Chemical, immunochemical, and structural studies of cross-reactive cell wall antigens of Streptococcus mutans

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Title:
Chemical, immunochemical, and structural studies of cross-reactive cell wall antigens of Streptococcus mutans
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ix, 92 leaves : ill. ; 28 cm.
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Brown, Thomas Allen, 1950-
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Streptococcus mutans   ( lcsh )
Bacterial antigens   ( lcsh )
Microbiology and Cell Science thesis Ph. D
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Thesis--University of Florida.
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Bibliography: leaves 87-91.
Statement of Responsibility:
by Thomas Allen Brown.
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Typescript.
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Vita.

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CHEMICAL, IMMUNOCHEMICAL, AND STRUCTURAL
STUDIES OF CROSS-REACTIVE CELL WALL
ANTIGENS OF Streptococcus mutans
















By

THOMAS ALLEN BROWN


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY








UNIVERSITY OF FLORIDA


1978


































To my wife, Mary















ACKNOWLEDGEMENTS


The author wishes to acknowledge the faculty, students, and staff

of the Department of Microbiology and Cell Science for their friendship,

advice, and encouragement during the preparation of this dissertation.

He wishes to express his deep appreciation to the chairman of

his supervisory committee, Dr. Arnold S. Bleiweis, for his valuable

guidance, criticisms and friendship throughout his graduate career.

He would also like to thank the other members of his committee,

Drs. Edward M. Hoffmann and L. William Clem for their advice and help

in direction of this work and preparation of this manuscript.

He would also like to thank Dr. R. W. King, Department of

Chemistry, for his generous assistance with mass spectrometry, and

Richard M. Vaught, Michael Taylor, and Steven F. Hurst for their

technical assistance.

Finally, the author would like to express his most loving

gratitude to his wife, Mary, whose love, support, and encouragement

made this dissertation possible.















TABLE OF CONTENTS


Page

ACKNOWLEDGEMENTS. . ... iii

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

LIST OF FIGURES ........ ............. vi

ABSTRACT................. .. .... viii

INTRODUCTION. . . 1

MATERIALS AND METHODS . .. 11

RESULTS . . . 24

DISCUSSION ...... ................... 79

LITERATURE CITED ................... .. 87

BIOGRAPHICAL SKETCH ................... 92















LIST OF TABLES


Table Pagi


1. SURVEY OF THE VARIOUS CLASSIFICATION SCHEMES
FOR Streptococcus mutans . 5

2. CHEMICAL COMPOSITION OF PURIFIED CELL WALLS OF
S. mutans AHT (a) and B13 (d). . 25

3. CHEMICAL COMPOSITION OF FORMAMIDE EXTRACTS OF
S. Mutans AHT (a) and B13 (d) PURIFIED CELL WALLS. 35

4. COMPOSITION OF LECTIN-PURIFIED ANTIGENS FROM
S. mutans AHT (a) and B13 (d) . ... 56

5. INHIBITION OF THE PRECIPITIN REACTION BETWEEN AHT
ANTIGEN AND ANTI-a SERUM ... .. 62

6. INHIBITION OF THE PRECIPITIN REACTION BETWEEN B13
ANTIGEN AND ANTI-d SERUM . .... .64

7. INHIBITION OF THE PRECIPITIN REACTION BETWEEN AHT
ANTIGEN AND ANTI-a-d SERUM . .. 66

8. INHIBITION OF THE PRECIPITIN REACTION BETWEEN B13
ANTIGEN AND ANTI-a-d SERUM . .... .67

9. METHYLATION ANALYSIS OF THE ART ANTIGEN. ... 68

10. METHYLATION ANALYSIS OF THE B13 ANTIGEN. ... 69














LIST OF FIGURES


Figure Page


1. Gel immunodiffusion experiments demonstrating
the reaction of identity between Lancefield
and formamide extracts of AHT and B13. ..... 27

2. Gel immunodiffusion experiments demonstrating
the patterns obtained between formamide extracts,
unabsorbed sera, and absorbed sera .. .. 30

3. Immunoelectrophoretic patterns obtained with formamide
and Lancefield extracts of AHT and B13 ...... 34

4. Gel filtration profile of AHT formamide extract on
Biogel P-150 . ..... ..... 37

5. Gel filtration profile of B13 formamide extract
on Biogel P-150. . . ... 39

6. Affinity chromatography column profile of B13
formamide extract. . ... 42

7. Gel immunodiffusion experiments using crude extracts
and purified antigens. . ... 45

8. Immunoelectrophoretic patterns obtained with crude
extracts and purified antigens . .. 47

9. Gel immunodiffusion experiments using purified antigens,
unabsorbed sera, and absorbed sera .. 49

10. Gel filtration profile of AHT purified antigen on
Biogel P-150 . .... 53

11. Gel filtration profile of B13 purified antigen on
Biogel P-150 . .... 55

12. Gel immunodiffusion patterns obtained using purified
antigens and cross-reacting sera . 59








Figure Page


13. Quantitative precipitin curves obtained using
the various absorbed sera and the purified
antigens . .... . 61

14. Immunoelectrophoretic study of the degradation
of Lancefield extracts of B13 cell walls at various
extraction times. . . ... 73

15. Immunoelectrophoretic study of the degradation of
Lancefield extracts of AHT cell walls at various
extraction times. . . ... 77

16. Schematic arrangement of the sugar residues in the
AHT and B13 polymers . . 84









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



CHEMICAL, IMMUNOCHEMICAL, AND STRUCTURAL
STUDIES OF CROSS-REACTIVE CELL WALL
ANTIGENS OF Streptococcus mutans


By

THOMAS ALLEN BROWN

June, 1978


Chairman: Arnold S. Bleiweis
Major Department: Microbiology and Cell Science

This dissertation is concerned with the isolation, purification,

and immunochemical characterization of two key polysaccharide antigens

of Streptococcus mutans, the principal etiologic agent of dental caries.

These cell-wall antigens were isolated from two strongly cross-reacting

serotypes (a and d) which have been confused by investigators. The in-

formation gathered here may aid in future reclassification or in the

development of more accurate identification procedures for these oral

pathogens. Also, the development of molecular vaccines composed of

important somatic antigens depends on knowledge of antigen composition

and structure.

The principal cross-reacting wall antigens of S mutans strains

AHT serotypee a) and B13 serotypee d) were extracted from purified cell

walls by hot formamide. The antigens were purified by affinity chro-

matography using the galactose-binding lectin from Ricinus communis.

The purified antigens appeared to be immunologically identical to crude

antigen extracts of whole cells or cell walls. The antigens were


viii








composed primarily of galactose and glucose in molar ratios of 3.4:1 for

AHT, and 2.1:1 for B13. Rhamnose, which is the major constituent of the

crude formamide extracts, was present in only trace amounts in the lectin

purified preparations. Hapten inhibition using absorbed antisera specific

for the unique antigenic determinants (a or d) and the cross-reacting

determinants (a-d) showed that the a specificity depended on an a-linked

galactose residue, possibly linked to glucose. In the case of the d

determinant both glucose and galactose allowed significant inhibition,

however, glucose gave slightly greater inhibition.

The cross-reacting a-d determinant appears to be galactose, possibly

linked 1-6 to glucose. Methylation analysis of the antigens using com-

bined gas-liquid chromatography-mass spectrometry showed that both antigens

possess identical branch point residues as well as three other identical

residues including terminal galactose moieties which may be responsible for

the cross reaction. The AHT antigen contains a 1-3 linked galactose residue

and B13 contains a 1-3 linked glucose residue which may determine the a and d

specificities respectively. Both antigens contain galactofuranose residues

which may contribute to the observed acid-lability of these antigens.

The data presented in this dissertation provide some clues concern-

ing the possible molecular structures of the a and d antigens. Due to the

complexity of each polymer, it is not yet possible to draw complete

structures. There does appear, however, to be a structure common to each

antigen which will be proposed in this dissertation. Finally, our

immunochemical studies of the a, d, and a-d haptenic sites (by precipitin

inhibition analysis) have failed to confirm published data or have

revealed contrary information.














INTRODUCTION


Streptococcus mutans, a Gram-positive coccus belonging to the

viridans group, is considered to be the principal etiological agent in

dental caries. Its ability to colonize tooth surfaces by the production

of a sticky dextran-like capsule and the ability to produce acid from

fermentable carbohydrates account for the cariogenicity of this organism.

Dental caries is the most prevalent disease of civilized populations and

poses serious health and financial problems. Gibbons (29) states "It has

been estimated that well over two billion dollars are spent each year in

the United States for the treatment of this disease, and thus tooth decay

ranks as one of the most expensive bacterial infections of mankind."

Accordingly, a great deal of work has been done on the classification of

S. mutans and toward the development of vaccines for these organisms.

One of the foremost ways of classifying S. mutans strains is serologically.

This, and the potential development of vaccines, necessitates a good

understanding of the antigens associated with the various strains.

This dissertation will present chemical, immunochemical, and structural

data on purified antigens of two cross-reacting strains of S. mutans of

the serotypes a and d.

Classification of S. mutans strains. The various cariogenic and

most other oral streptococci are classified as belonging to the viridans

group, which give greening on blood agar and are not classifiable by the

Lancefield grouping scheme.








Early investigations in 1960 by Fitzgerald and Keyes (27) and

Fitzgerald et al. (26) showed that cariogenic isolates from rats and

hamsters could be distinguished by their ability to ferment sorbitol,

but antigenic extracts failed to react with antisera used for the identi-

fication of Lancefield groups A-H and K-S (37). Zinner et al. (64) iso-

lated various cariogenic strains from human sources and also found them

to be non-reactive with Lancefield grouping sera.

S. mutans strains form a relatively homogeneous group when classi-

fied according to biochemical reactions. Most strains ferment sorbitol

and mannitol, as well as melibiose, salicin, and inulin, and produce

large amounts of non-dialyzable glucan from sucrose. They also hydrolyze

esculin, grow in 4% NaC1 broth, and on 10% bile agar. Using cluster

analysis, Colman and Williams (15) showed that among 216 non-hemolytic

streptococci the dectran-producing organisms formed a separate cluster

comprised of mostly S. mutans. Guggenheim (30) and Edwardsson (25) also

showed S. mutans to form a homogeneous group by biochemical tests.

Drucker and Melville (22, 23), and Carlsson (13) showed by numerical

taxonomy that S. mutans was a distinct group and could be distinguished

from other common oral streptococci: S. sanguis, S. mitis, and S.

salivarius.

When examined by serological and genetic methods, S. mutans as

a group appears more heterogeneous. Jablon and Zinner (36, 63) examined

eight strains of cariogenic streptococci using immunofluorescence and

were able to place them in four groups, I-IV. However, there were cross-

reactions with other streptococci making the technique less useful than

subsequently developed techniques for the identification of S. mutans.








Perhaps the most useful method to date of classifying strains of

5. mutans was the serological scheme developed by Bratthall (9, 10, 11)

utilizing diffusion in gel and comparative immunoelectrophoretic patterns

of extracts perpared according to Lancefield (37). Although cross-reactions

existed, as were seen by other workers, the use of comparative immuno-

electrophoresis allowed Bratthall to separate 70 strains of S. mutans into

five serotypes: a through e. The serotype e organisms cross-reacted using

anti-Lancefield group E serum. The serotypes a through d did not react

with any of the Lancefield grouping sera. In a subsequent study, Bratthall

(12) used antisera conjugated with fluorescein isothiocyanate (FITC) and

found that unabsorbed, conjugated antisera prepared against reference

strains of the five serotypes showed cross-reactions with members of the

other serological groups of S. mutans as well as members of other strepto-

coccal species. Most notably, anti-AHT or anti-3720 (a) serum showed a

strong cross-reaction (4+) with B13 (d). Anti-B13 serum showed a moderate

cross-reaction with the same serotype a organisms (2+). Anti-JC2 serum

(c) showed a strong cross-reaction (3+ to 4+) with LM7 (e), while anti-

SS9 serum, a Lancefield group E serum, reacted weakly (1+) with JC2 (c)

and B13 (d), and strongly (3+ to 4+) with LM7. Through various whole cell

absorption procedures Bratthall was able to eliminate cross-reactions with

non-S. mutans species and produce specific staining sera for all serotypes

except c, which still reacted with LM7 (e). The S. mutans serotype e

cells could be identified by absorbing antiserum against Lancefield group

E (strain SS9) with S. mutans LM7 (e). Then a S. mutans strain of sero-

type e will react with unabsorbed anti-SS9 serum, with anti-JC2 (c) serum,

but not with the absorbed anti-SS9 serum. On the other hand, Lancefield

group E strains react with both the absorbed and unabsorbed anti-SS9








serum, but not with anti-JC2 (c) serum. The serotype c organisms do not

react appreciably with either anti-group E serum.

de Stoppelaar (20) was able to divide various cariogenic strepto-

cocci into three groups by precipitin analysis. His groups MI, MII, and

MO correspond to Bratthall's serotypes c, a, and e respectively.

Further work by Perch et al. (48) demonstrated two more serotypes,

f and g, using absorbed fluorescine-labelled antisera.

In addition to the serological grouping schemes, four genetic

groups based on DNA-DNA hybridization and % guanine-cytosine content were

shown by Coykendall (16, 17, 19) to parallel four of Bratthall's five

serotypes. Coykendall (18) proposed the adoption of subspecies names for

his genetic groups I through IV: S. mutans var. mutans, var. rattus, var.

sobrinus, and var. cricetus respectively. Shklair and Keene (54) separated

S. mutans into five biotypes, a-e, which correspond directly with

Bratthall's serotypes. Biotypes were determined by fermentation patterns

using mannitol, mannitol + bacitracin, sorbitol, raffinose, melibiose,

and production of ammonia from arginine.

Table 1 summarizes the relationships among the strains of S. mutans,

determined by various workers, using serological methods, DNA-DNA re-

association (genetic groups), guanine-cytosine content (%GC), and bio-

chemical reactions (biotypes).

Immunochemical nature of the serotype antigens. In 1973 Van de

Rijn and Bleiweis (59) isolated a glycerol teichoic acid antigen from

S. mutans AHT (a) by cold 5% trichloroacetic acid (TCA) extraction.

The material was fractionated by agarose gel chromatography and the antigen

fraction contained glycerol and phosphorus in a 1:1 molar ratio. The

fraction also contained glucose, galactose, alanine, and fatty acids.












SURVEY OF THE VARIOUS CLASSIFICATION SCHEMES FOR

Streptococcus mutans


Genetic
Serotypes groups % GC Subspecies Biotypes
(10 )a (48 ) (20 ) (36) (17) (18) (54)

a MII I IV 42-44 var. circetus a

b II II 41-43 var. rattus b

c MI I 36-38 var. mutans c

d MII III 44-46 var. sobrinus d

e MO e

f

g



aSee Literature Cited


Table 1.







Partial alkaline hydrolysis yielded a glyceride containing a disaccharide

of glucose and galactose attached to the 2-hydroxyl group of glycerol.

Precipitin inhibition analysis showed that B-galactosides were the

greatest inhibitors (> 75%) while glucose and other glucosides inhibited

to a lesser extent (< 30%). Immunodifussion and immunoelectrophoresis

showed this antigen to be present in all six serotype a organisms tested

and absent in serotypes b, c, and d.

A short while later, in 1973, Mukasa and Slade (46) isolated a

polysaccharide antigen from lyophilized whole cells of a hamster strain

of S. mutans, HS6 (a), using boiling water. The ethanol precipitate of

the extract was purified by chromatography on diethylaminoethyl (DEAE)-

Sephadex A-25, Sephadex G-200, and Carboxymethyl (CM)-Sephadex C-25.

The antigen was primarily composed of galactose (54%), glucose (10.4%),

glucosamine (8.6%), and galactosamine (5.4%), with small amounts of

lipid, phosphorus, and 5% amino acids. The antigen reacted not only

with anti-HS6 serum but also with anti-B13 (d) serum. The cross-

reactivity could be eliminated by absorbing anti-HS6 serum with B13 whole

cells. Cross-reactive ("a-d") serum could be obtained by releasing the

antibody that bound to B13 cells during absorption. In subsequent

papers, Linzer et al. (39, 41) showed that the purified antigen possessed

two sites on a single molecule: the "a" site, unique to the a serotype,

and an "a-d" site, which was responsible for the cross-reaction with d

serotypes. Precipitin inhibition studies with abosrbed anti-HS6 serum

showed the "a" site to be dependent on a-1-4 and -1-6 glucosides,

however, free galactose or galactosides other than lactose were not tested

as potential inhibitors. The latter disaccharide gave less than 10%

inhibition. Maltose (glu a-1-4 glu) and gentiobiose (glu B-1-6 glu) each








gave approximately 50% inhibition, while a and 8-methylglucopyranosides gave

less than 10%. Inhibition studies with "a-d" serum showed 32% inhibition

with galactose, 36% with galactosamine, and 24% with melibiose. No other

inhibitors were tried. Since this dissertation deals with the relation-

ship of the antigens of serotypes a and d in great detail, including

structural analyses of the purified antigens, the reports outlined above

(41, 46) will be further discussed below (see Discussion).

In 1973, Mukasa and Slade (45) isolated two antigens from whole

cells of a serotpye b organism FA1, using cold 10% TCA. One (antigen 1)

was a polysaccharide and the other (antigen 2) was a mucoprotein. Both

antigens possessed the same immunological specificity. Both antigens

contained about 30% galactose and 3% galactosamine, while rhamnose was the

major component (47%) of the carbohydrate antigen (antigen 1) and present

only in traces in antigen 2. Antigen 2 contained about 40% protein while

antigen 1 contained 5% protein. When studied by immunoelectrophoresis,

the two antigens migrated as individual bands, with antigen 2 migrating

the farthest toward the anode. Galactose and galactosamine were the best

precipitin inhibitors (78%) of antigen 2 reactivity. Antigen 1 was not

studied for immunochemical specificity.

In 1974, Vaught and Bleiweis (60) isolated a glycerol teichoic

acid antigen from purified cell walls of S. mutans BHT (b) using cold 10%

TCA. The extract was fractionated by gel filtration and ion exchange

chromatography. Two fractions were obtained, each yielding a single

separate band on immunoelectrophoresis which migrated in the same manner

as the bands obtained from mild acid extracts of whole cells of serotype

(b) organisms. The faster migrating band, fraction C, was similar in

mobility to Slade's antigen 2 except fraction C contained primarily








galactose with glycerol and phosphorus in a molar ratio of 1.0: 1.4.

Only small amounts of rhamnose and glucose were measured. This fraction

resembled a glycerol teichoic acid. Fraction B was very similar to Slade's

antigen 1, both in mobility and in chemical content.

In 1974, Wetherell and Bleiweis (61) isolated a polysaccharide

antigen by hot formamide extraction from purified cell walls of S. mutans

GS5 (c). The major antigen, purified by gel filtration and ion exchange

chromatography, was composed mainly of rhamnose and glucose in a molar

ratio of 1.7: 1.0. Precipitin inhibition studies revealed the major

hapten to be an a-linked glucose moiety. In a subsequent study, Linzer

et al. (40) isolated a nearly identical polysaccharide from hot 5% TCA

extracts of whole cells of strain Ingbritt, another serotype c organism.

Linzer and Slade (42) isolated a polysaccharide antigen from whole

cells of S. mutans strain B13 (d) using cold 5% TCA. After the antigen

was purified by ion exchange and gel filtration chromatography, it was

found to be composed mainly of galactose and glucose in a 2:1 molar ratio.

The antigen was similar to the a antigen in that it was a single molecule

possessing two reactive sites: one specific for the d serotype (d site)

and one responsible for the cross-reaction with the a antigen (a-d site).

Anti-B13 serum could be abosrbed with HS6 (a) cells to yield a specific

d serum. Inhibition studies with anti-d serum (absorbed) showed the

hapten to be galactose, possibly a-linked. The nature of the a-d site

specificity in B13 was not investigated.

The serotype e antigen was purified after hot formamide extraction

of purified cell walls of V-100 by Wetherell and Bleiweis (62).

Two pclysaccharide antigens were resolved by ion-exchange chromatography:

one antigen (I) was specific for serotype e, and the other (II) cross-








reacted with serotype C antisera. Antigen I was composed mainly of

rhamnose and glucose in a 2.3:1 molar ratio and possessed a B-glucosyl

group as its antigenic determinant. Antigen II possessed identical

chemical components and its homologous reactivity with anti-e serum also

was inhibited primarily by B-glucosides. Although not proven by the

authors, it was speculated antigen II may possess a-glucosyl haptenic

moieties to account for cross-reactions with c serum. Hamada and Slade

(33) isolated a similar antigen from strain MT703 (e) by the autoclave

extraction method of Rantz and Randall (51). The major antigen, el was

composed mainly of rhamnose (56%) and glucose (37%) and possessed a

S-glucoside as a haptenic determinant. The e c cross-reactive antigen

was not found.

Hamada et al. (32) isolated a polysaccharide antigen with serotype

f specificity from whole cells of strains OMZ175 and M1557 using hot 5%

TCA. The antigen from OMZ175 was composed of rhamnose (49%) and glucose

(47%), and the antigen from MI557 also contained rhamnose and glucose,

60% and 39% respectively. The best inhibitor of precipitation was

isomaltose, a diglucoside with an l-~6 linkage. The antigen was cross-

reactive with the glucan ("mutan") produced by the same organism when

grown in sucrose.

A polysaccharide antigen from a pH 7.3 buffer-boiled extract of

S. mutans 6715 (g) was isolated by lacono et al. (35) in 1975. At the

time this strain was thought to belong to serotype d, but is now con-

sidered to be a serotype g organism (34, 48). The antigen, purified by

ethanol precipitation and gel filtration, was composed primarily of

galactose (60.6%) and glucose (10.3%) and amino acids (9.5%). The

composition was very similar to the a antigen of Mukasa and Slade (46)








and indeed serotype g strains have been shown to cross-react with both

serotype a and d strains. It is not known whether the molecular site

responsible for the a-a and d-g cross-reaction is the same as that for

the a-d cross-reaction. Precipitin inhibition tests showed that galactose

was the most effective inhibitor, although glucose also gave significant

inhibition. The immunodominant region is thought to involve the 1-6

linkage of galactose and glucose in either the a or B configuration.

The work presented in this dissertation began in 1973, prior to

the publication of work on the polysaccharide antigens of serotypes a

and d. Because of major discrepancies between the published data and our

early findings, and more importantly because of a desire to gain infor-

mation concerning the basic structural nature of these antigens, work was

continued. The findings in this dissertation agree in part with some of

the published data on the a, d, and a-d antigens, but they also refute,

or at least open to further debate, previous findings heretofore unveri-

fied. In addition, the structural data presented below which have not

previously been published, provide further insight into the chemical

structure of these antigens and may provide important information to

future investigators desiring to synthesize immunogens for anti-caries

vaccines or to more fully understand the immunogenicity and antigenicity

of surface polysaccharides of S. nmutans.














MATERIALS AND METHODS


Bacterial strains and growth conditions. S. mutans strain AHT

(a) was obtained from J. Jablon (University of Miami, Miami, Florida),

and strain B13 (d) was obtained from D. Bratthall (University of Goteborg,

Gbteborg, Sweden).

Cultures were maintained as lyophilized stocks. Samples to be

lyophilized were grown for 20 h at 37C in Todd-Hewitt broth (Difco

Laboratories, Detroit, Michigan) supplemented with 0.4% glucose (THG),

and adjusted to pH 6.5 with concentrated HC1 before autoclaving. The

pellet from 250 ml of broth was suspended in 2 ml of sterile 20% powdered

milk solution (Pet, Inc., St. Louis, Missouri), and 0.2 ml samples were

lyophilized in glass ampoules on a Virtis freeze drying apparatus (Virtis

Research Equipment Co., Gardiner, New York). The ampoules were sealed

under vacuum and stored at -200C.

Cells for antigen preparation were grown in ten liter batches in

THG. Growth was initiated by inoculating 10 ml of THG from lyophilized

stocks. The entire culture was transferred at 24 h intervals into 250 ml,

1000 ml, and finally 8750 ml of THG giving a total volume of 10 1.

Cells were harvested 24 h after the last transfer in a Delaval gyro-testor

(Delaval Separator Co., Poughkeepsie, New York). Autolysins were in-

activated by heating the cells at 600C for 20 min.

Extraction of antigens for routine identification. Lancefield-

type acid extracts were used for routine identification. Approximately

5 mg of whole cells or cell walls were suspended in 1 ml of saline and








adjusted to pH 2.5 with HC1. The suspensions were boiled for 10 min,

cooled, centrifuged at 1070 x g for 20 min to remove cell debris and the

supernate neutralized with 0.05 N NaOH.

Cell walls used for extraction were prepared by the method of

Bleiweis et al. ( 7) which involves breakage with glass beads in a Braun

tissue homogenizer (Bronwill Scientific, Rochester, New York), followed

by treatments with ribonuclease, deoxyribonuclease, and trypsin.

Extraction of antigens for purification. Antigens for further

purification were extracted by the hot formamide method of Fuller (28).

Cell walls (800 mg) were suspended in 40 ml formamide and heated at 180 C

for 30 min in a paraffin oil bath. The suspension was then washed by

adding 2 vol (80 ml) of an acidic ethanol solution containing 19 parts

absolute ethanol and 1 part 2N HC1 (vol/vol), and centrifuging at 7700 x g

for 20 min in glass centrifuge tubes. The supernate was decanted and

saved and the pellets were washed twice more with 30 ml of a mixture of

the above acid-ethanol solution and deionized water (2:1, vol/vol).

Acetone (900 ml) was added to the combined supernates and the resulting

solution was immediately poured into round bottom centrifuge bottles (250

ml capacity). One milliliter of 1M NaC1 was added to each bottle and the

bottles were stored at 40C for at least 8 h. The bottles then were

centrifuged at 530 x g for 20 min, the supernates were decanted and the

pellets were allowed to dry. The pellets were dissolved in deionized

water, centrifuged at 27,000 x g for 20 min, and the supernates were

dialyzed and lyophilized.

Isolation of castor bean lectin (CBL). The castor bean lectin

from Ricinus communis was isolated by the method of Nicolson and

Blaustein (47) for use in affinity chromatography. Extracts of castor








beans are extremely toxic and allergenic and, accordingly, care must be

taken in handling them and all glassware was detoxified using a dilute

solution of sodium hypochlorite.

R. communis beans, 100 g (kindly provided by the McNair Seed Co.,

Plainview, Texas), were blended in a model 1120 Waring blender (Waring

Products Div., Dynamics Corporation of America, New Hartford, Connecticut)

with 1 1 of 0.005 M sodium phosphate buffer pH 7.2 containing 0.2 M NaCI

and 10-4 M Mg+2, Ca+2, and Mnf2 as chlorides (PBS +). The beans were

blended into a smooth paste (ca. 30 min), then the extract was filtered

through cheesecloth and centrifuged at 10,000 x g for 30 min at 40C.

The supernate was carefully decanted and saved, without including the

floating lipid layer, and centrifuged again. The supernatant solution was

adjusted to 60% saturation with ammonium sulfate and slowly stirred at

40C overnight. After centrifugation at 10,000 x g for 20 min, the pellet

was dissolved in PBS+ and dialyzed against PBS+ to remove the ammonium

sulfate. The dialysate (ca. 50 ml) was then applied to a 4 x 40 cm column

of Sepharose 4B (Pharmacia Fine Chemicals Inc., Piscataway, New Jersey)

and eluted with PBS+ until the absorbance at 280 nm was less than 0.05.

Since Sepharose 4B contains polysaccharides with galactose-terminal groups,

it served as an affinity column to bind the lectin. The lectins ( a

mixture of two, RCA60 and RCA120) then were eluted with 0.2 M D-galactose

in PBS4+. The A280 peak was pooled, dialyzed against PBS++ and then

deionized water, and finally lyophilized.

Preparation of affinity columns with castor bean lectins. An

affinity chromatography gel was prepared using Affi-Gel 10 (Biorad

Laboratories, Richmond, California), an agarose gel containing a 10-A

aliphatic spacer arm terminating with an active carboxy-N- hydroxy-








succinimide ester group. Approximately 350 mg of the lectin was dissolved

in 50 ml of 0.1 M sodium phosphate buffer pH 7.0 containing 10 M metal

ions as before, and 0.05 M D-galactose, to prevent binding of the lectin to

the gel matrix. To each of two vials of gel (approximately 13 ml wet gel

volume per vial), 25 ml of the lectin solution was added and the vials

were gently agitated on a shaker for 24 h at 40C. The coupled gel was

packed into a 1.3 x 30 cm column and eluted with PBS+ plus 0.2 M D-galactose

until the absorbance at 260 nm reached baseline, indicating the N-hydroxy-

succinimide released during coupling was removed. The column was washed

with about 200 ml of PBS+ minus galactose before loading with a sample.

Purification of antigens by affinity chromatography. Samples of

crude formamide extract (50 mg) were loaded on the affinity column at 40C

and eluted with PBS+. The flow rate was 12 ml/h and 2 ml fractions were

collected. After 72 ml were collected to remove non-binding materials,

the elution system was changed to PBS+ plus 0.2 M D-galactose to remove

bound polysaccharides. The column was eluted with at least 100 ml of the

latter buffer before re-equilibrating with the starting buffer. [All

buffers used with the affinity columns were pre-filtered through a 0.45

um membrane filter to prevent clogging of the bed surface].

Fractions were monitored for carbohydrate by the phenol-sulfuric

acid method as described below and for antigenic reactivity by capillary

tube precipitation using the appropriate antiserum.

Gel filtration chromatography. Samples of crude formamide

extract (50 mg) were loaded on a 2.5 x 80 cm column of Bio-Gel P-150

(Biorad) 100-200 mesh and eluted with 0.85% NaCl at a flow rate of 10 ml/h.

Fractions (2 ml) were collected and analyzed for absorbance at 220 nm,

rhamnose as described below, and antigenic reactivity using capillary

precipitin tubes.








Samples of lectin-purified antigen were filtered as above except

on 2.5 x 40 cm columns. Fractions (2 ml) were collected and assayed for

sugars by the phenol-sulfuric assay and for antigenic reactivity by

precipitation in capillary tubes.

Quantitative chemical assays. Phosphorus in cell walls was

measured by the method of Chen et al. (14), and for the purified antigens

by the method of Lowry et al. (43). Both methods depend on the formation

of a blue phosphomolybdate complex, but differ in the ashing procedure

and in sensitivity.

Determination of glucose and galactose in cell walls was performed

using the Glucostat and Galactostat reagents (Worthington Biochemical Corp.,

Freehold, New Jersey). Samples were hydrolyzed in 3 ml of 2 N HC1 for

4 h at 1000C. Hydrolyzed samples were quantitatively transferred to a

10 ml volumetric flask and neutralized to a phenolpthalein endpoint with

5 N NaOH. The volume at this point was 7.6-7.8 ml. The sample was then

brought to 10.0 ml with 0.27 M sodium phosphate buffer, pH 7.0.

Rhamnose in cell walls and column fractions was measured by the

method of Dische and Shettles (21).

Glycerol was determined utilizing the Glycerol Stat-Pack

(Calbiochem, Atlanta, Georgia) according to the manufacturer's instructions.

Samples of cell walls (10 mg) were hydrolyzed for 3 h at 1050C in 1 ml of

2 N HC1. After hydrolysis, the acid was removed by lyophilization and

three 1.5 ml distilled water washes, each followed by lyophilization.

Free glycerol was released by treating the samples with 2 mg alkaline

phosphatase (Worthington) for 24 h at 370C in 3 ml of 0.08 M ammonium

carbonate buffer pH 9.3.








The phenol-sulfuric acid assay was performed according to Dubois

et al. (24) using glucose as a standard. The tubes were read at 485 nm.

Protein was determined by the method of Lowry et al. (44).

Details of the assay are given below.

Amino acids and amino sugars were determined using a JEOL model

JLC-6AH automated amino acid analyzer (JEOL, Inc., Cranford, New Jersey)

according to the method of Spackman et al. (58). Peaks were quantitated

using an Autolab System AA integrator (Spectra-Physics, Mountain View,

California). Samples (10 mg) were hydrolyzed in sealed ampoules with

either 5 ml of 4 N HC1 for 11 h at 1050C or with 5 ml of 6 N HC1 for 18 h

at the same temperature. The second method was necessary for the analysis

of cell walls of serotypes a and d in order to fully hydrolyze an acid

resistant component presumed to be a lysyl-threonine dipeptide (8).

After hydrolysis, acid was removed using a rotary evaporator (Buchler

Instruments, Fort Lee, New Jersey) and the samples were washed three

times with deionized water followed by evaporation. Hydrolyzed samples

were quantitatively transferred to a 25 ml volumetric flask and diluted

to volume using 0.01 N HC1.

Gas liquid chromatography of sugars. Sugars in the crude

formamide extracts and purified antigens were measured by gas liquid

chromatography. Samples (1-5 mg) were hydrolyzed in sealed ampoules with

1 N H2SO4 for 8 h at 100C. After hydrolysis, the samples were cooled,

opened, and 1.0 ml of a mannitol solution of a known concentration (usually

5.5 umole/ml) was added as an internal standard. The hydrolysates were

neutralized with BaCO3, and the precipitate of insoluble BaSO4, as well

as any excess BaC03, was removed by centrifugation at 1000 x g for 20 min.

Supernates were centrifuged again as above and then lyophilized.








Hydrolysates were converted to their trimethylsilyl derivatives using

TRI-SIL (Pierce Chemical Co., Rockford, Illinois) according to the

instructions in the Pierce handbook of silylation (49). The derivatives

were separated on 6 ft x 4 mm glass columns packed with SE-30 Ultraphase,

3%, on Chromosorb W (HP) 80/100 mesh (Pierce) on a Packard model 803 gas-

liquid chromatograph (Packard Instrument Co., Downers Grove, Illinois).

The operating temperature was 165 C isothermall), the carrier gas (nitrogen)

flow rate was 30 cc/min, and a hydrogen flame ionization detector was used.

Peaks were quantitated using an Autolab Minigrator electronic digital

integrator (Spectra-Physics, Santa Clara, California). Calibration curves

were constructed by injecting samples containing varying amounts of the

standard sugars deriviatized as above and a constant amount of the internal

standard, mannitol. The ratio of area mannitol/area standard was plotted

vs. moles mannitol/imoles standard.

Methylation analysis of polysaccharide antigens. Purified antigens

were methylated using the method of Hakomori (31) according to Bjorndal

et al. (4) and Lindberg (38). The procedure involves treating the poly-

saccharides with methylsulfinyl sodium in dimethyl sulfoxide (DMSO),

followed by methylation with methy iodide. The methylated product is

hydrolyzed, reduced, acetylated and analyzed by gas liquid chromatography

and mass spectrometry. All solvents were re-distilled and stored in glass-

stoppered bottles and care was taken to prevent contamination with plastics.

The methysulfinyl carbanion was prepared according to Sanford and

Conrad (53) and stored at -20 C under mineral oil.

Samples of antigen (1.5 mg) were placed in small serum vials and

capped with rubber septa. The vials were flushed with dry nitrogen through

an 18-gauge hypodermic needle and the samples dissolved in 1.0 ml of DMSO







0
which had been dried over 3 A molecular sieves (Davison Chemical,

Baltimore, Maryland). Methylsulfinyl sodium, 1.0 ml, was introduced into

the vials with a glass syringe and the mixture was treated for 1 h with

ultrasound at room temperature. The reaction was allowed to proceed for

an additional 6 h with stirring, and then methyl iodide (1.0 ml) was added

dropwise with cooling in an ice bath. The solution was agitated in an

ultrasonic bath for 30 min, diluted with 10 ml of deionized water, dialyzed

overnight against 10 1 of deionized water, and evaporated to dryness on a

rotary evaporator at 40 C. The above procedure was repeated using the

product of these reactions to ensure complete methylation.

The methylated polysaccharides were hydrolyzed in two steps.

The residue was dissolved in 3 ml of 90% formic acid and treated at 1000C

for 2 h. The samples were evaporated to dryness at 400C and then dissolved

in 1 ml of 1 N H 2SO4 and hydrolyzed for 8 h at 1000C. The hydrolysate

was neutralized with barium carbonate, clarified by centrifugation and

0 14
evaporated to dryness at 40 C.

The residues were dissolved in 5 ml of deionized water containing

15 mg sodium borohydride and allowed to stand at room temperature for 2 h.

To the mixture was added 1 ml (wet volume) of AG 50W-X8, H+ form, (Biorad).

The mixture was agitated for 5 min, filtered, and evaporated to dryness

at 400C. The residual boric acid was removed by codistillation with

methanol (5 x 5 ml) on the rotary evaporator. Finally, the mixture of

reduced, methylated sugars was acetylated with acetic anhydride-pyridine,

1:1, for 15 min at 1000C. The reagents were removed by distillation with

toluene (5 x 5 ml) on the rotary evaporator at 400C. The residue was

dissolved in 0.1-0.2 ml acetone for analysis.








Analysis of methylated sugar derivatives. The partially-methylated

alditol acetate derivatives were separated by gas liquid chromatography on

6 ft x 2 mm silanized glass columns packed with 3% OV-225 or 3% ECNSS-M

on Gas-Chrom Q,100-120 mesh (Applied Science Laboratory, Inc., State College,

Pennsylvania). Temperature for determination of relative retention times

was 1700C, and 155C for measurement of peak area. The nitrogen carrier flow

rate was 20 cc/min and a hydrogen flame ionization detector was used.

Analysis was carried out on a Packard model 803 gas-liquid chromatograph

(Packard Instrument Co.). Retention times relative to 1,5-di-O-acetyl-2,

3,4,6-tetra-O-methyl-D-glucitol were determined by interpolation between

known standards prepared from a mixture of 2,3,4,6-tetra-0-methyl-D-glucose

(Sigma Chemical Co., St. Louis, Missouri) and methylated, hydrolyzed, lactose

(Sigma) and oyster glycogen (Nutritional Biochemical Corp., Cleveland, Ohio).

This mixture provides standards of 1,5-di-0-acetyl-2,3,4,6-tetra-0-methyl-D-

glucitol, 1,5-di-0-acetyl-2,3,4,6-tetra-O-methyl-D-glactitol, 1,4,5-tri-O-

acetyl-2,3,6-tri-0-methyl-D-glucitol, and 1,4,5,6-tetra-0-acetyl-2,3-di-O-

methyl-D-glucitol.

Confirmation of peak identity was obtained by combined GLC-mass

spectrometry on a Pye model 104 gas chromatograph (Pye Unicam Ltd.,

Cambridge, England) interfaced with an Associated Electrical Industries

model MS 30 double-focusing mass spectrometer with a DS-30 data system

(AEI, Scientific Apparatus Ltd., Manchester, England). Peaks were detected

by following total ion current. Gas chromatographic conditions were as

described above, using OV-225 as the stationary phase and a column temper-

ature of 170 or 2000C. Helium was used as the carrier.








Preparation of antisera. Cells of each strain (AHT, B13) were

grown for 20 h in 250 ml of THG in plastic centrifuge bottles. Cells were

pelleted by centrifugation at 10,000 x g for 15 min and washed twice with

sterile saline. The cells were resuspended in 100 ml of saline containing

0.6% formalin and incubated for 24 h at room temperature. The cells were

washed twice with sterile saline and diluted in saline to a reading of 360

using a #66 filter on a Klett-Summerson model 800-3 colorimeter (Klett

Manufacturing Co., Inc., New York, New York). After noting the volume, the

cells were pelleted as above and resuspended in one-tenth of the original

volume in sterile saline. The suspensions were stored at -200C and used

as immunogens for antiserum production.

Male New Zealand white rabbits (3 kg) were injected intravenously

on Monday, Wednesday, and Friday according to the following regimen:

during the first week 0.1 ml was injected, the second week 0.2 ml, and

the third week 0.3 ml. Rabbits were given a prophylactic intramuscular

injection of 1 ml (50 mg/ml) of Liquamycin (oxytetracycline-HCl, Pfizer,

Inc., New York, New York) after each immunization. On Monday of the

fourth week animals were test-bled from the ear artery and the serum was

tested for ability to precipitate with the homologous, cell-wall

Lancefield extract in capillary tubes, and for cross-reactivity by

Ouchterlony gel-diffusion analysis. Animals with suitable titers were

anesthetized with pentothal sodium and exsanguinated by heart puncture.

Sera were stored in small aliquots at -200C with 0.01% sodium azide.

Gel diffusion and immunoelectrophoresis. For both gel diffusion

and immunoelectrophoresis (IE), gels containing 0.75% agarose (Sigma) in

0.05 M sodium barbital/HCl pH 8.2 were used. Slides for IE (7.6 x 3.8 cm)

were cleaned in chromic acid, dipped in 0.2% agarose in deionized water








and allowed to dry. The buffered, molten 0.75% agarose solution (3 ml)

was pipetted onto each slide and allowed to solidify. Wells and troughs

were cut in the slides using a Shandon gel cutter (Shandon Scientific Co.

Ltd., London, England). Electrophoresis was performed at 40C at 10 ma

per slide for 120 min using a VOKAM SAE 2761 power supply (Shandon) and

employing the above barbital buffer. After electrophoresis of the antigen,

the troughs were cleared of agarose, filled with the appropriate antiserum,

and the plates were incubated at 40C in a moist chamber.

For gel diffusion analysis, plastic petri dishes, 50 x 12 mm,

(Falcon No. 1006, Falcon Plastics, Oxnard, California) were filled with

3 ml of the same buffered agarose solution and the appropriate number of

wells (50 ul capacity) were cut. After addition of antigen and antiserum

preparations to the wells, plates were incubated at 20 C until development

of precipitin bands.

Absorption of antisera. Whole antisera were absorbed with an

equal volume of heat-killed, washed, THG-grown cells of the heterologous

strain for 4 h at 4 C with stirring. The cells were pelleted by centri-

fugation at 8000 x g for 20 min. The supernates contained antibodies

specific for the a (AHT) or the d (313) determinants and were found to be

free of the cross-reactive (a-d) antibodies by gel diffusion analysis.

These absorbed sera were designated "anti-a" and "anti-d", respectively.

The B13 (d) cells used to absorb the anti-AHT (a) serum were

used as a source of cross-reactive (a-d) antibodies. After absorption as

above, the cells were washed 3 times with cold 0.02 M sodium phosphate

buffered saline (PBS) pH 7.2, suspended in 3 ml PBS, adjusted to pH 2.2

with 1 N HC1 and stirred for 1 h at 40C. The cells were pelleted at 8000

x g for 20 min. The supernate was neutralized with 1 N NaOH, dialyzed







against one liter half-strength PBS overnight and lyophilized. The

residue was dissolved in one half of its original volume with deionized

water. This preparation was designated "anti a-d"serum.

Quantitative precipitin analysis. Quantitative precipitin

analyses were performed to determine equivalence for each antigen-antibody

system that was studied. A stock solution of each antigen (ca. 50 ug/ml)

was made in 0.85% saline containing 0.01 M disodium ethylenediaminetetraacetate

(sEDTA), adjusted to pH 7.0 with 4 N NaOH. Antisera (20 pl anti-d diluted

1:2 with sEDTA, 25 ul anti-a, or 20 ul anti a-d) were added to 13 x 100 mm

tubes. Because of the problem of insolubility of inhibitors at the higher

concentrations used in the a antigen-anti-a, and the d antigen-anti-d

systems, a volume of 100 ul of sEDTA (corresponding to the volume of inhib-

itor used) was added to the appropriate tubes, while a volume of 50 pl

of sEDTA was added to the a antigen-anti-a-d system, and to the d antigen-

anti-a-d system. Various dilutions of antigen (50 4l in sEDTA) were added

and the tubes were incubated at 370C for 1 h followed by incubation at

40C for 7 days. Following incubation, 200 ul of cold sEDTA was added to

each tube and the tubes were centrifuged at 1500 x g for 1 h at 40C.

The supernates were carefully decanted and the tubes were allowed to drain

for 10 min onto absorbent paper. The samples were washed twice as above

except with 200 4l of saline without EDTA. After thoroughly draining,

the samples were dissolved in 100 pl 1 N NaOH and held for 30 min at room

temperature. Folin reagent C (alkaline copper tartarate minus NaOH),

1.0 ml, was added and the tubes were incubated at 370C for 10 min.

Commercial Folin phenol reagent (Harleco, Gibbstown, New Jersey) was

diluted 1:2 (1 N after dilution) and 100 ul was added to each tube with

constant mixing. Tubes were incubated at 37 C for 30 min and read at








750 nm. A protein standard curve of rabbit gamma globulin (Calbiochem)

was used to quantitate antibody protein in samples. All tubes were done

in triplicate.

Quantitative precipitin inhibition. Inhibition of precipitation

was performed in the same manner as the quantitative precipitin analysis

except various concentrations of potential inhibitors in the above volumes

of sEDTA were incubated with the antiserum for 30 min at 370C prior to

the addition of the antigen solutions. All antigen-antibody systems were

reacted at the estimated equivalence point. Control tubes containing only

antiserum and the appropriate volume of sEDTA were run and protein values

subtracted from the readings of the experimental tubes. All inhibitions

were done in triplicate.

Synthesis of methyl furanosides. Two inhibitors, a and S-methyl-

D-galactofuranoside, were synthesized by the method of Augestad and Berner

(1, 2). Since it was not possible to crystallize the derivatives, their

concentrations were determined by the phenol-sulfuric acid assay, the

solutions were evaporated to the consistency of a syrup and then diluted

to the desired concentration in sEDTA.

Chemicals. Most common salts, organic solvents and reagents, and

acids were obtained from Mallinckrodt (Scientific Products, Chamblee,

Georgia). All carbohydrate inhibitors were obtained from Sigma Chemical

Co. (St. Louis, Missouri).















RESULTS


Composition of purified cell walls of S. mutns AHT and B13.

Purified cell walls of AHT and B13 were examined for chemical composition

and for purity as determined by the absence of non-peptidoglycan amino

acids (Table 2). The major sugars present in both walls are rhamnose,

glucose and galactose in molar ratios of 3.8:1.3:1 in AHT and 7.6:4.2:1

in B13. In both cases these three sugars make up nearly 50% of the wall

by dry weight. One unique feature of these walls is the presence of

threonine, which is not found in the walls of other S. mutans serotypes.

The threonine appears to be linked to lysine since upon hydrolysis with

4 N HC1 at 1050C for 11 h, an unknown dipeptide peak is present in the

amino acid profile; and upon employing harsher conditions (6 N HC1, 105 C,

18 h) this peak disappears, and the relative molar amounts of lysine and

threonine increase with respect to the other amino acids. The presence of

threonine, probably as an alanyl-threonine transpeptide was shown in the

peptidoglycan of these serotypes in previous investigations (5, 6, 8, 57).

Extraction of antigens. Preliminary studies showed that antigeni-

cally reactive extracts could be obtained with mild Lancefield-type acid

extractions of whole cells or cell walls, and by extraction with hot

formamide. Figure 1 shows a comparison by gel diffusion of Lancefield and

formamide extracts of AHT and B13 cell walls using unabsorbed, homologous

sera. In both cases identity is seen between the two extracts.

Gel diffusion analysis of the formamide extracts of each strain

against unabsorbed sera prepared against each organism shows a reaction

24















Table 2. CHEMICAL COMPOSITION OF PURIFIED CELL WALLS OF

S. mutans AHT (a) and B13 (d)


Major Componenta


Rhamnose

Glucose

Galactose

Glucosamine

Muramic Acid

Alanine

Lysine

Glutamic Acid

Threonine

Aspartic Acid

Glycerol

Phosphorus


Total Recovery


AHT
pmol/mg ug/mg


1.68 275.9

0.56 101.8

0.44 78.9

0.36 79.9

0.31 90.3

1.26 111.9

0.49 72.1

0.45 66.6

0.43 51.5

0.04 5.6

trb ---

0.18 5.7


940.0


B13
umol/mg ug/mg


1.44 236.1

0.80 143.5

0.19 33.3

0.33 71.9

0.27 79.6

1.02 91.1

0.40 58.5

0.41 60.4

0.36 42.7

tr --

tr --

0.31 9.6


826.7


aAmino sugars are reported as acetylated derivatives
b Trace amount not measured



































Gel immunodiffusion experiments demonstrating the
reaction of identity obtained between Lancefield
extracts of AHT and B13 (aLE and dLE) and formamide
extracts (aFE and dFE), and the homologous, unabsorbed
anti-AHT serum (Fig. la) and anti-B13 serum (Fig. lb).


Figure 1.

























Fig. la


Fig. lb








of partial identity in each case (Fig. 2a and 2b). Absorption of each

antiserum with the heterologous strain eliminates the cross-reaction

(Fig. 2c and 2d). The cross-reacting antibodies can be released from

the cells used for absorption by incubation at pH 2.2 for 1 h. Figure 2e

shows the reaction of antibodies released from B13 whole cells used to

absorb anti-AHT serum, showing a line of identity between the two extracts.

On immunoelectrophoresis, both extracts show single bands with

cathodic mobility (Fig. 3). Lancefield extracts of B13 generally show a

double-arc "gull-wing" pattern, particularly when extracted for longer

than 10 min (not shown here), but this and the apparent faster mobility

of formamide extracts may be due to size degradation or charge alteration

of the polymers as will be shown below.

For extraction of the antigens from purified cell walls, the

formamide technique was used because it yielded an antigenically reactive

product which was free of peptidoglycan components. Table 3 gives the

compositions of the formamide extracts of both strains. The extracts are

almost totally composed of carbohydrates with less than 1% phosphorus

suggesting the absence of lipoteichoic acid or teichoic acid polymers as

well as nucleic acid contaminants. Amino acid analysis (not shown) re-

vealed only trace amounts of amino acids and amino sugars indicating the

absence of peptidoglycan components or proteins. Routine yields of the

antigenic formamide extract were about 33% of the dry weight of the cell

walls used for extraction.

Purification of antigens. Initially, attempts were made to purify

the antigens by gel filtration of formamide extracts. Figures 4 and 5 show

the profiles of the formamide extracts on Bio-Gel P-150 which has an

































Gel immunodiffusion experiments demonstrating the
precipitin'patterns obtained between the formamide
(aFE or dFE) extracts and various antisera: unabsorbed
anti-AHT serum (Fig. 2a), unabsorbed anti-Bl3 serum
(Fig. 2b), anti-AHT serum absorbed with B13 cells (anti-a,
Fig. 2c), anti-B13 serum absorbed with AHT cells (anti-d,
* Fig. 2d), and immunoglobulins released from B13 cells used
to absorb anti-AHT serum (anti-a-d, Fig. 2e).


Figure 2.













Fig. 2a













Fig. 2b


FE. d FE



la"mA H T


FI dF













Fig. 2c













Fig. 2d


aFE dF


aFY








































Fig. 2e

































Figure 3. Immunoelectrophoretic patterns obtained with formamide (FE)
and Lancefield (LE) extracts of AHT (Fig. 3a) and B13 (Fig.
3b). Troughs were filled with unabsorbed anti-ART serum
(Fig. 3a) and unabsorbed anti-B13 serum (Fig. 3b).




































Fig. 3a


Fig. 3b














Table 3. CHEMICAL COMPOSITION OF FORMAMIDE EXTRACTS OF

S. mutans AHT (a) AND B13 (d) PURIFIED CELL WALLS



Major Component AHT B13


umol/mg Ug/mg Imol/mg ug/mg




Rhamnose 3.11 510.4 3.24 532.2

Galactose 1.39 250.3 0.34 62.0

Glucose 1.08 193.7 2.05 369.5

Phosphorus 0,27 8.4 0.25 7.8


Total Recovery 962.8 971.5










































caa








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exclusion limit of 150,000 daltons. The profiles were quite similar.

Fractions with antigenic reactivity emerged shortly after the void volume

and trailed into the large rhamnose-containing peak. It appeared that

the majority of these antigenic materials, because they emerged before

the rhamnose peak, did not contain a large amount of that common methyl

pentose. It was also apparent that it would be difficult to cleanly sep-

arate the antigenic materials from the non-antigenic rhamnose-containing

material by gel filtration. For this reason, an affinity chromatography

approach was devised employing the galactose-binding lectin from Ricinus

communis. Initial attempts to produce an affinity support using cyanogen

bromide-Sepharose 4B (Pharmacia) resulted in low capacity columns. The

best results were obtained using Affi-Gel 10 (Biorad) which contains a
o
10 A spacer arm. After loading 50 mg of formamide extract, the column was

eluted with approximately 70 ml of buffer to ensure elution of non-binding

material. Bound material was eluted using the staring buffer containing

0.2 M D-galactose and the antigen eluted as a sharp peak (Fig. 6). Results

obtained using ART crude FE (not shown) were identical to those depicted

in Fig. 6. The fractions under the peaks were pooled, dialyzed against

deionized water, and lyophilized. Some antigenic material passed through

the column initially without binding. This is probably due to overloading

of the column since these early fractions can be reloaded on the column

and substantial binding occurs. Initially-unbound fractions were routinely

recycled once in this manner and the bound material was pooled with the

initially-bound material.

Analysis of lectin-purified antigens. Analysis of the lectin-

purified antigens by gel diffusion indicates lines of complete identity

between the crude formamide and Lancefield extracts, and homologous










































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purified antigens of each strain (Fig. 7). Fig. 8 compares crude Lancefield

and formamide extracts of AHT and B13 cell walls with the homologous lectin-

purified antigen of each strain by immunoelectrophoresis. In each case a

single band with the same cathodic mobility is seen. As with the formamide

extracts, when the purified antigens are reacted with unabsorbed antiserum

by gel diffusion, partial identity between the two antigens is seen (Fig. 9a

and 9b). Absorption of the sera with the heterologous whole cells yields

specific anti-a or anti-d serum (Fig. 9c and 9d). Release of the immuno-

globulins absorbed from anti-AHT serum by B13 cells yields a serum giving

reaction of identity between the a and d antigens (Fig. 9e). Gel filtra-

tion profiles of each purified antigen are shown in Figs. 10 and 11.

A single, rather broad peak containing all of the antigen activity is seen

for each preparation. The broadness of the peaks may reflect the range of

polymer sizes which result from the rather harsh formamide extraction

procedure. The average molecular weight of the B13 antigen appears lower

than that for AHT and may reflect the increased susceptibility to hydrolysis

of B13 cell wall polysaccharides as discussed below. Chemical analyses

(Table 4) reveal that the antigens were composed primarily of galactose

and glucose in a molar ratio of 3.4:1 in AHT, and 2.1:1 in B13, with only

traces of rhamnose as shown by gas-liquid chromatography. Each antigen

contained less than 1% phosphorus. Both antigens were quite hygroscopic,

and although they were stored lyophilized and dessicated over CaSO4,

water of hydration collected over long periods of time and was not taken

into account in data shown in Table 4. Amino acid analyses did not reveal

any gross contamination with protein derived from the lectin-containing

affinity column used in the purification.
































Gel immunodiffusion experiments using crude extracts and
purified antigens. In Fig. 7a, a reaction of identity is
seen between AHT Lancefield extract (aLE), AHT formamide
extract (aFE), and AHT purified antigen (a), using unabsorbed
anti-ART serum (center well). In Fig. 7b, identity is also
seen using B13 Lancefield extract (dLE), B13 formamide
extract (dFE), and B13 purified antigen (d). Center well in
Fig. 7b contains unabsorbed anti-B13 serum.


Figure 7.















Fig. 7a
















Fig. 7b


LE,- ,dFE.,































Immunoelectrophoretic patterns obtained with crude extracts
and purified antigens. Fig. 8a: comparison of AHT formamide
extract (aFE), purified antigen (a), and AHT Lancefield ex-
tract (aLE). Troughs contain unabsorbed anti-AHT serum.
Fig. 8b: comparison of B13 formamide extract (dFE), purified
antigen (d), and B13 Lancefield extract (dLE). Troughs con-
tain unabsorbed anti-B13 serum.


Figure 8.






































Fig. 8a


Fig. 8b































Gel immunodiffusion experiments using purified antigens from
AHT (a) and B13 (d). Antisera used are: unabsorbed anti-AHT
(a-AHT, Fig. 9a), unabsorbed anti-B13 serum (a-B13, Fig. 9b),
absorbed anti-ART serum (anti-a, Fig. 9c), absorbed anti-B13
serum (anti-d, Fig. 9d), and immunoglobulins released from
B13 cells used to absorb anti-AHT serum (anti-a-d, Fig. 9e).


Figure 9.

























Fig. 9a


Fig. 9b

























Fig. 9c


Fig. 9d









































Fig. 9e














































o


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4.j



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0



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00


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41
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62 >

































i







r
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zt
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>-


O




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00

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Table 4. COMPOSITION OF LECTIN-PURIFIED ANTIGENS FROM

S. mutans AHT (a) and B13 (d)



Major Componenta AHT B13

umol/mg Pg/mg Pmol/mg Ug/mg




Rhamnose tra -- tr

Galactose 3.34 601.2 3.33 599.8

Glucose 0.97 175.4 1.58 284.8

Phosphorus 0.28 8.9 0.22 7.1


Total Recovery 785.5 891.7


a Trace amount not measured








Precipitin inhibition. Inhibition of antigen-antibody precipita-

tion was performed using absorbed sera specific for the a or d determi-

mants of each antigen, and serum specific for the cross-reactive (or a-d)

determinants of each antigen. The highest titers of anti-a-d reactivity

were obtained from anti-AHT serum and this was used in the inhibition

studies. Both absorbed a-d sera from anti-AHT or B13 showed the same

immunological reactivity (Fig. 12).

A precipitin curve was run in triplicate using varying amounts of

antigen and constant amounts of the absorbed antiserum (Fig. 13). For

the d-antigen, 1.25 ug was chosen as the approximate equivalence level

for studies using anti-d and anti-a-d sera, and 1.42 ug of the a antigen

was found optimally effective for precipitation of the anti-a and anti-

a-d sera.

A variety of monosaccharides, methyl glycosides and disaccharides

were used as potential inhibitors. All samples and controls were run in

triplicate. Table 5 shows the results of inhibition of the anti-a serum

reaction with the purified a antigen. Galactose at the 50 umol level

gives the greatest inhibition (44%) versus 29% for glucose. The a and 3-

methyl glucopyranosides and galactopyranosides show no significant differ-

ences in inhibition from their non-methylated parent sugars. Of possible

significance is the finding that 3-methyl galactofuranoside shows greater

inhibition than the a-methyl galactofuranoside, although they are both

substantially lower than the corresponding galactopyranoside derivatives.

Among the disaccharides tested, melibiose provides the greatest inhibition

(46%) while lactose gives 39%. Both of these galactose-glucose-containing

disaccharides yield greater inhibition than the three diglucosides (cello-

biose, maltose, and gentiobiose), suggesting that the a determinant involves

































Figure 12.


Gel immunodiffusion experiments demonstrating the reaction of
identity of cross-reacting antibodies (a-d) obtained from anti-
AHT or anti-B13 sera. Top well contains the purified antigen
from AHT; bottom well, the purified antigen from B13. The
left well contains cross-reactive antibodies from anti-ART
serum, and the right well contains cross-reactive antibodies
from anti-B13 serum.








































Fig. 12














































0










Qc
*T(

LW

M
























c4







4-1
CO













S04
C Q



cu 1-












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DCC~

CC~C
000CI
~ncncncn
>>>>
~tr 63 tT



iit~


z
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S uO L- ro d o-

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Table 5.


INHIBITION OF THE PRECIPITIN REACTION BETWEEN

AHT ANTIGEN AND ANTI- a SERUM


Inhibitor ymola % Inhibition


Glucose

Galactose

a-methyl glucopyranoside

B-methyl glucopyranoside

a-methyl galactopyranoside

8-methyl galactopyranoside

a-methyl galactofuranoside

B-methyl galactofuranoside

Melibiose

Lactose

Cellobiose

Maltose


Gentiobiose


aAmount in a final volume of 17511








a galactose-glucose sequence possibly in a 1-6 linkage. (Methylation

studies to be discussed below eliminate the possibility of the 1-*4

linkage being present in the antigen). The anomeric nature of the link-

age is not clear when one considers the results obtained with the methyl

glycosides (Table 5).

Table 6 shows the results of inhibition of the anti-d serum re-

action with the purified d antigen. In this case the results are not

nearly as clear as for the a-anti-a system. At the 100 pmole level,

glucose provides 59% inhibition of precipitation while galactose yields

47% inhibition. The B-methyl glucopyranoside derivative gives nearly

twice the inhibition provided by the a-derivative, while both galactopyrano-

sides give about the same inhibition as the parent sugar, D-galactose.

The galactofuranosides are considerably less effective. All of the disac-

charides provide substantial inhibition although the two diglucosides,

maltose and gentiobiose, allow slightly higher inhibition, 53% and 50%

respectively. Cellobiose was insoluble at the 40 mole level, however,

at the 20 pmole level it gave slightly less inhibition than the other two

diglucosides at the lower level. It appears that a glucose-glucose sequence

or a glucose-galactose sequence is involved in the d antigen specificity.

However, it is not possible to establish clearer indications of the nature

of the d-specific hapten, including linkage patterns due to the relative

insensitivity of specific antibody in these inhibition studies. However, as

also the case for the a antigen, 1-4 linkages (as in maltose) are absent (as

shown by methylation studies to be presented below) and thus could not con-

tribute to the specificity of the d-reactive site.











Table 6. INHIBITION OF THE PRECIPITIN REACTION BETWEEN

B13 ANTIGEN AND ANTI- d SERUM


Inhibitor


Glucose


Galactose


a-methyl glucopyranoside


B-methyl glucopyranoside


a-methyl galactopyranoside


S-methyl galactopyranoside


a-methyl galactofuranoside

B-methyl galactofuranoside

Melibiose


Lactose


Cellobiose


Maltose


Gentiobiose


umola

50
100

50
100

50
100

50
100

50
100

50
100

50

50

20
40

20
40

20
40

20
40

20
40


aAmount in a final volume of 170p1
blnsoluble


% Inhibition

36
59

27
47

11
25

24
48

20
44

15
46

19

18

28
44

25
46

27
ib

31
53

30
50








The results of the a-d specificity studies for both antigens were

quite similar, as would be expected (Tables 7 and 8). The general trends

are the same in both cases. Galactose gives greater inhibition than

glucose. The two 1-6 linked disaccharides, melibiose and gentiobiose,

allow the greatest inhibition. No conclusions concerning anomeric link-

age can be drawn from the use of methylated derivatives of glucose or

galactose since the inhibitions provided are quite similar. From these

data (Tables 7 and 8) one can only conclude that galactose plays a major

role in the a-d specificity and a 1-6 linkage may be involved.

Some of the conclusions derived from these precipitin inhibition

studies are at variance with those recently published for strain B13 (d)

(42) and another serotype a organism, strain HS6 (46). These differences

will be presented below in the Discussion.

Methylation analysis. Results of the methylation analysis of the

purified antigens are shown in Tables 9 and 10. These data give us specific

information on the types of linkages in which all constituent sugars are

involved, and the relative frequencies of each linkage type in each

polysaccharide.

To determine the exact position of each residue in the polymer

would necessitate isolation and characterization of several disaccharides

and oligosaccharides produced by partial hydrolysis of the antigenic

polysaccharide. The large amounts of material required prevented us from

taking this classical approach: however, much information on polymeric

structure can be gained from methylation analysis alone.

In each polymer there are four residues in identical molar ratios

with respect to the branch-point. Methylation of each polymer yields

1,5-di-0-acetyl-2,3,4,6-tetra-O-methyl-D-galactitol (2,3,4,6 Gal),












Table 7.


INHIBITION OF THE PRECIPITIN REACTION BETWEEN

AHT ANTIGEN AND ANTI- a-d SERUM


Inhibitor pmola % Inhibition

Glucose 25 21

Galactose 25 38

c-methyl glucopyranoside 25 26

s-methyl glucopyranoside 25 23

a-methyl galactopyranoside 25 29

8-methyl galactopyranoside 25 23

a-methyl galactofuranoside 25 18

B-methyl galactofuranoside 25 4

Melibiose 10 47

Lactose 10 11

Cellobiose 10 33

Maltose 10 24

Gentiobiose 10 47



aAmount in a final volume of 1201l













Table 8.


INHIBITION OF THE PRECIPITIN REACTION BETWEEN

B13 ANTIGEN AND ANTI- a-d SERUM


Inhibitor umola % Inhibition


Glucose


Galactose

a-methyl glucopyranoside

8-methyl glucopyranoside

a-methyl galactopyranoside

B-methyl galactopyranoside

a-methyl galactofuranoside

B-methyl galactofuranoside

Melibiose

Lactose

Cellobiose

Maltose

Gentiobiose


aAmount in a final volume of 120ul







68






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representing a terminal galactose residue, 1,5,6-tri-0-acetyl-2,3,4-tri-

O-methyl-D-glucitol (2,3,4 Glu), representing glucose linked at C-1 and

C-6, and 1,5,6-tri-0-acetyl-2,3,4-tri-O-methyl-D-galactitol (2,3,4 Gal)

which represents a galactose residue linked at C-1 and C-6. Finally,

both polymers yield the same branch-point derivative which has been

identified as either 1,3,5,6-tetra-0-acetyl-2,4-di-0-methyl-D-galactitol

(2,4 Gal) or 1,2,4,6-tetra-O-acetyl-3,5-di-0-methyl-D-galactitol (3,5 Gal).

These two derivatives are actually identical since they are symmetrical.

This derivative is obtained from the methylation of a galactopyranose

residue linked at C-1,3, and 6 or a galactofuranose residue linked at

C-1,2, and 6. These two particular galactose residues yield the same

derivative when methylated and reduced and can only be differentiated by

tagging C-l by reduction with sodium borodeuteride.

The four residues mentioned above may comprise a common sequence

of sugars in each polymer. It will be postulated that the terminal galac-

tose residues attached at the galactose branch-point compose part or all of

the a-d reactive site since these residues are common to both polymers, and

inhibition studies indicate that alactose may be the principal determinant of

a-d specificity (see Discussion).

There are two 1-3 linked moieties in each polymer in nearly equal

molar ratios with respect to the branch-point: a 1-3 linked galactose residue

in the AHT polymer (1.9:1), and 1-3 linked glucose in the B13 polymer (1.5:1).

The 1-3 linkage of these residues may produce pronounced "buckling" of the

polymeric chain allowing these and neighboring residues to be presented as a

sterically-accessible antigenic unit to antibody molecules. It will be pro-

posed (see Discussion) that these as well as neighboring residues constitute

the a and d determinants in the respective polymers. The AHT polymer








contains the 1-3 linked galactose residue and inhibition studies described

above show that a galactose-glucose sequence gives the greatest inhibition,

while the B13 polymer contains the 1-3 linked glucose residue and inhibition

studies suggest a glucose-glucose sequence is involved.

Another feature of both polymers that becomes evident from the

methylation studies is the presence of galactofuranose residues.

Methylation of the ART polymer yields 1,3,4-tri-0-acetyl-2,5,6-tri-0- methyl

galactitol (2,5,6 Gal) which represents a galactofuranose residue linked at

C-l and 3 to neighboring sugars. The B13 polymer yields 1,4,6-tri-O-acetyl-

2,3,5-tri-0- methyl galactitol (2,3,5 Gal) upon methylation, which represents

a galactofuranose residue linked at C-l and 6 in the native polymer.

Furanosidic linkages are known to be much more sensitive to acid hydro-

lysis than the corresponding linkages between pyranosides (3,50,52).

A study using immunoelectrophoresis was undertaken to demonstrate

the acid liability of these polymers and to compare the apparent greater

mobility of formamide extracts with products yielded by Lancefield acid

extractions of B13. Samples of B13 cell walls were hydrolyzed at pH 2.5

according to the procedure outlined in Methods, except that samples were

hydrolyzed for 10,20,40 or 60 min before neutralization. Fig. 14a and b

show the results of the hydrolysis of B13 cell walls at pH 2.5. After 10

min, essentially a single arc is seen. With increasing hydrolysis times,

however, a "gull-wing" type of precipitin band is obtained with the

faster-moving portion of the band becoming more evident and possessing

a mobility similar to that of the formamide extract shown in the center

well. To determine whether this represents the degradation of the polymer

or the extraction of a second antigen from the cell wall, the supernatant

































Figure 14.


Immunoelectrophoretic study of the degradation of Lancefield
extracts of B13 cell walls at various extraction times. In
Fig. 14a and Fig. 14b, cell walls were extracted for 10,20,40,
and 60 min and compared to a formamide extract (FE). In Fig.
14c the supernate from a 10 min Lancefield extract was hydro-
lyzed for an additional 30 min (10 + 30). In Fig. 14d the
supernate from a 10 min Lancefield extract was subjected to
formamide extraction (FE of LE). Unabsorbed anti-B13 serum
was used in all troughs.





































Fig. 14a


Fig. 14b






































Fig. 14c


Fig. 14d








from the 10 min extract which had been neutralized and centrifuged to

remove cell walls was readjusted to pH 2.5 and boiled for an additional

30 min. The appearance of the familiar "gull-wing" pattern upon additional

hydrolysis (Fig. 14c) suggests that this phenomenon is likely due to size

degradation or an alteration of the charge of the polymer and not due to

extraction of a second antigen from the wall. Likewise, if the neutralized

10 min supernatant is subjected to formamide extraction (Fig. 14d), further

degradation of the polymer is apparent.

The same type of experiment was repeated using AHT walls (Fig. 15).

Again, degradation can be seen with increasing hydrolysis time. When the

neutralized, cell wall-free supernatant of the 10 min acid extract is

subjected to formamide extraction, the conversion to a faster moving

component is seen as in the case of B13 (Fig. 15c).



























Figure 15. Repeat of the experiment shown in Fig. 14 using AHT
Lancefield cell wall extracts (10,20,40, and 60 min)
and AHT formamide extract (FE). Time degradation of
AHT antigen showing the appearance of a faster moving
component (Fig. 15a and Fig. 15b). Supernate from a
10 min Lancefield extract was further treated by
formamide extraction (FE of LE) and compared to a
formamide extract of cell walls (FE) and a 10 min
Lancefield extract (LE, Fig. 15c). All troughs were
filled with unabsorbed anti-AHT serum.





































Fig. 15a


Fig. 15b



















































Fig. 15c














DISCUSSION


Two antigenic polysaccharides were extracted and purified from

cell walls of the cross-reactive strains S. mutans AHT (a) and S. mutans

B13 (d). The antigens, extracted by the hot formamide method, were

purified by affinity chromatography on columns containing the galactose-

specific lectin from the castor bean (ricin) and found to be diheteroglycans

consisting primarily of galactose and glucose. Antigenic specificities

of both the specific and the cross-reactive sites on each polymer were

determined, and methylation analysis revealed structural similarities

that may reflect the nature of the cross-reactive sites and differences

that may reflect the natures of specific haptenic regions.

At the present time there are 5nly three papers dealing with the

immunochemistry of these two serotypes (41, 42, 46). Two points must be

considered when comparing the present study with the previously published

data: first, the starting materials (cell walls us whole cells) and the

methods of extraction and purification will undoubtedly affect not only

the size of the polymer extracted but also the amounts and types of

materials that may be associated with the purified products (peptidoglycans,

lipoteichoic acids, etc.) but which do not contribute to the antigenic

reactivity of the product. These differences should not, however, affect

the specificity of the combining sites with respect to antibodies when

measured by precipitin inhibition assays. Second, one is limited by the

availability of appropriate potential inhibitors particularly disaccha-

rides and higher oligosaccharides for use in precipitin inhibition studies.







The results of the inhibition of precipitation of the AHT antigen

with anti-a serum show that galactose is the best monosaccharide inhibitor

(44%), with melibiose and lactose also giving relatively high degrees of

inhibition. Since methylation analysis indicates the absence of 1-4

linkages in the purified AHT antigen, the inhibition by lactose is probably

not a function of its linkage pattern. But the presence of 1-6 linkages in

the antigen may explain the efficacy of melibiose in the inhibition (46%)

of the homologous reaction. Both disaccharides, however, contain galactose

which undoubtedly is located at the a-specific haptenic site. The use of

methylated galactopyranosides and galactofuranosides in inhibition studies

failed to reveal the probable anomeric linkages of galactose moieties at

the antibody binding site.

The a antigen isolated by Mukasa and Slade (46) from hot water

extracts of whole cells of HS6, differs in chemical composition from the

AHT antigen in the molar ratio of galactose to glucose (5.2:1 for HS6 (26)

us 3.4:1 for AHT) but appears to behave similarly in gel diffusion and

immunoelectrophoresis. Each product apparently possesses both an a and

a-d reactive site. Inhibition studies on the a site of the HS6 poly-

saccharide were carried out by Mukasa and Slade (46) with only a limited

number of inhibitors. Significant inhibition was obtained with glucose

(25%), and the diglucosides maltose (51%), and gentiobiose (49%). The

only galactose-containing inhibitor used, lactose, yielded less than 10%

inhibition. All inhibitors were compared on an equal weight rather than

equimolar basis. From these limited data they concluded that either

a-1-4 or a 0-1+6 configuration involving only glucose was responsible for

the a specificity in HS6. Their failure to employ galactose or galactosides

in their studies makes interpretation of their findings difficult.








Certainly, the present study of the AHT antigen fails to confirm the

immunochemical nature of the serotype a antigen presented by Mukasa and

Slade (46). Confidence in data presented in this dissertation is provided

by the method of antigen extraction (purified cell walls rather than whole

cells), the process of antigen purification (affinity chromatography) and

the wider range of monosaccharides and disaccharides tested in precipitin

inhibition analyses. It would appear, therefore, that further studies of

serotype a polysaccharides may be required to present a more coherent

picture of this important antigenic polysaccharide.

With respect to the specificity of the d site on the B13 antigen,

the results in Table 6 suggest that the major determinant may be glucopyrano-

side that is linked 1-6 to another glucose or galactose residue, possibly

by means of a B-linkage as indicated by the excellent inhibition obtained

using gentiobiose (50%) and B-methyl glucopyranoside (48%). Linzer and

Slade (42) meanwhile purified an antigen from cold, 5% trichloroacetic acid

(TCA) extracts of whole cells of B13. This antigenic polysaccharide had

about the same molar ratio of galactose: glucose (1.9:1) as the B13 antigen

isolated and purified in this study (2.1:1). Inhibition of the precipitation

of the Linzer-Slade antigen (42) with anti-d serum gave the following

results: galactose (31%), glucose (5%), rhamnose (0%), lactose (9%), and

melibiose (4%). Again, inhibitors were used on an equal weight (500 ug)

basis rather than an equimolar basis, despite the fact that a selection of

mono- and disaccharides with different molecular weights were being com-

pared. It was also reported that galactosamine, glucosamine, melibiose,

cellobiose, stachyose([Gal-a-l-6-] -Glu-a-1l2- fructofuranose), and

maltose inhibited less than 5%, again using 500 ug amounts of each. The

discrepancy between the published results and those of the present study







cannot readily be explained. There are differences in extraction techniques

(formamide on purified cell walls here vs 5% TCA on whole cells (42)),

purification (affinity chromatography here vs gel and ion-exchange chroma-

tography), and in the organisms used to absorb the whole B13 serum to yield

anti-d immunoglobulins (AHT cells here vs HS6 (42)). Again, it is concluded

there is much doubt as to the true nature of the d determinants, and further

studies with additional strains of the serotype are suggested to help re-

solve the discrepancies.

There is, however, agreement as to the apparent specificity of the

a-d site; namely galactose (probably in the pyranose configuration). This

specificity was found using both the AHT and B13 antigens in this study,

and in HS6 by Mukasa and Slade (46). (The specificity of the a-d site of

the B13 antigen purified by Linzer and Slade (42) was not determined in

that study). Tables 7 and 8 reveal pronounced inhibition by melibiose

(72% with B13 and 47% with AHT, each at 10 moless. These data suggest

that the galactose determinant of a-d reactivity may reside at the non-

reducing end of a branch of the main polymeric chain since melibiose con-

tains galactose linked a-1-6 to glucose.

The data provided by methylation analysis of purified polysaccha-

rides provide much useful information concerning the chemical composition

and linkage patterns of each antigen, and when considered with the inhi-

bition data, likely structures for each determinant can be postulated.

Although methylation studies do not provide information concerning the

sequential arrangement of sugar residues, nor data concerning anomeric

linkages, it is possible to derive plausible structures. Figure 16 shows

a schematic arrangement of sugar residues in the AHT and B13 antigens

taking into account the more important features suggested by the














































3






-Crp
0
r








4.Ju










cn~z



-4 c



h.J





84














''
3
0 "





*cZ



_ 2E









Cl,



q- l
iQ 0
-~ ~ ~
0c
\ I,
0n








methylation analyses and inhibition data. All residues present in each

polymer are not shown; only the suggested common (a-d) and specific (a

and d) haptenic areas are detailed.

Both polymers possess the same branch-point: a galactose residue

linked at C-1,3, and 6 (or a galactofuranose residue linked at C-1,2, and

6). Also, both polymers probably contain a terminal galactose moiety that

is always linked to this branch-point since they are present in equimolar

amounts. Two other common residues exist: almost 2 moles (relative to the

branch) of glucose linked at C-l and C-6 plus 1.4 moles of galactose linked

at the same carbon atoms. The sharing of a terminally-linked galactose

residue, and the fact that galactose was the best inhibitor of anti-a-d

activity, makes the terminal galactose linked to the galactose branch-point

a likely arrangement for the a-d reactive site. There may be a residue

or residues intervening between the terminal galactose and the branch-

point (perhaps the common non-terminal galactose linked at C-l and C-6),

however, this cannot be determined at present.

Another important feature found in each polymer is the presence of

1-3 linked galactose (AHT), and glucose (B13). D.A.R. Simmons (55, 56)

in studying molecular models of ShigZela flexneri 0-specific side chains

found that 1-2 and 1-3 linkages on rhamnose residues caused marked buckling

of the polymers, which "broke" the primary chains into sterically discrete

repeating sequences which correspond to the antigenic sequences determined

by serological procedures. It was also noted that these 1-2 or 1-3 linkages

were not involved in determining the antigenic specificity of these deter-

minants but only served to set them apart as sterically discrete and

accessible sequences. Simmons proposed that these determinants should be

called "apodeterminants" because "by conferring a distinctive shape on







the molecule, they determine the sequence and steric accessibility of the

more distant structures that compromise the antigenic determinant without

themselves forming an integral part of it" (56). He proposed that the

determinants themselves should be designated as "non-terminal determinants."

The presence of a galactose residue linked at C-1 and C-3 in the AHT polymer

and the fact that inhibition studies show that galactose (possibly linked

1-6 to glucose) is the more inhibitory hexose, support the arrangement shown

in Fig. 16. Likewise, the B13 polymer possesses a glucose residue linked

at C-l and C-3 and inhibition studies show that glucose is the better inhib-

itor of anti-d serum activity. Whether the d determinant involves a glucose-

glucose or a glucose-galactose sequence in a 1-6 linkage is not clear,

however both 1-6 linked glucose and galactose residues are found in the

813 polmer. In Fig. 16 the glucose-glucose (1-i6) linkage is suggested in

light of the fact that glucose residues linked at C-1 and C-6 are so common

in this polysaccharide.

Finally, each polymer possesses galactofuranose residues at some

location within the chain, and these residues may account for the acid

liability of these antigens, as was discussed previously.

More precise determinations of the actual sequence of sugars in

each polymer will necessitate isolation of oligosaccharides by partial

acid or enzymatic degradation, the determination of their structures, and

use of these authentic oligosaccharides as potential inhibitors.

Detailed studies of the fine structure of the S. mutans antigens

will aid in our greater understanding of the serological relationship

among the various strains, and may lead to further subdivision of strains

on a serological basis. The precise knowledge of these relationships also

will aid in epidemiological studies, and ultimately in the selection of

appropriate strains for use in future vaccination programs.














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