Journal of Dental Research

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Bacterial Adhesion to Oral Tissues: A Model for Infectious Diseases

R.J. Gibbons
J DENT RES 1989; 68; 750
DOI: 10.1177/00220345890680050101

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Bacterial Adhesion to Oral Tissues: A Model for Infectious Diseases
R.J. GIBBONS
Forsyth Dental Center and Harvard School of Dental Medicine, 140 Fenway, Boston, Massachusetts 02115
The majority of bacteria which colonize humans display sharp
host and tissue tropisms; consequently, relatively little is known
about how they initiate colonization on mucosal surfaces. The
mouth has a variety offeatures which have enabled it to serve as
a useful model for the discovery of basic principles of host-par-
asite interactions occurring in mucosal environments. Early stud-
ies demonstrated that indigenous bacteria attach to surfaces of
the mouth in a highly selective manner; attachment was often
observed to correlate with colonization. These studies led to the
recognition that bacterial attachment is an essential step for col-
onization in environments which contain surfaces exposed to a
fluid flow. Bacterial adhesion has subsequently grown into a ma-
jor area of infectious disease research. Many bacteria have been
found to possess proteinaceous components, called "adhesins ",
on their surfaces which bind in a stereochemically specific man-
ner to complementary molecules, or "receptors", on the tissue
surface. Adhesins are often lectins which bind to saccharide re-
ceptors, but some adhesins are thought to bind to proteinaceous
receptors. Studies of components of human saliva, which adsorb
to hydroxyapatite (HA) surfaces similar to those of teeth, and
promote the attachment of prominent plaque bacteria, have re-
vealed that the acidic proline-rich proteins (PRPs) promote the
attachment of several important bacteria. These include strains
of Actinomyces viscosus, Bacteroides gingivalis, some strains of
Streptococcus mutans, and others. The salivary PRP's are a unique
family of molecules. However, segments of PRPs are structurally
related to collagen. This may be significant, since B. gingivalis
and certain cariogenic streptococci bind to collagenous sub-
strata, and such interactions may facilitate their invasion into
gingival tissues, or into dentin or cementum, respectively. An-
other unexpected observation was that although A. viscosus and
other bacteria bind avidly to PRPs adsorbed onto apatitic sur-
faces, they do not interact with PRPs in solution. PRP molecules
evidently undergo a conformational change when they adsorb to
HA, and adhesins of A. viscosus recognize cryptic segments which
are only exposed in adsorbed molecules. This provides the bac-
teria with a mechanism for efficiently attaching to teeth while
suspended in saliva. It also offers a molecular explanation for
their sharp tropisms for human teeth. It has proven convenient
to refer to such hidden receptors for bacterial adhesins as "cryp-
titopes'" (from cryptic, meaning hidden, and topo, meaning place).
The generation of cryptitopes due to conformational changes or
because of enzymatic modifications appears to be involved in the
colonization of several bacteria on mucosal surfaces. In addition,
there is evidence which suggests that elevated levels of neura-
minidases and proteases associated with poor oral hygiene and
gingivitis may also generate cryptitopes which promote coloni-
zation of certain Gram-negative bacteria associated with destruc-
tive periodontal diseases. These enzymes concurrently destroy
receptors required for attachment of relatively benign species
such as S. mitis and S. sanguis. Thus, the elevated levels of
enzymes previously reported present in crevicular fluid and
liva of individuals with poor oral hygiene appear to have the
sa-
potential to modulate bacterial colonization.
J Dent Res 68(5):750-760, May, 1989
Received for publication October 11, 1988
Accepted for publication January 25, 1989
Modified from the 1988 Seymour J. Kreshover Lecture, National
Institute of Dental Research, September 26, 1988. Preparation of this
manuscript was supported by USPHS Research Grants DE-02847,
DE-07009, and DE-04881 from the National Institute of Dental Re-
search, National Institutes of Health, Bethesda, MD 20892.
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750
Introduction.
Most indigenous and pathogenic bacteria initiate colonization
on a mucosal surface. Yet, relatively little is known about this
process. One of the reasons for this lack of information is that
the majority of human pathogens display sharp host and tissue
tropisms; they do not colonize the mucosal surfaces of labo-
ratory animals when administered by natural routes. Conse-
quently, they have been mainly studied in intravenous or
intraperitoneal models which bypass their natural mode of col-
onization. As a result, a great deal is known about host-parasite
interactions in systemic situations, but relatively little is known
about such interactions in mucosal environments.
Studies of indigenous bacteria in the mouth have contributed
useful knowledge in this area. The present report summarizes
some highlights of studies concerning the adhesion of bacteria
to surfaces of the mouth, and of the role this plays in coloni-
zation of host tissues. These studies suggest that the mouth
may serve as a useful model for the discovery of basic prin-
ciples which are applicable to a variety of infectious diseases.
It is appropriate to point out that the bulk of the microbial
biomass on planet Earth grows attached to a surface. Thus,
the majority of bacteria in fresh-water streams, in marine en-
vironments, and in the soil colonize in the form of adhesive
biofilms (Paerl, 1980; Marshall, 1980). Bacteria also colonize
the surfaces of sand grains, plants, algae, and even on other
bacteria. In addition, the skin and mucosal surfaces of humans
and animals are heavily populated by adhesive masses of bac-
teria (Savage, 1980). However, despite the prevalence of bac-
terial surface colonization in many natural environments, it has
only been in recent years that the significance of this process
and the mechanisms involved have begun to be understood.
Researchers studying the etiology of dental caries and var-
ious forms of periodontal diseases have long recognized that
these diseases are infections caused by bacterial plaque accu-
mulations on the teeth. Efforts to understand the mechanisms
involved in the formation of such dental plaques revealed that
bacterial attachment to tissue surfaces is a remarkably specific
process, and attachment is often the first step required for
colonization of a host tissue (reviewed by Gibbons and van
Houte, 1975, 1980; Gibbons, 1980, 1984).
Features of the mouth for studying bacterial adhesion.-
Oral biologists have an opportunity to study basic principles
of host-parasite interactions by exploiting the unique features
of the mouth. Some of the features which make the mouth an
especially useful model for such studies are as follows:
a) The mouth contains several types of surfaces, including
keratinized and non-keratinized epithelium, those of the
teeth, and the surfaces of the bacteria themselves.
b) Bacteria display remarkable tropisms for colonizing oral
surfaces. Thus, organisms such as Streptococcus mutans,
Streptococcus sanguis, Actinomyces viscosus, and Bac-
teroides gingivalis mainly colonize the teeth, whereas
Streptococcus salivarius preferentially colonizes the tongue
dorsum. Streptococcus mitis is found in high proportions
on both buccal and tooth surfaces (Gibbons and van Houte,
1975, 1980).
Vol. 68 No. 5 BACTERIAL ADHESION TO ORAL TISSUES
c) Many surfaces of the mouth with their resident bacterial
populations are readily accessible for sampling.
d) Most oral bacteria are considered to be part of our "nor-
mal" flora, and they are relatively benign. It is therefore
often possible to study the adhesion of such organisms
directly in the mouths of volunteers, without involving
significant risks, and the observations made are directly
applicable to human host-parasite interactions.
e) Surfaces of the mouth are bathed by oral fluids. These
include from 0.5 to 1.5 liters of saliva estimated to be
produced by typical adults each day (Watanabe and Dawes,
1988; Mandel and Wotman, 1976), and gingival fluid,
which is a serum transudate (Cimasoni, 1983). Thus, it
is possible to study the influence of both the secretary
and the systemic immune systems on colonizing bacterial
populations. Because of the relative ease of collecting
oral fluids, this can often be done much more readily
than in most other body locations.
f) Surfaces in the mouth vary widely in their rates of des-
quamation. The gingival epithelium has the highest rate
of turnover, while buccal cells have a moderate rate
(Skougaard, 1970). Because the surfaces of teeth do not
desquamate, bacteria can attach and accumulate to form
thick biofilms, or plaques (Gibbons and van Houte, 1980).
Thus, the influence of desquamation on bacterial colo-
nization is easily observed in oral ecosystems.
g) It is easy to locate subjects for study.
It is interesting to consider that several species of bacteria
not only appear to colonize the teeth preferentially, but they
actually seem to require the presence of teeth in order to inhabit
the mouth. For example, S. mutans, S. sanguis, A. viscosus,
and B. gingivalis are not found in the mouths of infants prior
to tooth eruption (Ellen, 1976; Ellen et al., 1978; Gibbons,
1984; Slots and Gibbons, 1978; Slots and Genco, 1985; May-
rand and Holt, 1988). In addition, S. mutans and S. sanguis
have been shown to disappear from the mouth following ex-
traction of all teeth (Carlsson et al., 1969). As a biologist, I
find it especially intriguing to consider that the primary habitat
on planet Earth for these organisms appears to be the surfaces
of human teeth! Clearly, this is a remarkable tropism, and it
is analogous to the sharp tropisms which certain pathogenic
bacteria display for colonizing specific tissues of certain hosts.
Selectivity of bacterial adhesion to surfaces of the mouth
and its role in colonization. -To study dental plaque forma-
tion, laboratory models were devised for the study of the at-
tachment of bacteria to surfaces which mimicked those in the
mouth (reviewed by Gibbons and van Houte, 1975, 1980). A
variety of in vitro and in vivo experiments demonstrated that
the attachment of bacteria to oral surfaces occurred in a highly
selective manner (van Houte et al., 1970; Gibbons and van
Houte, 1971). For example, strains of S. sanguis were found
to attach better to the teeth than to tongue or cheek surfaces.
In contrast, S. salivarius attached best to the tongue dorsum,
while S. mitis attached well to both the cheek and the teeth
(Liljemark and Gibbons, 1972). It was also noted that there
often was a correlation between the ability of a bacterial spe-
cies to attach, and the extent to which it naturally colonized
an oral surface (van Houte et al., 1970; Gibbons and van
Houte, 1971; Gibbons and van Houte, 1975, 1980).
As a result of these studies, it became clear that bacteria
possess a highly developed recognition system which is ca-
pable of recognizing and interacting with specific macro-
molecules on tissue surfaces. Furthermore, the correlations
observed between attachment and colonization provided the
first convincing evidence that attachment was an essential step
for bacterial colonization of host tissues. Attachment is re-
quired to prevent the organisms from being washed away by
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751
oral fluids. These early experiments led to the simple reali-
zation that in environments which contain surfaces exposed to
a fluid flow, bacteria must attach to a surface in order to persist
and have opportunity to grow (Gibbons and van Houte, 1975,
1980; Gibbons, 1977, 1980). Examples of such environments
include the mouth, the naso-pharyngeal area, portions of the
intestinal canal, the eye, heart, bladder, etc. Furthermore, the
selectivity of bacterial attachment provides an explanation for
the differing susceptibilities of various tissues and hosts to
bacterial colonization and infection.
The selectivity of bacterial attachment to oral surfaces and
its correlation with colonization subsequently attracted consid-
erable interest. For example, this led Beachey (1980) to com-
ment that although the adhesiveness of bacteria for some
mammalian cells was recognized as early as 1908, and the
selectivity of this process was demonstrated by Duguid and
co-workers in the 1950s (for review, see Duguid and Old,
1980), an understanding of the role of microbial adhesion in
the initiation of infectious processes obtained a major stimulus
through studies of the attachment of oral bacteria to the sur-
faces of the mouth.
Bacterial adhesion has now developed into a major area of
infectious disease research. A Medline search of the biomed-
ical literature indicates that prior to 1970, there were five to
ten papers published per year which dealt with bacterial adhe-
sion (Fig. 1). These reports were mainly by marine and soil
microbiologists and by a few individuals interested in dental
plaque formation. Following the reports in 1970-1971 that oral
bacteria attached selectively to tissues and this was associated
with colonization (van Houte et al., 1970; Gibbons and van
Houte, 1971), the number of published reports dealing with
bacterial adhesion has shown rapid and continuous growth.
This growth received special stimulation by the observations
in 1972 that the virulence of the medical pathogen, Strepto-
coccus pyogenes, also correlated with its ability to attach to
epithelial cells (Ellen and Gibbons, 1972), and that IgA anti-
bodies in secretions may exert a protective function by inhib-
iting bacterial attachment (Williams and Gibbons, 1972). In
1987 there were more than 500 papers published which dealt
with bacterial adhesion, and the area is still growing (Fig. 1).
The reason for this interest is that knowledge of bacterial adhe-
sion promises to provide molecular explanations for the tro-
pisms of bacteria, and for the varying susceptibilities of different
hosts and tissues to infectious agents. Many investigators also
share the belief that information to be obtained will lead to
new methods for controlling infectious diseases in the future.
Molecular mechanisms of bacterial attachment to tissue sur-
faces. -The molecular mechanisms used by bacteria as a means
to attach to host tissues are beginning to be understood. Al-
though bacteria can become associated with tissue surfaces by
electrostatic or hydrophobic forces of low specificity, these are
usually not adequate to resist the cleansing forces present and
permit colonization. Rather, many bacteria have been found
to possess proteinaceous components, called "adhesins", on
their surfaces which bind in a stereochemically specific manner
to complementary molecules, or "receptors", on the tissue
surfaces (Jones and Isacsson, 1983; Gibbons, 1980, 1984).
Often, adhesins are associated with surface fibrils called "fim-
briae" or "pili". Many adhesins are lectins which bind to
saccharide receptors (Ofek and Perry, 1985; Ellen, 1985). Be-
cause saccharides can combine in so many ways, they can
contain an enormous amount of molecular information. Thus,
they are well suited to serve as a framework of a recognition
system. However, adhesins which bind stereochemically to
proteinaceous receptors have also been discovered, though they
appear to be less common.
Adhesion of bacteria to teeth. -The enamel surfaces of teeth
 752 GIBBONS
500T
450+
400+
7r
Number oou I..3
Attachment Selective;
.b
Papers 300
250 t Associated With Colonization
Published 200
Per Year 150.
100
50
0 N
I m
rz
III
N""N m L\:",l m
4
rx-"
66 6768697071 72737475767778798081 828384858687
Year
Fig. 1-Publications concerning bacterial adhesion in the biomedical literature based upon a literature search. The number of papers published per year
starting with 1966 is indicated. Note the growth of activity in this area following the reports in 1970-1971 that the attachment of oral bacteria to surfaces
of the mouth was selective, and it was associated with colonization. (4306 reports from 1966-1987)
are covered by a thin membranous film termed the "acquired
pellicle". This film is generally less than one ilm thick and is
formed by the selective adsorption of components from oral
fluids to the apatitic mineral of the enamel (Ericson, 1967;
Hay, 1967). Components of saliva, crevicular fluid, as well
as bacterial products may contribute to pellicle formation. The
attachment of bacteria to teeth therefore involves interactions
between bacterial adhesins and macromolecules comprising the
acquired pellicle.
Recently, we initiated collaborative studies aimed at eluci-
dating the nature of the salivary components which adsorbed
to apatitic surfaces and were responsible for promoting attach-
ment of prominent plaque bacteria (Gibbons and Hay, 1988).
The approach taken involved chromatographic fractionation of
parotid or submandibular saliva samples, and then hydroxy-
apatite (HA) beads were treated with the respective fractions
to form experimental pellicles. After any uncoated areas of the
mineral were blocked with albumin, the beads were incubated
with radiolabeled bacteria, and the number of bacteria which
attached was determined by scintillation counting.
Our initial studies showed that different species of oral bac-
teria exhibited remarkably different patterns of binding to HA
beads treated with fractions of submandibular saliva (Fig. 2).
For example, two groups of salivary fractions promoted at-
tachment of A. viscosus (Gibbons and Hay, 1988a, b). At-
tachment of S. mutans cells was also promoted by two groups
of fractions, but one of these was associated with high-molec-
ular-weight salivary mucins. Cells of B. gingivalis exhibited
yet a different binding profile (Gibbons and Hay, 1988b) (Fig.
2), whereas cells of S. sobrinus did not adsorb well to HA
treated with any of the salivary fractions (Gibbons et al., 1986)
(data not shown). The marked differences observed between
strains of S. mutans and S. sobrinus warrant special comment,
since these species have often been considered as simply dif-
ferent serotypes of a single cariogenic specie referred to as "S.
J Dent Res May 1989
0
L\M a N"N N'--N A 2 a LNM
mutans". An enormous literature has accumulated over the
past two decades which contains many seemingly conflicting
observations concerning the contribution of extracellular glu-
cans synthesized from sucrose on the adhesion of these organ-
isms to hard surfaces. Recognition that adsorbed salivary
components serve as receptors for S. mutans, whereas ad-
sorbed glucans/glucosyltransferases promote attachment of S.
sobrinus strains (Gibbons et al., 1986), helps to clarify this
situation. Collectively, the observations made to date illustrate
the remarkable specificity of bacterial interactions with sali-
vary components adsorbed on apatitic surfaces, and they in-
dicate that different components are responsible for promotion
of the attachment of different bacterial species.
The salivary components responsible for promotion of the
attachment of A. viscosus were studied further (Gibbons and
Hay, 1988a). Additional fractionation and electrophoretic anal-
yses showed that the first group of salivary adhesion-promoting
fractions contained the family of acidic proline-rich proteins
(PRPs), while the second group of fractions contained the pro-
tein statherin.
The salivary PRPs and statherin are unique phosphoproteins
which are characteristically present in saliva (Azen and Den-
niston, 1981; Bennick and Connell, 1971; Bennick, 1987; Hay,
1983). The PRPs comprise a family of closely related proteins
which possess several unusual characteristics. Three large PRPs,
designated PRP-1, PRP-2, and PIF-slow, have been isolated,
which are 150 amino acid residue proteins (Fig. 3a, b). These
proteins differ only in residues 4 and 50 (Fig. 3b). In PIF-
slow, residues 4 and 50 are asparagine and aspartate, while in
PRP-1 they are aspartate and asparagine. In PRP-2, both res-
idues are aspartate. Three small PRPs (PIF-fast, PRP-3, and
PRP-4) have also been isolated. These are 106 amino acid
residue proteins which are identical to the first 106 residues of
the larger proteins. They are believed to be derived from the
larger proteins by post-translational proteolysis, and the 44-
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VoL. 68 No. 5 BACTERIAL ADHESION TO ORAL TISSUES 753

10
9
8
7
Relative
6
Number
5
Bacteria
4
Attached
3
2
1
0
0 5 10 15 20 25 30 35
Saliva Fraction Used To Treat HA
Fig. 2-Attachment of bacteria to HA beads treated with fractions of submandibular saliva obtained by chromatography on columns of Trisacryl GF
2000. The absorption of the fraction at 220 nm (----) and the number of bacterial cells which attached are indicated. Note the different patterns of binding
exhibited by the three species. S. sobrinus 6715 did not bind effectively to HA treated with any of the salivary fractions (data not shown). Adapted from
Gibbons and Hay (1988a, b).

residue peptide anticipated from this cleavage has been de-
tected in human saliva (Isemura et al., 1980).
The PRPs are highly asymmetrical with respect to charge
(Fig. 3a). For example, the first 30 residues in the amino
terminal segment of PRP-1 contain 13 of the 15 negatively
charged amino acids in the protein, in addition to two phos-
phoserine residues. In contrast, the carboxy terminal segment
contains several basic amino acid residues. Statherin also pos-
sesses a marked charge asymmetry, and has two phosphoserine
residues at one end of the molecule (Hay, 1983). Several pre-
vious studies have established that the PRPs and statherin bind
with high affinity to HA surfaces, via the acidic amino terminal
segment (Bennick et al., 1979; Bennick, 1987; Hay, 1983;
Moreno et al., 1979, 1982).
Treatment of HA with 1-2 pg of pure PRP-1, PRP-2, or
PIF-slow was found to promote a level of attachment of A.
viscosus cells that was comparable with that of unfractionated
saliva (Gibbons and Hay, 1988a). Slightly higher concentra-
tions of each of the small PRPs were required to achieve this.
Statherin proved somewhat less effective, but maximal binding
occurred when HA beads were treated with 8-10 pg of this
protein. Using 3H-PRP-1 prepared by reductive methylation
with 3H-formaldehyde, we observed that maximal attachment
of A. viscosus cells occurred when only 10-15% of the HA
surface was covered with protein. This corresponds to approx-
imately 10,000 molecules of PRP-1 per square am of surface
area, which approximates one bacterial binding site (Gibbons
and Hay, 1988a).
One likely reason why maximal binding of A. viscosus cells
Downloaded from http://jdr.sagepub.com by on April 19, 2010
can occur when the substratum is only partially covered by
adsorbed PRP molecules is that the organism attaches by means
of spatially separated fimbriae. Studies by Cisar and co-work-
ers (1984) and Clark et al. (1986) have shown that A. viscosus
cells possess two types of fimbriae. Type 1 fimbriae mediate
binding of A. viscosus cells to salivary pellicles on apatitic
surfaces (Clark et al., 1986), whereas type 2 fimbriae are as-
sociated with a galactosyl-binding lectin which mediates at-
tachment of A. viscosus cells to certain mammalian cells and
bacteria (Ellen et al., 1980; Cisar et al., 1984). Specific fim-
briae-defective mutants have recently been isolated and char-
acterized by Cisar and co-workers (1988). In collaborative
studies, we have observed that actinomyces cells possessing
type 1 fimbriae attached to both adsorbed PRPs and statherin,
while cells with type 2 fimbriae did not (Gibbons et al., 1988).
Type 1 fimbriae have been isolated in pure form, and they are
reported to be free of carbohydrate (Wheeler and Clark, 1980;
Clark et at., 1984). Likewise, the PRPs and statherin are non-
glycosylated proteins (Hay, 1983; Bennick, 1987), and thus
the interactions between type 1 fimbriae and adsorbed PRPs
and statherin seem to represent examples of protein-protein
stereochemical interactions involved in bacterial adhesion.
PRPs promote attachment of a variety ofplaque bacteria. -
The ability of adsorbed PRPs to promote strong adhesion of
A. viscosus cells to apatitic surfaces prompted us to determine
the response of other oral bacteria to this family of proteins.
The adhesion of a variety of other species of bacteria was found
to be promoted by the presence of adsorbed PRP-1 on apatitic
surfaces (Gibbons and Hay, 1988b). Organisms in this cate-
754 GIBBONS J Dent Res May 1989
A)
-o2
1 5 1 10 15 20
ASH-PRAS-LUVARP-VA
PC-SPI-LEU m-SY-GIU-ASPVVAbETiSP-Gla-
mc-2
1 25 30 35 40
ASP-SERI-G1JJ-GIN-P-IIE-ASPGIIli-

45 50 55 60
GIN-FO-SER-AIA-GLY-ASP-GLY-ASN-GIN-ASN-ASPG- --

65 70 75 80
GLY-GLYGIN-GLN--GIN-GIN-GLY-I-E-HEHE-GIN--GLY-LSYS-IJ-GIN-GLY-L-I~LN-

85 90 95 100
GlNG- BS-PR,-}P:ROGNGY-ARG_
+ +

105 110 115 120

HIS 4SNI-
+ + + +

125 130 135 140

GL;Y-ER}-PROEE-PRD}PRO-PRDGY-IYS-RNPRRDG[YAEX
+ +

145 150
GIN-GLY-R-PR}EGN-GYIN-SER-PR-EN

B) 4 50 150
PRP1 -A
-r=v- -zumI
'IL
-AmAj _-
-
PIF-s
PRP-2
106
IEIx37-3
PIF-f
-Asp
~~~AFs
-Asxl=~Aq
PRP-4
Fig. 3-A. Primary structure of human acidic proline-rich protein-1 (PRP-1). Several other closely related PRPs have been identified and differ from
PRP-1 either by substitutions at residues 4 and 50, or by post-translational modification, as shown in Fig. 3B. PCA (residue 1) is pyrrolidone carboxylic
acid, which forms spontaneously when glutamate is present at the amino-terminus (from Gibbons and Hay, 1988b).
B. Structural variations in six human PRPs. The 106-residue proteins, PRP-3, PIF-fs and PRP-4, are considered to be formed by proteolysis of the
primary gene products, PRP-1, PIF-s, and PRP-2, respectively (Bennick, 1987; Hay et al., 1988; Schlesinger and Hay, 1979, 1986; Wong and Bennick,
1980.)

gory include strains of A. viscosus, A. israelii, and A. odon- this protein. Among black-pigmented Bacteroides, strains of
tolyticus. However, strains of A. naeslundii, which only possess B. gingivalis, B. loeschii and B. melaninogenicus displayed
type 2 fimbriae, did not respond. The adhesion of some S. enhanced adsorption to PRP-1-treated HA, whereas a strain of
mutans strains (i.e., JBP of serotype c) was also promoted by B. intermedius did not. S. sanguis Blackburn also exhibited
adsorbed PRP-1, but strains of S. sobrinus did not respond to strong adsorption to PRP-1-treated HA, but several other strains
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 Vol. 68 No. 5 BACTERIAL ADHESION TO ORAL TISSUES
of S. sanguis, a strain of S. mitis, and three strains of S.
pyogenes did not. On the basis of this limited survey, it seems
clear that a variety of bacteria resident in human dental plaque
possess adhesins which interact with adsorbed PRP molecules
on HA (Gibbons and Hay, 1988b).
Structural similarities between PRPs and collagens. -The
apparent possession of adhesins which react with adsorbed PRPs
by several plaque bacteria prompted us to determine whether
there were other proteins which are structurally related to the
PRPs. The acidic amino terminal segment of the PRPs is re-
sponsible for binding of the protein to apatitic surfaces (Ben-
nick et al., 1979; Hay, 1983; Bennick, 1987), but this segment
has not been found to promote adhesion of any of the bacteria
tested (Gibbons and Hay, 1988a, b). Therefore, we sought
structural similarities between the amino acid sequences of the
carboxy-terminal segment of PRP-1 and other proteins in the
data base of the National Biomedical Research Foundation Pro-
tein Identification Resource (Gibbons and Hay, 1988b). There
were 6418 sequences in this data base, but the only proteins
which showed a relatedness score of greater than 80 proved to
be human or rodent PRPs (Fig. 4), and no proteins had a score
between 71 and 80. This clearly illustrates the uniqueness of
the PRPs. However, the protein which exhibited the next high-
est degree of similarity to the carboxy-terminal segment of
PRP-1 proved to be the alpha chain of collagen (Fig. 4).
The structural relatedness between this segment of PRP-1
and collagen was between seven and eight standard deviations
greater than the mean of all protein matches in the data base
(Gibbons and Hay, 1988b). This structural relatedness is of
interest, considering that the matrices of dentin and cementum
are rich in collagen, and collagen fibers are major structural
elements of connective tissues, including the periodontal lig-
ament, as well as basement membranes. Furthermore, destruc-
>81 (17 - Human & Rodent PRPs)
80 (0)
(4 - Alpha 1 (1) Chain Of Collagen From
70 < Human, Mouse, Bovine; Plasmodium
(15) Circumsporozoite Precursor)
Relatedness 60
50 (45)
Score 40 (126)
30 (851)
20 [ N
10 (1,231)
i i
0 1000 2000
Number Of Proteins
Fig. 4.-Similarity search of the National Biomedical Research Foundation Protein Identification Resource of proteins related to residues 107-150 of
PRP-1. There were 6418 sequences in the data base. These showed a mean similarity score of 14.5 + 7.03 S.E. Note that after human and rodent PRPs,
collagens from human, mouse, and bovine sources showed a significant degree of structural similarity (from Gibbons and Hay, 1988b).
Downloaded from http://jdr.sagepub.com by on April 19, 2010
755
tion of collagen is a central feature of both periodontal disease
and advanced dental caries. The structural relatedness between
the PRPs and collagen assumes more relevance in light of our
recent observations that strains of B. gingivalis (Naito and
Gibbons, 1988) and certain "mutans" streptococci [especially
S. cricetus and S. rattus (Liu and Gibbons, unpublished data)]
bind avidly to collagenous substrata. It is possible that affinity
for collagenous elements plays a role in facilitating the inva-
sion of B. gingivalis cells through basement membranes and
into gingival connective tissues. Likewise, the affinity of var-
ious cariogenic streptococci for collagen may play a role in
the caries process by facilitating bacterial penetration into ce-
mentum and dentin following initial demineralization.
Importance of previously cryptic receptors (cryptitopes) in
bacterial adhesion. -One of the surprising observations made
during the course of these studies was that although A. viscosus
cells could bind avidly to adsorbed PRPs on apatitic surfaces,
they do not interact with PRPs in solution (Gibbons and Hay,
1988a). For example, the presence of PRP-1 or PRP-3 at con-
centrations as high as 1000 [Lg/mL does not affect the attach-
ment of A. viscosus cells to experimental pellicles prepared
from a few micrograms of PRP-1. Similarly, radiolabeled PRP-
1 does not bind to A. viscosus cells, nor is it degraded when
incubated with the organism. Yet A. viscosus cells attach equally
well to HA surfaces treated with either radiolabeled or unla-
beled PRP-1. The apparent explanation for this unexpected
difference in behavior between PRP molecules in solution vs.
PRP molecules adsorbed onto apatitic surfaces is that hidden
molecular segments of PRPs become exposed, as a result of a
conformational change, when the protein adsorbs to HA (Gib-
bons and Hay, 1988a). Previous studies had, in fact, suggested
that a major conformational change takes place when PRP
molecules adsorb to HA. This was suggested by differences in
[6,41 8 Sequences;
Mean Score = 1 4.5 (7.03)]
(4, 1 29)
~~~~~~~~~~~~~
~ ~ ~ i
3000 4000 5000
756 GIBBONS
their calcium ion binding properties (Bennick et al., 1981) and
by the thermodynamics of their adsorption (Moreno et al.,
1982). Adsorption of the protein to HA is an endothermic
process driven by an increase in entropy. Moreno et al. (1982)
suggested that this was consistent with the breaking of inter-
molecular bonds and the loss of ordered water, which would
occur during a change in the conformation of the protein as it
adsorbs onto the mineral. The high degree of asymmetry in
charged residues in PRP molecules is thought to contribute to
the folding of the polypeptide chain when in solution. Evi-
dently, when the negatively charged amino terminal segment
of the molecule interacts with calcium atoms on apatitic sur-
faces, the polypeptide chain unfolds.
The apparent ability of A. viscosus cells to recognize cryptic
segments of PRPs which are only exposed when the molecules
are adsorbed on surfaces provides a novel and highly efficient
mechanism for enabling the organism to attach to teeth (Gib-
bons and Hay, 1988a). The PRPs comprise as much as 30-
40% of the protein in saliva (Bennick, 1987; Hay, 1983), and
if A. viscosus cells could interact with PRP molecules in so-
lution, there would be little likelihood that many organisms
would attach to adsorbed PRP molecules on the tooth surface.
The ability of A. viscosus cells to recognize unique cryptic
segments in adsorbed PRP molecules provides a molecular
explanation for the predilection which this organism displays
for the teeth. It helps to explain why the surfaces of human
teeth are the primary habitat for A. viscosus on planet Earth!
It has proven convenient to refer to such previously hidden
receptors for bacterial adhesins as "cryptitopes". This word
is derived from "cryptic" (Gr. kryptos) meaning hidden, and
"topo" (Gr. topo) meaning place. It is easy to envision how
adhesins which recognize cryptitopes in surface-associated
molecules would evolve. They would provide a strong selec-
tive advantage for any bacterium which colonizes a mucosal
or tooth surface. The secretions which bathe these surfaces
contain components which are structurally related to those on
the surfaces of mucosal and other body cells. This molecular
mimickery is thought to contribute to the cleansing action of
secretions (Gibbons, 1982). When in solution, components of
secretions which bind to bacteria often cause aggregation, and
this is thought to facilitate bacterial clearance (Mandel, 1976).
However, if molecules on the surfaces of mucosal cells or teeth
became altered as a result of a conformational change, or be-
cause of an enzymatic modification, cryptitopes could be formed
which would promote bacterial attachment and colonization.
We believe that cryptitopes will probably be uncovered for
a variety of indigenous and pathogenic micro-organisms which
colonize mucosal surfaces. Some apparent examples are listed
in the Table. In addition to the binding of A. viscosus to ad-
sorbed PRPs, it has recently been reported that S. sanguis cells
bind to fibronectin which is complexed to collagen, but not to
fibronectin in solution (Lowrance et al., 1988). This appears
to be due to the recognition of a cryptitope created by a con-
formational change in the complexed fibronectin, and it has
been suggested that this may account for the ability of S. san-
guis cells to bind to damaged heart valves when the strepto-
cocci are circulating in serum which is rich in fibronectin.
Another apparent example concerns the relatively common
possession of galactosyl-binding adhesins by oral bacteria (Ta-
ble). Organisms which appear to possess such adhesins include
A. viscosus, A. naeslundii (Ellen et al., 1980; Cisar, 1986),
Leptotrichia buccalis (Kondo et al., 1976), Fusobacterium nu-
cleatum (Falkler et al., 1979), Eikenella corrodens (Yamazaki
et al., 1981), and Bacteroides internedius (Okuda and Kato,
1987). These organisms attach poorly, if at all, to unmodified
erythrocytes, epithelial cells, or experimental pellicles pre-
pared from salivary mucins. However, treatment of these sur-
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J Dent Res May 1989
TABLE
APPARENT INVOLVEMENT OF "CRYPTITOPES" IN BACTERIAL
ATTACHMENT
A. viscosus - binds to adsorbed PRPs on HA surfaces, not
to PRPs in solution (Gibbons and Hay, 1988a)
S. sanguis - binds to fibronectin complexed with colla-
gen; not affected by fibronectin in solution
(Lowrance et al., 1988)
A. viscosus, A. naeslundii, E. corrodens, F. nucleatum, L. buccalis, B.
intermedius
- bind to galactosyl receptors exposed after
neuraminidase treatment (Ellen et al., 1980;
Falkler et al., 1979; Kondo et al., 1976; Ya-
mazaki et al., 1981; Okuda and Kato, 1987)
- binds to trypsin-treated epithelial cells de-
P. aeruginosa
pleted of fibronectin (Woods et al., 1983)
B. gingivalis - binds in much higher numbers to oral epi-
thelial cells which have been mildly treated
with trypsin or papain (Childs and Gibbons,
1988a, b)
B. gingivalis - trypsin-treatment of fibronectin-collagen
complexes exposes collagen receptors which
promote attachment (Naito and Gibbons,
1988)
faces with neuraminidase removes terminal sialic acid residues
and exposes penultimate galactosyl residues. These now func-
tion as cryptitopes which interact with the galactosyl-binding
adhesins of these organisms.
It is interesting to note that treatment with certain proteases
may also create cryptitopes for bacteria. Trypsin and trypsin-
like enzymes cleave peptide bonds and expose arginine resi-
dues. B. gingivalis cells have been reported to bind to arginine-
containing receptors, and their attachment to certain host cells
is inhibited by arginine-containing peptides (Okuda et al., 1986).
We have observed that B. gingivalis cells show a greatly en-
hanced attachment to epithelial cells following trypsin treat-
ment (Childs and Gibbons, 1988a, b). This appears to represent
a clear example of an enzyme-generated cryptitope which pro-
motes bacterial adhesion to host tissues.
Still another example concerns Pseudomonas aeruginosa
(Woods et al., 1983) (Table). Early studies indicated that this
organism attaches rather poorly to untreated epithelial cells.
However, mild treatment with trypsin results in greatly en-
hanced attachment, presumably through the generation of cryp-
titopes. P. aeruginosa cells also attach in much higher numbers
to buccal and pharyngeal epithelial cells from acutely-ill pa-
tients in intensive care units, or from patients with cystic fi-
brosis, than to cells from healthy individuals. This enhanced
attachment has been associated with elevated levels of pro-
teases in the secretions from such patients. It has been sug-
gested that the elevated proteases remove fibronectin from the
epithelial cell surface and expose receptors, to which Pseu-
domonas attaches (Woods et al., 1983). This seems to rep-
resent a further example of the enzymatic creation of a cryptitope,
which promotes attachment and colonization of an infectious
agent.
Does poor oral hygiene generate cryptitopes?-The poten-
tial of neuraminidase and certain proteases to create cryptitopes
suggests that fluctuations in the concentrations of these en-
zymes in the fluids which bathe mucosal surfaces could mod-
ulate bacterial attachment and colonization. It is well-established
that the oral hygiene and periodontal status of an individual
correlates with the levels of a variety of enzymes in saliva and
in crevicular fluid. Thus, elevated levels of proteases (Berg et
al., 1947; Watanabe et al., 1981), including trypsin-like en-
Vol. 68 No. 5 BACTERIAL ADHESION TO ORAL TISSUES
zymes and collagenases (Golub et al., 1976; Iijima et al.,
1983), and of various glycosidases (Nakamura and Slots, 1983),
including neuraminidase (Perlitsch and Glickman, 1967; Ki-
tawaki et al., 1983), have been detected in saliva and in crev-
icular fluid of individuals with periodontal disease and/or poor
oral hygiene, as compared with healthy individuals. These en-
zymes are thought to be derived from inflammatory cells as-
sociated with the gingival inflammation, as well as from the
associated bacterial plaque accumulations.
These observations raise the intriguing possibility that alter-
ations in an individual's oral hygiene state, or state of gingival
inflammation, may result in elevated levels of enzymes with
the potential to modify local tissue surfaces and generate cryp-
titopes which affect the types of bacteria which colonize. In
fact, it is well established that poor oral hygiene almost in-
variably results in gingivitis (Lbe et al., 1965), and it fre-
quently is associated with the development of certain types of
periodontitis. However, several recent studies have suggested
that specific species of bacteria are associated with specific
types of periodontal diseases (Slots, 1979, 1986; Socransky et
al., 1988; Zambon, 1985; Moore, 1987; Slots and Listgarten,
1988). The question therefore arises as to how failure to brush
one's teeth properly predisposes to infection by a specific peri-
odontal pathogen. Our recent studies suggest that elevated lev-
els of enzymes, particularly neuraminidases and proteases, may
contribute to this apparently increased susceptibility to colo-
nization by periodontal pathogens (Childs and Gibbons, 1988a,
b). We have hypothesized that these enzymes modify the sur-
faces so as to destroy receptors for some bacteria, while cre-
ating cryptitopes for others. This could help to account for the
shift in the flora from one which is benign, and composed
primarily of streptococci and other Gram-positive organisms,
to one which is predominantly Gram-negative and associated
with periodontal destruction. We have begun to test this hy-
pothesis by studying the effect of treating oral epithelial cells
with neuraminidase or trypsin on the ability of target bacteria
associated with either gingival health or disease to attach (Childs
and Gibbons, 1988a, b). We have observed that treatment of
epithelial cells with neuraminidase greatly reduces the number
of S. sanguis and S. mitis cells which attach (Fig. 5). However,
attachment of potential periodontal pathogens such as B. gin-
givalis or B. intermedius was either unaffected or actually en-
hanced. Similarly, treatment of epithelial cells with low
concentrations of trypsin or papain also markedly decreased
attachment of these streptococci, while attachment of B. gin-
givalis was greatly promoted (Fig. 5). It appears significant
that treatment of epithelial cells with lysosomal enzyme prep-
arations, obtained from human PMNs, also markedly reduced
attachment of S. mitis, while it greatly enhanced attachment
of B. gingivalis and Actinobacillus actinomycetemcomitans
(Childs and Gibbons, 1988b). Thus, the elevated levels of
these enzymes reported present in crevicular fluid of patients
with poor oral hygiene and gingivitis appear to have the po-
tential to generate cryptitopes for periodontal pathogens.
The observations to date are consistent with the available
knowledge of the types of adhesins possessed by prominent
oral bacteria. Thus, strains of S. mitis and S. sanguis possess
adhesins which bind to sialic acid-containing receptors (McBride
and Gisslow, 1977; Murray et al., 1982, 1986). Treatment of
pellicles or epithelial cells with neuraminidase would be ex-
pected to impair their attachment (Gibbons and Etherden, 1982;
Gibbons et al., 1983). However, treatment with neuraminidase
increases exposure of galactosyl residues; this would be ex-
pected to promote the attachment of bacteria with galactosyl-
binding adhesins, including the actinomyces (Ellen et al., 1980;
Cisar, 1986) and the Gram-negative F. nucleatum (Falkler et
al., 1979), B. intermedius (Okuda and Kato, 1987), and Li-
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757
kenella corrodens (Yamazaki et al., 1981). The elevated levels
of trypsin-like enzymes in crevicular fluid would also be ex-
pected to increase exposure of arginine residues, and thereby
promote attachment of B. gingivalis cells which appear to pos-
sess an arginine-binding adhesin (Okuda et al., 1986).
It is of interest to note that some of the bacteria studied are
also known to elaborate enzymes appropriate for creating cyp-
titopes for themselves. For example, strains of A. viscosus and
A. naeslundii synthesize neuraminidases which can promote
their attachment to mammalian cell surfaces (Ellen et al., 1980),
while strains of B. gingivalis and other suspected periodontal
pathogens elaborate trypsin-like proteases which cleave argi-
nine peptides (Loesche et al., 1987). The highest concentra-
tions of these enzymes would likely be found around
actinomycete or B. gingivalis cells which had attached to cre-
vicular surfaces, and they would be expected to create appro-
priate cryptitopes on adjacent surfaces which would foster the
adhesion and colonization of their own progeny.
If our hypotheses are correct, then there should be differ-
ences in the quantity of sialic acid residues on the surfaces of
oral epithelial cells in individuals with different states of oral
hygiene and gingival health. Therefore, we have analyzed the
amount of sialic acid on the surfaces of buccal and gingival
epithelial cells from five healthy individuals, and from five
patients with gingivitis (Davis and Gibbons, 1988, unpublished
observations). Buccal epithelial cells had almost three times
as much sialic acid on their surface as did gingival epithelial
cells in both groups of individuals. This helps to explain why
S. mitis, which possesses a sialic acid-binding adhesin (Murray
et al., 1986), dominates on buccal mucosa. We further ob-
served that the amount of sialic acid on buccal and gingival
epithelial cells was much lower in persons with gingivitis as
compared with patients with healthy gingiva. For example,
gingival cells from patients with gingivitis averaged approxi-
mately 6 pLg of sialic acidper 104 epithelial cells, while cells
from healthy individuals averaged almost 40 pug sialic acid per
104 cells (Davis and Gibbons, unpublished data). These dif-
ferences were highly statistically significant. Thus, the ele-
vated level of neuraminidase reported present in oral fluids of
individuals with poor oral hygiene and gingivitis (Perlitsch and
Glickman, 1967; Kitawaki et al., 1983) does appear to modify
the surfaces of oral tissues.
Collectively, these observations offer an explanation as to
how poor oral hygiene and resulting inflammation can predis-
pose the gingival crevice area to increased colonization by
potential periodontal pathogenic bacteria. The resulting mod-
ification of oral surfaces by enzymes appears to result in the
creation of cryptitopes. This also may explain why persons
with poor oral hygiene tend to develop plaque more rapidly
than individuals with good oral hygiene and healthy gingivae.
These observations are also consistent with recent reports of
Harper et al. (1988) and Wolff et al. (1988) who have noted
that there are decreased proportions of streptococci, and ele-
vated proportions of black Bacteroides such as B. gingivalis
and B. intermedius, in periodontally-diseased sites associated
with elevated levels of lysosomal and other enzymes. How-
ever, further research is needed to confirm or negate these
hypotheses.
Acknowledgments.
I thank members of the selection committee for giving me
this opportunity to present the 1988 Kreshover Lecture. I would
also like to acknowledge the contributions of many individuals
who have collaborated in these studies. Particular acknowl-
edgment is due to Dr. J. van Houte, who collaborated in sev-
 758 GIBBONS
40
Mean 30
Number
Bacteria 20
Attached
Per Cell 10
0
None Neuraminidase
Epithelial Cell Treatment
Fig. 5.-Effect of treating buccal epithelial cells with neuraminidase, trypsin, or papain on the ability of bacteria to attach. Note that treatment with
these enzymes decreased the adhesion of S. sanguis and S. mitis, while adhesion of B. gingivalis was greatly enhanced (adapted from Childs and Gibbons,
1988b).
eral of the early studies, and to Dr. D.I. Hay, for his collaboration
in the recent ones. Thanks are also due to the NIDR for pro-
viding grant support for this work over several years.
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