Mechanisms of adsorption of actinomyces viscosus to hydroxyapatite surfaces

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Mechanisms of adsorption of actinomyces viscosus to hydroxyapatite surfaces
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Actinomyces   ( mesh )
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Thesis (Ph.D.)--University of Florida, 1980.
Statement of Responsibility:
by Timothy Thomas Wheeler.
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Vita.
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Photocopy of typescript. Ann Arbor, Michigan : University Microfilms International, 1981.--21 cm.

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MECHANISMS OF ADSORPTION OF Actinomyces viscosus
TO HYDROXYAPATITE SURFACES










BY

TIMOTHY THOMAS WHEELER


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


UNIVERSITY OF FLORIDA


1980



































For Liza













ACKNOWLEDGMENTS


I would like to sincerely thank my major advisor and

friend, Dr. William B. Clark, for his guidance, inspiration,

and support throughout my graduate studies. I also would

like to thank the members of my graduate committee,

Dr. Bernard Mansheim, Dr. Kenneth Berns, Dr. Catherine

Crandall, and Dr. William Hauswirth for their assistance in

planning and evaluating my dissertation research. Apprecia-

tion also goes to Dr. Dale Birdsell, Dr. Tom Grow, Dr. Dick

McCarron, Dr. Jim Powell, Dr. John Fitzgerald, Cynda

Crawford, Sandra Bragg and Elaine Beem for the numerous help-

ful discussions and many favors each has done for me. Spe-

cial thanks are extended to Lee Webb and Dave Brown for the

numerous tasks each has aided me in during this project. I

would also like to thank Dr. Werner Fischlschweiger for his

helpful suggestions on the electron microscopic aspects of

this paper and for the use of the facilities of the Electron

Microscope Laboratory. Thanks are also extended to

Katherine B. Williams for her concern and efforts in typing

my dissertation. I would like to especially thank Jim and

Judy Atkins and their son, Grant, and Tony and Pam Sandow

for the friendship, encouragement, and relief they provided

away from the laboratory.


iii







I would like to express my thanks and love for my

parents, Mr. and Mrs. Henry H. Wheeler, for their love,

guidance, and continuing support throughout my life. Spe-

cial gratitude goes to the rest of my family, Chris and

Deanna and their sons, Chris and Harrison, Bill, Jim, and

Rob, for their caring throughout my life.

Finally, I would like to express my deepest thanks and

love for my wife, Liza, for her understanding, support, in-

spiration and love during all phases of my studies as well

as for giving me a wonderful son, Timmy, and another child

soon to come. It is to her that I dedicate this disserta-

tion.















TABLE OF CONTENTS


Page


ACKNOWLEDGMENTS ...

LIST OF TABLES . . .

LIST OF FIGURES . . .

ABBREVIATIONS USED . .

ABSTRACT . . . .

INTRODUCTION . . .


. . . . .


* . iii

* . vii

.viii


Periodontal Disease


Role of Actinomyces viscosus in Periodontal
Disease . . . . . . . . .
General Considerations of Bacterial Attachment


to Solid Surfaces . . . . .
Theoretical Consideration of Bacterial
Attachment to Solid Surfaces . . .
Bacterial Polymers Promoting Attachment
to Surfaces . . . . .
Host Tissue Receptors for Bacteria . .

MATERIALS AND METHODS . . . . . .


Bacterial Strains . . . . . . .
Culture Conditions . . . . . . .
Electron Microscopy . . . . . . .
Acid Extraction of Whole Cells and
Cellular Components . . . . . .
Antisera Preparation . . . . . . .
Saliva Preparation . . . . . . .
Serum Preparation . . . . . . .
Bacterial Adsorption to Hydroxyapatite . .
Calculations of Parameters for Bacterial
Adsorption to HA Surfaces . . . . .
Influence of Environment on Adherence . .
Enzyme Treatment of A. viscosus Cells . .
Periodate Treatment of A. viscosus Cells . .
Isolation of A. viscosus T14V Surface Fibrils


* 17

S. 17
o 17
. 18


* *


. . . . . . .


. . . . . .


* * *


* *








Page


Cell Wall Preparation . . . .
Immunoelectrophoresis . . . .
Polyacrylamide Gel Electrophoresis
Column Chromatography . . . .
Chemical Analysis . . . . .
Competitive Inhibition Assay . .
Antisera Adsorptions . . . .
Antisera Inhibition Assay . . .
In Vivo Experiments . . . .


RESULTS . . . . . . . . . .


Bacterial Adsorption to Treated and
Untreated HA.... . ... . . . . . 35
Influence of Adsorbed Salivary Components to
HA Surfaces on Adsorption Isotherms . . .. .39
Adsorption of A. viscosus to Human
Teeth In Vivo . . . . . . . . . 42
Influence of Ionic Environmental Parameters
on A. viscosus Adsorption . . . . . .. .45
Effect of Heat, Proteolytic Enzymes, and
Periodate on Adherence of A. viscosus
Cells to Saliva-Treated HA ... ............ 52
Competitive Inhibition of Adherence . . . . 55
Adherence Inhibition by Specific Antisera . . 60
Adsorption of Antiserum Inhibition Activity . . 60
Adherence Inhibition with Antifibril Specific
Antibody and Antiserum . . . . . .. .60
Identification and Composition of
Isolated Fibrils . . . . . . . .. 64


DISCUSSION . . . . .

BIBLIOGRAPHY . . . .

BIOGRAPHICAL SKETCH ..


* . 78

* .101


. .118


. 35


* *














LIST OF TABLES



Table Page

1. Adsorption of A. viscosus to HA . . . .. .36

2. Estimates of Affinities and Adsorption Sites
of A. viscosus T14V and T14AV on HA . . .. .43

3. Adsorption of A. viscosus T14VJ1 and T14AVT1
to Human Tooth Surfaces . . . . . . 44

4. Influence of Ionic Strength on Adsorption
of A. viscosus to HA . . . . . .. .47

5. Influence of Calcium Concentration on Adsorp-
tion of A. viscosus to HA . . . . .. .49

6. Influence of Ions on Adsorption of A. viscosus
T14V to Saliva-Treated HA . .......... .. .50

7. Influence of Detergents on Adsorption of
A. viscosus to HA . . . . . . .. 51

8. Effect of Various Pretreatments of A. viscosus
Cells on Adherence to Saliva-Treated HA . . 53

9. Competitive Inhibition of A. viscosus T14V
to Saliva-Treated HA with Sugars . . .. .56

10. Competitive Inhibition of A. viscosus T14V
to Saliva-Treated HA with Whole Cell
Extracts . . . . . . . . .. 58

11. Adsorption of Goat Anti-Tl4V and Normal
Sera . . . . . . . . . . 62

12. Influence of Rabbit Serum on A. viscosus T14V
Adsorption to Saliva-Treated HA . . . .. .65

13. Amino Acid Composition of A. viscosus T14V
Fibrils Obtained from French Press Shearing
and Lysozyme Digestion . . . . . .. .70


vii














LIST OF FIGURES


Figure Page

1. Scanning electron microscopy of A. viscosus
to HA . . . . . . . . . .. 37

2. Adsorption isotherms of A. viscosus T14V
and T14AV to saliva-tFeated, untreated
and saliva/serum-treated HA . . . . 40

3. Scanning electron microscopy of various
concentrations of A. viscosus T14V ad-
sorbed to saliva-treated HA . . .. . .. 41

4. Influence of pH on adsorption of A. viscosus
T14V and T14AV to HA . . . . . .. .46

5. Transmission electron micrographs of uranyl-
acetate stained preparations of heat-
treated A. viscosus T14V . . . . .. 54

6. Competitive inhibition of A. viscosus T14V
adherence to saliva-treated HA with French
press crude supernatant, purified fibrils,
bovine serum albumin, and dextran . . .. .59

7. Titration of adherence inhibition activity
of A. viscosus T14V adherence to saliva-
treated HA . . . . . . . .. 61

8. Adsorption of goat anti-Tl4V serum with A.
viscosus T14V whole cells and Braun cell
walls and its subsequent effect on adher-
ence of strain T14V to saliva-treated HA . 63

9. Titration of adherence inhibition activity
of A. viscosus T14V adherence to saliva-
treated HA . . . . . . . .. 66

10. Transmission electron microscopy of uranyl-
acetate stained preparation of A. viscosus
T14V purified fibrils.... ............. 67


viii








Figure Page

11. Laurell rocket immunoelectrophoresis of
antigen preparations from A. viscosus
T14V . . . . . . . . . .. 68

12. Immunodiffusion of antigen preparations
from A. viscosus T14V . . . . . .. .70

13. Molecular seive chormatography of fibrils in
phosphate buffer . . . . . . . 73

14. Molecular seive chromatography of fibrils
in urea . . . . . . . . .. 74

15. Laurell rocket immunoelectrophoresis of
antigen preparations from A. viscosus
T14V . . . . . . . . . .. 76

16. Polyacrylamide gel SDS electrophoresis of
purified fibrils and high molecular weight
material from molecular sieve chroma-
tography .. . . . . . . . ... 77

17. Diagramatic representation of strain T14V
monolayer and multiple cell layer formation
and adsorption of fibrils to salivary re-
ceptor sites . . . . . . . .. 93














ABBREVIATIONS USED


CTMAC

EDTA

FSB

HA

IEP

IM

IP

IV

PAGE

PAGE-SDS

PAGE-urea

PBS

SDS

SEM

TC

TEM

TSB

VA


Cetyltrimethylammonium chloride

Ethylene diaminetetraacetic acid

Fibril-Sepharose bead

Hydroxyapatite

Immunoelectrophoresis

Intramuscular

Intraperitoneal

Intravenous

Polyacrylamide gel electrophoresis

Polyacrylamide gel electrophoresis in SDS

Polyacrylamide gel electrophoresis in urea

Phosphate buffered saline

Sodium dodecyl sulfate

Scanning electron microscopy

Top common

Transmission electron microscopy

Tryptic soy broth

Virulence associated













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


MECHANISMS OF ADSORPTION OF Actinomyces viscosus
TO HYDROXYAPATITE SURFACES

By

Timothy Thomas Wheeler

June 1980


Chairman: William B. Clark
Major Department: Immunology and Medical Microbiology

The purpose of this investigation was to examine the

mechanisms) by which the periodontopathogen Actinomyces

viscosus strain T14V adhered to the tooth surface. This

work was accomplished by utilizing an in vitro assay that

has been shown to mimic the adsorption of bacteria to teeth

in vivo. The problem was approached by comparing the in

vitro adsorption of the virulent human isolate strain T14V

with its avirulent variant, strain T14AV, to various saliva

and/or serum pellicles formed on hydroxyapatite (HA) beads.

In addition, the adherence receptor for this strain was

identified, isolated, and partially characterized.

Cell numbers of strain T14V adsorbed to saliva-treated

HA were greater than those of strain T14AV. Adsorption data








of strains T14V and T14AV to HA surfaces followed the

Langmuir adsorption model as judged by high correlation co-

efficients obtained for both strains to most of the treated

surfaces studied. The number of binding sites for strains

T14V and T14AV cells to human saliva-treated HA and untreat-

ed HA was similar. The affinity of strain T14V for saliva-

-treated HA was tenfold greater than the affinity of strain

T14AV for that surface.

To approximate the pellicle of the gingival crevice and

margin and to determine whether adherence by strain T14V was

to specific saliva or serum macromolecules, experimental

pellicles were formed on HA by saliva/serum mixtures. The

number of binding sites on the saliva/serum-treated HA re-

mained the same as for the saliva-treated surface. Although

the affinity of strain T14V cells for the saliva/serum HA

surface remained generally the same as the affinity for the

HA treated with saliva alone, the affinity of strain T14AV

cells decreased further as the serum content increased.

Strain T14V cell numbers adsorbed to serum-treated HA and

albumin-treated HA were less than those adsorbed to saliva-

treated HA, indicating that the adherence by strain T14V was

to specific saliva receptors. In vivo results from strepto-

mycin-resistant mutants of both strains T14V and T14AV con-

firmed in vitro results using saliva/serum pellicles.

Adsorption of strain T14AV to saliva-treated HA was

effected by pH and ion concentration of the suspending buffer

suggesting that hydrogen and ionic bonding played a role in


xii








strain T14AV adsorption to this surface. In contrast, strain

T14V adsorption to saliva-treated HA does not involve hydro-

gen bonding or electrostatic interactions. Pretreatment of

strain T14V with proteolytic enzymes and heat inhibited ad-

herence to saliva-treated HA, suggesting that the adherence

receptor(s) on the cell surface of strain T14V was protein

in nature.

Strain T14V possesses a fibrillar network on the cell

surface and it has been hypothesized that these mediate

bacterial adherence to surfaces. Fibril-mediated adherence

of strain T14V cells to saliva-treated HA was studied.

Fibrils were purified by ammonium sulfate precipitation and

differential centrifugation from the crude supernatant of

whole cells that were sheared by one passage through a French

pressure cell. Purified fibrils and crude supernatant in-

hibited strain T14V adherence to saliva-treated HA to similar

extents. However, anti-strain T14V serum, antifibril serum,

and antifibril specific antibody completely abolished strain

T14V adherence. The blocking immunoglobulin could be ad-

sorbed from anti-Tl4V serum by strain T14V whole cells and

purified fibrils. It was concluded that fibrils mediate

adherence of strain T14V cell to saliva-treated HA. In addi-

tion, fibril preparations were shown to contain more than 95%

protein and to be antigenically homogeneous by Laurell rocket

immunoelectrophoresis.


xiii













INTRODUCTION


The purpose of this study was to investigate the mech-

anism(s) by which the periodontopathogen Actinomyces

viscosus strain T14V adhered to the tooth surface. This

work was accomplished by utilizing an in vitro assay that

has been shown to mimic the adsorption of bacteria to teeth

in vivo (14, 174). The problem was initially approached by

comparing the in vitro adsorption of the virulent human

isolate A. viscosus strain T14V with its avirulent variant,

A. viscosus strain T14AV, to various saliva and/or serum

pellicles formed on hydroxyapatite (HA) beads. The strain

T14V adherence mechanism was further studied by identifying

the receptor(s) on the bacterial cell surface mediating

adherence. Finally, the bacterial cell surface receptor was

isolated and characterized as to its composition and the

role it assumes in adhering to specific salivary receptors.



Periodontal Disease

Periodontal disease is a general term that is used to

encompass various diseases ranging from gingivitis to

periodontitis. Both gingivitis and periodontitis begin at

the dentogingival junction in the gingival crevice. Gingi-

vitis refers to inflammation of gingival tissue whereas







peridontitis refers to the destruction and progressive

disease that affects the gingiva, alveolar bone, and perio-

dontal ligament. Gingivitis is an early stage of periodon-

titis; however, not all gingivitis cases progress to

periodontitis.


Role of Actinomyces viscosus in
Periodontal Disease

A. viscosus, a gram-positive rod, has been implicated

in the etiology of gingivitis (73, 75, 102, 163) and

periodontitis (74, 75). Colonization by this organism in

germfree animals maintained on a soft, high carbohydrate

diet results in plaque formation, root surface caries, and

bone loss characteristic of periodontal disease (72, 74, 75).

In humans it has been shown that accumulation of plaque on

the tooth surface initiates gingivitis (100, 167). In addi-

tion, Listgarten demonstrated that gingivitis and periodon-

titis are characterized by a quantitative increase in the

total mass of adherent organisms comprising plaque (98).

Loesche and Syed (102, 163) have shown in humans that in

experimental gingivitis, organisms from the Actinomyces

species become the predominate cultivable organism as plaque

accumulates. The predominate Actinomyces species isolated

from subjects with nonbleeding gingivitis and bleeding

gingivitis were A. israelii and A. viscosus, respectively.

The increase in Actinomyces was accompanied by a decrease

in Streptococcus species.







Immunological studies have shown that A. viscosus anti-

gens penetrate the gingival tissue and elicit a T-cell

response in various forms of periodontal disease (5, 63, 68,

89, 123, 150). Isolates of A. viscosus, A. naeslundii,

Streptococcus sanguis, Bacteroides melaninogenicus, Veil-

lonella, and Fusibacterium nucleatum were assayed for their

ability to elicit a T-cell blastogenesis in subjects who had

refrained from using oral hygiene (147). It was determined

that only antigens from A. viscosus significantly increased

the stimulation index as the gingivitis developed (147).

The T-cell response returned to normal values when oral

hygiene was reinstituted and gingivitis disappeared (147).

In addition, it has been reported that the T-cell response

to A. viscosus antigens decreased following treatment of

naturally occurring gingivitis (130).

It had been proposed that gingivitis developed as a

result of the nonspecific proliferation of the plaque

flora (99, 151). Therefore, treatment of periodontal dis-

ease has been directed toward debridement of the dentogingi-

val surfaces in order to eliminate large amounts of plaque

(101). However experiments by Loesche and Syed (102, 163)

suggest that if specific organisms are etiologically in-

volved in gingivitis and other forms of periodontal disease,

then antimicrobial treatment aimed at specific organisms

might be effective (101).







General Consideration of Bacterial
Attachment to Solid Surfaces

In the natural environment, many bacteria have a pre-

dilection for attaching to and colonizing surfaces. Solids

immersed in seawater or freshwater become colonized by

adherent microorganisms (178). In aquatic habitats, most of

the permanent bacterial population is found on solid sur-

faces (27). In addition, bacteria have been observed to

colonize the surfaces of sand grains (119), colloidal soil

particles (106), algae (16), plant tissues (32, 144, 145),

and other bacteria (71). The skin and mucous membranes of

humans and animals are also heavily colonized by adherent

indigenous bacteria (9, 59, 142). The initial step in the

development of a bacterial infection entails attachment and

colonization of the host tissue by the infecting organism

(148). A variety of pathogenic bacteria, including beta-

hemolytic streptococci (40), gonococci (173), Salmonella

(164), Shigella (87), enteropathogenic Escherichia coli

strains (149, 158), Vibrio cholerae (46), Clostridium

species (2), Corynebacterium diphtheriae (7), etc., have

been observed to attach to their host mucosal surfaces dur-

ing natural or experimentally induced infections. In addi-

tion, dental pathogens such as Streptococcus mutans and

Actinomyces species attach to and colonize the surfaces of

teeth (59).

Even though surface colonization appears to be wide-

spread in nature, only recently has the ecological signifi-

cance of attachment or the mechanisms by which bacteria




5



adsorb to surfaces been investigated. ZoBell (178) was one

of the first to appreciate the specificity of attachment of

marine organisms to glass. He noted that of 96 isolates

from sea water, 29 strains adhered strongly, 20 strains

adhered variably, and 47 strains did not adhere.

Zvyagintsev (179) further demonstrated that variation in

adsorption could occur by strains within the same genus.

The selectivity of bacterial adsorption to anion and certain

exchange resins, cellulose fibers, charcoal, kaolin, bento-

nite, sand, and a variety of other adsorbents also has been

shown (29).

Bacteria colonize different hosts and tissues with a

high degree of specificity. For example, lactobacilli

colonize the surface of nonsecreting keratinized stomach

epithelial cells of rats and mice, but not the surface of

secreting stomach epithelium (141). In addition, strepto-

cocci and other indigenous bacteria have been shown to

selectively colonize the tongue, cheek, or tooth surfaces of

the human mouth (52, 59, 81). Many pathogenic bacteria also

display a restricted range of hosts or tissues to which they

can attach and infect under natural conditions (49).

Studies of the ecology of indigenous bacteria in the

human mouth provided the first convincing evidence that

bacterial adherence is an ecological determinant of natural

colonization (49, 59). The mouth contains several surfaces

such as the teeth, tongue, buccal mucosa, palate and

gingiva which are available for colonization. There is







evidence that these sites are selectively colonized by

specific bacteria. For example, Streptococcus salivarius

accounts for about half of the streptococcal populations

found on the dorsal surface of the tongue; however, it is

not found on teeth (52, 81). In contrast, S. mutans may

comprise a significant proportion of the bacterial popula-

tion that colonize the teeth, but its proportion on oral

mucosal surfaces is generally low (59).

Several in vitro and in vivo assays have shown that

indigenous streptococci and other bacteria selectively

adsorb to specific oral surfaces, and that the experimental-

ly derived affinities of these organisms directly correlate

with their natural intraoral colonization (57, 59, 66, 94,

170, 171). Therefore, the bacterial cell surface apparently

contains a highly developed recognition system capable of

specifically interacting with specific host tissues. In

addition, the correlation found between adherence and

natural colonization strongly implies that adherence to host

tissues is an important determinant of colonization for oral

bacteria (49, 59).


Theoretical Considerations of Bacterial
Attachment to Solid Surfaces

Bacterial adsorption to solid surfaces has been studied

by several marine and soil microbiologists. ZoBell (178)

and others (29, 108) noted that marine bacteria first became

loosely associated with a surface and that firm attachment

was a time dependent process, frequently associated with







the synthesis of holdfast material. The adsorption of

bacteria to surfaces has been proposed to occur in two

phases (107, 108). The initial phase of adsorption is con-

sidered to be reversible and the organisms are in a state

of equilibrium. The bacteria are attracted to within 100 A

of the surface by Van der Waals forces; however, as the cell

moves closer to the surface, the net negative charge on both

the cell and the surface begin to exert a repulsive effect.

The second phase of attachment involves an "irreversible"

firm attachment of the bacteria to the surface through the

interaction of macromolecules which effectively bridge the

cell to the surface.

There are many important variables that could influence

the sorptive behavior of bacteria. Growth culture medium,

culture age, and bacterial cell concentration have been

shown to effect adsorption properties of bacteria (29). In

addition, environmental parameters such as the hydrogen ion

concentration, the ionic strength, the incubation time, the

incubation temperature and the degree of agitation are

important considerations for bacterial adherence (29).

Environmental parameters may affect the adherence of differ-

ent bacteria in various ways. Adsorption of Streptococcus

salivarius and S. pyogenes to human epithelial cells (40),

of Escherichia coli to urothelial cells (105), and of

lactobacilli to rat stomach epithelium (160) do not differ

significantly between pH 5 and 8. However, the adsorption

of S. mitior to saliva-treated HA shows a maximum at







pH 6 (96), and the adherence of gonococci to urothelial

cells increases with increasing acidity (105). Therefore,

it is evident that the optimal conditions for each bacterium-

host tissue interaction must be delineated.


Bacterial Polymers Promoting Attachment
to Surfaces

The ability of bacterial cells to interact with host

tissue in a specific manner is thought to be the primary

event by which most indigenous and pathogenic microorganisms

initiate colonization of a host (59). Although little is

known about the surface structures possessed by various

bacteria, which apparently must interact with host tissue

components to enable the microorganisms to become firmly

attached and thereby resist elimination by host defense

mechanisms (50), some information is becoming available.

For example, fibrils, fimbriae, and pili have been implicat-

ed in the adsorption of many gram-negative (15, 19, 35, 36,

122, 129, 139, 140) and gram-positive (67, 84, 85, 177)

microorganisms to host tissue. Pili have been shown to

mediate adherence of gonococci to tissue culture cells (161),

to erythrocytes (19), and to sperm cells (69) and of

Escherichia coli to intestinal epithelial cells (122) and

kidney cells (140). Streptococcus salivarius (60), S.

mitior (95), and S. pyogenes (39) cells possess a "fuzzy

coat" of thin, densely distributed surface fibrils that are

morphologically distinct from pili. The interaction of







these fibrils with surface components of epithelial cells

has been suggested from electron micrographs (39, 60).

Treatment of these cells with trypsin removed the fibrils

and greatly reduced the number of streptococci that attached

to epithelial cells (39, 60). This suggested that the

fibrils increased the strength of the adsorption bond be-

tween cell and surface. The S. pyogenes cell surface con-
k
tains a type-specific M antigen that is a trypsin sensitive

virulence factor (162). Further evidence that implicated

the M antigen of S. pyogenes as an adherence factor was that

M-negative mutants attached poorly and anti-M antibodies

inhibited attachment (39). However, it has been shown that

lipoteichoic acid extracted from heat-treated group A strep-

tococci bound to epithelial cells and blocked the attachment

of whole cells of the organism (10, 124). The observation

that lipoteichoic acids binds to cell membranes suggests

that these molecules may mediate bacterial adherence. How-

ever, the relative heat stability and trypsin resistance of

lipoteichoic acid suggested that it was not the only factor

promoting adherence of S. pyogenes to epithelial cell sur-

faces (10, 124).

In contrast to the observations with streptococci, it

had been suggested that attachment of lactobacilli to

chicken crop epithelium involved polysaccharide components

(47). Evidence supporting this possibility are that attach-

ment was not affected by heat or trypsin treatment, poly-

valent concanvalin A agglutinated the adherent strains of







lactobacilli and treatment of these strains with monovalent

concanavalin A inhibited attachment (47).

Theoretically, the chances for survival of a bacterial

species in nature would be substantially increased if the

organism had adapted to colonize many tissues and hosts dur-

ing evolution. However, this does not appear to have oc-

curred in view of the host and tissue tropisms of many

indigenous and pathogenic bacteria. Alternatively, multiple

surface components mediating adherence may be required for

successful bacterial colonization of a host tissue rather

than single surface receptors which might be easily altered

by mutation. Evidence supporting this theory has come from

studies involving the adherence of S. mutans. S. mutans

cells specifically interacted with high molecular weight

glycoproteins found in whole saliva (54) and in parotid

saliva (43). Components of these salivary secretions have

been shown to selectively adsorb to HA (43) and S. mutans

cellular adherence occurred via interactions with these

salivary components (59). Attachment of S. mutans cells to

the salivary pellicle has been shown to involve a protein-

aceous nonglucan receptor (25, 26, 86, 157). For example,

S. mutans cells have been shown to attach to saliva-treated

HA in the absence of sucrose (26). In addition, a glucosyl-

transferase defective mutant of S. mutans has been reported

to attach to rodent teeth in vivo and saliva-treated HA in

vitro (25). More recently, a lectin derived from Persea







americana seeds that is specific for protein side chains

(118) has been shown to inhibit the attachment of S. mutans

to saliva-treated HA (157). Accumulation of S. mutans on

the tooth or HA surface was dependent upon the presence of

glucan (48, 51, 114). Glucosyltransferase, a glucan synthe-

sizing (114) and binding enzyme (51), can be located bound

to the S. mutans surface where it synthesized glucan from

sucrose (114) and acted as a receptor for additional glucan

molecules located on other cells that resulted in cell to

cell aggregation (51). Cell free glucosyltransferase mole-

cules bound to the cell surface, bound glucan molecules and

served as additional glucan-binding receptors (48).

Furthermore, there appeared to be several additional cell

surface receptors, different from glucosyltransferase, that

bound glucan and allowed for additional mechanisms of S.

mutans aggregation (113, 176). Therefore, S. mutans cells

have many recognition molecules which are involved in its

adherence and colonization.

Attachment to plant tissue by certain pathogenic plant

bacteria also has been studied. Attachment of Agrobacterium

tumefaciens to pinto bean leaves is blocked by lipopolysac-

charides isolated from the organisms and therefore prevent-

ing the formation of tumors on the plant (97).

Lipopolysaccharides isolated from avirulent Agrobacterium

strains do not inhibit the attachment of the virulent

organism illustrating the high degree of specificity. In-

fection of clover root hairs by Rhizobium trifolii is







initiated by specific attachment of the organism to a lectin

made by the plant (4). This bacterium possesses a surface

polysaccharide antigen which is serologically cross-reactive

with a component on the root hairs (31). It has been sug-

gested that polyvalent lectin joins the bacterium to the

root hairs via these cross-reactive components (31).

A. viscosus may require specialized cell surface struc-

tures to promote adsorption and subsequent colonization on

teeth, epithelial cells and other bacteria in plaque.

Girard and Jacius (61) proposed that Actinomyces adsorption

was mediated by the surface fibrils they observed in elec-

tron photomicrographs. Fibrils have been implicated in the

coaggregation of A. viscosus (21, 116) or A. naeslundii (21,

37) with oral streptococci. In addition, A. naeslundii

strains that possessed fibrils adhered to buccal epithelial

cells whereas strains that had fibrils mechanically re-

moved did not adhere to epithelial cells suggesting that

fibrils mediated adherence of A. naeslundii to epithelial

cells (41). Brecher et al. (14) have shown differences in

virulence of A. viscosus strains T14V and T14AV in mono-

infected germfree rats and conventional rats. Furthermore,

these authors have shown that the prerequisite for virulence

of strain T14V is its ability to attach to and colonize the

teeth and gingival crevice of rodents, resulting in bone

loss characteristic of periodontal disease (14). In this

model, the failure of strain T14AV to result in alveolar

bone loss was attributed to its colonization pattern.







Strain T14AV colonizes pits and fissures, but lacks the

ability to colonize the gingival crevice region of teeth and

therefore cannot induce periodontal disease in these labora-

tory animals (14).

A. viscosus strain T14V possesses a virulence-associat-

ed antigen(s) identified by immunoelectrophoresis (63, 132)

or by immunodiffusion (23), which is not detectable in the

avirulent variant A. viscosus strain T14AV. In addition,

morphological and ultrastructural studies of strain T14V and

T14AV have shown a characteristic fibril structure on the

cell surface of strain T14V that is undetectable on the

avirulent strain (14, 132). Cisar and co-workers have

identified these fibrils isolated from cell walls as a

virulence-associated antigen (22, 23). Because fibrils are

numerous and predominant on the cell surface, it seems logi-

cal that they would be involved in adherence. However, a

priori role of fibrils of A. viscosus strain T14V in the

adherence to saliva-treated surfaces has not been demon-

strated.



Host Tissue Receptors for Bacteria

The adsorption of bacteria is also influenced by the

composition of the adsorbent surface (29, 50, 107). In vivo

the teeth are covered by an acquired pellicle, formed of

selectively adsorbed salivary components (42, 64, 110-112,

154). The presence of adsorbed salivary components on

enamel or HA surfaces alters the composition and surface







characteristics of those surfaces (135) and clearly influ-

ences the selectivity of bacterial adsorption (24, 26, 53,

66, 95, 125, 127). For example, oral bacteria adsorbed

differently to saliva-treated HA than to untreated HA (14,

24, 53, 66, 95, 174); the number of cells of some species

which adsorbed is increased by the salivary pellicle, where-

as the adsorption of other species is decreased. The role

of the adsorbed salivary components as receptors on host

tissues with which bacteria interact have not been well

characterized. However, data are available which suggest

that glycoproteins or glycolipids present on the teeth and

epithelial cells may serve as the receptors for some species

(175). Surface glycoproteins on human buccal epithelial

cells are associated with adsorption of S. mitis, S. sanguis

and S. salivarius (175). This was shown by masking the

adherence receptors on the epithelial cells with antibody

to specific blood group antigens or Concanavalin A. Blood

group reactive salivary glycoproteins are present in the

acquired pellicle on human teeth (152). Moreover, it has

been shown that the same sugars which inhibit human erythro-

cyte hemagglutination by Leptotrichia buccalis cells also

inhibit the organism's adsorption to saliva-treated enamel

(80) suggesting that the molecules involved in both inter-

actions possess similar binding determinants. In addition,

oral Streptococcus and Actinomyces species selectively ad-

sorbed blood group reactive glycoproteins from saliva and

mucin (55). It has been reported that different specific







salivary glycoproteins interact with various related and

non-related bacteria. Salivary constituents, particularly

high molecular weight glycoproteins, bound to and aggregat-

ed a variety of oral species (65). The observation that

salivary glycoproteins which aggregated strains of S. mitis

were different from those which reacted with strains of

S. sanguis provided evidence for specific interactions (76).

In addition, 0rstavik (126) provided evidence that different

strains within the same species may exhibit specificity for

host sites. Saliva pre-adsorbed with one of two strains of

S. sanguis was used to form a pellicle on enamel. It was

observed that S. sanguis cells would not adhere to pellicles

formed from saliva pre-adsorbed by the homologous strain;

however, S. sanguis cells would adhere to pellicles formed

from saliva pre-adsorbed by the heterologous strain.

Recent reports have suggested that the presence of

adsorbed salivary components influenced the adsorption of

oral bacteria by altering the strength of the adsorption

bonds formed between the organism and the surface, by chang-

ing the number of receptor or adsorption sites that the

surface provides for the organisms or by a combination of

the two (24, 53). A mathematical model has been described

which enables comparative estimates of these two parameters

to be made (53, 82). The model essentially describes a

Langmuir adsorption isotherm (90) which is often used in

studies of molecular adsorption. Gibbons, Moreno and

Spinell (53) found that the model adequately described the




16


adsorption of a strain of S. mitior to untreated and to

saliva-treated HA powder. Subsequently, the adsorption

model has been utilized to describe the adsorption of

several other streptococcal strains (1, 24) and two acti-

nomyces strains (24) to HA and of Rhizobium sp. to root

hairs (144, 145).













MATERIALS AND METHODS


Bacterial Strains

A. viscosus strains T14V and T14AV were kindly provided

by B. F. Hammond, University of Pennsylvania, Philadelphia.

A. viscosus strains T14VJ1 and T14AVTl are laboratory-

derived, streptomycin-resistant variants (200 pg/ml) of T14V

and T14AV, respectively, and were isolated as previously de-

scribed (171). All cultures were stored as lyophilized

stocks or as multiple frozen stocks at -80C in tryptic soy

broth (Difco Laboratories, Detroit, Mich.) containing 20%

glycerol.



Culture Conditions

Batch cultures were grown in tryptic soy broth with

dextrose (TSB; Difco). Tritium-labeled cells of A. viscosus

strains T14V and T14AV were prepared fresh from the frozen

stocks for each experiment by growing the organisms in TSB

containing 4 pCi of [3H]thymidine per ml (sp. act. 3 X 10-3

cpm per cell; Schwarz/Mann, Orangeburg, N.Y.). Cultures

were incubated under microaerophilic conditions (90% N2,

10% CO2) at 37C in a Psycrotherm (New Brunswick Scientific

Co., New Brunswick, N.J.) shaking incubator.








Electron Microscopy

HA beads with adsorbed bacterial cells were washed with

three volumes of 0.05 M KC1 containing 1 mM potassium phos-

phate (pH 7.3), 1 mM CaCI2, and 0.1 mM MgCl2 (buffered KC1)

and placed in a vial and dehydrated through a graded acetone

series. Beads were critical-point dried (Sorval, Newton,

Conn.) and mounted on studs by sprinkling the beads onto

double-coated tape (Scotch, No. 666). Studs were coated

with gold palladium for 3 min at 10 mA on a Hummer II

(Technics, Alexandria, Va.) plater and examined in a

Novascan 30 (Zeiss, New York, N.Y.) scanning electron micro-

scope (SEM).

For examination of unfixed whole cells or material

extracted from cells, one drop of washed material was placed

on carbon-stabilized Parlodion film. Excess material was

removed, and 1 drop of 1% uranyl acetate in water was added.

Excess stain was immediately removed, and the grids were

examined at 60 kV in a Zeiss EM9 S-2 transmission electron

microscope (TEM).


Acid Extraction of Whole Cells
and Cellular Components

The weak acid chemical extraction of Lancefield and

Perlmann (88) was used to solubilize antigens. Washed,

freshly cultivated whole cells (0.25, [wet weight] per ml)

or lyophilized extracted cellular components (0.25 mg [dry

weight] per ml) of A. viscosus strains T14V or T14AV were

suspended in 0.04 N HC1 in 0.85% saline and placed in a







boiling water bath for 15 min. After cooling, the suspen-

sions were titrated to neutrality by addition of 2.0 N NaOH

in saline. Insoluble material was removed by centrifugation

at 25,000 x g for 15 min and the supernatant lyophilized.



Antisera Preparation

Hyperimmune sera were prepared in adult New Zealand

rabbits by a series of intravenous (IV) injections of A.

viscosus T14V cells. Cells were grown to late exponential

phase in TSB without dextrose (Difco) supplemented with

0.1% yeast extract (Difco) and 1% glucose, harvested, and

washed with saline. Cells were suspended at 5 mg (wet

weight) per ml in saline and placed in a boiling water bath

for 15 min. Rabbits initially were injected with 0.1 ml of

killed whole-cell suspension. Beginning with week 2 and

continuing at weekly intervals, rabbits were injected with

1 ml of the cell suspension. Beginning with week 7, rabbits

were bled once per week from the marginal ear vein. The

sera was obtained and frozen at -30C until subsequent use.

No immune reactions were detected between antigen prepara-

tions and pre-immunization serum.

Rabbit immune serum to A. viscosus strain T14V purified

surface fibrils was prepared by immunizing New Zealand

rabbits with a series of intraperitoneal (IP) injections.

Fibrils were suspended at 2 mg per ml in saline and dis-

persed by mild sonication (Kontes, Vineland, N.Y.). One

milliliter of saline-fibril suspension and 1 ml of complete








Freund adjuvant (Difco) were mixed and injected IP twice a

week for 4 weeks. Beginning with week 5, rabbits were bled

from the marginal ear vein and reimmunized IP once a week.

Immune serum was prepared in a goat by a series of

intramuscular (IM) and IV injections of strain T14V cells.

Initially the goat was injected IM with 0.5 ml (10 mg) of

killed whole cell suspension mixed with 0.5 ml of complete

Freund adjuvant. A second 0.5 ml (10 mg) injection of

whole cells without Freund adjuvant was given IM during

week 2. At weeks 4, 6, 8, and 10, IV injections containing

1 mg of killed whole cells were given. Beginning with

week 12 and continuing at weekly intervals, the goat was

bled from the carotid artery and injected IV with a suspen-

sion containing 1 mg of killed whole cells.



Saliva Preparation

Whole paraffin-stimulated saliva (100 ml per collec-

tion) from one donor was collected in a container chilled in

ice and heated at 56C for 30 min to inactivate degradative

enzymes (65). Heat-treated saliva was clarified by centri-

fugation at 12,000 x g for 10 min (24). Sodium azide was

added at a final concentration of 0.04%, and this prepara-

tion was stored at -30C until subsequent use.



Serum Preparation

Blood was collected (100 ml per collection) from the

same saliva donor. The blood was allowed to clot overnight








at 4C. Serum was then obtained after centrifugation of the

clotted blood at 250 x g for 10 min. Sodium azide was added

to the serum at a final concentration of 0.04%, and this

preparation was stored at -30C for subsequent use.



Bacterial Adsorption to Hydroxyapatite

A. viscosus organisms labeled with [ H]thymidine were

harvested from 16-h cultures by centrifugation at 1,300 x g

for 10 min, washed twice and suspended in buffered KC1.

Clumped organisms were dispersed with medium power 10-s

pulses from a micro-ultrasonic cell disrupter (Kontes) be-

tween centrifugations and for three 10-s pulses before dilu-

tion to final concentrations. Final concentrations were

determined from a plot of optical density versus concentra-

tion made in a Spectronic 20 spectrophotometer (Bausch and

Lomb, Inc., Rochester, N.Y.). The plot was made from direct

microscopic counts of organisms at various concentrations,

using a hemacytometer (American Optical Corp., Buffalo,

N.Y.).

Forty milligrams of hydroxyapatite beads (BDH Biochemi-

cals Ltd., Poole, England) were washed twice and hydrated in

distilled water, equilibrated in buffered KC1 and treated

with saliva, serum, saliva/serum mixtures, albumin and

buffered KCl by the methods previously described by Clark et

al. (75). After 90 min of incubation of HA beads with bac-

terial suspensions, the HA beads were allowed to settle from

the mixture for 60 s. Aliquots, 100 pI each, were removed








from the supernatants, which contained unadsorbed organisms,

and placed in vials containing 10 ml of Aqueous Counting

Scintillant (Amersham/Searle, Arlington Heights, Ill.).

Microscopic examination of bacterial suspensions revealed

that cells were evenly dispersed during all phases of the

experiment. The samples were allowed to equilibrate for

2 h in the dark at 4C and were counted on a Searle Isocap

300 scintillation counter. Portions of known numbers of
3H-labeled cells were counted in a similar manner so that

counts per minute could be related to bacterial cell number.

Control bacterial suspensions were incubated without HA

beads and counted similarly to correct for cell loss due to

adsorption to the tubes. Direct counts of bacterial adsorp-

tion to HA surfaces by SEM confirmed that this was a reason-

ably sensitive and reliable method for studying bacterial

adherence to HA (26). Data are expressed as number of cells

adsorbed per 40 mg HA, percent of cells adsorbed of the

control (equation 1), or percent inhibition of the control

(equation 2).



Equation 1:

% Adherence of Control =

# cells experimental adsorbed 100
# cells control adsorbed







Equation 2:

% Inhibition of Control =

# cells control adsorbed # cell experimental adsorbed X 100
# cells control adsorbed

All experiments were done in duplicate and repeated at least

twice.


Calculations of Parameters for Bacterial
Adsorption to HA Surfaces

Adsorption isotherms obtained by direct measurements

were used to calculate the strength of the adsorption bond

(i.e. affinity) and the number of binding sites, using the

bacterial adsorption model described by Gibbons and co-

workers (53). The adsorption model is described by the

equation C/Q = l/KN + C/N, where C is the concentration of

free cells at equilibrium, N is the maximum number of bind-

ing or "receptor" sites, and Q is the total number of cells

adsorbed per unit of adsorbent. At equilibrium, the strength

of the adsorption bond between the bacterial cell and the

adsorbent surface is described by parameter K (ml per cell).

A plot of C/Q versus C yields a straight line if the experi-

mental data are adequately described by the mathematical

model.



Influence of Environment on Adherence

Various environmental parameters were altered within

the adherence assay to determine the subsequent effect on

bacterial adsorption to saliva-treated and untreated HA.







The buffered KCl parameters varied included pH (5.75-8.33),

ionic strength (KC1 M .001 M-2.0 M) and calcium concentra-

tion (0.0-10.0 mM). All other parameters within the

buffered KCl were exactly the same except where stated.

To determine the influence of certain ions on the ad-

sorption properties of both strain T14V and T14AV to HA,

separate 1 mM potassium phosphate buffers (pH 7.3) were made

to include: 1) 0.05 M KC1; 2) 1 mM CaCI2; 3) 0.1 mM MgCl2;

4) 0.05 M KC1, 1 mM CaCI2; or 5) 0.05 M KCl, 0.1 mM MgCl2.

Both bacteria and HA beads were washed with the appropriate

buffer prior to use in each experiment. In addition, con-

trols for each experiment included treatment of the cells

and HA with the normal buffered KC1.

The influence of various detergents on bacterial cell

adherence to saliva-treated and untreated HA surfaces was

examined. A non-ionic detergent, Tween-80 (Sigma Chemical

Co., St. Louis, Mo.); an anionic detergent, sodium dodecyl

sulfate (SDS; Sigma); and a cationic detergent, cetyl

trimethylammonium chloride (CTMAC; Sigma) were included in

various concentrations (0.001-1.0%) in the buffered KC1.

Both strain T14V and T14AV cells were washed and suspended

in these detergent containing buffers and used in the ad-

herence assay. In another experiment, cells were washed

once with buffered KCl containing 0.1% CTMAC or 0.01% SDS,

washed twice in buffered KC1 without detergent, resuspended

in buffered KC1 without detergent, and adherence to saliva-

treated and untreated HA determined.







Enzyme Treatment of
A. viscosus Cells

The influence of pretreating A. viscosus T14V or T14AV

cells with various enzymes on their subsequent binding to

saliva-treated HA was studied. Aliquots of cells (4 X 107

cells per ml) were treated with the following enzymes:

0.2% trypsin (type III, twice crystallized; Sigma Chemical

Co., St. Louis, Mo.) in phosphate-buffered saline (PBS)

adjusted to pH 8; 0.2 to 1.0% chymotrypsin (type II, Sigma)

in PBS, pH 7.8; 0.2% papain (type II, twice crystallized,

Sigma) in PBS, pH 6, containing 50 mM CaCI2; 0.2% protease

(type VIII, Sigma) in PBS adjusted to pH 7.8. The mixtures

were incubated for 1 h at 37C, except for the chymotrypsin

which was at 25C. After treatment, cells were collected

by centrifugation and washed twice with 0.5 M NaCl and twice

with buffered KC1, and inhibition of adherence was examined.

Control cells were treated similarly with each buffer but

without enzyme.



Periodate Treatment of A. viscosus Cells

Washed cells were suspended in 10 mM periodate in PBS

at pH 6.5 and incubated for 12 h at 4C. Cells were col-

lected by centrifugation and washed twice with buffered KC1

before the adherence assay to saliva-treated HA. Control

cells were incubated in PBS without periodate.








Isolation of A. viscosus T14V
Surface Fibrils

Washed whole cell suspensions of strain T14V were sub-

jected to sonication, ballistic disintegration (Braun homo-

genization) and French press extraction according to the

methods of Brown et al. (17). Whole cell and cell-wall free

supernatants from the various extracts were examined for

their ability to inhibit adherence as described below.

Supernatants obtained by French press extraction were fur-

ther purified as described below.

A modification of the method described by Buchanan (18)

for the purification of gonococcal pili was used to isolate

surface fibrils from A. viscosus strain T14V cells. Bac-

terial cells from 9 liters of TSB (100 g wet weight) were

washed in 0.05 M potassium phosphate buffer (pH 7.2) and

resuspended in the same buffer to form a thick paste. The

cell paste was passage once through a French pressure cell

(American Instrument Co., Silver Spring, MD) at
2
10,000 lb/in One passage resulted in less than 2% cell

disruption, as measured by release of material adsorbing at

260 nm. The product was centifuged at 48,000 x g for 20 min

to remove whole cells and cell walls. The crude supernatant

was centrifuged for 24 h at 160,000 x g. The pellet was

resuspended in 20 ml of 0.1 M tris hydroxymethyll) amino-

methane buffer (pH 7.5) and sonicated at full power for

1 min with a microultrasonic cell disrupter. After centrifu-

gation for 10 min at 23,700 x g, the supernatant was mixed







with an equal volume of 20% ammonium sulfate and left at

4C for 24 h. This solution was subsequently centrifuged

for 15 min at 30,900 x g. The supernatant remaining after

the first precipitation was reprecipitated by increasing the

ammonium sulfate concentration to 30%. This procedure was

continued up to an ammonium sulfate concentration of 60% in

10% gradations. Each resultant pellet was suspended in

10 ml of the appropriate concentration of ammonium sulfate,

and vortexed for 1 min. After centrifugation for 15 min

at 30,000 x g, the pellet was suspended in 10 ml of filtered

distilled water, sonicated for 1 min at full power, mixed

with the appropriate concentration of ammonium sulfate, and

again left at 4C for 24 h. The centrifugation process

through the suspension in ammonium sulfate was repeated.

The resulting pellet was suspended in filtered distilled

water, dialyzed against four changes of distilled water, and

lyophilized. Each purification step was monitored by TEM

and Laurell rocket immunoelectrophoresis, as described

below.



Cell Wall Preparation

Cell walls were kindly provided by David A. Brown,

University of Florida. A. viscosus strain T14V washed cells

were broken in a Braun cell homogenizer (Bronwill Scientific

Inc., Rochester, N.Y., Model MSK) and walls were isolated

and purified by the method of Bleiweis et al. (13).








Immunoelectrophoresis

The antigens present in the French press crude super-

natant and the various ammonium sulfate precipitates ob-

tained from the supernatant were detected by Laurell rocket

immunoelectrophoresis (IEP, 91) utilizing the Osserman

modification (83, 128) as described previously (132). To

increase the solubility of the antigens, the material was

suspended in 0.5% Triton X-100 (Sigma). In addition, 1.0%

Triton X-100 was included in the agarose.



Polyacrylamide Gel Electrophoresis

Disc gel electrophoresis of the isolated surface

fibrils was performed in 7.5% polyacrylamide gels containing

1% SDS (PAGE-SDS) according to the method previously de-

scribed (77). Electrophoresis was performed at a constant

current of 8.0 ma per tube until the tracking dye reached

5 mm from the bottom of the tube. Gels were stained with

a 0.05% Coomassie blue R stain (Sigma) with 25% isopropanol

and 10% glacial acetic acid overnight at 23C, overnight a

second time in a 0.025% Coomassie blue stain with 10% isopro-

panol and 10% glacial acetic acid, and a third night in

0.0125% Coomassie blue stain with 10% glacial acetic acid.

Gels then were destined electrophoretically for 10 min in

7% acetic acid with a Canalco destainer II (Miles Lab., Inc.,

Elkhart, Ind.).







Column Chromatography

Isolated surface fibrils (1 mg/ml) were dissolved in

8 M urea and then 0.5 ml chromatographed over an ultrogel

AcA 54 (LKB Instruments, Inc., Durham, N.C.) column

(1.5 cm X 30 cm) that had been equilibrated with 8 M urea

in 0.01 M tris hydroxymethyll) aminomethane buffer (pH 8.4).

The effluent was monitored by uv (280 nm), and 4 ml frac-

tions were collected. Single peak fractions were pooled,

extensively dialized against distilled water, and lyophil-

ized. Peaks were then examined by PAGE and IEP methods pre-

viously described. Hen egg albumin, chymotrypsinogen A, and

cytochrome C (Pierce Chemical Co., Rockford, Ill.) were

utilized to standardize the column.

The above column system also was used with a 0.01 M

potassium phosphate buffer (pH 7.2) solvent. Single peak

fractions were pooled, dialyzed, and examined by TEM.



Chemical Analysis

Isolated fibrils were assayed for protein by the method

of Lowry et al. (103), using bovine serum albumin (Sigma)

as a standard. Carbohydrate, as measured by reducing sugars,

was estimated by the phenol-sulfuric acid method (34), using

dextran (Sigma) as a standard. Amino acid and amino sugar

compositions of the purified fibrils were determined with an

amino acid analyzer (Model JLC-5AH, JOEL USA, Inc., Cranford,

N.J.). The fibril samples (2 mg) were hydrolyzed in 6 N HC1

(2 ml), phenol (10 vl), and mercaptoethanol (5 pi) at 110C







for 22 h in nitrogen-flushed, sealed vials. After lyophili-

zation, the samples were suspended in distilled water (4 ml),

filtered through glass wool, and analyzed.



Competitive Inhibition Assay

Various sugars and lyophilized preparations of purified

fibrils or crude supernatant obtained by shearing whole cells

as described previously were used in competitive inhibition

assays with strain T14V cells and saliva-treated HA. La-

beled strain T14V cells were suspended in buffered KC1 at a

concentration of 8 x 10 cells per ml. Equal volumes of

buffered KC1 containing various concentrations of sugars,

fibrils, or crude supernatant were used to dilute the

labeled cells to the standard working concentrations of

4 x 107 cells per ml. This mixture was assayed for adher-

ence to saliva-treated HA. Bovine serum albumin (Sigma)

and dextran (MW 18,400, Sigma) were used as controls to

examine non-specific competitive inhibition. Additional

controls consisted of strain T14V cells diluted with buf-

fered KC1 alone.



Antisera Adsorptions

In some experiments the goat and rabbit anti-strain T14V

serum was adsorbed with strain T14V whole cells, purified

fibrils, or cell walls as described below. Strain T14V cells

were grown in TSB, harvested, washed twice and suspended in







the buffered KC1 used in the adherence assay. Cell concen-

tration was determined with a Spectronic 20 as previously

described. A 5 ml aliquot of goat anti-Tl4V serum was

adsorbed with 0.25-18.3 mg of fresh strain T14V cells for

60 min at 37C. The adsorbent was removed by centrifugation

at 48,300 x g for 10 min. The adsorbed serum was stored at

-30C for subsequent use. In order to relate cell number

to dry weights, aliquots of known numbers of fresh washed

whole cells used for the adsorption were lyophilized and

weighed. An aliquot of goat anti-Tl4V serum was also

adsorbed with various amounts of strain T14V cell walls for

60 min at 37C. Cell walls were removed by centrifugation

at 48,300 x g for 20 min. The adsorbed serum was stored at

-30C.

Goat anti-Tl4V serum was adsorbed with purified fibrils

immobilized on Sepharose beads. Fibrils were convalently

linked to CNBr activated Sepharose beads (4B, Sigma) by

methods previously described (3, 28). With this procedure

approximately 75% of a 10 mg sample of fibrils attached to

5 g of beads as measured by optical density at 280 nm. A

5 ml aliquot of serum was incubated with fibril-Sepharose

beads (FSB) for 60 min at 37C. The FSB adsorbent was re-

moved by centrifugation at 225 x g for 1 min. The adsorbed

serum was stored at -30C for subsequent use. The FSB rea-

gent was reactivated by washing twice with 0.05 M potassium

phosphate buffer, pH 2.3, and twice with 0.5 M potassium

phosphate buffer, pH 7.2. Goat anti-Tl4V serum adsorbed with







Sepharose beads without fibrils behaved similarly to non-

adsorbed anti-Tl4V serum in all experiments.

Rabbit anti-Tl4V serum was also adsorbed with the FSB

reagent as described above. Due to the high titer of the

rabbit anti-Tl4V serum, the same 5 ml aliquot was adsorbed

3 times with the FSB reagent. After each adsorption, anti-

fibril specific antibody was eluted from the'immobilized

fibrils by two 5 ml washes of 0.05 M potassium phosphate

buffer, pH 2.3, and diluted immediately with 10 ml of 0.05 M

potassium phosphate buffer, pH 7.2. The anti-fibril anti-

body from the 3 adsorptions was pooled, concentrated to 5 ml

by ultrafiltration (PM-30 filter, Amicon Corp. Lexington,

Mass.), and stored at -30C.

Normal goat and rabbit sera (Grand Island Biologic Co.,

Grand Island, N.Y.) were adsorbed similarly by all the above

adsorbents and used as controls.



Antisera Inhibition Assay

Adherence assays with various antibody preparations

included in the cell-HA suspensions were done. Strain T14V

77
cells suspended at 8 X 10 cells per ml were diluted to the

standard cell concentration (4 X 10 cells per ml) with an

equal volume of buffered KC1 containing a 1:4 dilution of

one of the antibody preparations or normal serum. Micro-

scopic examination of the reaction mixtures revealed that

significant clumping had not occurred at the cell concentra-

tions and serum dilutions used. This serum dilution was




33


used in each antibody experiment unless indicated. Data

are expressed as percent adherence of control cells.



In Vivo Experiments

To determine whether in vitro bacterial adsorption

mimicked adsorption to human teeth in vivo, adsorption by

A. viscosus streptomycin-resistent strains T14VJ1 and

T14AVTl were compared for four human subjects. Both organ-

isms were cultured overnight in TSB. Organisms were harvest-

ed by centrifugation, washed twice, and suspended in saline

at a concentration of 4.0 X 109 cells per ml. Equal volumes

of each cell suspension were mixed together, and dilutions

of the mixture were plated in duplicate on triptic soy agar

(BBL Microbiology Systems, Cockeysville, Md.) plates con-

taining 200 pg of streptomycin per ml. These were incubated

at 37C. Proportions of colony-forming units of each strain

in the mixture were determined by colony morphology and

growth characteristics in TSB inoculated with one colony,

and further confirmed by antigen extraction by the method

of Lancefield and Perlmann (88). Antigens present in the

extracts were detected by Laurell Rocket Osserman immuno-

electrophoresis (91, 123) as modified by Powell et al. (132).

The six maxillary anterior teeth of four adult volun-

teers were cleaned by careful tooth brushing, and 1 ml of

the actinomyces mixture was placed into the mouth of each

subject. After 5 min, the mixture was expectorated, and the

subjects thoroughly rinsed their mouths with water. The







buccal surfaces of three individual teeth on the right side

were sampled by forceful rubbing with calgiswabs (Inolex

Corp. Glenwood, Ill.) 15 min later (172). The three anterior

teeth on the left side were sampled with calgiswabs after

4 h.

All swabs were immediately placed in 2 ml of saline

containing 1% Trypticase (Difco), and the actinomyces were

dispersed for 1 min by a Vortex mixer. Dilutions of the

resulting suspensions were plated in duplicate on tryptic

soy agar plates containing 200 pg of streptomycin per ml as

described above. The relative proportions of colony-forming

units of strain T14VJ1 to T14AVT1 recovered from teeth were

multiplied by the reciprocal of their proportions in the

mixture introduced into the mouth to reflect equal opportun-

ity of attachment (96, 171).














RESULTS


Bacterial Adsorption to Treated
and Untreated HA

The adsorption of A. viscosus strain T14V cells to

saliva-treated HA was greater than to untreated HA (Table 1).

This difference was not evident at low concentrations, but

at initial concentrations of 4.0 X 107 cells per ml substan-

tially more cells of strain T14V adsorbed to saliva-treated

HA. In contrast, the adsorption of strain T14AV cells to

saliva-treated HA was lower than to untreated HA at all

initial cell concentrations tested. The number of strain

T14V cells adsorbed to saliva-treated HA was 4- to 5-fold

higher than the number of strain TI4AV cells adsorbed to

saliva-treated HA at initial concentrations of 4.0 X 10

cells per ml. The number of cells of both strain T14V and

T14AV adsorbed to untreated HA was similar at all initial

cell concentrations. Because the maximum difference in

adherence to saliva-treated HA between strains T14V and

T14AV occurred at 4.0 X 10 cells per ml and a saturated

monolayer of cells was observed, 4.0 x 10 cells per ml was

utilized to study the strain T14V adherence mechanism.

Scanning electron micrographs (Fig. 1) of HA beads allowed

visual observation of the differences in the adherence of


I









Table 1

Adsorption of A. viscosus to HA


Cells adsorbeda
Concn of A. viscosus T14V added Concn of A. viscosus T14AV added
(cells/ml) b (cells/ml) b
HA pretreatment 4 x 105 4 x 106 4 x 107 2 x 106 4 x 106 4 x 107


Untreated 4.4 + 0.2 47.6 + 1.2 200 + 8 20.0 + 0.8 35.2 + 0.8 236 + 12

Saliva 4.0 + 0.1 43.2 + 0.8 375 + 12 8.0 + 1.2 8.4 + 2.0 86 + 10

80% Saliva + 20% serum 4.0 + 0.1 55.2 + 0.4 400 + 36 3.2 + 1.2 4.8 + 0.8 68 + 4

20% Saliva + 80% serum 4.4 + 3.6 48.8 + 1.2 256 + 4 0.8 + 0.8 1.6 + 1.6 24 + 4

100% Serum 94 + 10

20% Saliva + 80% buffer 393 + 1

0.5% Albumin 1.6 + 0.2 22.0 + 3.6 120 + 4

7.0% Albumin 3.2 + 1.2 16.0 + 2.4 124 + 4

a Values indicate cells (x 105) + standard error per 40 mg of HA.

bFixed concentrations indicated were added to tubes with total voluiie of 1.6 ml.


















































Figure 1. Scanning electron microscopy of A. viscosus to
HA. Strain T14V to A) saliva-treated HA and
B) untreated HA; Strain T14AV to C) saliva-
treated HA and D) untreated HA. Bar in each
micrograph represents 2 im.








both strains T14V and T14AV to saliva-treated and untreated

HA.

The pellicle in and around the gingival crevice may

contain serum components, which might serve as binding sites,

in addition to salivary constituents (58). Therefore,

saliva/serum mixtures or serum alone were also used to form

experimental pellicles. The addition of high concentrations

of serum to saliva further reduced the number of strain T14AV

cells which adsorbed to HA compared with HA treated with

saliva alone (Table 1). The number of strain T14V cells ad-

sorbed to HA treated with a mixture of 80% saliva/20% serum

was similar to the number of cells adsorbed to HA treated

with saliva alone. Moreover, HA treated with 20% saliva in

buffered KCl adsorbed a similar number of strain T14V cells

to that adsorbed by HA treated with saliva alone. However,

HA treated with a 20% saliva/80% serum mixture adsorbed a

fewer number of cells, suggesting that serum masks or blocks

the salivary binding sites. When HA was treated with

100% serum, strain T14V adsorption was substantially reduced.

These experiments were repeated with saliva and serum from

different collection periods with identical results. Ad-

sorption of strain T14V cells was inhibited to a similar

extent by treatment of HA with either 0.5 or 7.0% albumin

(Table 1). The number of strain T14V cells adsorbed to

serum or albumin treated HA was dramatically less than the

number of strain T14V cells adsorbed to saliva-treated HA,








jesting that these cells interact specifically with ad-

rbed salivary components rather than serum components.


Influence of Adsorbed Salivary Components
to HA Surfaces on Adsorption Isotherms

The differences in adsorption of strain T14V and T14AV

to treated and untreated HA were also apparent when data

were plotted as isotherms (Fig. 2). Isotherms of strain

T14V to saliva-treated and 80% saliva/20% serum-treated HA

appeared nearly identical (Fig. 2A and B) and followed

Langmuir kinetics. Adsorption of strain T14V to untreated

or to 20% saliva/80% serum-treated HA and of strain T14AV

to untreated or saliva-treated HA also follows Langmuir

kinetics (Fig. 2A and B). Adsorption of strain T14V to

albumin-treated HA (Fig. 2B) was dramatically less than its

adsorption to any other treated or untreated HA surface. It

was confirmed by SEM that adsorption of strain T14V to

saliva-treated HA was limited to a monolayer over the range

used to generate the isotherms. At initial concentrations

equal to or less than 4.0 X 107 cells per ml, SEM observa-

tion demonstrated that monolayer adsorption had occurred

8
(Fig. 3A and B). At concentrations of 4.0 X 10 or greater,

cell to cell interactions and the formation of multiple

layers of cells were observed (Fig. 3C and D).

Graphic plots of C/Q versus C, derived from the adsorp-

tion isotherms, generally resulted in straight lines for

the various HA treatments (data not shown). The bacterial











1000


10-


I
1000


100




I0


100


1000


CELLS NOT ADSORBED/ML x 10s


Figure 2.


Adsorption isotherms of A. viscosus T14V and
T14AV to saliva-treated, untreated, and saliva/
serum-treated HA. (A) Strain T14V adsorption to
saliva-treated (0) and untreated (U) HA. Strain
T14AV adsorption to saliva-treated (0) and un-
treated (0) HA. (B) Strain T14V adsorption to
80% saliva/20% serum (0), 20% saliva/80% serum
(M), and to 0.5% (i*) and 7.0% (*) albumin-treat-
ed HA. Strain T14AV adsorption to 80% saliva/
20% serum (0) and 20% saliva/80% serum (0)-
treated HA.


A








.:'

/
**
// ,~
-~
B


100
















































Figure 3.


Scanning electron microscopy of various
concentrations of A. viscosus T14V adsorbed to
saliva-treated HA. (A) Initial concentration,
4.0 X 106 cells/ml; (B) initial concentration,
4.0 X 107 cells/ml; (C) initial concentration,
4.0 X 108 cells/ml; (D) initial concentration,
4.0 X 109 cells/ml. The bar represents 1 pm
on all micrographs.







affinity for a surface as well as the number of binding sites

available on the surface for that bacterium can be calculated

from the slope and y-intercept of the line. The generally

high correlation coefficients of experimentally derived data

indicate that the previously described mathematical model

delineates the adsorptive behavior of the organisms studied

to a satisfactory degree (Table 2). This did not hold true

when a high percentage of cells did not adsorb to the HA, as

was observed for adsorption of strain T14AV to saliva/serum-

treated HA (Table 2).

The calculated number of binding sites (parameter N)

for both strains T14V and T14AV cells to saliva-treated HA

were similar to those for untreated HA (Table 2). The

strength of the bacterial adsorption bonds (parameter K) to

untreated HA was similar for both strains T14V and T14AV.

The affinity of strain T14V for saliva-treated HA was

10-fold greater than the affinity of strain T14AV for the

same surface. The affinity of strain T14V for HA treated

with saliva/serum mixtures was more than 100-fold greater

than that calculated for strain T14AV cells to those sur-

faces.



Adsorption of A. viscosus to Human
Teeth In Vivo

Higher cell numbers of strain T14VJI than strain T14AVT1

adsorbed to human teeth in vivo (Table 3). There was an

average of 6.3-fold more cells of strain T14VJ1 recovered








Table 2
Estimates of Affinities and Adsorption Sites of A. viscosus T14V and T14AV on HA



Na Kb Correlation coefficients

T14V T14AV T14V T14AV T14V T14AV

Untreated 2.4 3.4 2.0 1.0 0.97 0.97

Saliva 3.3 3.3 1.0 0.11 0.88 0.93

80% Saliva + 20% serum 5.0 12.0 2.0 0.016 0.96 0.67

20% Saliva + 80% serum 3.3 Nc 2.0 N 0.96 N

0.5% Albumin 1.0 1.0 0.85


a Each value (x 10 7) indicates number of adsorption sites per 40 mg of HA.

bEach value (x 10- 7) indicates affinity constant in milliliters per cell.

CN, Not determinable due to low percentage of adsorption.








Table 3


Adsorption of A. viscosus T14VJ1 and T14AVT1 to Human Tooth Surfaces


Adsorption

0.25 h 4.0 h
Subject
Strain Strain Strain Strain
T14VJ1 T14AVTl T14VJ1 T14AVT1


1 2.18 + 0.17 0.47 + 0.20 0.46 + 0.24 0.098 + 0.074

2 1.80 + 0.20 0.50 + 0.01 0.66 + 0.24 0.050 + 0.010

3 4.6 + 2.0 0.66 + 0.24 3.2 + 1.8 0.24 + 0.19

4 6.0 + 3.0 0.06 + 0.10 3.3 + 1.7 0.43 + 0.37


aEach value indicates number of cells (x 10 ) + standard error and is the mean of
three teeth sampled in each subject, at the indicated times after organisms were
introduced.








from all teeth as compared with strain Tl4AVTl 15 mmin after

introduction into the mouth of a bacterial mixture contain-

ing equal amounts of organisms. An average of 10-fold more

strain T14VJ1 cells than strain T14AVT1 cells were isolated

from all tooth samples taken at 4 h. Therefore, during the

4-h time period, the numbers of avirulent strain T14AVT1

cells, which were adsorbed to the teeth, decreased to a

greater extent than did the numbers adsorbed virulent strain

T14VJ1 cells.



Influence of Ionic Environmental Parameters
on A. viscosus Adsorption

The influence of the buffered KC1 pH on adsorption of

strains T14V and T14AV to saliva-treated and untreated HA

is shown in Figure 4. Varying the pH of the buffered KC1

did not dramatically influence strain T14V adsorption to

saliva-treated HA. However, adsorption of strain T14AV to

saliva-treated HA was enhanced by approximately 80% as the

pH of the suspending buffer decreased from 7.0 to 5.7.

Adsorption of both strains to untreated HA decreased slight-

ly as the pH increased to 7.1. Varying the pH of the saliva

(pH 6.0-8.0) prior to the formation of the pellicle on HA

did not alter the adsorption of either strain to saliva-

treated HA (data not shown).

Increasing the ionic strength of the buffered KC1,

decreased the adsorption of strain T14V to both saliva-

treated and untreated HA (Table 4). Adsorption of strain












-J
0

02

00

U.\
0-


zIO __ _
w

0:13
UJ

a 5 6 7 8 9

pH






Figure 4. Influence of pH on adsorption of A. viscosus
strain T14V to A) saliva-treated TO) and
B) untreated HA (0) and of strain T14AV to
C) saliva-treated (0) and D) untreated HA (0).














Table 4

Influence of Ionic Strength on
of A. viscosus to HA


Adsorption


% Adherence of Controla

T14V T14AV

KC1(M) Saliva- Saliva-
treated Untreated treated Untreated


0.001 150 + ib 167 + 10 112 + 13 63 + 1

0.01 136 + 1 160 + 11 133 + 7 102 + 2

0.10 96 + 8 109 + 6 86 + 10 103 + 1

0.50 81 + 8 96 + 10 58 + 11 119 + 9

1.00 73 + 4 52 + 2 55 + 7 103 + 1

2.00 77 + 5 57 + 4 36 + 2 76 + 1


control is buffered KC1.

bMean + standard deviation.








T14AV to saliva-treated HA diminished as the KCl molarity

increased. However, adsorption of strain T14AV to untreat-

ed HA was enhanced as the ionic strength increased up to

0.50 M, but then decreased when cells were suspended in

higher salt buffers.

Adsorption of strain T14V to saliva-treated HA dimin-

ished slightly as the calcium concentration of the buffered

KCl increased (Table 5). Strain T14AV adsorption to saliva-

treated HA was greater when cells were suspended in 5 x 10

M calcium than at lower molar concentrations. Adsorption

of both strains T14V and T14AV to untreated HA was not sig-

nificantly altered by variations in the calcium concentra-

tion. Pretreatment of strain T14V and T14AV with 0.1 M

ethylenediamine-tetraacetic acid at pH 8 had no effect on

adherence in reaction mixtures containing PBS without Ca2+
2+
or Mg

The effect of various ions on the adsorption of strain

T14V to saliva-treated HA is shown in Table 6. The addition

of K Ca 2+, or Mg2+ singly or in any combination to the

phosphate buffer significantly decreased the adsorption to

a similar extent.

Strain T14V and T14AV adsorption to untreated HA in-

creased when the cationic detergent CTMAC was included in

cell-HA suspensions or when cells were pretreated with CTMAC

(Table 7). Strain T14V adsorption to saliva-treated HA

drastically decreased when CTMAC was included in the cell-

HA suspensions whereas pretreatment of this strain with the














Table 5

Influence of Calcium Concentration on Adsorption
of A. viscosus to HA



% Adherence of Control a

T14V T14AV

Ca 2+(mM) Saliva- Saliva-
treated Untreated treated Untreated


0.0 111 + 6b 95 + 3 NDc ND

0.5 97 + 5 92 + 8 111 + 10 77 + 1

1.0 100 + 1 100 + 1 100 + 3 100 + 3

2.0 88 + 1 94 + 7 ND ND

5.0 68 + 5 84 + 4 171 + 25 94 + 5


aControl is buffered KC1.

bMean + standard deviation.

cND, not done.














Table 6

Influence of Ions on Adsorption of
A. viscosus T14V to Saliva-Treated HA


Ions included in buffer


None (Control)

50 mM KC1

1 mM CaCl2

0.1 mM MgC12

50 mM KC1, 1 mM CaCl2

50 mM KC1, 0.1 mM MgCl2


% Adherence of Control


100 + ib

60 + 1

64 + 1

59 + 9

67 + 1

57 + 4


a1 mM potassium phosphate buffer, pH 7.2.

bMean + standard deviation.














Table 7

Influence of Detergents on Adsorption
of A. viscosus to HA


% Adherence of Control

T14V T14AV

Cell treatment Saliva- Saliva-
treated Untreated treated Untreated

Untreat-
ed (Control) 100 + 1a 100 + 1 100 + 1 100 + 1

CTMACb 4 + 2 184 + 3 126 + 5 154 + 1

CTMAC-Bufferc 158 + 1 236 + 1 188 + 7 126 + 4

SDSd 140 + 4 82 + 4 107 + 4 52 + 8

SDS-Bufferc 128 + 14 128 + 1 118 + 1 90 + 4

Tween 80e 99 + 4 105 + 3 82 + 1 89 + 6


Mean + standard deviation.

bCTMAC, cells suspended in 0.10% cetyltrimethylammonium
chloride.
CCTMAC-Buffer, SDS-Buffer, cells suspended in detergent,
then washed with buffered KC1 and resuspended in buffered
KC1.
dSDS, cells suspended in 0.01% sodium dodecylsulfate.

eTween 80, cells suspended in 1.0% Tween 80.








same detergent produced a marked increase in cell numbers

adsorbed over control cell numbers. Strain T14AV adsorption

to saliva-treated HA increased slightly in the presence of

CTMAC and after pretreatment of cells with the detergent.

Strain T14V adsorption to saliva-treated HA increased

when SDS was included in the cell-HA suspension or when

cells were pretreated with the detergent (Table 7). Ad-

sorption of strain T14V to untreated HA or of strain T14AV

to saliva-treated HA was not influenced by the SDS treat-

ments. Pretreatment of strain T14AV did not influence ad-

sorption to untreated HA whereas SDS included in the cell-HA

suspension decreased adsorption to that surface.

Neither strain T14V nor strain T14AV adsorption to

saliva-treated or untreated HA was altered by the non-ionic

detergent Tween-80 (Table 7).



Effect of Heat, Proteolytic Enzymes, and Periodate
on Adherence of A. viscosus Cells
to Saliva-Treated HA

Heating A. viscosus of either strain T14V or T14AV

cells at 100C for 15 min reduced their ability to adhere to

saliva-treated HA (Table 8). However, adherence of both

strains was not influenced by 60 or 80C heat for 15 min.

When examined by TEM (Fig. 5), strain T14V cells that had

been heated to 60 or 80C appeared similar to control cells.

In contrast, strain T14V cells heated to 100C lacked the

surface fibrils present on the control cells.













Table 8

Effect of Various Pretreatments of A. viscosus
Cells on Adherence to Saliva-Treated HA



% Adherence of Controla
Pretreatment
T14V T14AV


None (Control) 100 + 1b 100 + 1

60C, 15 min 100 + 2 100 + 1

80C, 15 min 97 + 1 100 + 2

100C, 15 min 29 + 6 47 + 6

0.2% Papain 178 + 1 136 + 3

0.2% Protease 8 + 1 0 + 2

0.2% Trypsin 29 + 2 NDc

0.2% Chymotrypsin 68 + 1 12 + 5

0.4% Chymotrypsin 47 + 2 ND

0.6% Chymotrypsin 29 + 1 ND

1.0% Chymotrypsin 18 + 1 ND

10 mM Periodate 53 + 1 12 + 2


control cells were treated separately for each enzyme.

bMean + standard deviation.

cND, not done.
















































Figure 5.


Transmission electron micrographs of uranyl-
acetate stained preparations of heat-treated
A. viscosus T14V. A) Control, 23C, B) 60C,
C) 80C, and D) 100C.


61A\ *^







Pretreatment of strain T14V or T14AV cells with pro-

tease, trypsin, and chymotrypsin inhibited their subsequent

adherence to saliva-treated HA (Table 8). Pretreatment of

strain T14V or T14AV cells with papain increased their

adherence to saliva-treated HA, indicating that enzyme may

remain bound or alter the cell surface and therefore promote

adherence. Pretreatment of strain T14V with higher concen-

trations of chymotrypsin inhibited its adherence by as much

as 82%, indicating that it also was sensitive to this

enzyme, as was strain T14AV. Pretreatment of both bacterial

strains with periodate inhibited adherence (Table 8). This

suggests that carbohydrate moieties present on the bacterial

cell surface could be associated with adherence. However,

periodate could also cause protein alterations which could

influence adherence. Electron microscopy of the enzyme- and

periodate-treated strain T14V cells did not reveal any

morphological changes from control cells (data not shown).



Competitive Inhibition of Adherence

Various sugars were included into the bacteria HA

suspension to examine direct inhibition of strain T14V

adherence to saliva-treated HA (Table 9). There was little,

if any, inhibition by any of the various hexoses and disc-

charides tested. Fructose, glucosamine and N-acetyl glucos-

amine did inhibit slightly at 0.1 M concentrations.

However, when glucosamine and N-acetyl glucosamine concen-

trations were increased to 0.5 M, percent inhibition of














Table 9

Competitive Inhibition of A. viscosus T14V to
Saliva-Treated HA with Sugars



Sugar % Inhibition of Control


None (Control) 0 + 1

0.1 M Glucose 0 + 1

0.1 M Galactose 6 + 1

0.1 M Mannose 2 + 1

0.1 M Sucrose 5 + 1

0.1 M Maltose 0 + 1

0.1 M Lactose 0 + 1

0.1 M Rhamnose 7 + 1

0.1 M Fructose 13 + 1

0.1 M Galactosamine 0 + 1

0.1 M Glucosamine 16 + 2

0.5 M Glucosamine 20 + 2

0.1 M N-acetylglucosamine 11 + 1

0.5 M N-acetylglucosamine 23 + 1


aMean + standard deviation.







strain T14V to saliva-treated HA did not increase signifi-

cantly.

Crude supernatants obtained by sonication, Braun

homogenization, or French press extraction of whole strain

T14V cells were included into the bacterial cell-HA sus-

pension to examine direct inhibition of strain T14V ad-

herence to saliva-treated HA by cell-surface components

released by those techniques (Table 10). At a concentration

of 1 mg per ml, the French press extract of strain T14V in-

hibited adherence to a greater degree than did the extracts

obtained by sonication or Braun homogenization. Therefore,

the French press extract was studied further for the pres-

ence of a specific adherence inhibiting structure. An

inhibiting component, isolated from the extract, was subse-

quently identified as strain T14V cell-surface fibrils.

Purified fibrils or crude French press supernatant were

included in the bacterial cell-HA suspension to examine

direct inhibition of strain T14V adherence to saliva-treated

HA (Fig. 6). Increasing the amount of purified fibrils or

crude supernatant inhibited adherence of whole cells up to

a maximum of 30%. To assess the influence of nonspecific

inhibition of adherence, bovine serum albumin and dextran

were included in bacterial cell-HA suspensions in quantities

similar to those tested for fibrils and crude supernatant.

At the highest concentration of BSA or dextran examined

(2 mg/ml), inhibition was approximately 10%. When the cell














Table 10

Competitive Inhibition of A. viscosus T14V to
Saliva-Treated HA with Whole Cell Extracts


Extract (1 mg/ml)


% Inhibition of Control


Control 0 + 2'

Sonic 16 + 6

Braun Homogenization 17 + 12

French Press 36 + 1


aMean + standard deviation.













100


80


60


40


20


0 I 2


MG/ML



Figure 6. Competitive inhibition of A. viscosus T14V
adherence to saliva-treated HA with French press
crude supernatant (*), purified fibrils (G), bo-
vine serum albumin (0), and dextran (0).







concentration was decreased to 4 X 105 cells per ml, fibrils

(1 mg/ml) inhibited whole cell adherence by 47%.



Adherence Inhibition by Specific Antisera

Goat anti-Tl4V serum inhibited adherence of strain T14V

to saliva-treated HA (Fig. 7). Adherence decreased as the

concentration of antiserum in the bacterial cell-HA suspen-

sion increased. At antiserum dilutions equal to or less

than 3:10, adherence was completely abolished. Normal goat

serum did not diminish adherence of strain T14V cells.



Adsorption of Antiserum Inhibition Activity

The inhibition activity of the goat anti-Tl4V serum

could be adsorbed completely with strain T14V cells or iso-

lated fibrils and to a lesser degree with strain T14V cell

walls (Table 11). Adherence inhibition activity was dimin-

ished in specific antisera as the number of strain T14V

cells used to adsorb the serum increased (Fig. 8). However,

adsorption of antisera with strain T14V cell walls did not

reduce inhibition to the same extent as did whole cells.

Adsorption of normal serum with whole cells or fibrils had

little influence on adherence.


Adherence Inhibition with Antifibril
Specific Antibody and Antiserum

Inhibition of strain T14V adherence to saliva-treated

HA with rabbit anti-T14V serum was similar to goat anti-Tl4V














_J
0
it
I-
z
0
0

U-
0

w
bd
0
z


w
Ll



NO
0-
0U


100



80


60



40



20



0:


2:10


ANTISERA


Figure 7.


4:10

DILUTION


Titration of adherence inhibition activity of
A. viscosus T14V adherence to saliva-treated HA.
Normal goat serum (@); goat anti-Tl4V serum (m).
Undiluted serum contained 133 mg protein per ml.
Dilutions are expressed as ratios of antiserum
volume to total volume.


10


I I I I
I._____ -






- a

U
-



S -

S U -
I I .L I.


&














Table 11

Adsorption of Goat Anti-Tl4V and Normal Sera


Adsorbent


% Adherence of Controla

Normal Anti-Tl4V
Serum Serum


None (Control)b 100 + 2c 12 + 1

T14V Whole Cells (7.5 mg) 87 + 3 95 + 3

Fibrils (7.5 mg) 107 + 1 93 + 1

T14V Braun Cell Walls (7.5 mg) NDd 29 + 3


aSerum was diluted 1:4.

controls with and without normal serum in cell-HA sus-
pensions adhered similarly.
CMean + standard deviation.

dND, Not done.
















0
I.-
M 100
z
0



0
#o.
0

... 50


2 4 6 8
MG ADSORBENT


I0


Figure 8. Adsorption of goat anti-Tl4V serum with A viscosus
T14V whole cells (U) and Braun cell walls (*) and
its subsequent effect on adherence of strain T14V
to saliva-treated HA.








serum inhibition (Table 12). A volume of rabbit antifibril

specific antibody equivalent to the volume of anti-Tl4V

serum inhibited strain T14V adherence to saliva-treated HA.

Undiluted rabbit anti-Tl4V serum contained 73 mg of total

protein per ml whereas undiluted rabbit antifibril specific

antibody contained 3.2 mg of total protein per ml. Adsorbed

or unadsorbed normal serum did not alter adherence. In

addition, inhibition of strain T14V adherence to saliva-

treated HA with rabbit antifibril serum occurred at very

high dilutions of the antiserum (Fig. 9).



Identification and Composition
of Isolated Fibrils

Electron photomicrographs of negatively stained prepara-

tions of purified fibrils (Fig. 10) indicated the relative

morphological homogeneity obtained with the fibril purifica-

tion procedure. Rocket immunoelectrophoresis of purified

fibril preparations demonstrated only one antigen reacting

with rabbit anti-Tl4V serum (Fig. 11). The fibril-specific

antigen was found in French press crude supernatant prepara-

tions in small amounts. The fibril-specific antigens in

both preparations showed identity with one of the virulence-

associated antigens (VA 1) in the whole cell Lancefield

extract described previously by Powell et al. (132). In

addition to the fibril-specific antigen, the crude superna-

tant contained two additional antigens detected by the

anti-T14V serum. One of these showed identity with the other




65








Table 12

Influence of Rabbit Serum on A. viscosus TI4V
Adsorption to Saliva-Treated HA



Seruma % Adherence of Control


Normal b (control) 100 + ic

Anti-Tl4V 10 + 3

Antifibril Specific Antibody 9 + 3


aSerum was diluted 1:4.

controls with and without normal serum in cell-HA suspen-
sions adhered similarly.
cMean + standard deviation.






































Figure 9.


1:1


S2:10
DILUTION


Titration of adherence inhibition activity of
A. viscosus T14V adherence to saliva-treated HA.
Normal rabbit serum (0); rabbit antifibril
serum (W). Dilutions are expressed as ratios
of antiserum volume to total volume.


100


50


0:10
ANTISERA


















































Figure 10. Transmission electron microscopy of uranyl-
acetate stained preparation of A. viscosus
T14V purified fibrils.
































3.


Figure 11.


Laurell rocket immunoelectrophoresis of antigen
preparations from A. viscosus T14V. (1) 250 ug
of Lancefield extract; (2) 200 pg of purified
fibrils in 0.5% Triton x-100; (3) 200 pg of
French press crude supernatant. Rabbit anti-
T14V whole cell serum was used at 50 pl/ml of
agarose. The reference antigen in the Osserman
trough was French press crude supernatant. TC,
top common; VA 1 and VA 2, virulence-associated
antigens 1 and 2, respectively.


T-C




69


virulence-associated antigen (VA 2) of the whole cell as

described by Powell et al. (132). A rapidly migrating

antigen was also found in the crude supernatant, but it

migrated off the gel.

Immunodiffusion experiments in which crude supernatant

and purified fibrils were used as antigen sources also

showed a reaction of identity between one antigen in the

crude supernatant and the purified fibril antigen when devel-

oped against rabbit anti-Tl4V serum or rabbit antifibril

specific antibody (Fig. 12). In addition to the fibril-

specific precipitin line, three additional precipitin lines

were evident when the crude supernatant and anti-Tl4V serum

were reacted. However, only the single precipitin line was

observed in the fibril-antifibril reaction or in the crude

supernatant-antifibril reaction.

Chemical analysis of purified fibrils revealed prepara-

tions composed of 95.2% protein and less than 2% carbo-

hydrate as measured by reducing sugars. Amino acid analysis

(Table 13) showed that fibrils contain high quantities of

aspartic acid, threonine, glutamic acid, and alanine. Basic

amino acids were present in a small quantity (15.8%) whereas

acidic, polar uncharged, and nonpolar amino acids were pres-

ent in approximately equal quantities (25-30%). N-acetyl

muramic acid and N-acetyl glucosamine, as well as other

hexosamines, were not detected in the preparation, indicat-

ing that cell wall material did not contaminate the fibril















































Figure 12. Immunodiffusion of antigen preparations from
A. viscosus T14V. (1) 200 pg of French press
crude supernatant; (2) 200 ig of purified
fibrils; (3) Rabbit antifibril specific anti-
body; (4) Rabbit anti-Tl4V serum.














Table 13

Amino Acid Composition of A. viscosus
T14V Fibrils Obtained from French Press
Shearing and Lysozyme Digestion


Residues per 1000a
Amino Acid
French press Lysozymeb


Acidic (26%) (24%)
Aspartic Acid 137 139
Glutamic Acid 120 102

Basic (16%) (12%)
Lysine 76 95
Arginine 70 20
Histidine 12 8
Ornithine 0 NDc

Polar uncharged (29%) (29%)
Threonine 114 136
Glycine 86 83
Serine 74 35
Tyrosine 23 33
Cystine 0 ND

Non polar (29%) (35%)
Alanine 102 95
Leucine 74 75
Valine 51 62
Isoleucine 28 35
Phenylalanine 23 19
Methionine 11 1
Proline trace 62


aNot corrected for losses.
bObtained from Cisar and Vatter (22).

cND, not done.








preparation. A minimum molecular weight between 15,000-

25,000 for the fibrils was calculated from the amino acid

data.

Molecular sieve chromatography of isolated fibrils

suspended at 1 mg per ml in phosphate buffer produced two

peaks (Fig. 13). The first peak eluted at the void volume

of the column and the second peak to a component correspond-

ing to a molecular weight of approximately 12,500. The high

molecular weight material was observed by TEM to contain

fibrils whereas no structures resembling fibrils could be

visualized by TEM from the low molecular weight fraction

(data not shown). In order to determine if the aggregation

of fibrils was dependent upon the fibril concentration, the

concentration was decreased and then chromatographed. It

was observed that the high molecular weight peak decreased;

however, the low molecular weight peak actually increased

(Fig. 13) suggesting that less aggregation had occurred in

the dilute sample.

Molecular sieve chromatography in the presence of 8 M

urea was used in an attempt to dissociate aggregates of

the fibrils (Fig. 14). Two peaks were observed with the high

molecular weight material eluting at the void volume and the

low molecular weight material corresponding to a molecular

weight of approximately 12,500. The material from these

peaks was examined by IEP. Both peaks showed identity with

each other as well as with the fibrils and the VA 1 antigen
















.10



.05


.10


.05


.20


.10


20 40 60


ML ELUENT


Figure 13.


Molecular sieve chromatography of A) I. Blue
dextran (mw 2 X 106); II Bovine serum albumin
(mw 67,000); III Hen egg albumin (mw 45,000);
IV Chymotrypsinogen A (mw 25,000); V Cytochrome
C (mw 12,500); B) Isolated fibrils (1 mg/ml)
and C) Isolated fibrils (0.33 mg/ml). Samples
were chromatographed over an ultragel-54 column
and eluted with 0.01 M potassium phosphate
buffer (pH 7.2). One ml fractions were col-
lected and absorbance at 280 nm read.


0
CO
N
0
0























A

II -I


B ~ NA- >./\ / ~I./ I


20


40


60


ML ELUENT


Figure 14.


Molecular sieve chromatography of A) I Hen egg
albumin (mw 45,000); II Chymotrypsinogen A
(mw 25,000); and III Cytochrome C (mw 12,500);
and B) Isolated fibrils (1 mg/ml). Samples
were chromatographed over an ultragel-54 column
and eluted with 0.01 M Tris buffer (pH 8.4)
containing 8 M urea.


.20


.10


0
N
0
0


.10


.05








of strain T14V Lancefield extracts when the antigen prepara-

tions were subjected to electrophoresis into gel containing

either anti-Tl4V serum (Fig. 15).

Fibril preparations subjected to PAGE-SDS produced gel

patterns that contained numerous bands (Fig. 16A). The high

and low molecular weight fractions obtained from molecular

sieve chromatography were subjected to PAGE-SDS. The high

molecular weight fraction (Fig. 16B) produced a pattern

similar to the pattern produced by the fibrils. The low

molecular weight fraction produced a pattern of very light

bands that also was similar to the pattern of the fibrils

(data not shown). However, the low molecular weight frac-

tion did not contain the large dark band seen at the top of

the PAGE gels containing the fibril or high molecular weight

preparations.






































- 41


Figure 15.


Laurell rocket immunoelectrophoresis of antigen
preparations from A. viscosus T14V. (1) 250 ug
Lancefield extract; (2) High molecular weight
peak I from urea molecular sieve column; (3) Low
molecular weight peak II from urea molecular
sieve column; (4) 200 pg fibrils in 0.5% Triton
X-100. Rabbit anti-Tl4V whole cell serum was
used at 50 pi/ml of agarose. The reference
antigen in the Osserman trough was Lancefield
extract.


















































Figure 16. Polyacrylamide gel SDS electrophoresis of
(A) 50 pg of purified fibrils and (B) 50 pg
of high molecular weight material from
molecular sieve chromatography.














DISCUSSION


Adsorption properties of the virulent A. viscosus strain

T14V were observed to be very different from those of its

avirulent variant strain T14AV. The adsorption of strain

T14V to all surfaces studied appeared to follow Langmuir

kinetics, as has been previously reported for other oral

bacteria (1, 24, 53). The observation that adsorption iso-

therms of strain T14V to saliva-treated, untreated, and 20%

saliva/80% serum-treated HA were generally similar indicated

that adsorption to these three different surfaces followed

similar kinetics. In addition, strain T14AV adsorption to

untreated and saliva-treated HA also followed Langmuir

kinetics, although the adsorption kinetics to saliva-treated

HA was of a lower order. Strain T14AV exhibited poor ad-

sorption to HA treated with mixtures of saliva/serum, and

the data exhibited a poor fit to the adsorption model, as

judged by the low correlation coefficient. Application of

the adsorption model requires that a significant percentage

of the cells initially available adsorb to the surface (53).

Since only 6% or less of strain T14AV cells initially avail-

able adsorbed to the saliva/serum HA surface, it is not

likely that these data adequately described its adsorption.








It is important to note that Langmuir kinetics are limited

to monolayer adsorption to a surface (90). We have con-

firmed by scanning electron microscopy that a monolayer of

strain T14V cells adsorbed to these HA surfaces from initial
7
cell concentrations up to and including 4 X 10 cells per ml.

However, at concentrations of 4 X 10 cells per ml and great-

er, cell to cell interactions were observed which lead to

the formation of multiple cell layers on the HA surface.

Dispersed cells of A. viscosus, A. naeslundii strains, and

other oral strains are known to reaggregate rapidly (41, 58,

121, 132). Aggregation between homologous or heterologous

bacterial cells has been implicated in the accumulation

phases of developing plaque (58). However, the mechanisms

by which cells interact with adsorbed salivary components

are not necessarily the same as those involved in cell to

cell aggregation (50). The series of photomicrographs

(Fig. 3) illustrate that at saturating cell densities of

strain T14V, cell to cell aggregation can occur. These

aggregates may not be distinguishable in the in vitro ad-

herence assay from initial attachment of the cell to the HA

surface. Although the degree of cell to cell aggregation

may vary from strain to strain, this possibility should not

be overlooked when studying the kinetics of the initial

attachment of cells to HA or other surfaces.

Calculations made from adsorption isotherms showed

that HA treated with saliva or saliva/serum mixtures pro-

vided a slightly greater number of binding sites for strain








T14V than did untreated HA. Although the strength of the

adsorption bond between cells of strain T14V and the saliva

or saliva/serum-treated surfaces was' equal to or half that

calculated for the untreated HA surface, similar numbers of

strain T14V cells adsorbed to untreated and saliva/serum-

treated HA from fixed cell concentrations of 104 (data not

shown) and 10 cells per ml. Substantially more cells ad-

sorbed to saliva or saliva/serum-treated HA surfaces from

concentrations of 10 cells per ml. This is consistent with

a previous observation that cell adsorption at higher

initial cell concentrations was more closely related to the

maximum number of binding sites, whereas at low concentra-

tions the cell adsorption was influenced by the affinity of

the cell for the surface (24). Although the numbers of

binding sites for strains T14V and T14AV are similar, the

affinity of strain T14AV for the saliva-treated surface was

10-fold less than that calculated for strain T14V. This

suggests that the feeble adsorption observed for strain

T14AV is due to its relatively poor affinity for the saliva-

treated HA surface. Assuming that both the virulent parent

strain and the avirulent variant strain compete for similar

binding sites on the saliva-treated HA surface, the adsorp-

tion model predicted that a decrease in adsorption would

result from a reduced affinity between strain T14AV and the

saliva surface rather than an alteration in the number of

adsorption sites on the HA surface. The data support this

prediction. Strain T14V also adsorbed as well to HA treated








with a mixture of 80% saliva/20% serum as to HA treated with

20% saliva in buffer, but did not adsorb well to serum or

albumin-treated HA. This suggests that strain T14V cells

interact specifically with adsorbed salivary components and

not serum components.

It is significant that relative in vitro adsorption

specificites exhibited by A. viscosus strains T14V and T14AV

to saliva and saliva/serum HA surfaces were confirmed in

vivo in humans by determining recoverable cell numbers from

teeth after introduction of streptomycin-resistant organisms

of both strains, indicating that this in vitro model mimics

the adsorption specificities of human teeth in vivo. In vivo

results showed that cell numbers of strain T14V averaged

10-fold higher than those for strain T14AV. In addition,

the recovery of strain T14AV was less than that observed for

strain T14V, indicating that strain T14AV exhibited a weaker

association with the tooth surface. Thus, observations based

on both the in vitro adsorption model and in vivo studies in

humans indicate that strain T14AV exhibits a feeble affinity

for adsorbed salivary components relative to strain T14V.

The adsorption properties of strain T14V to saliva-

treated HA were very different from those of strain T14AV

for the same surface when the pH of the suspending buffer

was varied. The pH did not alter the adsorption of strain

T14V to saliva-treated HA whereas strain T14AV adsorbed much

better under acidic conditions. Several authors have pro-

posed that plaque formation and plaque cohesion occur








via hydrogen bonding between the hydroxyl groups of bacterial

polysaccharides and the acidic proteins in the salivary

pellicle (78, 134). However, no direct evidence is avail-

able to support this mechanism. It has recently been re-

ported that under specific growth conditions, strain T14AV

cells produced large amounts of extracellular heteropoly-

saccharide and possessed a microcapsule composed of the

heteropolysaccharide (14, 132). The increase in strain T14AV

adsorption to saliva-treated HA in an acidic environment

could be attributed to hydrogen bonding between the poly-

saccharide capsule and the salivary pellicle. In contrast,

an acidic environment does not effect the adsorption of

strain T14V to saliva-treated HA because strain T14V neither

produces the large amounts of extracellular polysaccharide

nor possesses a microcapsule (132). In addition, isolated

strain T14AV extracellular heteropolysaccharide inhibited

strain T14V adherence to saliva-treated HA (14, 131) indicat-

ing that the polysaccharide material does appear to have some

affinity for the salivary pellicle.

The mechanism of bacterial adsorption to surfaces could

involve electrostatic interactions. Adsorption to hydroxy-

apatite of polyanions such as negatively charged bacteria

has been observed to be inhibited by phosphate or fluoride

(137) and to be enhanced by cations such as calcium (12, 137).

The negatively charged surfaces of bacteria maintain electro-

neutrality by sodium and potassium serving as counter ions








(135). Calcium, the most abundant divalent cation in human

saliva (104), can displace the monovalent potassium and so-

dium ions and create a situation which favors the adsorption

of bacteria to the negatively charged pellicle. Therefore,

the negatively charged salivary pellicle and bacterium are

bridged together by divalent calcium cations. It was ob-

served that adsorption to saliva-treated HA by strain T14AV

was increased by high concentrations of calcium as would be

predicted. It has been observed that sucrose-grown S.

mutans strains exhibit an increased negative charge (78, 120,

136) due to insoluble polysaccharides completed with extra-

cellular teichoic acid and that these sucrose-grown strains

bond high amounts of calcium (78, 120). This mechanism has

been used to explain, in terms of electrostatic interaction,

the selective adsorption of S. mutans to teeth (79). As

discussed previously, strain T14AV possesses a heteropoly-

saccharide microcapsule (132) which could allow for an in-

creased negative charge and thus more completing with calcium

cations. This would result in more strain T14AV cells bound

to the salivary pellicle.

Contrary to strain T14AV, strain T14V adsorption to

saliva-treated HA decreased in the presence of high concen-

trations of calcium. Recently, it has been reported that

elevated levels of calcium inhibited S. mutans adherence to

saliva-treated HA (157). In addition, Fischman et al. (45)

reported that cations which bind to both bacteria and the

tooth surface will inhibit bacterial adsorption to teeth.








Therefore, strain T14V adsorption could be inhibited as a

result of calcium binding to both the salivary pellicle on

the HA and to the bacterial cell surface. The result that

calcium cations could inhibit the adsorption of strain T14V

to saliva-treated HA was supported by the observation that

high concentrations of potassium also inhibited the adsorp-

tion. In addition, it was observed that the presence of

potassium, calcium, or magnesium cations significantly re-

duced the adsorption of strain T14V to saliva-treated HA

compared to a buffer (PBS) that did not contain any of the

ions.

Since EDTA in the suspending buffer did not alter strain

T14V adsorption to saliva-treated HA, calcium ions do not

appear to be required. Several studies concerning the bio-

chemistry of plaque formation have suggested that calcium

cations mediate an important role in plaque formation. It

has been reported that EDTA is capable of deaggregating

dental palque (78). In addition, calcium ions have been

shown to increase adsorption of mucins to glass and to HA

(70, 115). Calcium ions appear to be required for the aggre-

gation of both S. mitis and S. sanguis since the addition of

EDTA inhibits aggregation (56, 76). Moreover, it has been

reported that coaggregation of A. viscosus/A. naeslundii

strains with streptococcal strains (21) and hemagglutination

by A. viscosus/A. naeslundii strains (38) are dependent on

calcium. However, the mechanism of A. viscosus coaggregation








with streptococci must differ from the mechanism of strain

T14V adsorption to saliva-treated HA since calcium does not

appear to be necessary for adsorption to saliva-treated HA.

The use of detergents in adsorption assays produced

additional data concerning electrostatic interactions of

A. viscosus with the salivary pellicle. Detergents contain

molecules which possess covalently linked lipophilic and
h
hydrophilic moieties. When these agents are in solution,

they concentrate at available surfaces, including bacteria,

with the lipophilic portion away from the polar solvent and

the hydrophilic portion toward the solvent. The ionization

of the hydrophilic portion classifies these detergents into

cationic, anionic and non-ionic. The non-ionic detergents

carry both an anion and a cation. The adsorption of strain

T14V to saliva-treated HA was not influenced by the presence

of the non-ionic detergent, Tween-80. However, the anionic

detergent, SDS, increased strain T14V adsorption. The reason

for this is unknown; however, the SDS could increase the

bacterial surface charge and provide new binding sites to

which divalent cations such as calcium could then utilize

to bridge the bacterium to the salivary pellicle. To

answer this question, assays in SDS containing buffers both

with and without calcium present, as well as EDTA, need

to be done.

The cationic detergent, CTMAC, may provide the most

useful and interesting detergent data. Cationic detergents

have been known to possess bactericidal properties (20, 92).








Cationic detergents are also of interest because of potential

reversible binding to anionic groups on oral muscosal sur-

faces (11, 109, 143, 153, 155) which could permit prolonged

oral retention of cationic antiseptics. The quarternary

ammonium compounds such as CTMAC are permanent cations due

to substitution of all four hydrogens of the ammonium cation.

In addition, the long lipophilic group (16 carbons) of CTMAC

increases the antibacterial activity of the agent (20).

Recent studies have demonstrated that several quarternary

ammonium detergents, such as chlorohexidine, possess in vivo

and in vitro antiplaque activities (8, 33, 62, 159, 168).

In an aqueous environment such as the oral cavity, the

lipophilic portion of the molecule would force these drugs

to any available non-aqueous surface such as bacteria or

saliva-treated HA (6). The cationic moiety would then bind

the molecule to the negatively charged surfaces electro-

statically. This model would fit that proposed by Rolla and

Melsen (138) for the action of chlorohexidine. It was ob-

served that the presence of CTMAC in strain T14V and saliva-

treated HA suspensions completely obliterated any bacterial

adsorption. However, when strain T14V cells were pretreated

with the same detergent prior to their use in the adherence

assay, adsorption of the organism increased. These data fit

the model discussed above (6) such that the CTMAC, when in-

cluded in the bacterial-HA suspension, bound to both the

bacterial and saliva-treated HA surface to inhibit any ad-

sorption. However, when the bacteria were pretreated with








the detergent, the detergent bound in a fashion that exposed

the hydrophilic cationic end of the molecule and allowed

the organism to adhere in greater numbers to the negatively

charged pellicle. Recent reports have shown that another

cationic detergent, cetyltrimethylammonium bromide, will

inhibit in vitro plaque formation of A. viscosus, A.

naeslundii, S. mutans and S. sanguis (165). Therefore,

cationic detergents such as CTMAC may prove useful in selec-

tively eliminating periodontopathogenic as well as cariogenic

microorganisms. In contrast to strain T14V, the presence

of CTMAC in the strain T14AV-HA suspension enhanced adsorp-

tion to saliva-treated HA. The reason for this is not known

at this time. However, the polysaccharide capsule of strain

T14AV provides a strong negative charge to the organism

which may bind to the exposed cations on the HA surface.

Efforts were then directed toward further understanding

the mechanism of adherence and differential affinity between

strains T14V and T14AV. The observation that heat or pro-

teolytic enzymes inhibit the adherence of both strains to

saliva-treated HA suggests that the bacterial receptor

molecules are associated with proteins or glycoproteins on

the cell surface. Examination by TEM and negative staining

of heat-treated cells which did not adsorb well to HA re-

vealed that fibrils had been destroyed by the treatment.

Recent reports have also shown that TSB cultures of strain

T14AV possess fewer surface fibrils than those of strain

T14V (23, 132). The number of cell surface receptor




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