Purification and chemical characterization of a polysaccharide antigen and a lipoteichoic acid from streptococcus mutans AHT

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Title:
Purification and chemical characterization of a polysaccharide antigen and a lipoteichoic acid from streptococcus mutans AHT
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x, 85 leaves : ill. ; 28 cm.
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Craig, Ronald Albert, 1946-
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Streptococcus mutans   ( lcsh )
Bacterial antigens   ( lcsh )
Microbiology and Cell Science thesis Ph. D
Dissertations, Academic -- Microbiology and Cell Science -- UF
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non-fiction   ( marcgt )

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Thesis:
Thesis--University of Florida.
Bibliography:
Bibliography: leaves 80-84.
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Typescript.
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Vita.
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by Ronald Albert Craig.

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Table of Contents
    Title Page
        Page i
    Acknowledgement
        Page ii
        Page iii
    Table of Contents
        Page iv
    List of Tables
        Page v
    List of Figures
        Page vi
        Page vii
    Glossary of abbreviations
        Page viii
    Abstract
        Page ix
        Page x
    Introduction
        Page 1
        Page 2
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
    Materials and methods
        Page 8
        Page 9
        Page 10
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        Page 20
        Page 21
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        Page 24
        Page 25
        Page 26
    Results
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
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        Page 33
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    Discussion
        Page 70
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    Literature cited
        Page 80
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        Page 84
    Biographical sketch
        Page 85
        Page 86
Full Text













PURIFICATION AND CHEMICAL CHARACTERIZATION OF A POLYSACCHARIDE ANTIGEN AND A LIPOTEICHOIC
ACID FROM STREPTOCOCCUS MUTANS ALIT









By

RONALD ALBERT CRAIG




















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 1979















ACKNOWLEDGMENTS


I would like to express my most sincere and deepest

appreciation to Dr. Arnold S. Bleiweis, chairman of my supervisory committee, for his unfailing support and faith in me and for his excellent guidance and understanding as a teacher, advisor, and friend.

I would like to thank the members of my committee,

Dr. Edward P. Previc, Dr.* Dinesh 0. Shah, and Dr. Dale C. Birdsell for their advice and scientific insight in my research and preparation of this dissertation.

Dr. Edward M. Hoffmann was extremely helpful in trying to correct and augment my deficiencies in the area of immunology specifically and in science in general. Dr. bonnie 0. Ingram was of invaluable assistance in suggesting the use of artificial liposomes in the purification of lipoteichoic acid. I would also like to thank the rest of the faculty of the Department of Microbiology and Cell Science for the excellent education given to me by being associated with them and for their assistance with all phases of my research.

I would like to express my deepest appreciation to

Dr. Sylvia E. Coleman for doing the electron muicroscopy associated with this work as well as sharing her expertise in this filid with me; Lo Steve Hurst for his technical assistance in the laboratory, for his assistance in all phrases of the




Ii













preparation of this dissertation, and for his friendship; and to Dr. Louis J. Silvestri for his assistance in working out the liposoine-purification technique.

Finally I would like to thank my wife, Gail, for all

her assistance, understanding, and patience with me and my work. I would like to dedicate this dissertation to her and to my son, Andrew Bryon.















































Iff

















TABLE OF CONTENTS


PACE

ACKNOWLEDGEMENTS ............................................ ii

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

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

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

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

INTRODUCTION ......................... 66 ....................... I

MATERIALS AND METHODS .................................. 6 ..... 8

RESULTS .................................................... 27

DISCUSSION .................................................. 70

LITERATURE CITED ............................................ 80

BIOGRAPHICAL SKETCH ......................................... 85


























iv


















LIST OF TABLES




TABLE PAGE

1. CHEMICAL COMPOSITION OF VARIOUS
PREPARATIONS OF THE POLYSACCHARIDE
ANTIGEN OF S. MUTANS AHT............................... 35

2. EXCRETION OF THE POLYSACCHARIDE
ANTIGEN AND PROTEINS INTO SYNTHETIC
MEDIUM MC............................................... 44

3. CHEMICAL COMPOSITION OF VARIOUS
PREPARATIONS OF LTA FROM S. MUTANS MIT................. 56

4. HEMAGGLUTINATION TITERS OF LTA
AND POLYSACCHARIDE ANTIGEN PREPARATIONS ................62

















LIST OF FIGURES


FIGURE PAGE

1. Purification of a polysaccharide antigen from
the acid extract of 5 g of S. mutans AHT whole
cells by Bio-Gel A-5M gel filtration chromatography ....................................... ...29

2. Immunodiffusion patterns of the extracellular
(CHOx) and intracellular (CHOi) or extracted
polysaccharide antigens from S. mutans AHT,
the S. mutans AHT LTA (AHTLTA), and the BioGel A-5M chromatographed phenol-water extract
(4OH) reacting with antiserum #27 ..................31

3. Purification of a polysaccharide antigen from
the acid extract of 5 g of S. mutans AHT whole
cells by Sepharose CL-4B gel filtration chromatography. ...........................................34

4. Immunoelectrophoresis pattern of the polysaccharide antigen extracted from S. mutans
AHT (CHOi) and a Lancefield extract of S.
mutans AHT (LE) reacting with antiserum #27........37

5. Bio-Gel A-5M gel filtration chromatography of
20 g (original volume 6.6 1) of lyophilized
S. mutans AHIT culture fluid ........................40

6. Growth of S. mutans AHT and excretion of the
polysaccharide antigen in synthetic medium MC ......43

7. Radial immunodiffusion (RID) standard curve........47

8. Bio-Gel A-5M gel filtration chromatography of
the phenol-water extract of 10 g of S. mutans
AHT whole cells ....................................50

9. Bio-Gel A-5M gel filtration chromatography of
the phenol-water extract of 10 g of S. mutans
AHT whole cells previously extracted with acid.....53






vi











FIGURE PAGE

10. Immunoelectrophoresis pattern of the L. casei
LTA (Lc LTA), the AHT LTA (AHT LTA), and the
Bio-Gel A-5M chromatographed phenol-water
extract of AHT (@OH) reacting with antiserum
#27. ................................................60

11. Electron micrograph of S. mutans AHT treated
with horseradish peroxidase conjugated to goat
antirabbit immunoglobulins, with glutaraldehyde,
with H202 and DAB, and then stained with OsO ......65

12. Electron micrograph of S. mutans AHT treated
with a 1/5 dilution of antiserum #26, with
horseradish peroxidase conjugated to goat
antirabbit immunoglobulins, with glutaraldehyde,
with H202 and DAB, and then stained with s04 ......67

13. Electron micrograph of S. mutans AHT treated
in the same manner as in Fig. 12, except that
the dilution of antiserum #26 was 1/20............. 68

14. Electron micrograph of S. mutans ART treated
in the same manner as in Fig. 12, except that undiluted antiserum to S. mutans BHT was used
instead of antiserum #26 ...........................69





























vii
















GLOSSARY OF ABBREVIATIONS



DAB: 3, 3'-Diaminobenzidine

HA: Hemagglutination

IHA: Inhibition of hemagglutination

IU: International units

LTA: Lipoteichoic acid

OD: Optical density

PHA: Passive hemagglutination

PBS: Phosphate buffered (0.05 M, pH 7.2) saline (0.85% NaCl)

PC: DL a phosphatidylcholine

RID: Radial immunodiffutsion

SRBC: Sheep red blood cells TA: Teichoic acid

THG: Todd Hewitt broth supplemented with glucose

Tris: Tris (hydroxymethyl) aminomethane TC: Tris-carbonate, 0.01 M, pH 6.8


















vil













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

PURIFICATION AND CHEMICAL CHARACTERIZATION OF A
POLYSACCHARIDE ANTIGEN AND A LIPOTEICHOIC
ACID FROM STREPTOCOCCUS M UTANS AHT By

RONALD ALBERT CRAIG

March, 1979


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

A polysaccharide antigen was extracted in good yield from Streptococcus mutans AHT whole cells by boiling in pH 4.0 sodium acetate buffer for 10 minutes and was then purified by 6% agarose gel filtration chromatography. This antigen is a heteropolysaccharide containing galactose and glucose in a molar ratio of 3.6:1 respectively and, when tested by immunodiffusion and immunoelectrophoresis, appears to have characteristics identical to the S. mutans serotype a specific antigen as described by Bratthall. Electron micrographs using indirect horseradish peroxidase labeling of this antigen shows it to be a fluffy coat (capsule) surrounding the cell. Analysis of spent culture fluid indicates that this antigen is released into the medium; during exponential growth release is proportional to the cell mass of the culture. A second surface antigen, a membrane-associated lipoteichoic acid (LTA) was extracted from whole cells with hot phenol-water. The phenol-water extract contained both the polysaccharide antigen and the LTA in



ix













close association such that complete separation of the two by current purification techniques could not be achieved. This association allows erythrocytes to be passively sensitized with the polysaccharide antigen when erythrocytes and the crude phenol-water extracted material are mixed; however the purified polysaccharide antigen can not passively sensitize erythrocytes. The association of LTA and polysaccharides (and proteins) has been shown in organic solvents but this is the first report of their association in an aqueous solution. Since LTA can bind to other mammalian cells besides erythrocytes, the association of LTA and the polysaccharide antigen could provide a mechanism for the transfer of bacterial antigens to the surface of certain mammalian tissues during an infection resulting in immunopathological lesions. A new technique is described which uses phosphatidylcholine liposomes to adsorb the LTA which then can be separated from almost all of the polysaccharide antigen by centrifugation. The LTA from S. mutans

AH-T purified by this technique was shown to contain only glycerol, phosphorus, alanine, and glucose in molar ratios of 1:1:0.5:0.14 respectively. This corresponds to a LTA with a glycerol phosphate backbone with up to 50% of the glycerols substituted with alanyl ester groups, no carbohydrate substitution and a glucose containing glycolipid. This structure differs from one previously published and the difference is attributed to the greater purity of the present product.








X
















INTRODUCTION


The role of Streptococcus mutans in the production of smooth surface and pit-fissure caries has been established in numerous studies. The major emphasis of research in this area is on finding a cure for S. mutans caused caries. In a workshop in January 1976 sponsored by the National Caries Program of the National Institute of Dental Research, on the selection of iminunogens for a caries vaccine and cross reactivity of antisera to oral microorganisms with mammalian tissue, some of the most respected immunologists and immunochemists working with oral microbes were brought together to discuss the complexities of anti-caries vaccine. Two conclusions were reached at this workshop: 1) that work should continue toward the development of an anti-caries vaccine, and 2) that immunization against S. mutans is a valid goal (14).

Given the goal of an anti-caries vaccine, the question is what type of vaccine should be employed? Should whole cells or specific polymers be used as iminunogens? The immunogen used determines effectiveness and possible toxicity of the vaccine. Since Van de Rijn, Bleiweis, and Zabriskie have reported the possibility of an antigen or antigens in S. mutans which share antigenic determinants with human sarcolemmal sheaths (45,46), the safety of using a whole cell vaccine or some mixed antigen vaccine is questionable without first eliminating this cross-reacting antigen(s) from the vaccine. Fundamental to this goal is the













isolation and characterization of these cross-reactive antigens. This task has been extremely difficult with S. pyogenes and in finding its role in rheumatic fever. Secondary to this is to isolate possible immunogens from the various S. mutans serotypes and once they are purified to check for possible cross-reactions

with human tissue.

The search for an anti-caries vaccine would be easier if a single antigen could be found that would protect equally against all S. mutans strains. One possibility is the glucosyltransferase, the extracellular enzyme that produces dextran from sucrose. There are two problems with this; one is that there seem to be differences in the glucosyltransferase system between the different strains, and the other is the apparent lack of immunogenicity of the purified enzyme preparation (10). The problem with using surface polymers is that there are at least 8 major surface antigens in the various strains of S. mutans and an undetermined number of minor ones, so that use of one antigen would not necessarily confer immunity to all the strains. A mixed antigen preparation is the only possibility, however the choice of antigens must be carefully considered. To be able to make this choice all of the major surface antigens of the various S. mutans serotypes must be completely characterized and available for testing.

S. mutans was divided into 7 serotypes by Bratthall (8) and Perch et al. (35) based upon the comparative immunoelectrophoretic patterns of surface antigens extracted from a large number








3




of strains. These major serotypes are designated a to R, with b having two major antigens and all others having one. Although placed into different serotypes a, d, and & contain a common cross-reacting antigen as do serotypes c, e, and f. A vaccine using the cross-reacting antigens would only have to contain a mixture of 3 antigens to be effective against all of the S. mutans. This vaccine would contain the a-d-& antigen, the c-e-f antigen, and one of the two b antigens (9).

The focus of this report will be on the serotype a

antigen isolated from strain AHT. This antigen is a polysaccharide composed of specific (a) and cross-reacting (a-d-,) determinants on the same molecule. Initially, there had been some disagreement as to the nature of this antigen. Van de Rijn and Bleiweis reported that the serotype a antigen of AliT was a membrane-associated glycerol teichoic acid substituted with alanine (10-15%) and a disaccharide of D-glucose and D-galactose (40-50%) with the haptenic moiety being s-linked D-galactose (44). A major discrepancy was that the serotype a antigen migrates slowly toward the cathode while the structure of the teichoic acid indicates that it should migrate toward the anode. Mukasa and Slade reported that the serotype a cross-reacting antigen of strain HS6 was a polysaccharide containing galactose, glucose, glucosamine, and galactosamine with molar ratios of 5.5:1:0.7:0.5 respectively. The immunological specificity of the serotype a antigen depended on a glucose moiety while D-galactose is found at the cross-reactive (a-d-_) site. They also reported a protein












associated with the polysaccharide, antigen which they say could have attached this antigen to the cell wall (33). A major discrepancy was that Bleiweis et al. reported finding no galactosamine in S. mutans AHT, HS-6, or other serotype a organisms (2,3). Also at question is the nature of the protein and its association with the polysaccharide, which could be important in a vaccine in relation to immunogenicity, toxicity, and cross-reactivity with human tissue.

Linzer and Slade purified and characterized a polysaccharide from a serotype d organism (B-13) that was 96% carbohydrate containing only galactose and glucose in a 2:1 molar ratio. This molecule contained both the d specific site and the a-d-g cross-reactive site, with the latter being a terminal Dgalactose moiety (28). These results have been partially confirmed by Brown (T. A. Brown, Ph.D. dissertation, University of Florida, Gainesville, 1978). Brown found the specific (d) determinant to

be D-glucose while Linzer and Slade reported D-galactose for the same antigenic site. There was no disagreement about the a-d-R site.

Iacono et al. isolated, purified, characterized, and localized a polysaccharide antigen from a serotype g organism (strain 6715) (20,21). This antigen contained mainly galactose and glucose in a 5.9:1 molar ratio and about 10% protein. This antigen

also was shown by peroxidase labeling to be a capsule surrounding the surface of the cell.







5



Since LTA is a major antigenic component of most grampositive membranes (52) and considering its possible roles in the adhesion of S. mutans to teeth and in the pathogenesis of rheumatic fever or glomerulonephritis (4), a further discussion of this unique molecule is warranted. LTA is an amphipathic molecule containing a hydrophilic portion, the negatively charged glycerol-phosphate backbone, and a hydrophobic portion, the glycolipid. The glycerol-phosphate backbone usually contains

25-30 subunits and the glycerol molecules can be substituted with mono-, di-, or trisaccharides or with alanine. The glycolipid portion is usually a di- or trisaccharide containing phospholipid which is usually identical to the free glycolipid of the plasma membrane (24,52).

While LTA is found associated with the membrane of most

gram-positive organisms, there are a few exceptions: 1) Micrococcus lysodeikticus contains a lipomannan associated with its membrane instead of LTA (37); 2) Actinomyces sp. contain no LTA; however,

a fatty acid substituted heteropolysaccharide was found associated with the membrane of A. viscosus (50); 3) LTA has been found to be absent in the oral microorganism, Streptococcus mitis (39). Even though it is associated with the plasma membrane, LTA is a surface antigen and is the group antigen of the streptococcal Lancefield groups D and N, and the lactobacillus groups A and F.

The degree of participation of LTA as a surface antigen depends on the length of the LTA backbone and the thickness of the wall








6



polysaccharide-peptidoglycan network for each organism as shown in lactobacilli (47).

Most organisms containing LTA excrete it into the medium and some organism, such as S. mutans, excrete large quantities, up to 320 pg/mi of culture fluid (32). This property, coupled with the long known ability of LTA to bind to erythrocytes and the more recent findings that it will bind to other mammalian cells

(34) thereby allowing the adherence of group A streptococci to human oral epithelial cells (1), makes it possible to visualize how LTA could play roles in the primary aspects of pathogenesis (adherence of whole cells) and in the secondary aspects (adherence of LTA-molecular aggregates to tissue) causing autoimmune responses or immune complex reactions either in gingival

tissue to cause gingivitis or in heart tissue to cause symptoms of rheumatic fever.

LTA may not be the sole antigen responsible for an autoimmune response but it could act as a carrier of other bacterial antigens. This interaction between LTA and other antigens could occur in any number of ways due to the amphipathic nature of this molecule. It can interact with both hydrophilic and hydrophobic molecules. The large negative charge produced by the phosphate groups of the backbone gives the molecule the possibility of ionic interactions with positively-charged antigens.

The ability to form large aggregates, or "micelles," with other amphipathic molecules or itself could allow it to trap neutral













antigens in these aggregates. An interaction has been found between LTA and polysaccharides (and certain proteins) in solutions containing 50-97% ethanol or other short chain alkyl alcohols (12).

The objectives of this study were:

1) to disclose the nature and cellular localization

of the serotype a cross-reacting antigen of

S. mutans AlIT.

2) to isolate, purify, and characterize the LTA from

S. mutans ART and show its relationship to the

-- cross-reacting antigen. 3) and to probe any possible molecular interactions

between the AIT LTA and other antigens of S.

mutans ART.















MATERIALS IAND METHODS

Bacterial strains and growth conditions. Streptococcus mutans AHT, a serotype a organism (8) isolated from a human by Jablon and Zinner (22), served as the major subject of this study. Another strain, also isolated by Jablon and Zinner, S. mutans BHT, serotype b, was used as a source of lipoteichoic acid (LTA). These organisms were maintained in a lyophilized state in 20% skim milk under a vacuum and stored at -200C. When lyophilized stock cultures were opened, the organisms were

transferred to broth and subsequently streaked on plates. Orgnaisms growing in broth were transferred every 24 hours and were transferred at least 3 times in the appropriate broth before use. Large cultures were grown after making a 1:100 dilution of an exponentially growing culture into fresh broth.

Whole cells needed for chemical extractions were obtained by growing cultures at 370C for 24 hours (late stationary phase) in carboys containing 10 liters of Todd-lewitt broth (Difco, Detroit, MI) supplemented with 0.4% glucose and adjusted to

pH 6.5 prior to autoclaving (THG). Harvesting of the cells was done on a DeLaval. gyro-testor (DeLaval Separator Co., Poughkeepsie,
-O
NY). The harvested cells were heated to 60 C for 20 minutes to destroy autolysins, washed 3 times with water and then lyophlilized.

For prowth curves and thie experiments dealing with the excretLon of maci n-oolecol es Into Chi.' md Illul, two types of growth h




8













media were used: THG and synthetic medium MC as described by Terleckyj et al. (42).

Crude extracellular material was obtained by growing S. mutans AHT or BHT in THG prefiltered through a PTGC Pellicon molecular filter (nominal molecular weight limit:10,000 daltons) on a Pellicon cassette system (Millipore Corp., Bedford, MA). After prefiltering, the TUG medium was filter sterilized using a

0.45 pm filter, inoculated, and then incubated at 37'C for 24 hours. After harvesting the cells as above, the culture fluid was filtered on the Pellicon cassette system using a 0.2 pm filter to remove all cells. The crude extracellular material that was retained when the cell free culture fluid was passed through the PTGC filter was lyophilized. This retentate contained the large (>10,000 daltons) macromolecules released by the streptococci during growth.

Acid extraction of whole cells. Ten grams of lyophilized whole cells were added to 150 ml of 0.1 M sodium acetate buffer, pH 4.0, preheated to 100*C. After 10 minutes at 100C the suspension was cooled to 5oC in ice water, centrifuged at 10,000 g for 10 minutes to remove the whole cells, and the supernate was brought to pH 7.0 with 1.0 M NaOH. The neutralized supernate was filtered (0.45 Pm) and the volume reduced to 15 ml on a Buchler rotary evaporatory (Buchler Instruments, Fort Lee, NJ). The acid extract was ready for gel filtration chromatography.

Phenol-Water extraction of whole cells (49). To 1 o f I yoph1 i ied whole cellis 150 ml of preheated (68%C) water was








10



added, followed by the addition of 150 ml of preheated (68 0C) 90% liquid phenol. The temperature was maintained at 68 0C for 30 minutes with stirring. After cooling in an ice bath, the cell suspension was transferred to 30 ml Corex centrifuge tubes and centrifuged at 10,000 X g for 10 minutes. The water layer was removed carefully and the phenol layer reextracted with an equal volume of water which then was added to the original water layer. The pellet was reextracted with phenol-water and that water layer added to the others. Flash evaporation of these combined water layers reduced the volume to 50-100 ml which was applied to a Bio-Gel P-6 (Bio-Rad Laboratories, Richmond, CA) gel column to remove the phenol. The material coming off with the void volume was lyophilized or applied directly to an agarose gel column.

Quantitative assays. Total phosphorus was determined by the method of Lowry et al. (29). This assay, as well as absorbance at 260 nm for nucleic acids and the phenol-sulfuric acid assay for carbohydrates (13), was used to assay column fractions.

Three different protein assays were employed. The method of Lowry et al. (30) was used to determine the protein content of whole cells during growth. A sample from a growing culture (10 Ml for the early growth points but only 1.0 ml for the later points)

was centrifuged and the cells were washed 3 times with water to remove media contaminants. The pelleted cells were dissolved in

0.3 ml of 1.0 N NaOlI by boiling for 30 minutes. This material was then assayed by the imthod of Lowry et al. The Blo-Pad protein











assay (Bio-Rad) was used to determine the amount of extracellular proteins in culture fluids. This method was used since short polypeptides, amino acids and other organic molecules do not interfere with this assay (7). Amino acid analysis using a JEOL Model JLC-6AH amino acid analyzer (JEOL, Inc., Cranford, NJ) was done on purified final products. Samples, 5-10 mg, were
0
hydrolyzed in 5 ml of 6.0 N HCl for 11 hours at 105 C in sealed ampoules. The acid was removed on a Buchler rotary evaporator and the sample was washed 3 times with 5 ml of water. The residue was brought to a final volume of 5.0 ml in 0.01 N HCl. The amino acid analysis of the liposome-purified LTA was done using 1.0 mg of LTA which was brought to a final volume of 1.5 ml, and only the neutral and acidic amino acids were analyzed.

Sugars were assayed by gas-liquid chromatography. Samples, 1-2 mg, were hydrolyzed in 2 ml of 1.0 N H 2SO4 for 8 hours at 1056C in sealed ampoules. Upon opening the ampoules exactly 1.0 or 0.2 mg of mannitol in 0.2 ml, depending on the amount of carbohydrate in the sample, was added as an internal standard. Neutralization with 0.5 g of BaCO3 was followed by centrifugation to remove BaSO4 and any excess BaCO 3. The supernate was lyophilized and then treated with 0.2 or 1.0 ml of Tri-Sil Z (Pierce Chemical Co., Rockford, IL) (41) to convert free sugars to their trimethylsilyl derivatives. The analysis was carried out on a Packard Model 803 gas-liquid chromatograph using a 0.4 x 153 cm glass column packed with 3% SE-30 Ultraphase on Chromosorb W(HP), 80-100 mesh, solid support (Pierce). The








12



analysis was carried out isothermally with a column temperature of 160 C and the flame ionization detector at 195 C. Nitrogen at a flow rate of 30 cc/min was used as the carrier gas. The retention times and peak areas from the samples were compared with those of

standards in order to identify and quantitate the carbohydrates present in the sample. An Autolab Minigrator electronic digital integrator (Spectra-physics, Santa Clara, CA) was employed for data collection and computation.

Glycerol was determined using the Glycerol Stat-Pack

(Calbiochem, Atlanta, GA). Samples were hydrolyzed in 2.0 N HCI for 4 hours at 1050C in sealed ampoules. The acid was removed by lyophilization, followed by 3 washings with water and lyophilizations after each washing. The sample then was treated with alkaline phosphatase (Worthington Biochemical, Freehold, NJ) in

(NHQ)2CO3 (0.05 M pH 9.3) in sealed ampoules for 24 hours at 370C to yield free glycerol. This material was used for the glycerol determination according to Calbiochem Information Leaflet 3056.

Gel filtration chromatography. Bio-Gel P-6 (Bio-Rad), a polyacrylamide gel, in a 2.8 x 50 cm column was used to desalt and to remove phenol from samples. This column has an exclusion limit of 10,000 daltons, a void volume of 100 ml and a total volume of 300 ml. The sample was eluted with water at a flow rate of 50 ml/h in 5 ml fractions using a Gilson Mini-Escargot fraction collector (Gilson Medical Electronics Inc., Middleton, WI).

A Bio-Gel A-5M, 200-400 mesh, 6% agarose gel (Bio-Rad) columil measuring 2.5 x 100 cm was used In purifying the







13



polysaccharide antigen found in the crude acid extract of whole cells of AHT and in culture fluid. It was also used to partially purify the LTA from the phenol extract of whole cells. This column had a void volume of 190 ml, as determined by Blue Dextran (Pharmacia Fine Chemicals, Piscataway, NJ), a total volume of 530 ml, and an exclusion limit of about 1 X 106 daltons. A pressure head of 30 cm was used to obtain a flow rate of 5-10 ml/h. The sample was loaded on the column pre-equilibrated with

0.25 M NaCl containing 0.05% NaN3 and was eluted at room temperature with the same solution.

A 2.5 X 100 cm Sepharose CL-4B (Pharmacia) gel filtration column was also used to purify the acid extract of whole cells. This gel with an exclusion limit of 5 X 106 daltons was run with exactly the same conditions and buffers as the Bio-Gel A-5M column.

Hydrophobic interaction chromatography. The use of Octyl-Sepharose CL-4B (Pharmacia) for hydrophobic interaction chromatography to purify LTA was suggested to me by Drs. A. J. Wicken and K. Knox (personal communication). The glycolipid end of the LTA molecule should interact with the hydrophobic n-octyl groups which are covalently bound to the Sepharose. This interaction is enhanced by a high salt concentration; therefore, the

column (2.25 X 25 cm) was equilibrated with 0.01 M tris(hydroxymethyl)aminomethane (Tris) -carbonate (Sigma Chemical Co., St. Louis, MO), pH 6.8, containing 1.0 M NaCl. The sample was

loaded in this buffer and the column was eluted with 150 ml (1.5 column volumes) of this high salt buffer. The column was then







14



eluted with 100 ml of buffer, 0.01 M Tris-carbonate pH 6.8, without the NaCI. Finally the column was eluted with a 250 ml gradient of 10-70% n-propanol (by volume) in 0.01 M Tris-carbonate pH 6.8. As the amount of n-propanol in the eluting buffer increases, the interaction between the buffer and the glycolipid end of the LTA molecule should increase and the LTA would be eluted from the column.

Sephadex LH-20 chromatography. Removal of the n-propanol from the LTA fraction after chromatography on octyl-Sepharose CL-4B was performed on the organic solvent stable Sephadex LH-20 (Pharmacia). The gel was equilibrated with water and poured into a 2.5 X 50 cm column. Samples, 15-20 ml, previously concentrated by flash evaporation were loaded on the column and eluted with water in order to separate salts as well as the n-propanol from the LTA.

Phosphatidylcholine (PC) liposome purification of LTA. In collaboration with Dr. L. Silvestri, a new method for the purification of LTA was devised which uses the same principles as hydrophobic interaction chromatography. LTA was mixed with PC liposomes, the lipid end of the LTA molecule intercalating into the outer lipid bilayer of the liposome. Contaminants then were removed by washing the LTA-liposome complexes. Pure LTA was recovered by dissolving the liposomes in a mixture of chloroform and methanol. The LTA, insoluble in this solvent, was collected on a membrane filter. This technique has been used to purify the








15



extracellular LTA from the culture fluid of S. mutans BHT (40) and is employed below for the purification of intracellular LTA

derived from AHT cells.

Artificial membrane vesicles, liposomes (43), were prepared from dipalmitoyl DL-3-phosphatidylcholine (PC) (Sigma) by a modification of the method of Hill (19). One milliliter of chloroform containing 40 mg PC was placed into 30 ml Corex centrifuge tubes. The chloroform was evaporated by rotating the tube in a 500C water bath so as to coat the bottom of the tube with PC. Any residue of solvent was removed under a vacuum at room temperature. One milliliter of the 0.01 M Tris-carbonate, pH 6.8, buffer (TC) was added to each tube and alternately heated in a 50 C water bath and vortexed (Vortex Genie Mixer, Scientific Industries Inc., Bohemia, NY) until a milky emulsion was formed. After the addition of 15 ml of the TC buffer, the liposome suspension was centrifuged at 27,000 X g for 30 minutes. Decanting the supernate allowed only the large liposomes to remain in the pellet which upon resuspension in

1.0 ml of the TC buffer were used to absorb LTA from the crude phenol-water extract.

Two milliliters of crude phenol-water extract, containing 2.5 mg/ml, were added to a 1 ml suspension of liposomes in TC buffer. After incubation at 37 C for 2 hours in a shaking water bath, 13 ml of the TC buffer were added and the suspension was centrifuged at 27,000 X g for 45 minutes. The supernate was discarded and the pellet redissolved in 1.0 ml of the TC buffer. Fifteen milliliters of the TC buffer were added to the 1.0 ml suspension, mixed, then







16



centrifuged. The pellet was washed 3 times in this way. The washed pellet finally was dissolved in 5 ml of chloroform-methanol (3:1, v/v) and allowed to remain at room temperature for 1 hour. This solution was then filtered through a 3.0 pm fluoropore membrane filter (Millipore) prewashed with 10 ml of the chloroform-methanol solvent. The centrifuge tube was washed twice with 2 ml of chloroform and these washings filtered. The filter was then rinsed with 5 ml of chloroform and allowed to air dry. The centrifuge tube and the filter funnel were rinsed with water. The dry filter was placed into the water used to rinse the centrifuge tube and filter funnel. The resulting solution was passed through a 5.0 pm membrane filter (Millipore) and then lyophilized. Eight tubes processed in this manner yielded 3-5 mg of LTA. This represents a final yield of approximately 6-12% by

dry weight.

Growth curves and production of extracellular macromolecules by S. mutans AHT. Growth curves were made of S. mutans AHT grown in THG and MC media. Two methods were used to determine growth with both methods giving the same results. First, 1 ml of exponentially growing cells, A660 of 0.5, was transferred to 100 ml of fresh broth perheated to 370C. Sambles, 1.5 ml, were removed every 30 minutes and the absorbance read immediately at 660 nm on a Gilford spectrophotometer. The second method used involved the addition of 20 i of 3.7% formalin (final concentration of 0.05%) to the 1.5 ml sample immediately after its removal








17



from the growing culture. The samples were allowed to stand at room temperature for at least 30 minutes before the absorbance was read. Samples were also removed, centrifuged, and then treated as described above to determine the amount of intracellular protein present.

To determine the amount of extracellular material

excreted by the organism, 200 ml of media were inoculated with

2 ml of exponentially growing cells. The absorbance was followed and when the culture reached a given absorbance, 2.7 ml of 3.7% formalin (final concentration of 0.05%) were added. The cells were removed by centrifugation and the supernate was filtered through a 0.45 p.m membrane filter. The cell-free culture fluid was dialyzed for 2 days against 4 changes of 10 1 of wzter at

4 0 C. Ten milliliters of this undiluted, dialyzed, cell-free culture fluid were frozen, while 2 other samples, 40 ml and 150 ml, were taken and concentrated to 4 ml (l0X) and 1.5 ml (lOOX) respectively by flash evaporation at 40 0 C. These concentrates were frozen and tested later for the presence of protein and polysaccharide antigen.

Production of rabbit antisera. One antiserum employed

(AVD) was made against S. mutans AlIT whole cells by Dr. Ivo Van de Rijn and was used in a previous study (44). Antiserum made against Lactobacillus casei LTA and specific for the glycerolphosphate backbone was provided by Dr. K. Knox, Institute of Dental Research, Sydney, Australia.








18



Antisera (25-29) were made against S. mutans AIT grown in THG and formalin killed (0.6% formalin). The cells were then washed 5 times with water. The washed, formalin-killed cells were used for injection or were broken with glass beads in a Braun tissue homogenizer (5). The broken cells were centrifuged at 30,000 g and the supernate and pellet were lyophilized separately. Three New Zealand white male rabbits (#25, 26, and 27) were injected intraveneously with 2.5 mg of pelleted material on alternate days for a week. At the same time rabbits #28 and #29 received 1 mg of supernate material mixed with Freund's complete adjuvant and injected subcutaneously in 5 different places in the area of the trapezius muscle. Both rabbits received 1 mg of supernate material on alternate days for a week. Five days after the last injections all 5 rabbits were test bled and their sera tested by capillary precipitin and Ouchterlony gel diffusion against a Lancefield extract (26) of AHT whole cells. All sera showed no reaction at this time. One week after the test bleed all the rabbits on alternate days received 1 ml of formalinkilled whole cells at 10, 20, 40, then 60 IU (10 IU= 9 X 108 cells/ml) intraveneously. Following the last injection the rabbits were test bled, but again their sera gave no reaction. The rabbits were rested for 1 week then injected intraveneously again with formalin killed whole cells using the same regimen. After the last injections all the rabbits were test bled and this time all sera showed a positive reaction with the Lancefield extract.







19



All 5 rabbits were then exsanguinated and their sera frozen separately.

Immunological techniques. Ouchterlony gel diffusion was

done in small, 50 X 12 mm, petri dishes (Falcon Plastics, Oxnard, CA). Three milliliters of 0.05 M sodium barbitol, pH 8.2, containing 0.02% NaN3, and 0.8% agarose were placed into each petri dish. Holes, 2 mm in diameter, were cut in the resulting gel just prior to use. After filling the wells the dishes were
0
incubated, usually 24-48 hours, at 4 C until precipitin bands were visible.

Immunoelectrophoresis was done on acid cleaned microscope slides, 38 X 76 X 1 mm. The acid cleaned slides were dipped into a 0.2% agarose solution which was allowed to harden before 3 ml of a 0.8% agarose solution in the samebuffer used for gel diffusion was added to the surface of each slide. Wells and troughs were cut into the gel just prior to use with a Shandon gel cutter (Colab, Chicago, IL). After the addition of the antigen solution to the wells, the slides were run at a
0
constant current of 5 ma/slide or 10 ma/slide for 1 hour at 4 C. After electrophoresis the gel in the troughs was removed and antiserum was added. The slides were incubated at 4 0C for 24 hours or until precipitin bands developed.

Radial immunodiffusion (RID) was used to quantitate the amount of polysaccharide antigen in concentrated culture fluids. This technique, although used mainly for the quantitation of immunoglobulins (11,15,31), worked well with this polysaccharide







20.



antigen. A timed diffusion was used rather than the limited diffusion because of the tendency of the polysaccharideantibody precipitin bands to become diffuse with time resulting in very broad bands. Tubes containing 1% agarose in 0.05 M sodium barbitol, pH 8.2, plus 0.02% NaN3 were melted in a boiling water bath then tempered in a 50C water bath. To each milliliter of this agarose solution, 0.02 ml of rabbit antiserum #26 was added. This agarose-antiserum mixture (7 ml/slide) was cast between two microscope slides (51 X 76 X 1 mm) separated by a thickness of 2 m. The bottom slide was acid cleaned and predipped in 0.2% agarose while the top slide was coated with silcone oil to facilitate its removal. After the agaroseantiserum mixture hardened, the top slide was removed and twelve 2 mm diameter holes were cut into the gel with a 14-gauge cannula. Exactly 5 i of various concentrations of the polysaccharide antigen, 25-1,000 pg/ml, or concentrated culture fluids were placed into the wells with a 10 pi Hamilton syringe. After incubation at room temperature for 18 hours the outside diameter of all the precipitin rings was measured to the nearest 0.1 mm. A standard curve was made by plotting the logarithm of the antigen concentration versus the diameter of the precipitin band. The diameters used in the standard curve were the average of triplicate samples.

A modification of the Laurell rocket electrophoresis technique or electroimmunoassay (27,48) was used to try to







21



quantitate the amount of LTA in concentrated culture fluids. Instead of adding antiserum to the agarose gel, cetyltrimethylammonium bromide (cetavlon) was added. Cetavlon is a positively charged amphipathic molecule which forms precipitates with many large, negatively charged molecules, such as: DNA, RNA, proteins, and also LTA (Dr. A. J. Wicken, personal communication). Although cetavlon does not possess the specifiity of antibodies, it can be helpful under some circumstances. Cetavlon can also be used in place of antiserum in gel diffusion and other immunoelectrophoretic techniques. For this modified electroimmunoassay, slides were cast in the same way as for the RID using 1% agarose, 0.05% NaN3, and 0.004% cetavlon in

0.025 M sodium barbitol, pH 7.0. Ten to eleven holes were punched along the bottom of the narrow side of the slide. Exactly 2 p1 of a solution containing 20-500 pg LTA/ml were placed into the wells. Other wells received 2 p1 of concentrated culture fluid from synthetic medium MC taken at various points during growth. The solutions were run toward the positive electrode at 2.5 ma/slide for 2-3 hours.

Hemagglutination techniques. LTA can be bound passively to sheep erythrocytes (SRBC) by the following technique

(18). Five milliliters of 0.85% saline was added to 0.5 ml of SRBC in Alsever's solution. The SRBC were washed 4-5 times with

0.85% saline and after the last centrifugation the SRBC were brought up to a suspension of 4 X 108 cells/ml. To 3 ml of this







22



suspension, 3 ml of LTA in 0.85% saline (20-25 pg LTA/ml) were added dropwise with mixing. The mixture was incubated of 30 min at 370C then cilled in an ice bath for 15 min. The sensitized SRBC were washed 5 times with 0.85% saline and the volume adjusted to give a suspension of 2 X 108 cells/ml. This cell suspension was used in hemagglutination studies in U-shaped

Cooke microtiter plates using the 25 pi system (Cooke Engineering Co., Alexandria, VA). Antiserum was diluted using

0.85% saline containing 0.2% bovine serum albumin; then the sensitized SRBC were added to each well. The titers were read
0
after incubation for 2 hours at 37 C and again after 12 hours at room temperature.

Although the polysaccharide antigen will not

passively bind to SRBC, it can be attached using a modification of Poston's CrCl technique (36). One milliliter of 0.27 M
3

piperazine buffer, pH 6.5, containing 100lig/ml of polysaccharide
9
antigen was added to 2.4 X 10 washed and pelleted SRBC. To this cell suspension 1.0 ml of a 0.025% CrCl3 solution was added slowly with mixing. After exactly 5 min at room temperature, with frequent mixing, 5 ml of 0.85% saline were added and the cells centrifuged. After washing the sensitized cells 4 times

with 0.85% saline the cell concentration was adjusted to
8
2 X 10 cells/ml. The hemagglutination assays were done in the same manner as with the LTA sensitized cells.

Hemagglutination inhibition was done in the same

25 pI microtiter system. After diluting the antiserum, either








23



0.85% saline or a possible inhibitor in 0.85% saline was added to each well. The plates were incubated for 1 hour at 37 0C. After incubation sensitized SRBC were added to each well and the plates were incubated and read as in the other hemagglutination assays.

Mixing of the polysaccharide antigen with S. mutans ANT LTA. In order to try to find conditions under which the

purified polysaccharide antigen could be reassociated with purified LTA, a number of different experiments were tried. Doyle et al. have reported the formation, in various organic solvents, of soluble macromolecular complexes composed of polysaccharides and TA or LTA (12). To see if stable complexes would form under their conditions, 5 ml of cold 100% ethanol were added to 1 ml of an aqueous solution containing 50 jig of LTA and 500 ipg of the polysaccharide antigen. The mixture was held for 24 hours at 4 0C. After centrifuging at 15,000 X g for 5 min the supernate was removed, flash evaporated to dryness and then brought up in

2 ml of 0.85% saline to be added to SEEC for hemagglutination assays.

Since the complexes were observed when LTA was

extracted from whole cells with phenol, 1 ml containing 50 p.g LTA and 500 iig polysaccharide antigen was added to 1 ml of 90% liquid phenol. The mixture was heated to 680C for 30 min, cooled in an ice bath, and then centrifuged for 10 min at 10,000 X g. The aqueous phase was removed and dialyzed against 0.85% saline to remove the phenol. The volume of this material was adjusted to 2 .0 ml for hemagglutination (HA).







24



Incubation of 1 ml of an aqueous solution containing 50 pg LTA and 500 pg polysaccharide antigen for 12 hours at 370C was also tried. After the incubation 1 ml of 1.7% saline was added and the solution was used for HA. Lyophilized and flash evaporated aqueous mixtures also were tested.

Immunoelectron microscopy. The procedure used was

similiar to that described by Lai et al. (25). Cells from a 24 hour culture of S. mutans AHT grown in THG were washed 3 times and then resuspended in 0405 M PBS, pH 7.2. The suspension was adjusted to an absorbance of 0.5 at 660 nm, which is approximately 1 mg of cells (wet weight)/ml. Two milliliters of this suspension were placed into 10 ml centrifuge tubes and pelleted. Four tubes of cells were incubated with 0.1 ml of 4 different dilutions of antiserum #26 (1. 1/5. 1/10, and 1/20) at room temperature for 30 min. The cells were then washed 3 times with 1 ml of PBS and incubated for 30 min at room temperature with 0.1 ml of horseradish peroxidase conjugated to goat anti-rabbit immunoglobulins (Cappel Labs., Inc., Downingtown, PA). Again the cells were washed 3 times with 1 ml PBS and then fixed in 0.2 ml of 5% glutaraldehyde in 0.075 M phosphate buffer, pH 7.2, for 1 hour at 40C. The fixed cells were pelleted, resuspended, and held overnight in 0.05 M phosphate buffer containing 4.5% maltose. The peroxidase label was developed by incubating the cells for I hour at 40C in 1 ml of 3,3'-diaminobenzidine (DAB), 80 mg DAB/100 ml 0.05 M Tris, p11

7.6, followed by incubation in 1 ml of the same DAB solution








25



but containing 0.005% H202 for 30 min at room temperature. After washing the cells with water they were given to Dr. Sylvia E. Coleman who fixed the cells with 2% OsO4 and processed them for electron microscopy.

In control A the cells were incubated with normal (nonimmune) rabbit serum then treated as above. Other controls were not treated with rabbit antiserum and were done as follows:

Control B--The cells were fixed with glutaraldehyde,

treated with the DAB solution, then treated with the

DAB and H202 solution.

Control C--The cells were fixed with glutaraldehyde,

then treated with the DAB solution.

Control D--The cells were fixed with glutaraldehyde,

then treated with the DAB and H202 solution.

Control E--The cells were treated with uncoupled horseradish peroxidase (10 mg/ml), fixed with glutaraldehyde, treated with the DAB solution, and then

treated with the DAB and H202 solution.

Control F--The cells were treated with uncoupled

horseradish peroxidase then fixed with glutaraldehyde.

Control G--The cells were treated in the same manner as

in Control E except the initial treatment was with horseradish peroxidase coupled to goat anti-rabbit

immunoglobulins.







26



Control H--The cells were treated in the same manner as

in Control F except that the initial treatment was with horseradish peroxidase coupled to goat antirabbit immunoglobulins.

The cells from Controls B-H were washed with water after their respective treatments and given to Dr. Coleman for processing.

Chemicals. Most common salts, organic solvents and reagents, and acids were obtained from Mallinckrodt (Scientific Products, Chamblee, GA). Amino acids, amino sugars, and sugars were purchased from Sigma.














RESULTS


Extraction and purification of a polysaccharide surface antigen from S. mutans AHT. Because of the variety of methods used to extract surface antigens, a number of different extraction methods were tried in preliminary studies. Whole cells of S. mutans AHT were extracted using the autoclave method of Rantz and Randall (38). This method was used extensively by Bratthall and Pettersson for the serological comparison of the antigens of the several different S. mutans serotypes (9). A polysaccharide similar in composition to that found by Mukasa and Slade (33) was obtained in very low yield using this method. The harsher Lancefield extraction (26) next was tried in order to improve the yield. This method improved the yield but apparently degraded the polysaccharide resulting in a polymer of a much lower molecular weight. A mild acid extraction at pH 4.0 (Materials and Methods) finally was found to provide the best yields with minimal degradation of the polysaccharide antigen. This technique was adopted for the present studies and is referred to as "acid extraction."

Gel filtration on BioGel A-5M, a 6% agarose, of the acid extracted material from 5 g of whole cells showed a broad polysaccharide peak (Fig. 1) which reacted strongly with antisera made against S. mutans AHT whole cells (Fig. 2). The entire antigenic peak from Kave 0.1-0.6 was pooled, desalted on a BioCel P-6 column, and lyophilized. A yield of 4 mg of


27



















Fig. 1. Purification of a polysaccharide antigen from the acid extract of 5 g
of S. mutans AHT whole cells by Bio-Gel A-5M gel filtration chromatography. Curve designations: unbroken lines carbohydrate (CHO)
as determined by the method of Dubois et al. (13) and read at 485 nm;
dashed lines phosphorus (Pi) as determined by the method of Lowry et al. (30) and read at 820 nm; dotted lines absorbancy at 260 nm
(A260); and the hatched box indicates positive antigenic reactions
by immunodiffusion analysis employing anti-AHT serum. The ordinate
applies to direct readings at each wave length. The abscissa was
calculated from the formula: KAVE = (Ve Vo)/(Vt Vo). Ve is the elution volume of the peak, Vo is the void volume of the column, and
Vt is the total volume of the column.









BIO-GEL A-5M: ACID EXTRACT OF AHT WHOLE CELLS


1.0 ..

CHO A260
0.9 .


0.8..

N9
0.7 g


0.6
S S

0. .5
0 A

0.4 I


0.3- /
r/ 1


0.2- / '


0.1 *


0.0 ** *. *. ... *

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
K
AVE

































Fig. 2. Immunodiffusion patterns of the extracellular
(CHOx) and intracellular (CHOi) or extracted
polysaccharide antigens from S. mutans AHT,
the S. mutans AHT LTA (AHT LTA), and the BioGel A-5M chromatographed phenol-water extract
(@OH) reacting with antiserum #27.






31











































CHO x
0

CHO, 0 0 AHT LTA

0

(POH 0 0 (POH








32



polysaccharide antigen/g of lyophilized whole cells was obtained by this method.

Gel filtration on Sepharose CL-4B, a crosslinked 4% agarose, of the same acid extracted material showed a similar broad peak indicating extreme molecular-size heterogeneity for this polysaccharide (Fig. 3). The antigenic polysaccharide peak was pooled from Kave 0.2-0.55, desalted on a Bio-Gel P-6 column,

and lyophilized.

Analysis of the polysaccharide surface antigen. Chemical

analysis of the pooled peaks from the Bio-Gel A-5M and the Sepharose CL-4B columns showed a polysaccharide which was 96%

(by weight) glucose and galactose. Amino acids and glucosamine accounted for less than 4% of the peak from the Bio-Gel A-5M column and no galactosamine was found. Table 1 shows that the molar ratio of galactose:glucose was 3.6:1. Methylation analysis of this material by Dr. Thomas A. Brown yielded the same products, qualitatively and quantitatively, as from a lower molecular weight polysaccharide obtained by hot formamide extraction of purified cell walls of S. mutans AHT (personal communication and T. A. Brown, Ph. D. dissertation, University of Florida, Gainesville, 1978).

Immunoelectrophoresis of this polysaccharide (Fig.4) reveals an antigenic band that migrates slowly to the cathode with the same mobility as the major antigenic band described by Bratthall as the serotype a antigen (8,9).























Fig. 3. Purification of a polysaccharide antigen from the acid extract of 5 g
of S. mutans AHT whole cells by Sepharose CL-4B gel filtration chromatography. Curve designations are the same as in Fig. 1.








SEPHAROSE CL-4B: ACID EXTRACT OF AHT WHOLE CELLS


2.0-

.*
*

CHO "

1.5-- A260

Ag g
*
*S



1.0-- "
-S





0.5.
1 *





-: ".k\.
P* *
.






0.0

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
KAVE








TABLE 1

CHEMICAL COMPOSITION OF VARIOUS PREPARATIONS OF THE POLYSACCHARIDE ANTIGEN OF S. MUTANS AHT


Acid Extract Acid Extract Culture Fluid
Bio-Gel A-5M Sepharose CL-4B Bio-Gel A-5M
Chromatographed Chromatographed Chromatographed
Compound
moles (molar umoles (molar moles (molar
mg ratio) mg ratio) mg ratio)
Glucose 0.954 (1) 0.966 (1) 0.516 (1)

Galactose 3.424 (3.59) 3.430 (3.55) 2.397 (4.64)

Ribose <0.07 (<0.07) 0.070 (0.07) <0.05 (<0.10)

Phosphorus 0.151 (0.16) 0.151 (0.16) 0.117 (0.23)

Glucosamine 0.032 (0.03) NDa 0.649 (1.26)

Glutamic Acid 0.041 (0.04) ND 0.027 (0.05)

Aspartic Acid 0.038 (0.04) ND 0.008 (0.02)

Alanine 0.034 (0.04) ND 0.045 (0.09)

Threonine <0.01 (<0.01) ND 0.134 (0.26)

Proline <0.01 (<0.01) ND 0.104 (0.20)

Serine <0.01 (<0.01) ND 0.076 (0.15)

Other Amino Acids <0.01 (<0.01) ND <0.01 <(0.02)
a
Not Determined

































Fig. 4. Immunoelectrophoresis pattern of the polysaccharide antigen extracted from S. mutans
AHT (CHOi) and a Lancefield extract of S.
mutans AHT (LE) reacting with antiserum #27.
Electrophoresis was at 10 ma/slide at 4C
for 1 h.






37










































0 CHO 0 LE



0 CHO Antiserum #27








38



Isolation and purification of the polysaccharide

antigen from culture fluid. S. mutans AHT was grown to late stationary phase in prefiltered THG. Most of the cells were removed from the culture fluid by a DeLaval gyro-tester and the remainder were removed by filtration through a 0.2 urm filter on the Pellicon cassette system (see Materials and Methods). The cell-free culture fluid was concentrated by a PTGC filter (nominal molecular weight limit: 10,000 daltons) on the Pellicon cassette system and then lyophilized. Of the 30g of crude material obtained from 10 1 of broth 20 g were loaded on the Bio-Gel A-5M

column (Fig. 5). A broad "peak" containing polysaccharide reactive with anti-AHT immunoglobulins was eluted at the K ave
observed for the antigen extracted from whole cells (Fig. 1). The antigenic material from K 0.1-0.5 was pooled, desalted on
ave
a Bio-Gel P-6 column, and lyophilized.

Analysis of the polysaccharide antigen from the culture fluid. The chemical analysis of the extracellular antigen (Table 1) shows a similarity between this material and the polysaccharide antigen extracted from whole cells. The extracellular antigen was 75% glucose and galactose, 18% glucosamine and 7% protein. It had a slightly higher galactose:glucose molar ratio (4.6:1) as shown in Table 1. The extracted and extracellular polysaccharides were shown to be antigenically identical by Ouchterlony gel diffusion (Fig. 2).






















Fig. 5. Bio-Gel A-5M gel filtration chromatography of 20 g (original volume
6.6 1) of lyophilized S. mutans AHT culture fluid. Curve designations are the same as in Fig. 1.









BIO-GEL A-5fl: AHT CULTURE FLUID

2.0





A1:


1. o 26






I..

1.5 0. 0. . . . .
K
_ _ _ _ _ _ _ _/AV EI








41




Quantitation of excretion of the polysaccharide antigen into the culture fluid. Growth curves of S. mutans AlIT were obtained with two different media, THG and MC. The growth curve obtained with medium MC is shown in Fig. 6. This curve is very similar to the growth curve obtained with TuIG (not shown). The doubling time for AHT in the MG medium was 56+2 min (4 runs) and in the THG medium the doubling time was 63+5 min (7 runs). Excretion of macromolecules into the medium was analyzed at specific points along these curves. A more complete profile was obtained for the MC medium (Table 2) and only these data are presented. S. mutans AlIT was grown in the MC medium to various given absorbancies at 660 nm; the cells were then removed by centrifugation and filtration to provide cell-free culture fluid. This fluid was dialyzed and frozen directly; or concentrated by flash evaporation and then frozen.

The concentration of the polysaccharide antigen in the

cell-free culture fluids was determined by two ways. One method, the inhibition of hemagglutination (lIHA), can detect very low concentrations of antigen but is not very accurate. A standard curve was made by sensitizing SRBC in the presence of CrCl with the polysaccharide antigen extracted from whole cells and using this same material at various known concentrations as an inhibitor of agglutination. When culture fluid was used as an inhibitor, the reduction in titer was directly related to the concentration of antigen in the culture fluid. Estimates of





























Fig. 6. Growth of S. mutants AHT and excretion of the
polysaccharide antigen in synthetic medium MC
(42). Curve designations: open circles are the points where samples were removed from a
growing culture and the absorbance read at
660 nm; the X indicates the absorbance to
which a 250 ml culture was grown for study of
the culture fluid (Table 2); and the ooen
squares indicate the concentration of polysaccharide antigen, measured by RID (Table 2),
at points (X) on the growth curve.






43


GROWTH OF S. MUTANS AHT AND EXCRETION OF THE POLYSACCHARIDE ANTIGEN IN SYNTHETIC MEDIUM MC



i. 00------/I0
1.00 10
A660










CHO
U x
O Xo

>I
0
p.4



0.01 1.0





0.05 0.5 1/K =56 + 2 min













0.1
0.01-1 I 1 1 1 $lb I Imm~mm
0 1 2 3 4 5 6 7 8 9 10 18 24

Time (h)









TABLE 2

EXCRETION OF THE POLYSACCHARIDE ANTIGEN AND PROTEINS INTO SYNTHETIC MEDIUM MC Polysaccharide
Antigen Protein d
Turbidity Time from RIDa IHA Intracellularb Extracellular c
A660 Transfer Pg/mi pg/mi g/ml p g/ml
0.052 2.0 h 0.25e 0.25 3.2 0.1 3

0.156 3.5 h 0.6 0.5-1 10.2 0.3 3

0.500 5.3 h 1.87 1-2.5 32.5 1.0 3

0.930 8.6 h 3.35 2.5 60.1 2.3 4

1.224 10.0 h 4.88 5.0 79.1 3.7 5

1.500 18.0 h 8.29 5-10 96.9 2.6 3

1.548 24.0 16.0 10-25 100.4 6.9 7

a RID on concentrated culture fluids (50-100x) b Total intracellular protein by the Lowry Protein Assay c Total protein in concentrated culture fluids (50-100x) by the Bio-Rad Protein Assay d Extracellular protein as percent of intracellular protein

e Approximated from nonlinear portion of standard curve








45




extracellular antigen concentration made by IHA are presented in Table 2. Another way of determining the antigen concentration in the culture fluid is by radial immunodiffusion (RID). RID is not nearly as sensitive but is much more accurate then IHA. The polysaccharide antigen extracted from whole cells was used to construct the RID standard curve (Fig. 7). Concentrated culture fluids were run at the same time as the standard curve so as to minimize the effects of temperature. Estimates of excreted antigen made by the RID assay are presented in Table 2. The results of both of these methods agree very well indicating that there was probably very little loss of antigen during the concentration of the culture fluids for the RID assay.

To complete the analysis, the amounts of intracellular and extracellular proteins were compared for each growth point. Throughout the growth curve, except for the 24 h point, the ratio of intracellular to extracellular proteins remained the same. The small and relatively constant percentage of extracellular protein compared to intracellular protein (Table 2) indicates little apparent autolysis during the course of this growth study.

Figure 6 shows that the polysaccharide antigen is released into the media at the same exponential rate as the increase of the cell mass during exponential growth of the culture. This relation between cell mass and excretion of the polysaccharide antigen does not hold during late stationary phase where the

































Fig. 7. Radial imiunodiffusion (RID) standard curve.
Each point was run in triplicate using 0.2 ml
of antiserum #26/mi of gel and 5.0 jil of
sample per well. The diameters of all the
precipitin rings for both the standards and
samples were measured after 18 h (timed diffusion) at room temperature.






47




RADIAL IMMUNODIFFUSION STANDARD CURVE





1000






















<100
m

4



W G

0
c












10
7
D.Lameter (mm)







48



amount of extracellular polysaccharide antigen increases rapidly. This increase may be due to some autolysis since the amount of extracellular protein also increaces at this time.

Although the concentration of extracellular protein

excreted into the medium could not be measured for cells growing in THG because of its complex nature, the concentration of polysaccharide antigen in culture fluid from cells grown in THC

was measured by RID for 3 points on the growth curve: A 0.152 0.4 Vg/ml, A 0.902 1.87 pg/ml, and A 1.680 (24 h) 6.16 pg/ml. These concentrations were about 1/2 to 1/3 the concentrations found in the MC medium for the same growth points.

Extraction and gel filtration of LTA from S. mutans AHT.

Van de Rijn and Bleiweis used trichloroacetic acid to extract LTA

from high-speed (78,000 X g) supernates of disrupted cells of AHT

(44). This method not only cleaves the glycolipid portion from the LTA, it may also cleave the glycerol-phosphate bonds of the LTA backbone and degrade some polysaccharides. A much milder extraction technique which removes the intact LTA from whole cell with minimal degradation and protein contamination using hot aqueous phenol was used (49,51).

When the phenol extract of 10 g of cells was applied to a Bio-Gel A-5M column, two high molecular weight peaks were observed (Fig. 8). A sharp phosphorus peak, containing LTA is situated within a broad polysaccharide peak, containing the polysaccharide antigen described previously. Only the tubes under the first phosphorus peak from K 0.2-0.4 were pooled, desalted
ave























Fig. 8. Bio-Gel A-5M gel filtration chromatography of the phenol-water extract
of 10 g of S. mutans AHT whole cells. Curve designations are the
same as in Fig. 1.






BIO-GEL A-5M: PHENOL EXTRACT Or AHT WHOLE CELLS



2.5

CHO



2 .0 .
I \A260





PI
1.0
0.5 I I I *'"









1.000
I \I l
SI I





0.5 I 1 1l



0.0-- ** 7 i/0/I|*! *j% \

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.3 0.9 1.0
K AVE








51



on a Bio-Gel P-6 column, and then lyophilized. This material is a mixture of LTA (30% by weight) and polysaccharide antigen (70%). As will be discussed later this does not seem to be a simple case of co-elution due to molecular weight similarities, but instead a definite interaction between the two molecules will be shown below. The second phosphorus peak contained teichoic acid (TA), deacylated LTA, non-antigenic polysaccharide, and polynucleotides and was discarded.

The amount of polysaccharide antigen contamination of the LTA could be greatly reduced by first extracting the whole cells by the acid extraction technique described above prior to the phenol extraxtion. Figure 9 shows the profile of material extracted in this way and then chromatographed on the Bio-Gel A-5M column. This method reduced the polysaccharide antigen contamination to 25% by weight; however, the yield of LTA was reduced by half due to degradation during the initial acid extraction.

Hydrophobic interaction chromatography. To attemp the separation of the LTA and polysaccharide antigen, hydrophobic interaction chromatography on octyl Sepharose-4B was tried. Drs. Wicken and Knox have used this technique successfully to separate protein and polysaccharide contaminants from preparations of extracellular LTA from S. mutans BHT (personal communication). However work done in collaboration with Dr. L. Silvestri with similar LTA preparations of BHT showed that most of the






















Fig. 9. Bio-Gel A-5M gel filtration chromatography of the phenol-water extract
of 10 g of S. mutans AHT whole cells previously extracted with acid.
Curve designations are the same as in Fig. 1.







A-5M: PHENOL EXTRACT OF ACID EXTRACTED AHT WHOLE CELLS


2.5 Ii
II

1P.

2.0 I

L~gAg


1.5 -/

1l i

1.0o ~1
I I \



0.5I- I






A260 / ../CHO L
0.0 I I I I* *
5 I I I
1. -I I I I
I I I





0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0



KAVE
I l
o -I I ,\I \
If
A260 ,"'.....CH


0.0 0.1 0.2 0.3 0.4 0.3 0.6 0.7 0.8 0.9 1.0
KAVE r








54



protein contamination could be removed by this method but that very little of the contaminating polysaccharide was removed (40).

The first phosphorus peak from the Bio-Gel A-5M column fractionation of the phenol extract of AHT whole cells (Fig. 8) was applied to an octyl Sepharose-4B column in the high salt buffer. Elution with high salt buffer (I M) and then the low salt buffer (0.01 M) removed only a small amount of the polysaccharide antigen from the column. Upon elution with 50% n-propanol in the

0.01 M Tris-carbonate buffer, pH 7.0, both the LTA and polysaccharide antigen were eluted simultaneously. This method was not used further.

Phosphatidylcholine (PC) liposome purification of LTA.

After the failure of gel filtration and of hydrophobic interaction chromatography to separate LTA from the polysaccharide antigen, I decided to use the ability of LTA to bind to cell membranes or to artificial membranes as a purification approach. In collaboration with Dr. L. Silvestri, a method for the purification of LTA

using PC liposomes was developed.

Preliminary results showed that the extracellular LTA from S. mutans BHT would bind readily to PC liposomes. Only 2 h at 370C or overnight at room temperature was needed for maximal uptake of LTA. The LTA uptake was 100% up to a maximum of 15 pg LTA/mg PC. Contaminants were removed by repeated washings of the LTA-liposome complexes. The LTA was recovered by adding a 3:1 mixture of chloroform-methanol to the final pellet. The PC which







55




is very soluble in this mixture was dissolved while the LTA precipitated. At first the LTA recovery was low since centrifugation would not pellet the flaky precipitate because of the high density of the solvent system. The best recovery method was to collect the precipitate on a solvent resistant membrane filter, such as Fluoropore or Mitex, and then to dissolve the LTA trapped on the filter in water. By using 14C-PC in the purification and following the radioactivity at each step of the purification, it was determined that less than 0.005% of the total counts or less than 3 pg PC/mg LTA remained in the final product (40).

Since this technique worked so well in purifying the

extracellular LTA from S. mutans BHT, it was used to separate the LTA and the polysaccharide antigen found in the phenol extract of S. mutans AHT whole cells. The crude phenol extract (80 mg), obtained from 10 g of whole cells, was mixed with 640 mg of PC liposomes in 16 separate tubes and treated as described in the Materials and Methods. This procedure yielded 5 mg of AHT LTA which is 6% of the crude extract employed and about 60% the recovery calculated from the maximum binding capacity of the PC liposomes for the extracellular BHT LTA.

Chemical and immunochemical characterization of the various LTA preparations. Table 3 compares the chemical compositions of the 3 LTA preparations. The crude phenol extract contained LTA (as indicated by the glycerol and phosphorus contents); nonantigenic, cell-wall polysaccharide (as indicated by'the rhamnose);









TABLE 3

CHEMICAL COMPOSITION OF VARIOUS PREPARATIONS OF LTA FROM S. MUTANS AHT



Crude Phenol Bio-Gel A-5M Liposome
Compound Extract Chromatographed Purification
Compound
moles (molar moles (molar moles (molar
mg ratio) mg ratio) mg ratio)

Glycerol 0.475 (1) 1.51 (1) 2.47 (1)
Phosphorus 1.35 (2.84) 1.60 (1.06) 2.48 (1.00)
Glucose 0.522 (1.10) 1.01 (0.67) 0.344 (0.14)
Galactose 0.988 (2.08) 2.43 (1.61) <0.02 (<0.01)
Rhamnose 0.099 (0.21) <0.02 (<0.01) <0.01 (<0.01)
Ribose 0.180 (0.38) <0.01 (<0.01) <0.01 (<0.01)
Alanine 0.466 (0.98) 0.79 (0.52) 1.22 (0.49)
Glutamic Acid 0.010 (0.02) <0.05 (<0.03) 0.016 (<0.01)
Glycine 0.020 (0.04) <0.05 (<0.03) 0.023 (<0.01)
Aspartic Acid <0.01 (<0.02) <0.05 (<0.03) 0.013 (<0.01)
Serine <0.01 (<0.02) <0.05 (<0.03) 0.014 (<0.01)
Other Amino Acids <0.01 (<0.02) <0.05 (<0.03) <0.01 (<0.01)







57



antigenic polysaccharide (indicated by the large amounts of glucose and galactose) and ribonucleic acid (RNA) (indicated by the significant amount of ribose). Fortunately, little protein appeared to be associated with this extract as shown by the amino acid analysis (alanine is likely all part of the LTA). After Bio-Gel A-5M chromatography, the non-antigenic cell-wall polysaccharide and the RNA were removed. A glycerol:phosphorus molar ratio of unity is characteristic of LTA, while the glucose and galactose content is due to the polysaccharide antigen. After purification of the crude phenol extract on PC liposomes, the LTA obtained was free also of the polysaccharide antigen contamination. This simple method removed RNA and both polysaccharides without the need for column chromatography. All that remained as product was the AHT LTA. This LTA has a typical glycerol-phosphate backbone (molar ratios; 1:1), a glucose-containing glycolipid, and the backbond glycerol-phosphate moieties are 50% substituted with alanine and are unsubstituted with sugars or amino sugars. Assuming a chain length of 24 glycerol-phosphate groups (24,44), a glucose disaccharide in the glycolipid, and no sugar substitutes on the backbone, the molar ratios of glycerol:phosphate:glucose should be 1:1:0.08 respectively which is very close to the values obtained. Since there are 3.4 molecules of glucose per LTA molecule, the glycolipid could contain a glucose trisaccharide or the extra glucose could be a backbone substitutent.

By using antiserum #27, which contains antibodies against both the LTA and the polysaccharide antigen, two separate bands







58



form in gel diffusion (Fig. 2). The precipitin bands formed from the intracellular (i.e., acid-extracted) and extracellular polysaccharides and the polysaccharide antigen found in the Bio-Gel A-5M chromatographed phenol extract (first phosphorus peak) are all identical. The LTA bands show identity between the liposome purified material and the Bio-Gel A-5M partiallypurified material. The results of the gel diffusion and the chemical data agree that the Bio-Gel A-5M chromatographed phenol extract contains both the LTA and the polysaccharide antigen. Immunoelectrophoresis of the S. mutans AHT LTA (liposome-purified), the Bio-Gel A-5M chromatographed phenol extract, and the L.casei LTA using antiserum #27 showed all LTA polymers migrating toward the anode and the polysaccharide antigen which slowly migrates toward the cathode (Fig. 10). Both the gel diffusion (Fig. 2) and the immunoelectrophoresis (Fig. 10) of the liposome-purified AHT LTA show a very faint polysaccharide antigen band, even though the chemical analyses indicate the absence of galactose, with only a small amount of glucose present in the final product. This could be due to the use of very high titer antisera which is reacting with minute amounts of polysaccharide antigen still present in the liposome-purified AHT LTA. Also, it is possible that small amounts of glucan (e.g. mutan) remain closely associated with the LTA.

Quantitation of extracellular LTA. The addition of neutralized, cell-free culture fluid from S. mutans MIAHT grown to late stationary phase (24 h) to SRBC sensitizes them so that
































Fig. 10. Immunoelectrophoresis pattern of the L.
casei LTA (Lc LTA),the AHT LTA (AHT LTA),
and the Bio-Gel A-5M chromatographed
ohenol-water extract of AHT (MOH) reacting with antiserum #27. Electrophoresis
was at 5 ma/slide at 4*C for 1 h.






60












































0 4)OH



0 Lc LTA



0 AHT LTA Antiserum #27







61



they agglutinate when added to antiserum made against LTA. This indicates the presence of at least 1-2 pg LTA/ml culture fluid

(32). However, attempts to better quantitate the amount of LTA

in concentrated culture fluids by rocket immunoelectrophoresis using cetavlon or to isolate the LTA by gel chromatography (Fig. 5) showed an apparent absence of LTA. The LTA could have been rendered non-active during the concentration of the culture fluid due to deacylation or have been degraded in other ways. In any event, studies of extracellular LTA production were not pursued for these technical reasons and due to extensive reports from other laboratories (23,32).

The interaction of LTA and the polysaccharide antigen of S. mutans AHT. The basis for the assumed interaction of LTA and the polysaccharide antigen in the phenol-water extract comes

from the passive hemagglutination experiments presented below (Table 4). What is important here is: a) the specificity of the antisera used, b) the ability of LTA to passively bind to SRBC, and c) the inability of the polysaccharide antigen to bind to SRBC except in the presence of CrC13.

Three different types of antisera were used. Antiserum made against L. casei LTA (provided by Drs. Wicken and Knox) which reacts with the LTA glycerol-phosphate backbone. Six antisera (AVD, #23-26, 28, and 29), all made against S. mutans AHT whole cells, react mainly with the polysaccharide antigen while they react only very weakly with LTA. One antiserum, #27, also made







62







TABLE 4

HEMAGGLUTINATION TITERS OF LTA AND POLYSACCHARIDE ANTIGEN PREPARATIONS



SRBC Antisera
Sensitized Anti-LTA Anti-AHT Anti-AHT
with L. caseia (6 sera) #27

Uncoated <10 <10 <10

L. casei
LTA (20 pg/ml)a 1280 20 640

AHT LTA (20 pg/ml) 320 20 640

AHT Polysaccharide Antigen (100 pg/ml) <10 20 20

AHT Polysaccharide Antigen (100 pg/ml) plus CrCl3 <10 2560 2560

AHT Phenol Extract (25 pg/ml) 160 80 320

AHT Phenol Extract Lyophilized
(25 pg/ml) 160 1280 1280

AHT Phenol Extract after Bio-Gel A-5M Chromatography
(25 pg/ml) 320 2560 2560

a Both purified L. case LTA and specific antiserum were kindly provided by Drs. Wicken and Knox.








63




against S. mutans AHT whole cells, reacts with both LTA and the polysaccharide antigen.

Both the LTA from L. casei and the LTA extracted from S.

mutans AHT bind readily to SRBC upon mixing and incubation at 37C for 30 min (Table 4). The polysaccharide antigen when mixed with SRBC under the same conditions did not bind to them, however in the presence of CrCI3 at room temperature for 5 min the polysaccharide antigen sensitized the SRBC.

When 25 pg of the crude phenol-water-extracted material were added to SRBC both the LTA and a small amount of the polysaccharide antigen were present on the SRBC, as detected by hemagglutination with the appropriate antiserum. If this crude phenol-water extract was a) lyophilized or b) partially purified on the Bio-Gel A-5M column, the resulting titers with the various antisera indicate that both the LTA and the polysaccharide antigen are able to bind to the SRBC membrane without CrCI When the purified polysaccharide antigen (10 pg/ml) was used as an inhibitor in an IHA assay with SRBC sensitized with the Bio-Gel A-5M chromatographed phenol-water extract, the titers using anti-L. casei LTA remained unchanged (320); however the titers obtained using anti-AHT #26 and anti-AHT #27 antisera dropped from 2560 to 20 and from 2560 to 640 respectively.

These data tend to indicate that LTA mediates the binding of the polysaccharide antigen to membranes. It was speculated that complexes of LTA and polysaccharide may be formed upon extraction, perhaps in the form of mixed micelles.








64



Mixing of purified LTA and the polysaccharide antigen. The purified LTA and polysaccharide antigen from S. mutans AHT were mixed under various conditions (see Materials and Methods) to determine if LTA-polysaccharide complexes could be formed spontaneously in vitro. The formation of such complexes would explain the enhanced sensitization of SRBC by the polysaccharide antigen in the presence of LTA. However, the presence of both purified LTA and polysaccharide antigen yielded sensitization of the SRBC by the LTA only. For all of the several conditions tried (see Materials and Methods), when the mixtures were added to SRBC only the LTA was detected on the surface of the SRBC by passive hemagglutination.

Immunoelectron microscopy. To localize the polysaccharide antigen on the cell surface, the immunoelectron microscopy technique of indirect peroxidase labeling was used. Antiserum #26 was added to S. mutans AHT cells grown to late stationary phase. Addition of horseradish peroxidase conjugated to goat anti-rabbit immunoglobulins to the antiserum-treated cells yields a complex of microbial antigen-rabbit antibody-goat antibody-horseradish peroxidase. Development of the horseradish peroxidase with H202 and DAB allows the ultrastructural localization of the antigen on the cell. Of all the controls that were done (see Materials and Methods), only two showed slight, non-specific reactions (Fig. 11). They were cells treated with normal rabbit serum and those treated with horseradish peroxidase conjugated to






65



































Fig. 11. Electron micrograph of S. mutans AHT treated
with horseradish peroxidase conjugated to
goat antirabbit immunoglobulins, with glutaraldehyde, with H202 and DAB, and then stained
with OsO 4 (Control G). Similar results were
obtained with Control A (Materials and
Methods). All of the other controls showed
the complete lack of any label on the cell
surface. X20,000.








66



goat anti-rabbit immunoglobulins and then fully developed, Controls A and G respectively. The cells treated with antiserum #26 showed a very thick coating of label completely surrounding

the cells (Figs. 12 and 13).

When antiserum to S. mutans BHT was used with S.,mutans

AlIT cells, there was very little label found deposited on the cell surface (Fig. 14). This indicates the absence of the LTA as a surface antigen since the antiserum to BHT contains antibodies specific for the glycerol-phosphate backbone of LTA (23).






67



































Fig. 12. Electron micrograph of S. mutans AHT treated
with a 1/5 dilution of antiserum #26, with
horseradish peroxidase conjugated to goat antirabbit immunoglobulins, with glutaraldehyde, with H202 and DAB, and then stained
with OsO4. X20,000.






68








































Fig. 13. Electron micrograph of S. mutans AHT treated
in the same manner as in Fig. 12, except that
the dilution of antiserum #26 was 1/20.
X2 0 ,000.






69





















bI















Fig. 14. Electron micrograph of S. mutans AHT treated
in the same manner as in Fig. 12, except that undiluted antiserum to S. mutans BHT was used
instead of antiserum #26. X20,000.














DISCUSSION


A polysaccharide antigen was extracted from Streptococcus mutans AHT, a serotype a organism, with mild acid and purified by 6% agarose gel filtration chromatography. This antigen contained galactose and glucose (3.6:1 molar ratio) as the only carbohydrate, about 1% glucosamine, and less than 3% protein. This polysaccharide antigen contains less than 0.1% contaminating LTA as indicated by the hemagglutination data (Table 4). Although 20-25 pg/ml of crude LTA preparations were normally used to sensitize SRBC, this was done because some of the LTA preparations contained only 20-25% LTA, the rest being polysaccharide and other contaminants. Markham et al. (32) have shown that only 1-2 pg/ml of purified LTA is needed to fully sensitize SRBC, which would give a titer of 160-320. Since titers of < 10 were observed with 100 vg/ml of polysaccharide antigen and since only 1% contamination by LTA would have yielded fully sensitized cells, it is apparent LTA contamination of the purified polysaccharide was less than 0.1%.

This polysaccharide antigen is similar to the one isolated by Mukasa and Slade (33) from HS6, also a serotype a organism. Their antigen also contained galactose and glucose (5.2:1 molar ratio) as the only sugar constituents. However their antigen contained 8.6% glucosamine, 5.4% galactosamine, and

5.0% protein. The Mukasa-Slade antigen appears to be more




70







71



closely related chemically to the extracellular antigen isolated from the culture fluid of AHT and purified by 6% agarose gel filtration chromatography. This extracellular antigen contained galactose and glucose (4.6:1), 18% glucosamine, and 7% protein. No galactosamine was detected, however, in the AHT product. Further purification of this polysaccharide was not attempted since it was shown that the extracellular antigen was similar in structure to the acid-extracted polysaccharide antigen, as indicated by comparative methylation analysis studies, and that both antigens were identical immunologically, as shown by gel diffusion (Fig. 2), immunoelectrophoresis, and hemagglutination

(not shown).

The two polysaccharide antigens of AHT studied possessed immunoelectrophoretic mobilities identical to the serotype a antigen described by Bratthall (8,9); and, being composed mainly of galactose and glucose, are similar to the serotype d crossreactive antigen described by Linzer and Slade (28), and the serotype g cross-reactive antigen described by lacono et al.

(20). The presence of galactosamine in the HS-6 polysaccharide antigen can not be explained since analysis of purified cell walls of 2 serotype a organisms, AHT and HS-1, and 3 serotype d organisms, B-13, SL-1, and OMZ-176, showed an average of about 7% glucosamine but no galactosamine (6). The major cross-reacting antigen in all 3 serotypes is very similar. All 3 antigens are polysaccharides containing galactose and glucose as the main







72




components with the only variations being the molar ratios of the 2 sugars and the amount of protein present in each preparation. It is generally agreed that the a-d- cross-reacting hapten is a terminal D-galactose on these polysaccharides.

Two methods, radial immunodiffusion and inhibition of hemagglutination, were used to show that this a-d-g crossreacting antigen is excreted into growth media in direct proportion to the cell mass of an exponentially growing culture (approximately 3.7 pg/ml/O.D. unit at 660 nm) and that this excretion increases in late stationary phase. This terminal increase is probably due to cell lysis since the ratio of extracellular to intracellular protein also increases at this time. The concentration of polysaccharide reached 16 pg/ml after 24 h, when AHT was grown in synthetic (MC) medium. The excretion seems to be 2-3 times higher in synthetic medium than in complex medium although the growth rates in both were similar.

This polysaccharide was shown by electron microscopy using indirect peroxidase labeling to be a fluffy coating surrounding the cell, similar to that found by Iacono et al. (21) in strain 6715, serotype &, and to that found by Grenier et al. on most serotypes (16). Since absorbed antiserum was not used, there is a possibility that the label is due to other surface antigens. But, this is unlikely since the polysaccharide antigen was shown to be the only antigen present in Lancefield extracts of whole cells by immtnoelectrophoresis (Fig. 4) and








73




because of the observation that good labeling still occurs when the antiserum is diluted 1/20. It would appear that the acidextractable antigen coats the cell surface as a thick capsule and is released into the growth medium as cell growth proceeds. The two polysaccharide antigens isolated from AHT (acidextractable and excreted), therefore, are probably identical polysaccharides although the extracellular product may contain accessory polymers (e.g. proteins) required to bind the antigen to the bacterial cell surface. The roles of this capsule in cell-cell cohesion or cell-enamel adhesion remain obscure. Also, the ability of the growing cells to continuously generate soluble antigenic polysaccharide poses questions about the roles of these molecules in the immunopathology of the host (e.g. gingivitis) or in the induction of specific, protective salivary antibodies.

A LTA from AHT was extracted and purified by a new technique using phosphatidylcholi ne liposomes. Using the standard hot phenol-water extraction of whole cells, both LTA and the polysaccharide antigen were extracted together and could not be separated by 6% agarose gel filtration chromatography (Fig. 8). Preextraction of cells with acid reduced the amount of polysaccharide antigen present but also reduced the recovery of LTA (Fig. 9). Separation of the polysaccharide antigen from the LTA proved to be extremely difficult. A number of separation techniques were tried including hydrophobic interaction chromatography using octyl-Sepharose which has been found







74



useful in removing protein contaminants from other LTA preparations (A. J. Wicken, personal communication). Although there was minimal protein contamination even in the crude extract, it was hoped that this technique would be able to remove the polysaccharide antigen contaminants. Some of the polysaccharide antigen was removed by this technique; however, about 50-75% of the polysaccharide originally present was eluted with the LTA. The LTA was finally purified free of galactose, the haptenic sugar of the polysaccharide antigen, by adsorption of the crude LTA onto large phosphatidylcholine liposomes, pelleting the LTA coated-liposomes by centrifugation, and, after repeated washings, dissolving the phosphatidylcholine in chloroform/methanol before collecting the purified LTA on a membrane filter. The phenolwater extracted LTA prepared in this manner contained glycerol, phosphorus, alanine, and glucose in a 1:1:0.5:0.14 molar ratio. If the LTA were assumed to be 24-30 glycerol-phosphate subunits long, then there would be only 3-4 glucose molecules associated with the molecule. Since the glycolipid portion is usually a di- or trisaccharide, 0-2 glucose molecules would be left as possible side groups substituting the backbone. Also, this LTA molecule would appear to have 50% of the glycerols substituted with alanine. Even though no galactose could be detected by gas-liquid chromatography, a very faint polysaccharide antigen band was still visible upon gel diffusion or immunoelectrophoresis of the liposome purified LTA. This could be due to the very high







75




titer of the antiserum against the polysaccharide antigen being able to detect quantities of the polysaccharide antigen below the detection limit of the chemical analysis. However, the hemagglutination assay does not indicate any of this polysaccharide antigen passively sensitizing the SRBC. With the liposome purification technique only small amounts of LTA can be prepared; however the ability to start with the very crude material from phenol-water extraction and to obtain the high degree of purification reported here, more than compensates for the small amounts of LTA which can be produced.

The 6% agarose purified phenol-water extract contains both the polysaccharide antigen and LTA as demonstrated by gel diffusion (Fig. 2) and by immunoelectrophoresis (Fig. 10). The liposome purified LTA contains a very faint polysaccharide antigen band migrating slightly towards the cathode and a LTA band migrating towards the anode. This is a rapid anodic migration while the serotype a antigen has a slow cathodic migration. The difference between LTA and the serotype antigen, therefore, is easily determined. Thus, the conclusion (44) that the AHT LTA is the serotype antigen appears not to be the case. The difference in mobilities between the L. case LTA and the AHT LTA (Fig. 10) is probably due to the neutralization of the negative charge by the high degree of alanine substitution, 50%, on the AHT LTA. This reduction in charge would slow down the migration of the AHT LTA due to a decrease in the charge:mass ratio. These LTA







76



bands are present whether antiserum #27, made against AHT, anti-LTA from L. casei, or cetavlon is used.

LTA is not a major surface antigen of AHT since only 1 of 7 rabbits immunizied with AHT responded to the LTA immunogen. Also, when anti-BHT serum, which contains anti-LTA antibodies specific for the glycerol-phosphate backbone, was used in indirect peroxidase labeling of AHT cells, very little label was

seen on the cells. The amount of label was not more than was seen on cells treated with normal (non-immune) rabbit serum. This indicates that the LTA is being masked by other surface polymers, such as the thick polysaccharide coat.

Attempts to isolate and quantitate the extracellular LTA present in the culture fluids of AHT failed. The 6% agarose gel filtration chromatography of the lyophilized culture fluid showed no high molecular weight phosphorus peak (Fig. 5) like that found with the phenol-water extracts of whole cells (Figs. 8 and 9). Rocket immunoelectrophoresis using cetavlon also failed to detect the presence of LTA in concentrated culture fluids of AHT. It is likely that the LTA was deacylated by our procedures, which include lyophilization. Certainly, Markham et al. (32) have amply demonstrated the excretion of LTA into the growth medium of AHT.

One of the more interesting and less easily explainable

results was the ability of the phenol-water extracts to sensitize SRBC

both with LTA and the polysaccharide antigen (Table 4). Purified







77



polysaccharide antigen, extracted or extracellular, could not passively sensitize SPBC unless CrCl3 was used. However, the 6% agarose purified phenol-water extract, which contains only the polysaccharide antigen and LTA (Table 3), could be used to sensitize SRBC with both polymers. It is conceivable that some of the polysaccharide antigen extracted by phenol-water could possibly be bound to a lipid, like the lipomannan of micrococci (37) or the amphophile (a heteropolysaccharide containing 16.8% O-esterified fatty acids) of Actinomyces viscosus

(50). It has been shown by Hammerling and Westphal (17) that polysaccharides which cannot passively sensitize SPBC can be esterified with stearoyl chloride to yield a fatty acid substituted polysaccharide which can then bind to membranes. Another explanation is that the LTA is "carrying" the polysaccharide antigen to the surface of the SRBC. This "carrier" effect of LTA was shown by Doyle et al. (12). They showed that LTA could solubilize polysaccharides and some proteins in high concentrations of alkyl alcohols in which the polysaccharides and proteins were normally insoluble. Mixing the purified polysaccharide antigen and the LTA from AHT under various conditions (see Materials and Methods) failed to reproduce this "carrier" effect, even under the conditions of the initial extraction. Also, the inability to observe this effect in neutralized culture fluids which contain both LTA and polysaccharide antigen does not argue for the possibility of the in vivo production of these "carrier" complexes.







78




The discrepancies between this work and the work of Van de Rijn and Bleiweis with the same organism (44) could be explained if their antigen contained a mixture of polysaccharide and teichoic acid. In their gel diffusion and immunoelectrophoresis assays only one band that corresponded to the serotype a antigen was observed. That precipitin band was most likely due to the polysaccharide described above. A LTA band would not have been observed because the antiserum used contained very few if any anti-LTA antibodies (Table 4). Also for this reason, all their immunochemical data would describe the polysaccharide not the LTA. Their material, however, would still react with antiLTA serum (i.e. L. casei anti-LTA) because it did contain some teichoic acid. The only result which is hard to rationalize is the glycerol-glucose-galactose degradation product obtained upon

alkaline hydrolysis of their "purified" antigen. One explanation is the possibility of more than one LTA being present and associated with the cell membrane--one LTA substituted with a glucose-galactose disaccharide and the other unsubstituted with carbohydrate. The latter type of LTA structure would correspond to that reported in the present study of AHT. Although slight variations in fatty acid content and percentage of backbone substitution have been reported between intracellular and extracellular LTA, there have been no reports of two completely different LTA molecules occurring in the same organism. A more intriguing possibility is that the degradation product was from the polysaccharide antigen and not the teichoic acid. This would







179





indicate that the polysaccharide antigen could have contained a lipid portion and thus be amphipathic. If the bacterial membrane contains a polysaccharide-lipid intermediate precursor of the surface polysaccharide antigen, it would be extracted with the LTA by phenol-water and would be almost impossible to separate from the LTA due to mixed micelle formation and their similar chemical properties. This polysaccharide-lipid intermediate would explain the sensitizing of SRBC with the polysaccharide antigen from the phenol-water extract but not the acid extract.

Until this ability of the polysaccharide antigen to

sensitize SRBC is completely explained, both LTA and any possible polysaccharide-lipid intermediate should be excluded from a caries vaccine, since either one would offer the possibility of adherence of bacterial antigens to tissue which could lead to an immunopathologic lesion. The acid-extracted polysaccharide antigen from any of the serotype a, d, or organisms, however, appears to be a good possibility for a mixed polymer vaccine. The polysaccharide antigen extracted in this way is free of tissue sensitizing material and can easily be tested for heart cross-reactivity. The only problem may be immunogenicity which has yet to be determined and which is never great in the case of highly-purified polysaccharides.














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BIOGRAPHICAL SKETCH


Ronald Albert Craig was born August 22, 1946, in Abington, Pennsylvania. In 1956, his family settled in Pensacola, Florida. After graduating from Escambia High School, Pensacola, Florida, in June 1964, he attended Pensacola Junior College for two years. He transferred to the University of Florida where in March, 1969, he received the degree of Bachelor of Science in chemistry. After working as a technologist for two years for Dr. A. S. Bleiweis, he returned to the University of Florida as a graduate student in organic chemistr y working under Dr. M. A. Battiste. In September, 1973, until December, 1977, he had been working toward the degree of Doctor of Philosophy in the Department of Microbiology and Cell Science, University of Florida. In January, 1978, he took the position of research assistant at the Dental Research Institute, University of Michigan under Dr. D. B. Clewell while finishing the writing of his dissertation.

Ronald Albert Craig was married to Gail Ann Nichols on

August 31, 1968, -in Miami, Florida. His son, Andrew Bryon was born on April 20, 1978, in Ann Arbor, Michigan.















85













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.



Aold S. Bleiweis, Ch~airman
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.


( <((Lr A(I K

Edward P. Previc
Associate 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.



Dale C. Birdsell
Associate Professor of Basic Dental Science

This dissertation was submitted to the Graduate Faculty of the Department of Microbiology and Cell Science in the College of Liberal Arts and Science and to the C'raduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy.

March 1979



Dean, Graduate School