Journal of Dental Research - Universidad NacMost indigenous and pathogenic bacteria initiate colonization on a mucosal surface. Yet, relatively little is knownabout this process. Oneofthe

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Journal of Dental Research

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

R.J. Gibbons Bacterial Adhesion to Oral Tissues: A Model for Infectious Diseases

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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 sharphost and tissue tropisms; consequently, relatively little is knownabout how they initiate colonization on mucosal surfaces. Themouth 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 ofthe mouth in a highly selective manner; attachment was oftenobserved to correlate with colonization. These studies led to therecognition 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 beenfound to possess proteinaceous components, called "adhesins",on their surfaces which bind in a stereochemically specific man-

ner to complementary molecules, or "receptors", on the tissuesurface. Adhesins are often lectins which bind to saccharide re-

ceptors, but some adhesins are thought to bind to proteinaceousreceptors. Studies of components of human saliva, which adsorbto hydroxyapatite (HA) surfaces similar to those of teeth, andpromote the attachment of prominent plaque bacteria, have re-

vealed that the acidic proline-rich proteins (PRPs) promote theattachment of several important bacteria. These include strainsof Actinomyces viscosus, Bacteroides gingivalis, some strains ofStreptococcus mutans, and others. The salivary PRP's are a uniquefamily of molecules. However, segments ofPRPs are structurallyrelated to collagen. This may be significant, since B. gingivalisand certain cariogenic streptococci bind to collagenous sub-strata, and such interactions may facilitate their invasion intogingival tissues, or into dentin or cementum, respectively. An-other unexpected observation was that although A. viscosus andother bacteria bind avidly to PRPs adsorbed onto apatitic sur-

faces, they do not interact with PRPs in solution. PRP moleculesevidently undergo a conformational change when they adsorb toHA, and adhesins of A. viscosus recognize cryptic segments whichare only exposed in adsorbed molecules. This provides the bac-teria with a mechanism for efficiently attaching to teeth whilesuspended in saliva. It also offers a molecular explanation fortheir sharp tropisms for human teeth. It has proven convenientto 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 thecolonization ofseveral bacteria on mucosal surfaces. In addition,there is evidence which suggests that elevated levels of neura-

minidases and proteases associated with poor oral hygiene andgingivitis may also generate cryptitopes which promote coloni-zation ofcertain Gram-negative bacteria associated with destruc-tive periodontal diseases. These enzymes concurrently destroyreceptors required for attachment of relatively benign speciessuch as S. mitis and S. sanguis. Thus, the elevated levels ofenzymes previously reported present in crevicular fluid and sa-

liva of individuals with poor oral hygiene appear to have thepotential to modulate bacterial colonization.

J Dent Res 68(5):750-760, May, 1989

Received for publication October 11, 1988Accepted for publication January 25, 1989Modified from the 1988 Seymour J. Kreshover Lecture, National

Institute of Dental Research, September 26, 1988. Preparation of thismanuscript 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.

750

Introduction.

Most indigenous and pathogenic bacteria initiate colonizationon a mucosal surface. Yet, relatively little is known about thisprocess. One of the reasons for this lack of information is thatthe majority of human pathogens display sharp host and tissuetropisms; 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 orintraperitoneal models which bypass their natural mode of col-onization. As a result, a great deal is known about host-parasiteinteractions in systemic situations, but relatively little is knownabout such interactions in mucosal environments.

Studies of indigenous bacteria in the mouth have contributeduseful knowledge in this area. The present report summarizessome highlights of studies concerning the adhesion of bacteriato surfaces of the mouth, and of the role this plays in coloni-zation of host tissues. These studies suggest that the mouthmay 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 microbialbiomass 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 adhesivebiofilms (Paerl, 1980; Marshall, 1980). Bacteria also colonizethe surfaces of sand grains, plants, algae, and even on otherbacteria. In addition, the skin and mucosal surfaces of humansand 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 hasonly been in recent years that the significance of this processand 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 thatthese diseases are infections caused by bacterial plaque accu-mulations on the teeth. Efforts to understand the mechanismsinvolved in the formation of such dental plaques revealed thatbacterial attachment to tissue surfaces is a remarkably specificprocess, and attachment is often the first step required forcolonization of a host tissue (reviewed by Gibbons and vanHoute, 1975, 1980; Gibbons, 1980, 1984).

Features of the mouth for studying bacterial adhesion.-Oral biologists have an opportunity to study basic principlesof host-parasite interactions by exploiting the unique featuresof the mouth. Some of the features which make the mouth anespecially useful model for such studies are as follows:

a) The mouth contains several types of surfaces, includingkeratinized and non-keratinized epithelium, those of theteeth, and the surfaces of the bacteria themselves.

b) Bacteria display remarkable tropisms for colonizing oralsurfaces. Thus, organisms such as Streptococcus mutans,Streptococcus sanguis, Actinomyces viscosus, and Bac-teroides gingivalis mainly colonize the teeth, whereasStreptococcus salivarius preferentially colonizes the tonguedorsum. Streptococcus mitis is found in high proportionson both buccal and tooth surfaces (Gibbons and van Houte,1975, 1980).



BACTERIAL ADHESION TO ORAL TISSUES

c) Many surfaces of the mouth with their resident bacterialpopulations 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 thereforeoften possible to study the adhesion of such organismsdirectly in the mouths of volunteers, without involvingsignificant risks, and the observations made are directlyapplicable to human host-parasite interactions.

e) Surfaces of the mouth are bathed by oral fluids. Theseinclude from 0.5 to 1.5 liters of saliva estimated to beproduced by typical adults each day (Watanabe and Dawes,1988; Mandel and Wotman, 1976), and gingival fluid,which is a serum transudate (Cimasoni, 1983). Thus, itis possible to study the influence of both the secretaryand the systemic immune systems on colonizing bacterialpopulations. Because of the relative ease of collectingoral fluids, this can often be done much more readilythan in most other body locations.

f) Surfaces in the mouth vary widely in their rates of des-quamation. The gingival epithelium has the highest rateof turnover, while buccal cells have a moderate rate(Skougaard, 1970). Because the surfaces of teeth do notdesquamate, bacteria can attach and accumulate to formthick 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 theyactually seem to require the presence of teeth in order to inhabitthe mouth. For example, S. mutans, S. sanguis, A. viscosus,and B. gingivalis are not found in the mouths of infants priorto 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. sanguishave been shown to disappear from the mouth following ex-traction of all teeth (Carlsson et al., 1969). As a biologist, Ifind it especially intriguing to consider that the primary habitaton planet Earth for these organisms appears to be the surfacesof human teeth! Clearly, this is a remarkable tropism, and itis analogous to the sharp tropisms which certain pathogenicbacteria display for colonizing specific tissues of certain hosts.

Selectivity of bacterial adhesion to surfaces of the mouthand 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 themouth (reviewed by Gibbons and van Houte, 1975, 1980). Avariety of in vitro and in vivo experiments demonstrated thatthe attachment of bacteria to oral surfaces occurred in a highlyselective manner (van Houte et al., 1970; Gibbons and vanHoute, 1971). For example, strains of S. sanguis were foundto 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 thereoften was a correlation between the ability of a bacterial spe-cies to attach, and the extent to which it naturally colonizedan oral surface (van Houte et al., 1970; Gibbons and vanHoute, 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 correlationsobserved between attachment and colonization provided thefirst convincing evidence that attachment was an essential stepfor bacterial colonization of host tissues. Attachment is re-quired to prevent the organisms from being washed away by

oral fluids. These early experiments led to the simple reali-zation that in environments which contain surfaces exposed toa fluid flow, bacteria must attach to a surface in order to persistand have opportunity to grow (Gibbons and van Houte, 1975,1980; Gibbons, 1977, 1980). Examples of such environmentsinclude the mouth, the naso-pharyngeal area, portions of theintestinal canal, the eye, heart, bladder, etc. Furthermore, theselectivity of bacterial attachment provides an explanation forthe differing susceptibilities of various tissues and hosts tobacterial 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 somemammalian cells was recognized as early as 1908, and theselectivity of this process was demonstrated by Duguid andco-workers in the 1950s (for review, see Duguid and Old,1980), an understanding of the role of microbial adhesion inthe initiation of infectious processes obtained a major stimulusthrough studies of the attachment of oral bacteria to the sur-faces of the mouth.

Bacterial adhesion has now developed into a major area ofinfectious disease research. A Medline search of the biomed-ical literature indicates that prior to 1970, there were five toten papers published per year which dealt with bacterial adhe-sion (Fig. 1). These reports were mainly by marine and soilmicrobiologists and by a few individuals interested in dentalplaque formation. Following the reports in 1970-1971 that oralbacteria attached selectively to tissues and this was associatedwith colonization (van Houte et al., 1970; Gibbons and vanHoute, 1971), the number of published reports dealing withbacterial adhesion has shown rapid and continuous growth.This growth received special stimulation by the observationsin 1972 that the virulence of the medical pathogen, Strepto-coccus pyogenes, also correlated with its ability to attach toepithelial 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). In1987 there were more than 500 papers published which dealtwith 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 differenthosts and tissues to infectious agents. Many investigators alsoshare the belief that information to be obtained will lead tonew 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 meansto attach to host tissues are beginning to be understood. Al-though bacteria can become associated with tissue surfaces byelectrostatic or hydrophobic forces of low specificity, these areusually not adequate to resist the cleansing forces present andpermit colonization. Rather, many bacteria have been foundto possess proteinaceous components, called "adhesins", ontheir surfaces which bind in a stereochemically specific mannerto complementary molecules, or "receptors", on the tissuesurfaces (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 tosaccharide receptors (Ofek and Perry, 1985; Ellen, 1985). Be-cause saccharides can combine in so many ways, they cancontain an enormous amount of molecular information. Thus,they are well suited to serve as a framework of a recognitionsystem. However, adhesins which bind stereochemically toproteinaceous receptors have also been discovered, though theyappear to be less common.Adhesion ofbacteria to teeth.-The enamel surfaces of teeth

Vol. 68 No. 5 751



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500T450+

400+7r I..3Number oou

Papers 300

250Published

200

Per Year 150.100

50

0

Attachment Selective;t Associated With Colonization

4IIIrz

66 6768697071 72737475767778798081 828384858687Year

Fig. 1-Publications concerning bacterial adhesion in the biomedical literature based upon a literature search. The number of papers published per yearstarting 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 surfacesof the mouth was selective, and it was associated with colonization. (4306 reports from 1966-1987)

are covered by a thin membranous film termed the "acquiredpellicle". This film is generally less than one ilm thick and isformed by the selective adsorption of components from oralfluids to the apatitic mineral of the enamel (Ericson, 1967;Hay, 1967). Components of saliva, crevicular fluid, as wellas bacterial products may contribute to pellicle formation. Theattachment of bacteria to teeth therefore involves interactionsbetween bacterial adhesins and macromolecules comprising theacquired pellicle.

Recently, we initiated collaborative studies aimed at eluci-dating the nature of the salivary components which adsorbedto apatitic surfaces and were responsible for promoting attach-ment of prominent plaque bacteria (Gibbons and Hay, 1988).The approach taken involved chromatographic fractionation ofparotid or submandibular saliva samples, and then hydroxy-apatite (HA) beads were treated with the respective fractionsto form experimental pellicles. After any uncoated areas of themineral were blocked with albumin, the beads were incubatedwith radiolabeled bacteria, and the number of bacteria whichattached was determined by scintillation counting.Our initial studies showed that different species of oral bac-

teria exhibited remarkably different patterns of binding to HAbeads 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 groupsof fractions, but one of these was associated with high-molec-ular-weight salivary mucins. Cells of B. gingivalis exhibitedyet a different binding profile (Gibbons and Hay, 1988b) (Fig.2), whereas cells of S. sobrinus did not adsorb well to HAtreated with any of the salivary fractions (Gibbons et al., 1986)(data not shown). The marked differences observed betweenstrains 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.

mutans". An enormous literature has accumulated over thepast two decades which contains many seemingly conflictingobservations concerning the contribution of extracellular glu-cans synthesized from sucrose on the adhesion of these organ-isms to hard surfaces. Recognition that adsorbed salivarycomponents serve as receptors for S. mutans, whereas ad-sorbed glucans/glucosyltransferases promote attachment of S.sobrinus strains (Gibbons et al., 1986), helps to clarify thissituation. Collectively, the observations made to date illustratethe remarkable specificity of bacterial interactions with sali-vary components adsorbed on apatitic surfaces, and they in-dicate that different components are responsible for promotionof the attachment of different bacterial species.The salivary components responsible for promotion of the

attachment of A. viscosus were studied further (Gibbons andHay, 1988a). Additional fractionation and electrophoretic anal-yses showed that the first group of salivary adhesion-promotingfractions 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 proteinswhich 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). Theseproteins differ only in residues 4 and 50 (Fig. 3b). In PIF-slow, residues 4 and 50 are asparagine and aspartate, while inPRP-1 they are aspartate and asparagine. In PRP-2, both res-idues are aspartate. Three small PRPs (PIF-fast, PRP-3, andPRP-4) have also been isolated. These are 106 amino acidresidue proteins which are identical to the first 106 residues ofthe larger proteins. They are believed to be derived from thelarger proteins by post-translational proteolysis, and the 44-

I m N""N m L\:",l m rx-" 0 L\M a N"N N'--N A 2 a LNMN

J Dent Res May 1989

. b




10

9

8

7Relative

Number

Bacteria

6

5

4Attached

3

2

1

00 5 1 0 1 5 20 25 30 35

Saliva Fraction Used To Treat HAFig. 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 bindingexhibited by the three species. S. sobrinus 6715 did not bind effectively to HA treated with any of the salivary fractions (data not shown). Adapted fromGibbons 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 aminoterminal segment of PRP-1 contain 13 of the 15 negativelycharged amino acids in the protein, in addition to two phos-phoserine residues. In contrast, the carboxy terminal segmentcontains several basic amino acid residues. Statherin also pos-sesses a marked charge asymmetry, and has two phosphoserineresidues at one end of the molecule (Hay, 1983). Several pre-vious studies have established that the PRPs and statherin bindwith high affinity to HA surfaces, via the acidic amino terminalsegment (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, orPIF-slow was found to promote a level of attachment of A.viscosus cells that was comparable with that of unfractionatedsaliva (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 bindingoccurred when HA beads were treated with 8-10 pg of thisprotein. Using 3H-PRP-1 prepared by reductive methylationwith 3H-formaldehyde, we observed that maximal attachmentof A. viscosus cells occurred when only 10-15% of the HAsurface was covered with protein. This corresponds to approx-imately 10,000 molecules of PRP-1 per square am of surfacearea, which approximates one bacterial binding site (Gibbonsand Hay, 1988a).One likely reason why maximal binding of A. viscosus cells

can occur when the substratum is only partially covered byadsorbed PRP molecules is that the organism attaches by meansof spatially separated fimbriae. Studies by Cisar and co-work-ers (1984) and Clark et al. (1986) have shown that A. viscosuscells possess two types of fimbriae. Type 1 fimbriae mediatebinding of A. viscosus cells to salivary pellicles on apatiticsurfaces (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 andbacteria (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 collaborativestudies, we have observed that actinomyces cells possessingtype 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 arereported 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 thusthe interactions between type 1 fimbriae and adsorbed PRPsand statherin seem to represent examples of protein-proteinstereochemical interactions involved in bacterial adhesion.PRPs promote attachment of a variety ofplaque bacteria. -

The ability of adsorbed PRPs to promote strong adhesion ofA. viscosus cells to apatitic surfaces prompted us to determinethe response of other oral bacteria to this family of proteins.The adhesion of a variety of other species of bacteria was foundto be promoted by the presence of adsorbed PRP-1 on apatiticsurfaces (Gibbons and Hay, 1988b). Organisms in this cate-

753VoL. 68 No. 5



754 GIBBONS

A)-o2

1 5 1 10 15 20PC-SPI-LEUASH-PRAS-LUVARP-VAm-SY-GIU-ASPVVAbETiSP-Gla-

mc-21 25 30 35 40

ASP-SERI-G1JJ-GIN-P-IIE-ASPGIIli-

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

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

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

+ +

105 110 115 120HIS 4SNI-

+ + +

125 130 135 140GL;Y-ER}-PROEE-PRD}PRO-PRDGY-IYS-RNPRRDG[YAEX

+ +

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

4 50

-AmAj

106-Asp ~~~AFs-Asxl=~Aq

Fig. 3-A. Primary structure of human acidic proline-rich protein-1 (PRP-1). Several other closely related PRPs have been identified and differ fromPRP-1 either by substitutions at residues 4 and 50, or by post-translational modification, as shown in Fig. 3B. PCA (residue 1) is pyrrolidone carboxylicacid, 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 theprimary 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-tolyticus. However, strains of A. naeslundii, which only possesstype 2 fimbriae, did not respond. The adhesion of some S.mutans strains (i.e., JBP of serotype c) was also promoted byadsorbed PRP-1, but strains of S. sobrinus did not respond to

this protein. Among black-pigmented Bacteroides, strains ofB. gingivalis, B. loeschii and B. melaninogenicus displayedenhanced adsorption to PRP-1-treated HA, whereas a strain ofB. intermedius did not. S. sanguis Blackburn also exhibitedstrong adsorption to PRP-1-treated HA, but several other strains

+

B)PRP1

PIF-s

PRP-2

IEIx37-3PIF-fPRP-4

150-A

J Dent Res May 1989

-r=v-'IL-

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of S. sanguis, a strain of S. mitis, and three strains of S.pyogenes did not. On the basis of this limited survey, it seemsclear that a variety of bacteria resident in human dental plaquepossess adhesins which interact with adsorbed PRP moleculeson HA (Gibbons and Hay, 1988b).

Structural similarities between PRPs and collagens. -Theapparent possession of adhesins which react with adsorbed PRPsby several plaque bacteria prompted us to determine whetherthere were other proteins which are structurally related to thePRPs. 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 segmenthas not been found to promote adhesion of any of the bacteriatested (Gibbons and Hay, 1988a, b). Therefore, we soughtstructural similarities between the amino acid sequences of thecarboxy-terminal segment of PRP-1 and other proteins in thedata base of the National Biomedical Research Foundation Pro-tein Identification Resource (Gibbons and Hay, 1988b). Therewere 6418 sequences in this data base, but the only proteinswhich showed a relatedness score of greater than 80 proved tobe human or rodent PRPs (Fig. 4), and no proteins had a scorebetween 71 and 80. This clearly illustrates the uniqueness ofthe PRPs. However, the protein which exhibited the next high-est degree of similarity to the carboxy-terminal segment ofPRP-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 deviationsgreater than the mean of all protein matches in the data base(Gibbons and Hay, 1988b). This structural relatedness is ofinterest, considering that the matrices of dentin and cementumare rich in collagen, and collagen fibers are major structuralelements of connective tissues, including the periodontal lig-ament, as well as basement membranes. Furthermore, destruc-

tion of collagen is a central feature of both periodontal diseaseand advanced dental caries. The structural relatedness betweenthe PRPs and collagen assumes more relevance in light of ourrecent observations that strains of B. gingivalis (Naito andGibbons, 1988) and certain "mutans" streptococci [especiallyS. cricetus and S. rattus (Liu and Gibbons, unpublished data)]bind avidly to collagenous substrata. It is possible that affinityfor collagenous elements plays a role in facilitating the inva-sion of B. gingivalis cells through basement membranes andinto gingival connective tissues. Likewise, the affinity of var-ious cariogenic streptococci for collagen may play a role inthe caries process by facilitating bacterial penetration into ce-mentum and dentin following initial demineralization.

Importance of previously cryptic receptors (cryptitopes) inbacterial adhesion. -One of the surprising observations madeduring the course of these studies was that although A. viscosuscells 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 preparedfrom a few micrograms of PRP-1. Similarly, radiolabeled PRP-1 does not bind to A. viscosus cells, nor is it degraded whenincubated with the organism. Yet A. viscosus cells attach equallywell to HA surfaces treated with either radiolabeled or unla-beled PRP-1. The apparent explanation for this unexpecteddifference in behavior between PRP molecules in solution vs.PRP molecules adsorbed onto apatitic surfaces is that hiddenmolecular segments of PRPs become exposed, as a result of aconformational change, when the protein adsorbs to HA (Gib-bons and Hay, 1988a). Previous studies had, in fact, suggestedthat a major conformational change takes place when PRPmolecules adsorb to HA. This was suggested by differences in

>81

80

70

Relatedness 60

Score 40

30

(17 - Human & Rodent PRPs)

(0)(4 - Alpha 1 (1) Chain Of Collagen From

< Human, Mouse, Bovine; Plasmodium(15) Circumsporozoite Precursor)

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(126)

(851)

[6,41 8 Sequences;Mean Score = 1 4.5 (7.03)]

20 [ N (4, 1 29)

10

0 1000

(1,231)i i ~ ~ ~ ~~~~~~~~~~~~~ii

2000 3000 4000 5000Number Of Proteins

Fig. 4.-Similarity search of the National Biomedical Research Foundation Protein Identification Resource of proteins related to residues 107-150 ofPRP-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).

Vol. 68 No. 5 755



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their calcium ion binding properties (Bennick et al., 1981) andby the thermodynamics of their adsorption (Moreno et al.,1982). Adsorption of the protein to HA is an endothermicprocess 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 wouldoccur during a change in the conformation of the protein as itadsorbs onto the mineral. The high degree of asymmetry incharged residues in PRP molecules is thought to contribute tothe folding of the polypeptide chain when in solution. Evi-dently, when the negatively charged amino terminal segmentof 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 moleculesare adsorbed on surfaces provides a novel and highly efficientmechanism 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), andif A. viscosus cells could interact with PRP molecules in so-lution, there would be little likelihood that many organismswould attach to adsorbed PRP molecules on the tooth surface.The ability of A. viscosus cells to recognize unique crypticsegments in adsorbed PRP molecules provides a molecularexplanation for the predilection which this organism displaysfor the teeth. It helps to explain why the surfaces of humanteeth are the primary habitat for A. viscosus on planet Earth!

It has proven convenient to refer to such previously hiddenreceptors for bacterial adhesins as "cryptitopes". This wordis derived from "cryptic" (Gr. kryptos) meaning hidden, and"topo" (Gr. topo) meaning place. It is easy to envision howadhesins which recognize cryptitopes in surface-associatedmolecules would evolve. They would provide a strong selec-tive advantage for any bacterium which colonizes a mucosalor tooth surface. The secretions which bathe these surfacescontain components which are structurally related to those onthe surfaces of mucosal and other body cells. This molecularmimickery is thought to contribute to the cleansing action ofsecretions (Gibbons, 1982). When in solution, components ofsecretions which bind to bacteria often cause aggregation, andthis is thought to facilitate bacterial clearance (Mandel, 1976).However, if molecules on the surfaces of mucosal cells or teethbecame altered as a result of a conformational change, or be-cause of an enzymatic modification, cryptitopes could be formedwhich would promote bacterial attachment and colonization.We believe that cryptitopes will probably be uncovered for

a variety of indigenous and pathogenic micro-organisms whichcolonize mucosal surfaces. Some apparent examples are listedin the Table. In addition to the binding of A. viscosus to ad-sorbed PRPs, it has recently been reported that S. sanguis cellsbind to fibronectin which is complexed to collagen, but not tofibronectin in solution (Lowrance et al., 1988). This appearsto be due to the recognition of a cryptitope created by a con-formational change in the complexed fibronectin, and it hasbeen 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 commonpossession of galactosyl-binding adhesins by oral bacteria (Ta-ble). Organisms which appear to possess such adhesins includeA. viscosus, A. naeslundii (Ellen et al., 1980; Cisar, 1986),Leptotrichia buccalis (Kondo et al., 1976), Fusobacterium nu-cleatum (Falkler et al., 1979), Eikenella corrodens (Yamazakiet al., 1981), and Bacteroides internedius (Okuda and Kato,1987). These organisms attach poorly, if at all, to unmodifiederythrocytes, epithelial cells, or experimental pellicles pre-pared from salivary mucins. However, treatment of these sur-

TABLEAPPARENT INVOLVEMENT OF "CRYPTITOPES" IN BACTERIAL

ATTACHMENT

A. viscosus

S. sanguis

- binds to adsorbed PRPs on HA surfaces, notto PRPs in solution (Gibbons and Hay, 1988a)

- 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 afterneuraminidase treatment (Ellen et al., 1980;Falkler et al., 1979; Kondo et al., 1976; Ya-mazaki et al., 1981; Okuda and Kato, 1987)

P. aeruginosa

B. gingivalis

B. gingivalis

- binds to trypsin-treated epithelial cells de-pleted of fibronectin (Woods et al., 1983)

- binds in much higher numbers to oral epi-thelial cells which have been mildly treatedwith trypsin or papain (Childs and Gibbons,1988a, b)

- trypsin-treatment of fibronectin-collagencomplexes exposes collagen receptors whichpromote attachment (Naito and Gibbons,1988)

faces with neuraminidase removes terminal sialic acid residuesand exposes penultimate galactosyl residues. These now func-tion as cryptitopes which interact with the galactosyl-bindingadhesins of these organisms.

It is interesting to note that treatment with certain proteasesmay 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 cellsis 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 representa 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 thisorganism 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 numbersto 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 enhancedattachment 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 theepithelial 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 infectiousagent.Does poor oral hygiene generate cryptitopes?-The poten-

tial of neuraminidase and certain proteases to create cryptitopessuggests 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-establishedthat the oral hygiene and periodontal status of an individualcorrelates with the levels of a variety of enzymes in saliva andin crevicular fluid. Thus, elevated levels of proteases (Berg etal., 1947; Watanabe et al., 1981), including trypsin-like en-

J Dent Res May 1989




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 poororal 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 theassociated bacterial plaque accumulations.

These observations raise the intriguing possibility that alter-ations in an individual's oral hygiene state, or state of gingivalinflammation, may result in elevated levels of enzymes withthe potential to modify local tissue surfaces and generate cryp-titopes which affect the types of bacteria which colonize. Infact, 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 ofperiodontitis. However, several recent studies have suggestedthat specific species of bacteria are associated with specifictypes of periodontal diseases (Slots, 1979, 1986; Socransky etal., 1988; Zambon, 1985; Moore, 1987; Slots and Listgarten,1988). The question therefore arises as to how failure to brushone'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, maycontribute 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 theshift in the flora from one which is benign, and composedprimarily of streptococci and other Gram-positive organisms,to one which is predominantly Gram-negative and associatedwith periodontal destruction. We have begun to test this hy-pothesis by studying the effect of treating oral epithelial cellswith neuraminidase or trypsin on the ability of target bacteriaassociated with either gingival health or disease to attach (Childsand Gibbons, 1988a, b). We have observed that treatment ofepithelial cells with neuraminidase greatly reduces the numberof 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 lowconcentrations of trypsin or papain also markedly decreasedattachment of these streptococci, while attachment of B. gin-givalis was greatly promoted (Fig. 5). It appears significantthat treatment of epithelial cells with lysosomal enzyme prep-arations, obtained from human PMNs, also markedly reducedattachment of S. mitis, while it greatly enhanced attachmentof B. gingivalis and Actinobacillus actinomycetemcomitans(Childs and Gibbons, 1988b). Thus, the elevated levels ofthese enzymes reported present in crevicular fluid of patientswith 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 prominentoral bacteria. Thus, strains of S. mitis and S. sanguis possessadhesins which bind to sialic acid-containing receptors (McBrideand Gisslow, 1977; Murray et al., 1982, 1986). Treatment ofpellicles or epithelial cells with neuraminidase would be ex-pected to impair their attachment (Gibbons and Etherden, 1982;Gibbons et al., 1983). However, treatment with neuraminidaseincreases 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 etal., 1979), B. intermedius (Okuda and Kato, 1987), and Li-

kenella corrodens (Yamazaki et al., 1981). The elevated levelsof trypsin-like enzymes in crevicular fluid would also be ex-pected to increase exposure of arginine residues, and therebypromote 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 arealso known to elaborate enzymes appropriate for creating cyp-titopes for themselves. For example, strains ofA. viscosus andA. naeslundii synthesize neuraminidases which can promotetheir attachment to mammalian cell surfaces (Ellen et al., 1980),while strains of B. gingivalis and other suspected periodontalpathogens elaborate trypsin-like proteases which cleave argi-nine peptides (Loesche et al., 1987). The highest concentra-tions of these enzymes would likely be found aroundactinomycete 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 theadhesion 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 oforal epithelial cells in individuals with different states of oralhygiene and gingival health. Therefore, we have analyzed theamount of sialic acid on the surfaces of buccal and gingivalepithelial cells from five healthy individuals, and from fivepatients with gingivitis (Davis and Gibbons, 1988, unpublishedobservations). Buccal epithelial cells had almost three timesas much sialic acid on their surface as did gingival epithelialcells in both groups of individuals. This helps to explain whyS. mitis, which possesses a sialic acid-binding adhesin (Murrayet al., 1986), dominates on buccal mucosa. We further ob-served that the amount of sialic acid on buccal and gingivalepithelial cells was much lower in persons with gingivitis ascompared 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 cellsfrom healthy individuals averaged almost 40 pug sialic acid per104 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 ofindividuals with poor oral hygiene and gingivitis (Perlitsch andGlickman, 1967; Kitawaki et al., 1983) does appear to modifythe surfaces of oral tissues.

Collectively, these observations offer an explanation as tohow poor oral hygiene and resulting inflammation can predis-pose the gingival crevice area to increased colonization bypotential periodontal pathogenic bacteria. The resulting mod-ification of oral surfaces by enzymes appears to result in thecreation of cryptitopes. This also may explain why personswith poor oral hygiene tend to develop plaque more rapidlythan individuals with good oral hygiene and healthy gingivae.These observations are also consistent with recent reports ofHarper et al. (1988) and Wolff et al. (1988) who have notedthat there are decreased proportions of streptococci, and ele-vated proportions of black Bacteroides such as B. gingivalisand B. intermedius, in periodontally-diseased sites associatedwith elevated levels of lysosomal and other enzymes. How-ever, further research is needed to confirm or negate thesehypotheses.

Acknowledgments.I thank members of the selection committee for giving me

this opportunity to present the 1988 Kreshover Lecture. I wouldalso like to acknowledge the contributions of many individualswho have collaborated in these studies. Particular acknowl-edgment is due to Dr. J. van Houte, who collaborated in sev-

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758 GIBBONS

40

Mean 30

Number

Bacteria 20

Attached

Per Cell 10

0None Neuraminidase Trypsin Papain

Epithelial Cell TreatmentFig. 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 collaborationin 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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J Dent Res May 1989



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