956. Hemolysis
and hemagglutination by normal serums of erythrocytes treated
with a non-species specific bacterial substance. Infect. Dis.
98:211.
112. Fiedler, F., and L. Glaser. 1974. The attachment of poly-
ribitol phosphate to lipoteichoic acid carrier. Carbohvdr.
Res. 37_:37.
113. Fiedler, F., J. Mauck, and L. Glaser. 1974. Problems in cell wall
assembly. Ann. N. Y. Acad. Sci. 2_3 5:198.
114. Chatterjee, A. N., and W. Wong. Isolation and characterization of
a mutant of Staphylococ.cus aureus deficient in autolyti.c activity.
J. BacterioL 125:961.
115. Holtje, J. W., and A. Tomasz. 1975. Lipoteichoic acid: A specific
inhibitor of autolysin activity in pneumococcus. Proe. Natl. Acad.
Sci. U. S. A. 72:1960.
116. Cleveland, R. F. .1. V. Holtje, A. J. Wicken, A. Thomasz, L. Dar.eo-
Moore, and G. I). Shockman. 1975. Inhibition of bacterial wall
lysins by lipoteichoic acids and related compounds. Biochem.
Biophys. Res. Comm. 67:1128.
117. Cleveland, R. F., A. J. Wicken, L. Daneo-Moore, and G. P. Shockman.
1976. Inhibition of wall nutoy Is is In St re p t ococcus facea Us by
lipoteichoic acids and lipids. .1. Bacteriol. 126:192.
118. Cleveland, R. F., L. Daneo-Moore. A. .1. Wicken, and G. D. Shockman.
1976. Effect of lipoteichoic acid and lipids on Ivsis of intact
cells of Streptococcus faecal is. .1. Bacteriol. 12_7:13S2.
119. Simmons, D. A. R. 1971. Immunochemistry of Shlgel la f le.xneri 0-
antigens: A study of structural and genetic aspects of the bio
synthesis of cell-surface antigens. Bacteriol. Rev. 3_5 : 11 7 .
120. Wicken, A. .1., and K. W. Knox. 1973. Characterization of group N
streptococcus lipoteichoic acid. Infect. Imimm. 11:973.
121. Doyle, R. J., A. N. Chatterjee, V. N. Streips, and F. K. Young.
1975. Soluble mneromoI ocular cenplexes involving bacterial
teichoic acids. .1. Bacteriol. 124:34 1 .
122. Driel, I). V., A. .1. Wicken, M. R. Dickson, and K. W. Knox. 19/3.
Cellular location of the lipoteichoic acids of l,ac_tobac_i_l 1 us
casei NCTC 6375. Ultra. Rsh. 43:483.
123. Frederick, G. T., and F. W. Chorponning. 1974. Characterization
of antibodies specific for polvglyceroL phosphate. -I. Immunol.
113:489.

1 2f)
124. Daugherty, !1. D. R. R. Martin, and A. White. 1969. Reaction of
sera and nasal secretions with staphylococcal antigens. .1.
Lab. Clin. Med. 73: LOU.
125. Markham, J. L., K. W. Knox, R. G. Schamschula, and A. .1. Wicken.
1973. Antibodies to teichoic acids in humans. Arch. Oral. Biol.
IS:313.
126. Jackson, R. W., and U. Muskowitz. 1966. Mature of a red cell
sensitizing substance from streptococci. J. Bncteriol. 91:2205.
127. Carlsson, J. 1967. Presence of various types of non-hemolvtic
streptococci in dental plaque and in other sites of the oral
cavity in man. Odontol. Revy. 18:55.
128. Zinner, D. D. J. M. Jablon, A. R. Aran, and If. S. Sas.lnw. 1965.
Experimental caries induced in animals by streptococci of human
origin. Proc. Soc. Exp. Biol. Med. 118:766.
129. Guggenheim, B. 1968. Streptococci of dental plaques. Caries Res.
2:147.
130.Fitzgerald, R. J. and P. H. Keyes, i960. Demonstration of the
etiological role of streptococci in experimental caries in the
hamster. J. A.mer. Dent. Asso. 61:9.
131.Hoffmann, E. M. 1969. Inhibition of complement by a substance
isolated from human erythrocytes. 1. Extraction from human
erythrocytes stromata. Immunochemis try. 6:30]..
132.Nelson, R. A., J. .Jensen, I. Gigli, and N. Tamura. 1966. Methods
for the separation, purification, and measurement of nine com
ponents of hemolytic complement in guinea pig serum. I inmuno-*
chemistry. 3:11.
1.33.
Ruddv, S., and K. F. Austen. 1967. A stoichiometric assay for the
Cl
fourth component of complement in whole human serum using EACl
and functionally pure human second component. J. Immunol. 99:1162.
134.Ruddy, S., and K. F. Austen. 1969. C3 inactivator of man. I. Hemo
lytic measurement bv the inactivation of cell-hound C3. J. Immunol.
102:533.
135.Rabat:, E. A., and M. M. Mayer. 1961. Comp lenient and complement fix
ation. In Kxper ¡mental f.mmnnochem ist rv. Charles C. Thomas,
Springfield, 111. p 149.
136.Bersos, T., and II. J. Rapp. 1967. Immune homolvsis: A simplified
method for preparation of F.AC4 with guinea pig or with human
complement. J. Immunol. 99:263.

137. Hoffmann, E. M. 9 f3 9. Inhibition of complement by a substance
isolated from human erythrocytes. II. Studies on the site and
mechanism of action. Immunochemistry. 6:403.
138. Wic.kon, A. J., J. W. Gibbons, and K. W. Knox. 1973. Comparative
studies on the isolation of membrane lipoteichoic acids from
Lactobacillus fermenti 6991. J. Bacterio!. 113:365.
139. Rapp, J. J., and T. Borsos. 1970. Molecular Basis ojf Complement
Action. Appleton-Century-Crofts, N. Y., N. Y. p 105.
140. Hill, M. W. 1974. The effect of anaesthetic-1 ike molecules on
the phase transition in smectic mesophases of dipalmitoyl-
lecithn. I. The normal alcohol up to C--9 and three inhala
tion anaesthetics. Riochem. Biophys. Acta. 356:117.
141. Lowry, 0. H., N. R. Roberts, K. Y. Leiner, M. Wu, and A. L. Farr.
1954. The quantitative histochemistry of the lira in. J. Biol.
Chem. 207:1.
142. Dubois, M., K. A. Giles, J. K. Hamilton, P. A. Rebers, and F. Smith
1956. Colorimetric method for the determination of sugars and
related substances. Anal. Chem. 28:350.
143. Grabar, P., and P. Burt in. 1964. Immunoelec trophoretic Analys is.
Elsevier, N. Y., N. Y.
144. Yonemasu, K., R. M. Stroud. 1971. Clq: Rapid purification method for
preparation of monospecific antisera and for biochemical studies.
J. Immunol. 106:304.
145. Bryant, R. E., D. E. Jenkins, Jr. 1968. Calcium requirements for com
plement dependent hemolytic reactions. J. Immunol. 10_1:664.
146. Fine, D. P. 1977. Comparison of e.tliy lonegl yco 11 et rancet ic acid and
its magnesium salt as reagents for studying alternative comple
ment pathway function, rnfcct. Immun. 1_6:124.
147. Fine, D. P., S. R. Mamey, Jr., D. G. Colley, J. S. Sergent, and R.
M. Des Prez. 1972. G3 shunt activation in hitman serum chelated
with EGTA. J. Tmmunol. 109: <307.
148. Becker, E. L. I960. Concerning the mechanism of complement action.
V. The early steps in immune hemolysis. J. Immunol. 84:299.
149. Wilkinson, S. G. 1968. Clycosyl diglyeoridos from Pseudomonas
ruj 11 use e ns B BA. 1 6 4 : 14 8.
150. Schmit, A. S. D. D. Ploss, and W. I. Lennatz. 1974. Some aspects
of the chemistry and biochemistry of membranes of gram-positive
bacteria. Annals of the N. Y. Academy of Sciences. 235:91.

131
151. Bladen, II. G. Hagenge, R. Harr, and F. Pol.lurk. 1972. l.ysls of
certain organisms by the synergistic action of complement and
lysozyme. J. Dent. Res. 52:371.
152. Mill ler-Eberhard, H. J., M. J. Policy, and M. A. Calcott. 1967.
Formation and functional, significance of a molecular complex
derived from the second and the fourth component human complement.
J. Exp. Med. 125:359.
153. Kingsley, G. R. 1939. The determination of serum total protein,
albumin and globulin by the biuret reaction. J. Biol. Chem. 1_31:19
154. Lowry, 0. H., N. J. Rosegrough, A. L. Farr, and R. .1. Randall. 1951.
Protein measurement with the folin phenol reagent. J. Biol. Chem.
193:265.
155. Agnello, V. R. J. Winchester, and 11. G. Kunkel. 1970. Precipitin
reactions of the Clq component of complement with aggregated y
globulin and immune complexes in gel diffusion. Immunology. 1.9: in11
156. Agnello, V. R, 1. Carr, D. Doffler, and H. G. Kunkel. 1969. Gel
diffusion reactions of Clq with aggregated y globulin, DNA, and
various anionic substances. Fed. Proc. 23:696.
157. Borsos, T., H. J. Rapp, and C. J. Crisler. 1965. The interaction
between carrageenan and the first component of complement. J.
Immunol. 94:662.
158. Harhoe, M. 1964. interactions between trace labeled cold agglu
tinin, complement, and red cells. Brit. J. Hnemat. 10:339.
159. Harboe, M. 11. .1. MU l ler-Eberhard, 11. Fudenherg, M. J. Policy,
P. L. Mollison. 1963. Identification oc components of complement
participating in the antiglobulin reaction. Immunology 6:412.
160. Sweet, C., and J. K. Full. 1970. The binding of serum albumin to
phospholipid liposomes. BBA. 219:253.
161. Weissmann, G., A. Brand, and F. C. Franklin. 1974. Interaction of
immunoglobulins. .1. C.lin. Invest. 4:V3G.
162. Shin, M. L., W. A. Paznekas, A. S. Ahranevits. and M. M. Mayer.
1977. On the mechanism of membrane damage bv complement: Expo
sure of hydrophobic sites on activated complement. .1. Immunol.
119:L 358.

BIOGRAPHICAL SKETCH
Louis (Loui) Silvestri was born in Peckville. PA on January II,
1952. He spent most of his years in Archbald, PA.
Loui attended a parochial grade school (St. Thomas of Aquinas),
a Jesuit preparatory high school (Scranton Preparatory School) and a
college heavily influenced by Augustinian Catholicism (Villanova
University).
Loui's higher education was continued at the University of Florida
(Gainesville, FL) where, under the tutelage of Dr. Edward Hoffmann, he
received his Ph.D. However, earning that degree became more of a
challenge than originally anticipated.
Loui is currently employed at the University of Alabama (Birmingham.
AL) as a post doctoral fellow under the direction of Dr. Robert Stroud.

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
Edward M. Hoffmann, Ich^aijhnan
Professor of Microbiology and Cell Science
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
L. William Clem
Professor of Immunology and Medical
Microbiology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
Briah" Gebhardt/
Associate Professor of Pathology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
Arnold S. Bleiweis
Professor of Microbiology and Cell Science

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
L. 0. Ingram fj
Associate Professor of Microbiology and
Cell Science
This dissertation was submitted to the Graduate Faculty of the
Department of Microbiology and Cell Science in the College of Arts
and Sciences and to the Graduate Council, and was accepted as
partial fulfillment of the requirements for the degree of Doctor of
Philosophy.
December 1977
Dean, Graduate School

f
FÂ¥3 9 17 8. 0 3 8.5.
C' ^V\Cc_ /Jj/ / / J



1 4
Phosphatidyl choline vesicle (PVC) purification of LTA using C
h 14
labelled phosphatidyl, choline. Approximately 5 X 10 DPM of C
labelled phosphatidyl choline were added to 40 mg of phosphatidyl
choline dipalmitovl. Phosphatidyl choline vesicles (PVC) were pre
pared as described in Materials and Methods. Three test tubes contain
ing identical volumes and concentrations of non-labelled PCV were pre
pared simultaneously and 2.0 ml of LTAppx (1.5 mg/ml) were added to each
test tube. Fifty microliter samples from the ^4C containing test tube
were removed at various steps during the purification process and an
alyzed as described (Materials and Methods). A standard chloroform
quench curve was constructed and all reported counts represent corrected
1 4
DPM values. Table 5 depicts the distribution of C counts at various
steps in the purification procedure. Utilizing this procedure as des
cribed, essentially no contaminating phospholipid could be detected in
the final product. The typical mass yield of product via PVC purifica
tion was about 10-15%. Percent recovery of LTA at various steps in the
procedure is summarized in Table 6.
Comparison and summary of LTApcx versus LTAosx. As indicated in
Table 7, both methods of LTA purification removed the majority of pro
tein as compared to the total amount ahailabie in the LTAppx. Both
methods ostensibly recovered > 80% of the original LTA. However, the
major difference between the two products is reflected in the percent
total, mass recovery and the concomitant increase in percent carbohydrate
in the final LTAosx product. This latter difference can he most readily
discerned by observing the composite gas chromatograph tracings in
Figure 15. The carbohydrate standard (CHO-STD) depicts the typical
chromatograph of glucose and galactose after preparing trimethvlsiIv1


RECIPROCAL OF HUMAN C2 DILUTION
Ln
NJ


RESIDUAL CHc UNITS/ml
600
500
CONCENTRATION OF LTAcx(/g/ml) USED IN
PREINCUBATION WITH WHOLE HUMAN
COMPLEMENT(Chu)


TABLE 3
Comparing Several Different Methods of Measurement
LTA
Source
PHAg
Protein
X 10~3
Carbohydrate
X 10~3
A260
X 10-3
Pi
X 10~3
Mass
X 103
LTAcx
400
(3.4)
H. D.b
(0.3)
(4.6)
(1.4)
LTAppv
2,400
(96)
(35.7)
(26,667.0)
(1,116.0)
(1,333.3)
LIAosx
3,000
(1,600)
(347.8)
(87,912.0)
(2,222.2)
(5,128.2)
LTApcx
32,000
(12,300.0)c
(6.400)c
(344,086.0)
(5,203.2)
(21,333.3)
1 Protein,
( tiXD TdSS
carbohydrate, phosphorus (Pi)
ed on the reciprocal of PHAg t
and mass calculations were made
iter) per mg.
on the basis
of activity
Not determined.
C These values are estimations since the amounts of material present were below the Limits of
detection for the methods.


A 260
£
CO
O)
o
E
TEST TUBE NUMBER


added to each well and the plate was Incubated at 37C for 15
minutes on a Cordis Micromixer. The plate was removed from the
mixer and the cells were allowed to settle for two hours at 37C,
followed by three hours at room temperature.
Inhibition of complement mediated lysis. E A coated with
in DGVB and 0.4 ml of DGVB diluted HuC. The HuC was diluted so
that a maximum of 80 percent lysis was produced in EA which had
not been treated with LTAcx. The mixture was incubated at 37C
with continuous shaking for 60 minutes. One milliliter of ice
cold EDTA-GVB was added, the mixture was centrifuged for 5 minutes
at 500 g at 0C and the superna tent fluid was recovered. The
optical density of the supe run tent fluid was determined at a wave
length of 414 nm. Inhibition of hemolysis was calculated for each
concentration of LTAcx used by comparing the extent of lysis in
each assay with a control reaction mixture which contained EA
that had not been treated with LTAcx.
Ef fee t of LTAcx on t_he_ t it or of antibodies spo c_i f ic for _shee_p
erythrocyte stromata. Because LTA associate with some proteins (138)
it was necessary to perform a hemolytic antibody titration to
determine if the ability of the immunoglobulins to fix complement
at the cell surface was being affected by LTAcx treatment. The
possibility of similar ,mt igens in LTAcx and sheep erythrocyte
stromata was also considered. Equal volumes of LTAcx (500 ug/ml
in VBS) and rabbit anti-sheep E stromata were incubated together
at 37C for 20 minutes. A control consisted of incubating an


i 6
A typical component titration in serum treated with I.TAox is
depicted in Figure 3. In this example, the LTAcx treated serum was
serially diluted in DGVB. Next F.AC142, C5, 6, /, and 08-9 were added
sequentially to the dilutions. Since all components were added in
excess, C3 became the limiting factor in contributing to the hemolysis
of the target cells. Percent lysis in each test tube was mathematically
converted to Z (the average number of SAC1423 sites per cell) and this
was plotted against the reciprocal of the serum dilution. Percent inhi
bition of site forming units (SFU) was then calculated from 7.-1 values
or percent inhibition of CHr units was determined from values asso-
.30
dated with Z= 0.69. Figure 4 represents a composite of multiple com
ponent. titrations from whole human sera treated with I.TAcx. As can be
seen in this figure, Cl and C4 activities were consumed to some degree,
however, more than 50% inhibition of C2 activity was observed. As
indicated, C.3 activity was al.so consumed during preincubation of com
plement with LTAcs, but incubation with purified C3^~ produced no inhi
bition of C3 hemolytic potential. No C3 consumption occurred if the
incubation was performed in the presence of the chelator ethvlenodiamine
tetra acetic acid (KD'i'A) and less than 7 if incubated in the presence
of EOTA-Mg ions. The above results indicated the necessity for divalent
cations as cofactors mediating the consumption of C3 in the presence of
LTAcx. in addition, there? appeared to be a requirement for other serum
factors (possibly natural, AB and/or components of the alternative path
way' since purified (13 activity remained unaffected hv incubation with
LTAcx.


Figure 19. Immunodiffusion and precipitation analysis of various
steps in the purification of human Clq. Purification
was achieved by repeated fractional precipitations of
whole human sera in buffers varying in ionic, strength,
pH, and concentrations EGTA or EDTA (144) .
Well designations:
(A) Whole human sera (starting material);
(B) Supernate from first precipitation;
(C) Supernate from second precipitation;
(D) Supernate from third precipitation;
(E) Material from resuspended pellet prior to
final precipitation.
(F) Final product (purified Clq).
Plate designation: (1) Center well contains nnti-lgC;
(2) Center well, contains Anti-IgA; (3) Center well
contains anti-whole human sera: (4) Center well
contains anti-IgM: (5) Center well contains anti-Clq.


Carbohydrate analysis was performed
Gas Liquid Chromatography.
after treatment of the samples with 1.0 N F^SO, in sealed ampules
for 8 hours at 105aC. Upon cooling, the seal was broken and exactly
0.2 ml of mannitol (either at 5.0 mg/ml or 1.0 mg/ml depending on
carbohydrate concentration of the sample) was added as an internal
standard. The contents of each vial were quantitatively transferred
to 15 ml centrifuge tubes (Corning Glass Works) containing 0.5 g
BaCO^. Each centrifuge tube was heated in a boiling water bath
and alternately vortexed until the pH approached neutrality as
indicated by full-range pH paper (Micro Essential Laboratory,
Brooklyn, NY). All tubes were centriufged at 500 g for 5 minutes
and the supernates were removed and collected in appropriately labeled
13 mm screw cap tubes fitted with teflon lined lids. The centrifuge
tubes containing BaCO^ were washed once with one ml of deionized
water and the supernates were appropriately pooled.
After lyophilization, the hydrolyzed carbohydrates were con
verted to trimethylsilyl ester (IMS) derivatives by the addition
of 0.2 or 1.0 ml (depending upon carbohydrate concentration) of
TRI SIL Z (Pierce Chemical Go.). Samples were warmed to approx
imately 60C in a water hath for 15-30 minutes before use and
assayed using a Packard 800 series gas chromatograph equipped with a
flame ionization detector. The gas chromatographic column (153 cm X
4 cm) was packed wi th SE-40 ULTRAP1IASK 33 on Chromosorb W (IIP) 80/L00
mesh matrix (Pierce Chemical Co., Rockford, TL). Column and detector
temperatures were set at lf)0C and 1F5"C respectively. The N,, carrier
gas was set at approximately 30 co/minute.


Figure 9.
Effect of LTAcx treatment on the lysis of EAC142.
Various limiting concentrations of C2 were used to
prepare EAC142 cellular intermediates. The cells
were then treated with LTAcx (250 yg/ml) and lysis
was developed using procedures described in Materials
and Methods. Symbols: (o) EAC142 incubated with
LTAcx; () EAC142 incubated with buffer.


Figure 7.
Effect of LTAcx treatment on the lysis of various
complement component, intermediates. Each cellular
intermidiate was prepared and then treated with
LTAcx (125 ug/ml). Lysis was developed using
procedures described in Materials and Methods.
Percent inhibition was calculated by comparison
against buffer treated controls.


EAC14 (10 cells/ml in DGVB). The mixture was incubated at 30'JC for JO
minutes and cooled to 0C in an ice bath for 2.0 minutes. Three tenths
of a milliliter of C-EDTA (1:37.5 in 0.04 M EDTA-GVB") were then added to
each test tube and the mixtures were incubated at 37C for AO minutes.
At the end of the incubation period, 1.0 ml of cold EDTA-GVB was added,
the tubes were centrifuged, and the supernates read for release of oxv-
hemaglobin at a wave length of 414 nra. External controls consisted of C2
with no Cls nor LTApcx, C2 with Cls but not LTApcx, and C2 with LTApcx
but no Cls. The usual internal controls (spontaneous lysis, color cor
rection, no C2, and total lysis) were included at all times. Results were
expressed as percent inhibition of C2 consuming ability compared with a
control containing only Cls and C2.
The ability of Cls to hydrolize the synthetic substrate p-Tosy1-1-
arginine methylester (TAMe) was determined as described hv Nagaki and
Stroud (38). Inhibition assays were performed by incubating equal volumes
of Cls (approximately 8.0 X 1.0^ SFU/ml) and LTApcx (approximately 100 g/m
at 37 for 10 minutes. Residual Cls activity was then determined as des
cribed (38-40).
Clq inhibition assays. The effect of LTApcx on the ability of puri
fied Clq to bind to antibody sensitized sheep erythrocytes was determined
by methods described by Loos et al. (39) and Raepple et al. (40). Equal
8
volumes of Clq (approximately L.3 X 10 SFU/ml) and LTApcx (10 hg/ml)
were incubated at 37 for 10 minutes. Residual Clq activity was then
determined as indicated above.


70
60
50
40
30
20
! 0
CELLULAR INTERMEDIATES TREATED
WITH LTApcx (100 g/ml)
CO


67
All test tubes containing greater than 25.0 n-moles Pi/ml were
pooled. The entire peak (approximately 22 ml) was loaded on to a
65.0 cm X 3.0 cm column packed with LH-20 equilibrated with deionized
water. Four and two tenths milliliter of effluent were collected per
test tube a a flow rate of approximately 30.0 ml/hour. The results
of this procedure, which simultaneously removed salt and propanol, are
shown in Figure 14. The column effluent was monitored at a x-zave length
of 220 nm and was also screened for LTA by PHA (-H-H-) using a single
dilution sample. In addition column fractions were tested for the pre
sence of chloride ions by placing one drop of a saturated AgNO^ solution
on a coverslip containing one drop from each test tube. Any resulting
precipitation was evaluated on a +1 to +5 basis and plotted accordingly.
It was empirically determined that not only Cl reacted with the AgNO^
resulting in insoluble AgCl, but the NaN^ and tris carbonate in the
buffers reacted as well. The presence of propanol was monitored indi
rectly by changes in test tube volume. Since LTA. azide, and tris
carbonate all absorb at a wave length of 220 nm tin? combination of ultra
violet light screening, the AgNO^ precipitation test, and visual inspec
tion of volume changes per test tube proved to be invaluable for rapidly
discerning the location and separation of LTA from contaminating salts
and solvents. The entire contents of peak I were pooled, frozen, and
lyophilixed. The final product was referred to as LTAosx (extracellu
lar lipoLeiehoic acid purified by Octyl Sepharose hydrophobic affinity
gel chromatography). The typical mass yield from such a procedure
was about 60-70. Percent recovery of LTA at various points in the
procedure is summarized in Table 4.


AVERAGE NUMBER OF SACI4/CELL
103
RECIPROCAL OF HUMAN C I DILUTION