Oral Microbiol Immunol 1992: 1: 198-203

Inhibition o\ Actinomyees viscosus - Porphyromonas gingivaiis coadhesion by trypsin and other proteins

R. P. Ellen, M. Song, I. A. Buivids Faculty of Dentistry. University of Toronto. Ontario, Canada

Ellen RP Song M, Buivids IA. Inhibition o/ Actinomyees viscosus - Porphyromonas gingivalis coadhesion by trypsin and other proteins. Oral Microbiol Immunol 1992: 7: 198-203. Protease activity is associated with the coadhesion of Actinomyees visco.sus and Porphyromonas gingivalis. To try to distinguish whether the recognition/adhesion or degradative futictions of proteases are more crucial for coadhesion, we determined the effect of trypsin and other purchased proteases and proteins on coadhesion when they were incorporated in the coadhesioti assay buffer or when A. viscosus cells were pretreated with trypsin. Coadhesion was tneasured by the decrease in turbidity caused by the absorption of A. viseosus cells frotn aqueous suspension by P. gingivalis-coated hexadecane droplets. Pretreatment of A. viscosus with trypsin had no obvious effect on the kinetics of coadhesioti. Likewise, trypsinization of A. viscosus failed to aid or enhance coaggregation by chemically induced, trypsin activity-deficient mutants of B. gingivalis. In contrast, incorporating trypsin in the buffer during the coadhesion assay yielded a concentration-dependent inhibition of coadhesion greater than the inhibition found with the same concentration of other proteases. Coadhesion was also impaired to a greater extent by similar wt/vol concentrations of nonproteolytic proteins (bovine serum albumin (BSA), defatted BSA, gelatin, and casein), by antisera against whole P. gingivatis cells and fimbriae, by preitntnune serum, and by the amino acid arginine but not lysine. These findings suggest that the role of proteases in coadhesion is not solely to enzytnatically "prime" A. vi.sco.sus for more avid coadhesion and that their role as potential protein or peptide seeking adhesins should be considered.

The highly proteolytic periodontai pathogen Porphyromonas (Bacteroides) gingivalis is initially retained on the teeth by avidly adhering to bacteria that have colonized previously (30). Actinomyees viscosus is likely to be significant among these, because it is ubiquitous in plaque at the gingival tnargin, it is often cocultivated with P. gingivalis, it coaggregates with P. gingivalis, and it produces by-products that enhance the growth of P gingivalis (5, 6, 21, 32). Our laboratory has developed quantitative, sequential coadhesion assays with which to explore the molecular tnechanisms by which, these species recognize and adhere to each other and the surface structures that bear the significant adhesins (5, 6, 27). We found that P gingivalis coadheres with A. viseosus more avidly than do the other species once

classified together in the getius Bacteroides (18). Because it is the most proteolytic of these species and because its proteolytic surface vesicles bind readily to A. vi.seo.sus (4), we began to study the possibility that P. gingivali.s's proteolytic activity might contribute to its coadhesion with A. viseosus. We found that trypsin activity seemed to be associated with coadhesion, based on evidence that 1) chemically induced mutants deficient in trypsin activity failed to coaggregate or coadhere with A. vi.sco.sus; 2) assay conditions known to enhance P. gingivatis trypsin activity enliaticed coadhesion; and 3) conditions known to diminish enzyme activity, including the incorporation of protease inhibitors iti the assay buffer, impaired coadhesion (19). Such finditigs compelled us to raise two hypotheses, which are not necessarily

Key words: coadhesion: Porphyromonas gingivaiis: Actinomyees viscosus: protease: inhibition assay R. R Ellen, University of Toronto, Facuity of Dentistry, 124 Edward Street, Toronto M5G 1G6, Canada Accepted for publication January 22, 1992

mutually exclusive (19): I) Through their substrate-specific recognition domains, P. gingivalis's cell-associated proteases foster coadhesion by functioning as adhesins. 2) As implied in some of Gibbons and coworkers' recent reports (8), through their degradative activities, P. gingivalis proteases "pritne" A. viscosus cells for more avid coadhesion by tnodifying or uncovering cryptic dotnains. Rosenberg et al. recently developed a rapid assay for investigating the kinetics of coadhesion by tneasuring the decrease iti turbidity caused by the partitioning of A. vi.scosus from aqueous suspension onto P. gingivalis-coated nhexadecane droplets (27). They proposed a model in which initial coadhesive interactions would occur pritnarily through stereochemical associations.

A. viscosus - P. gingivalis coadhesion frotn C. I. Hoover, University of California, San Francisco (Hoover Cl, Felton JR. J Dent Res 1988; 67: 368, abstr 2044); NG4B19 atid NG5B2 proved to be coaggregation-negative with A. visco.sus (19). They will be referred to in this article as Bacteroides strains. Stocks of the Porphyromonas and Bacteroides strains were lyophilized. Working cultures were tratisferred every second week on laked blood agar no. 2 (Oxoid, Basingstoke, Hampshire, UK), suppletnented with 7%) laked sheep's blood and I /ig/tnl each filter-sterilized hemin and tnenadione. The plates were incubated anaerobically as above. For experiments, P. and B. gingivatis strains were grown in a modified Trypticaseyeast extract broth containing Trypticase-peptone (BBL Microbiology Systems, Cockeysville, MD) suppletnented with 3 g yeast extract (Difco Laboratories), 5 g NaCl, 2.5 g K,HPO4, 2.5 g Material and methods glucose, 5 tngfilter-sterilizedhemin, and Bacterial strains and culture conditions 0.5 tng filter-sterilized menadione (10). A. vi.scosus WVU 627, which represents The cells were washed, dispersed by the typical A. viscosus strains of hutnan passage through a 25-gauge tieedle, and isolates in the nutnerical taxonomy clus- suspended in phosphate buffer to an opter 1 of Fillery et al. (7), was originally tical density of 2.0 for preparing P. ginobtained frotn M. A. Gerencser of West givati.s-coa\.ed hexadecane droplets for Virginia Utiiversity. A. viscosus T14V- coadhesion assays and 1.0 for coaggreJl atid its fitnbrial tnutants 5519 (lack- gation experiments. ing type 2 fimbriae) and 5951 (lacking type 1 fimbriae) were originally obCoadhesion and coaggregation assays tained frotn J. Cisar, National Institute of Dental Research (3). A. vi.seosus Titncd coadhesion assays were used to strains were tnaintained as lyophilized determine the degree of adhesion of A. stocks atid brain heart infusion agar viscosus cells in suspension to P. ginslants (Difco Laboratories, Detroit, givalis cells coated on n-hexadecane MI), which were subcultured tnonthly droplets by the tnethods of Rosenberg and stored at 4 C. For coadhesion ex- et al. (27). Briefiy, P. gingivalis 2561periments, WVU 627 was cultivated in coated hexadecane droplets (PCHD) tryptic soy broth (Difco Laboratories) were prepared by high-speed vortexing at 37"C for 48 h in an attnosphere of of aqueous suspensions of P. gingivalis 80% Nj, 10% CO2 and 10% H,. Bacteria with n-hexadecane, followed by phase were harvested by low-speed centrifuga- separation. To 1,0 ml A. viseosus sustioti, washed three titnes, and then sus- pension in bofosilicate glass test tubes pended in phosphate-buffered saline, (11 tnm inner diatneter) were added 0.5 pH 7.2 (PBS), to an optical density of ml of PBS, with or without inhibitors, 2.0 at 550 nm in a model 350 spectro- and 0.5 tnl of PCHD. For sotne experiphotometer (G. K. Turner Associates, metits, the volutnes were doubled but Palo Alto, CA) (27). the ratio of phases and ingredients was P gingivalis 2561 (ATCC 33277, type held constant. The tubes were rotated strain) was originally obtained frotn ,1. at a fixed incline of 40' at atnbient temSlots, Utiiversity of Southern Califor- perature in a Multi-purpose Rotator nia, when he was at the State University (Scientific Industries, Springfield, MA). of New York at Buffalo. Chemically iti- At 2-tnin ititervals, phases were allowed duced tnutants of a Macaca fascicutaris to separate and the turbidity of the isolate were received along with their aqueous A. viscosus suspension was paretit strain as Bacteroides gingivatis tneasured. Cotitrol tubes cotitained hexNG4B19 and NG5B2 (trypsin activity adecane rather than PCHD. deficient [trypact"], NG5A2 (collagenThe effects of various proteases, proase deficient), and 3079.03 (parent) teins, chromogenic substrates, and ami-

with subsequent lateral adhesion between accutnulating like cells being stabilized by hydrophobic interactiotis. Bovine serum albutnin (BSA) was found to partially inhibit and alter the kinetics of coadhesion. Their finding that defatted BSA was a more potent inhibitor than BSA suggests that, in addition to some potential differetices iti hydrophobic effects, it might be the exposure to proteins or specific protein dotnains that inhibits coadhesion more specifically. The purpose of this investigation was to compare the effects of various proteolytic and nonproteolytic proteitis on A. viscosus - P. gingivalis coadhesion atid thereby generate tnore evidence to distinguish between the two hypotheses explaining the function of proteases in coadhesion; adhesin or degradative etizyme?

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no acids on coadhesion were tested by additig them to the PBS coadhesioti buffer. The sources of these reagents were as follows; trypsin (type III bovine pancreas, Sigtna T8253, Sigma Chetnical Co., St. Louis, MO), chymotrypsin (pchymotrypsin, bovine pancreas, Sigtna Chemical Co. C4629), and protease K (type XI, Tritiraehium album. Sigma Chetnical Co. P0390); BSA and defatted BSA (Sigma Chemical Co.); gelatin and casein (Difco Laboratories); L-arginine and L-lysine (J. T. Baker). Proteases and nonproteolytic proteins were tested at final concentrations ranging frotn 1.0 //g/tnl to 1.0 tng/ml. Arginine atid lysine were used at fitial concentratiotis of 0.1 tnM, 1.0 mM, atid 10.0 tnM. The experiments with trypsin and arginine were repeated, cotnparitig their effects when A. viscosus cells were heated at 70 C for 30 tnin. The effects of peptide chrotnogenic substrates often used for tneasuring trypsin-like and chymotrypsiti-like activity were tnonitored by incorporating N-a-benzoyl-DL-arginine-pnitroanilide (BAPNA) or N-succinyl-Lalanyl-L-alanyl-L-prolyl-L-plienylalanine-p-nitroanilide (SAAPNA) (Sigtna Chemical Co.) respectively in the buffer at concentrations from 0.05 to 1.0 tnM. As dimethyl sulfoxide (DMSO, Sigma Chetnical Co.) was used in preparing the solutions, a DMSO control without substrates was iticluded at a fitial concentration of 10% vol/vol, the same concentration as in the tubes with the peptides. Iti addition to detertnining the effects of inhibitors in the assay buffer, the effect on coadhesion of pretreating the A. viscosus cells with trypsin was tested. A. viscosus suspensions iti phosphate buffer, pH 8.0, containing 1.0 tng/tnl trypsin were incubated at 37 C for either 1 h, 3 h, 5 h or overnight and washed twice before preparing the final suspension for coadhesion assays. The assays were run in the absence or presence of trypsin over the concentration range 1.0 //g/tnl to 1.0 tng/ml, or run in the absetice or presence of arginine or lysitie at 10.0 mM. To detettnine whether P. gingivalis cells coated on hexadecane droplets were proteolytically active under the coadhesion assay conditions, PCHD were rotated with coadhesion buffer containing 1.0 tnM of either BAPNA or SAAPNA. Trypsin at 5.0 /ig/tnl to 20 //g/tnl final concentration was used as a standard for degradative activity. Enzyme activity was detertnined by tneasuring the change in ab-

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sorbance of the aqueous phase at 405 nm. The trypact" mutants partition on hexadecane droplets, but they do not form coatings over much of the droplet surface area. Therefore, the effect of trypsin pretreatment of A. viscosus strains was also tested by coaggregation as described by Li et al. (19). Washed suspensions of trypsin-treated (1.0 mg/ ml, pH 8.0, 2 h) or untreated A. viseo.sus cells were mixed on fiocculation slides with B. gingivalis or P. gingivatis suspensions and rotated at 90 rpm for 5 min. The degree of coaggregation was scored visually from 0 (no visible aggregates) to 4 (maximum aggregation yielding one clump). Antiserum against whole P. gingivalis 2561 cells was raised in female New Zealand white rabbits. Fortnalinized cells stored at 4 "C were washed twice in sterile PBS, pH 7.2, resuspended to 1.0 x 10' cells/ml and mixed in equal volume with Freund's complete adjuvant. The rabbits were inoculated with 0.5 ml at four subcutaneous sites. Four weeks later, 4 additional subcutaneous injections were given, using Freund's incomplete adjuvant. After 2 weeks, 0.5 ml of formalinized cells was administered intravenously. The rabbits were bled 2 weeks later. The anti-fimbrial antiserum raised against isolated fimbrial preparations was described previously (11). Unabsorbed anti-fimbrial antiserutn was used for this investigation. These antisera and preimmune rabbit serum were adjusted to contain an equivalent concentration of protein (6.4 mg/ml) and dilutions were used to pretreat PCHD prior to the coadhesion assay. PCHD were rotated with 1/5, 1/10, and 1/50

phosphate buffer dilutions of the sera for 1 h and allowed to separate from the aqueous phase. They were washed twice and then rotated with aqueous suspensions of A. viseosus WVU 627. Results Trypsin pretreatment versus trypsin in the assay buffer

Pretreating A. vi.seosus WVU 627 with trypsin had very little effect on its subsequent coadhesion with P. gingivalis 2561 (Fig. 1). Only the longer pretreatments, 5 h atid overnight, caused the slope of the turbidity curves to change slightly, if at all, and the change was in the direction of partial inhibition. In contrast, incorporating trypsin in the assay buffer yielded a concentration-dependent inhibition of codhcsion within the first few tninutes (Fig. 2). Incorporating trypsin also inhibited coadhesion with A. viscosus cells which had been pretreated with trypsin (data not shown). Atnong the proteases tested, trypsin had the greatest inhibitory effect (Fig. 3). As we have published previously, heated A. viscosus cells still coadhere with P. gingivalis (5). Trypsin in the assay buffer yielded equivalent concentration-dependent inhibition of coadhesion with heated A. viscosus ceWs (data not shown). As found previously (19), the trypact" mutants did not coaggregate with any of the A. viscosus strains tested. Trypsin pretreatment of the A. viscosus cells did not render them agglutinable by these mutants (Table 1). For the parent and collagenase-deficient mutant, some coaggregation scores for trypsin-treated A. viscosus were lower than for untreated A. visco.sus. Simi-

0

2

4

6

Tlmo{min.)

Fig. L The effect of pretreating A. viscosus WVU 627 with 1 tng/ml trypsin for various titne periods on its subsequent adhesioti to P. gingivalis 2561-coated hexadecane droplets. Data are expressed as the log percentage decrease in turbidity as a function of mixitig time, where "at" is the absorbance for titrte t and "ao" is the absorbance for tirne zero, just before mixing. Sytnbols: - • - , 1 h pretreatment; - O - , 3 h; - • - , 5 h; - D - , overnight; - • - , control (no trypsin pretreatment).

larly, trypsin pretreatment o( A. viscosus WVU 627 did not enhance its negligible coadhesion with the trypact" B. gingivatis strains coated on hexadecane (data not shown). Inhibition by trypsin relative to other proteins and amino acids

Like trypsin, the nonproteolytic proteins BSA, gelatin, and casein impaired coadhesion when incorporated in the assay buffer, and itihibition was concentration-dependent. On a weight/volume basis, the nonproteolytic proteins were more effective inhibitors than trypsin and the other proteases (Fig. 4). There was also a great difference in the degree of inhibition by the proteins, with casein being most effective. Arginine yielded inhibition of coadhesion at 10 mM final

Tahle J. Coaggregation with trypsin-treated and control cells of A. viscosus strains

Coaggregation score*

A. viscosus strain (treatment)

P gingivalis or B. gingivalis strain 2561

3079.03 parent

NG4B19 trypact"

NG5B2 trypact"

NG5A2 collagenase"

PBS

3 2 4 . 3

0 0 0 0

WVU 627

control trypsin

4 4

3 3

0 0

0 0

T14V-J1

control trypsin

4 4

4 4

0 0

0 0

5519

control trypsin

4 4

4 2

0 0

0 0

4

1

0 0

control trypsin

4 4 1

4 3 0

0 0 1

0 0 0

4 :2 0

0 0 0

5951 PBS control

,

•

* Coaggregation score: 0 = no aggregation detected; 4 = maximum aggregation yielding a single clump of cells.

0

2 4 TIME(mln.)

Fig. 2. The effect of various concentrations of trypsin in the assay buffer on the adhesion of A. vi.scosiis WVU 627 to P gingivati.s 2561coated hexadecane droplets, expressed as in Fig. 1. Symbols: -O-, 1 /jg/ml trypsin; - • - , 10 //g/ml; - A - , 100 //g/ml; - D - , 1 mg/ml; - • - , control (no trypsin added).

A. viscosus - P. gingivalis coadhesion concentration, both for trypsin-treated and control A. viseosus cells (Fig. 5), but it had no inhibitory effect when the A. viscosus cells were pretreated by heating. Lysine had no effect. BAPNA and SAAPNA had very little effect except at the highest concentration, 1.0 mM (data not shown). The DMSO control and SAAPNA actually enhanced coadhesion slightly; BAPNA at 1.0 mM yielded ~ 20% decrease in the degree of coadhesion relative to the DMSO control. Determination of peptidase activity by PCHD in the absence of A. viseosus yielded positive results with BAPNA as the substrate. Over 10 min, 0.5 tnl PCHD had the equivalent BAPNA degrading activity as 34.4 //g/ml trypsin. No SAAPNA-degrading activity was detected. As expected, antisera raised against whole P. gingivalis cells and against fimbriae impaired coadhesion to a greater extent than the preimmune serum, when adjusted to the same protein concentration (Fig. 6). All three sera were inhibitory relative to the serutn-free control when diluted 1 /5 (1.28 mg/tnl protein) and 1/10 (640 //g/tnl) but not 1/50 (128//g/ml).

4

6 8 TIME (min)

10

12

Fig. 4. The effect of I mg/tnl nonproteolytic proteins in the assay buffer on the adhesion of A. vi.scosus WVU 627 to P gingivatis 2561-

coated hexadecatie droplets, expressed as in ftgure 1. Trypsiti 1 tng/tnl is included as a cotnparisoti. Symbols (frotn top): - • - , casein; - • - , gelatin; - D - , defatted BSA; - O - , BSA; - • - , trypsin; - D - , control (no protein).

A. viscosus cells with trypsiti had little effect on their subsequent adhesion, especially not an enhaticing effect. Trypsinization of A. viseosus strains did not retider thetn agglutinable by mutant B. gingivalis strains defective in trypsin activity. Such data imply that the futiction of P. gingivalis's proteases in coadhesion with A. viscosus might not derive solely from their degradation of A. viseosus surfaces to foster tnore avid interaction Discussion with P. gingivalis adhesins. Although In these experiments the tnost effective trypsin proteases were likely enzyinhibitors of coadhesion between A. vi.s- matically active during the brief assay eosus and P. gingivalis coated on hexade- period, as indicated by the degradation cane were the common proteins casein, of BAPNA, it is not clear what effect gelatin, and defatted BSA. Among the their degradative properties had on the proteolytic enzymes tested, trypsin was rather immediate coadhesion reaction. the only consistent inhibitor, and its inWhy would P. gingivalis proteases be hibitory activity was slightly less than involved in coadhesion with A. viscosus that of the nonproteolytic proteins on a or be significant in other P. gingivalis weight/volume basis. Pretreattnent of adhesion functions? Probably because

;S

201

of its need to transport peptides for energy, P. gingivatis has evolved a variety of proteases which allow it to degrade a wide range of proteitis iti its envirotitnetit, iticluditig tissue tnatrix proteins, collagen, fibrinogen, imtnunoglobulins, and a host of other significant intermediates in inflatnmation and clotting pathways (17, 20, 24, 33). Moreover, it has evolved to contain tnuch of its proteolytic activity iti its outer tnembrane, which sheds surface vesicles and thereby increases the effective substrate contact area (13, 31). We have shown previously that P. gingivalis vesicles bind avidly to A. viscosus (4). Indeed, they seetn to adhere to many types of bacteria with little apparent selectivity (1, 4, 13). Our previous work has cast doubt that outer membrane LPS is significant in coadhesion with A. viseosus (5). The P. gingivalis adhesin(s) is heat, glutaraldehyde (unpublished data) and pH 2.0 sensitive (5), and therefore likely a protein itself. However, it is relatively stable to proteolysis (5). Coadhesion is itnpaired by protease inhibitors and running the assay at 4"C, and it is enhanced under reduced conditions and at 37°C (19). Sitice coadhesion was inhibited by excess protein "substrate" in the buffer, it is conceivable that the tnany proteases concentrated in P. gingivalis vesicles comprise a group of relatively protease-resistant adhesins which are available to interact with surface proteins on several species of bacteria. This raises the possibility that trypsin in the assay buffer was inhibitory because it is a protein and not necessarily because it is an enzyme.

t.6 0 TIME(mln.)

10 TIME (min.)

Fig. 5. The effect of 10 mM L-arginine on the Fig. 6. The effect of pretreatment of P. giiiadhesion of trypsin-preated and untreated A. givati.s-coaled hexadecane droplets (PCHD) Fig. 3. The effect of including 1 tng/tnl pio- vi.scosus WVU 627 cells to P. gingivalis 2561- with atitisera against whole P. gingivalis cells teases in the assay buffer on the adhesion of coated hexadecane droplets, expressed as in or fttnbriae and preimtnune serum, at 1.28 A. viscosus WVU 627 to P. gingivalis 2561Fig. I. Sytnbols: With no arginine: - • - , no mg/tnl protein, on the adhesion of ^. viscosus coated hexadecane droplets, expressed as in ttypsin pretreatment; - O - , 1 b trypsin; -EI-. WVU 627 to PCHD, expressed as in Fig. 1. Fig. 1. Symbols: - 0 - , trypsin; - O - , chytno- 3 h trypsin; - A - . 5 h trypsin; - + -, overnight Symbols: - • - , control (no PCHD); - • - , trypsin; - • - , protease K; - • - , control (no trypsin; With arginine in the buffer: - x -, no anti-whole cell antiserum: - O - , anti-fimbrial protease); - D - , BSA included as a comtrypsin pretreatment; - 0 - , 1 h trypsin; - • - , antiserutn; - D - , preitnmune serutn; - • - , parison. 3 h trypsin; -EB-, overnight trypsin. conttol (no serum).

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The protein BSA is often added to viously cryptic receptors for coadheadhesion assay buffers to prevent non- sion. Trying to distingush the recognition specific interactions from masking the stereospecific interactions being from the degradative functions of P. ginstudied. Both BSA and defatted BSA givalis surface enzymes is also very diffiaffected the coadhesion of A. viseosus cult using inhibition assays based on and P. gingivalis, but was the inhibition proteins, peptides, amino acids, and a nonspecific? We have shown previously wide variety of protease inhibitors. In that P. gingivalis 2561 fimbriae, distitict each case, effective arguments can be structures which bear adhesins for A. raised that activity was inhibited by viseosus, do not bind appreciably to im- either blocking access to specific submunobilized BSA (11). However, con- strate recognition domains or intersidering potential adhesins that might fering with the function of the catalytic be borne on P. gingivalis vesicles, at least domain of the enzyme, or both. Perhaps 7 proteins have been shown to degrade in the case of specific arginine inhi(and therefore bitid) BSA as a substrate bition, which is common to some coad(12). Thus it is possible that the inhi- hesion and hemaggiutination activities bition of coadhesion by BSA might of P gingivalis (1, 15, 19, 22, 26), it is have involved its combination with any more likely that access to the enzyme's of several surface enzymes. If so, a simi- recognition domain by substrate was inlar scenario is conceivable for casein, hibited cotnpetitively than if inhibition gelatin, and many other potentially in- was due to blockage of functional prohibitory proteins. Consistent with this teolysis. Such reasoning assumes that a concept is the finding that preimtnune major P. gingivalis adhesin is an enzyme serum adjusted to contain at least 0.64 and its significant target adhesin/submg/ml protein was inhibitory compared strate on the A. viscosus surface is at with the serum-free control. Recent least, in part, an arginine-containing studies on P. gingivatis-Streptococcus peptide or protein. The evidence that it mitis coaggregation led to rather similar is a protein is weak. Neither heating A. findings. Hutnan plasma, saliva, and a viseosus cells (5) nor fixation by glutarfew of the plasma proteins which were aldehyde (unpublished data) has any eftested inhibited their coaggregation fect on their subsequent coadhesion (22). Adherence of P. gingivatis to types with P. gingivalis. However, heating A. I and IV collagen is also inhibited sub- viseosus renders coadhesion no longer stantially by these coUagens and by susceptible to inhibition by arginine, gelatin, fibronectin, and fibrinogen (23). which might not eliminate peptides as Moreover, a few of P. gingivalLs's pro- potential adhesins. Trypsinization of A. teolytic enzymes have already been im- vi.seosus cells had little effect on subsequent coadhesion, and lysine was plicated as adhesins (17, 25). Because technical difficulties cur- noninhibitory, which might not have rently limit the engineering of isogenic been the case if the A. viscosus adhesin P. gingivalis mutants, segregatioti of the were a trypsin-degradable protein. recognition and the degradative func- Coadhesion with P gingivalis also tions of its proteases has not yet been showed no association with A. viscosus achieved experimentally. In substituting fitnbriation or fimbrial type in this or the use of chemically induced trypact" a previous study (27); yet its fimbriae, mutants, there is a risk that the strains which are virtually pure proteins, are might also be deficient in other ad- significant for coaggregation with other hesion-related defects. Our finding that bacteria (2) and for adhesion to apatitethey were less able than most P. gin- adsorbed proline-rich proteins and stagivalis strains to coat hexadecatie drop- therin (9). A. viscosus - P. gingivatis lets and previous reports that pleio- coadhesion is not very sensitive to the tropic mutants were protease-deficient, incorporation of common sugars or less hydrophobic, and weak hemaggluti- sugar amities in the assay buffer (1, 5); nators (14, 29) caution against strong yet both of these species coaggregate by conclusions based solely on the use of lectin-like functions with other species such mutants. However, from our ex- (2, 16). periments it seems unlikely that the inCurrently, the target adhesin(s) on ability of the two trypact" mutants to the A. viseosus surface retnains a puzzle. coaggregate with A. viseosus derived ex- It tnight be similar to a short peptide, clusively from their inability to degrade without a trypsin-accessible arg-lys site, trypsin-sensitive substrates on the A. vi- and possibly bound to a polysaccharide scosus surface and thereby "prime" pre- or other heat-resistant polymer. It

would still be sensitive to inhibition by arginine or the proteins used. P. gingivalis probably has more than one type of adhesin and adhesin-bearing structure. At least one type would be associated with fimbriae but not necessarily identical to fitnbrillin (11). Others would be associated with the outer membrane, most likely outer membrane and vesicle proteins. Some of these adhesitis might coincidently be identical to the trypsin-like and perhaps other proteases which this species has evolved to help satisfy its energy requirements. If so, their adhesive functions would probably be inhibitable by trypsin and other protein substrates. Acknowledgements

This investigation was supported by grant MT-5619 frotn the Medical Research Council of Canada. We thank C. I. Hoover and J. R. Felton of UCSF and J. O. Cisar of NIDR for contributing the original cultures of their valuable tnutant strains. References 1. Bourgeau G, Mayrand D. Aggtegation of Actinomyees strains by extracellular vesicles produced by Bacteroides gingivalis. Can J Microbiol 1990: 36: 362-365. 2. Cisar JO, Brennan MJ, Sandberg AL. Lectin-specific interaction of Actinomyees fttnbriae with oral streptococci. In: Mergenhagen SE, Rosan B, ed. Molecular basis of oral microbial adhesion. Washington, DC: Am Soc Microbiol, 1985: 159-163. 3. Cisar JO, Vatter AE, Clark WB, Curl SH, Hurst-Calderone S, Sandbetg AL. Mutants of Actinomyees viscosus T14V lacking type 1, type 2, or both types of fimbriae. Infect Imtnun 1988: 56: 2984-2989. 4. Ellen RP, Grove DA. Bacteroides gingivalis vesicles bind to and aggregate Aclinomyces viscosus. Infect Immun 1989: 57: 1618-1620. 5. Ellen RP, Schwarz-Faulkner S, Grove DA. Coaggregation among periodontai pathogens etnphasizing Bacteroides gingivalis - Actinomyees viscosus cohesion on a saliva-coated tnineral surface. Can J Mierobiol 1988: 34: 299-306. 6. Ellen RP, Segal DN, Grove DA. Relative proportions of Actinomyees viseosus and Aetinomyces naeslundii in dental plaque eollected from single sites. J Dent Res 1978: 57: 550. 7. Fillery ED, Bowden GH, Hardie JM. A comparison of strains of bacteria designated Aetinomyces viscosus and Acti-

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