Vol. 38, No. 2INFECTION AND IMMUNITY, Nov. 1982, p. 637-6440019-9567/82/110637-08$02.00/0Copyright 0 1982, American Society for Microbiology

Hydrophobic Interactions and the Adherence of Streptococcussanguis to Hydroxylapatite

WARREN E. NESBITT,1t RONALD J. DOYLE,'* AND K. GRANT TAYLOR2Department of Microbiology and Immunology' and Department of Chemistry,2 Health Sciences Center,

University of Louisville, Louisville, Kentucky 40292

Received 23 April 1982/Accepted 1 July 1982

Streptococcus sanguis demonstrated a high affinity for hydrocarbon solvents.When aqueous suspensions of the organism were mixed with either hexadecane ortoluene, the cells tended to bind to the nonaqueous solvent. Increases intemperature resulted in a greater affinity of cells for hexadecane. Interactionbetween the cells and hexadecane was also enhanced by dilute aqueous sodiumchloride and by low pH (pH < 5). The results suggest that the cell surface of S.sanguis has hydrophobic properties. Isolated cell walls also tended to partitioninto the nonaqueous solvent. Amino acid analyses of the walls revealed thepresence of several amino acids which possess hydrophobic side chains. It islikely that the hydrophobic amino acids associated with the cell wall contribute tothe hydrophobicity of intact S. sanguis. When the adherence of S. sanguis tosaliva-coated hydroxylapatite was measured, it was found that hydrophobic bond-disrupting agents, such as the Li' cation, the SCN- anion, and sodium dodecylsulfate, were capable of inhibiting the cell-hydroxylapatite union. In addition, itwas observed that both urea and tetramethylurea were inhibitors of the adher-ence, although the latter reagent was the superior inhibitor. The results suggestthat the adherence of S. sanguis to saliva-coated smooth surfaces is at leastpartially dependent on the formation of hydrophobic bonds between the cell andadsorbed salivary proteins. Hydrophobic bonding may contribute to cooperativeinteractions involving S. sanguis and saliva-coated hydroxylapatite (Nesbitt et al.,Infect. Immun. 35:157-165, 1982).

The genesis of many bacterial diseases occurswhen the organisms adhere to host tissues. Theadherence is usually specific in that a givenbacterium will adhere only to tissues which havereceptors capable of forming a productive unionwith that organism. When the adherence of oralstreptococci to hard surfaces of the oral cavity isconsidered, it appears that Streptococcus san-guis has the highest affinity (1, 3, 11). S. sanguishas been considered to be important in cariogen-esis because it is an early colonizer of freshlycleaned tooth surfaces and because it is fre-quently isolated from carious lesions (2, 28).

It has been difficult to describe the adherenceof S. sanguis to saliva-coated smooth surfaces inmolecular terms. Several reports suggest that S.sanguis has cell surface-associated proteinswhich may be important in adherence. Rosanand Appelbaum (25) and Liljemark and Schauer(15) have reported that the adherence of S.sanguis is destroyed by protease treatment ofthe cells. Bloomquist et al. (C. G. Bloomquist,

t Present address: Department of Microbiology, Universityof Alabama, Birmingham, AL 35294.

W. F. Liljemark, and L. Fenner, J. Dent. Res.,61:190, 1982) have isolated a protein from cellwalls of S. sanguis which has the ability to blockthe adherence of the intact bacteria. Further-more, Murray et al. (P. A. Murray, M. J. Le-vine, and L. A. Tabak, J. Dent. Res. 61:215,1982) have isolated a cell surface-associatedprotein from S. sanguis which could bind sialicacid residues of salivary mucins. McBride andGisslow (17) have suggested that S. sanguis canbind the sialic acid residues of certain salivaryproteins, thereby promoting aggregation of thebacteria. When adherence data are treated byuse of Scatchard and Hill plots, curves sugges-tive of positive cooperativity are observed (20).We have suggested (R. J. Doyle, W. E. Nesbitt,and K. G. Taylor, FEMS Microbiol. Lett., inpress) that cell surface-pellicle complexes, pos-sibly involving protein-protein interactions andstabilized by hydrophobic bonds, could accountfor the adherence of S. sanguis to saliva-coatedsurfaces. In this report, we provide evidencethat the cell surface of S. sanguis exhibits char-acteristics of hydrophobicity and that hydropho-bic bonds may stabilize the cell-pellicle union.

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638 NESBITT, DOYLE, AND TAYLOR

(Preliminary results of this work have beenpresented previously [W. E. Nesbitt, R. J.Doyle, and K. G. Taylor, J. Dent. Res. 61:215,1982; and R. J. Doyle, ASM Conference onBacterial Adhesion in Pathogenesis, Atlanta,Ga., 1981].)

MATERIALS AND METHODSBacterial strains and growth conditions. S. sanguis

Challis was originally obtained from R. Kolstad (Bay-lor University, Waco, Tex.). S. sanguis MJM 19 hasbeen described previously (20). Streptococcus salivar-ius 13419 was obtained from R. Arnold (University ofLouisville, Louisville, Ky.). Streptococcus mutans6715 has been described in a previous study (21). Thecharacteristics of Bacillus subtilis hpr 10 have beenreported previously (13). Stock cultures of all strainsof streptococci were maintained on brain heart infu-sion agar slants (Difco Laboratories, Detroit, Mich.)supplemented with a few milligrams of calcium car-bonate. Inoculations into 10 ml of brain heart infusionbroth (Difco) were made from the stock cultures. Afterovernight incubation at 37°C, the contents of the entireculture were transferred into 500 ml of the samemedium supplemented with 2 ,Ci of [3H]thymidine(specific activity, 82 Ci/mmol) (ICN, Irvine, Calif.) perml. These radioactive cells were used for adherencemeasurements. The labeled thymidine was deletedfrom the growth medium when cells were needed forhydrophobicity assays. In some experiments, Todd-Hewitt broth was used as the culture medium. Afterovernight inoculation, cells were harvested by centrif-ugation and washed twice in cold distilled-deionizedwater before suspension in water or in buffered KCI(1.0 mM potassium phosphate buffer [pH 6.0], 50 mMKCI, 1.0 mM CaCl2, and 0.04% [wt/vol] sodium azide)to the appropriate cell density. All experiments wereconducted with freshly harvested cells.

Partition assay. Washed cells suspended to a finalvolume of 3 ml in distilled-deionized water were addedto glass culture tubes (12 by 75 mm). The opticaldensity (500-nm wavelength, 1-cm path length) of thecell suspension was adjusted to 0.5 before the additionof the appropriate volume of hexadecane or toluene(26). The contents of each tube were mixed on aVortex mixer for exactly 30 s. After the contents of thetube had settled for 20 min, a Pasteur pipette wascarefully inserted below the nonaqueous phase, and asmall sample of the aqueous phase was transferred to acuvette. Control samples consisted of suspensions ofcells without added hydrocarbon. It was necessary tomake modifications of the procedures to accommodatechanges in ionic strength and hydrogen ion concentra-tion. In these measurements dilutions of salt wereadded to the cell suspensions to yield the appropriatefinal solute concentration or pH. A volume of 0.6 ml ofhexadecane was then added to all samples. For parti-tion assays in the presence of different hydrogen ionconcentrations, cells were suspended in prepared 0.5M buffer solutions. The buffer substances were chosento encompass a range of pH values from 3 to 10.Hexadecane (0.6 ml) was added to all samples. Assaysconducted under conditions of varying temperaturewere performed as described above, except all materi-als were allowed to equilibrate to the appropriatetemperature before the assays were conducted.

Adherence assay. The adherence assay used is basedupon that originally described by Clark et al. (3).Hydroxylapatite beads (40 ± 2 mg) (BDH ChemicalsLtd., Poole, England) were added to 4-ml plasticscintillation vials (Beckman Instruments Inc., Fuller-ton, Calif.). After one wash with distilled-deionizedwater to remove fines, the beads were allowed toequilibrate by constant inversion at a rate of 12 timesper min in 1.5 ml of buffered KCl for 2 h. Parafilm-stimulated saliva was collected on ice from a singleindividual for saliva-coated hydroxylapatite (SHA)bead preparation. The saliva was clarified by centrifu-gation (at 12,000 x g for 10 min) and heated to 60°C for30 min. After recentrifugation, 0.04% (wt/vol, finalconcentration) sodium azide was added. After aspira-tion of the buffer, 1.5 ml of prepared saliva wasallowed to mix with the beads for 2 h. The beads werethen washed three times with 3.0-ml volumes of KClbuffer to remove unadsorbed saliva. For assays mea-suring the effects of urea and 1,1,3,3-tetramethylurea(TMU), S. sanguis Challis was suspended to a densityof 542 ,ug of cells (dry weight) per ml in buffer. Thereagent was diluted in buffer to the appropriate con-centration before addition to the cell suspension. Themixture was subsequently added to saliva-coatedbeads. Similarly, in a direct comparison of the effect ofsalts and chaotropes on adherence, cells suspended toa density of 236 ,ug/ml in buffer were mixed with thereagent before addition to saliva-coated beads. Allincubations with cells were conducted for a period of1.5 h. Unadsorbed bacteria and reagents were re-moved with three washes of buffer. After aspiration ofthe final buffer wash, the beads were dried at 60°Covernight before being assayed for radioactivity in ascintillation spectrometer. Adventitious adherence tothe plastic vials was negligible. All materials for tem-perature studies were preequilibrated at the appropri-ate temperature before the assays were conducted.

Cell wall preparation. The procedure used for cellwall preparation has been described previously (21).Cells were ruptured in a French pressure cell (Ameri-can Instrument Co., Inc., Rockville, Md.) at approxi-mately 20,000 lb/in2. After differential centrifugation at39,000 x g for 10 min, the walls were extensivelywashed with water before the extraction procedure toremove loosely bound molecules. Crude walls weresubjected to boiling 3% (wt/vol) sodium dodecyl sul-fate (SDS) for three 0.5-h intervals. The detergent-treated cells, after being washed in distilled-deionizedwater several times, were then extracted thrice with5.0 M lithium chloride for 0.5 h. The walls were againwashed twice in water before being stirred overnight in8.0 M urea. Residual urea was removed from the wallsby extensive washing with water before freeze-drying.Amino acid analyses. Amino acids were analyzed on

columns packed with Durrum resin. Wall sampleswere hydrolyzed in 6 N HCI at 110°C for 24 h. Nocorrections were made for loss of amino acids duringhydrolysis.

Chemicals and reagents. All salts, toluene, and ureawere obtained from Fisher Chemical Co., Cincinnati,Ohio. Hexadecane and Tris were purchased fromSigma Chemical Co., St. Louis, Mo. SDS, imidazole,and TMU were products of Aldrich Chemical Co.,Milwaukee, Wis. Citric acid was provided by J. T.Baker Chemical Co., Phillipsburg, N.J. Potassiumphthalate was purchased from Allied Chemical Co.,

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ADHERENCE AND HYDROPHOBICITY 639

IJ 110

L 1004z

0L. 9000 80

H 700m 60

H 50

! 40

LU 30

A----- v--A-v---a- -v---_AA\-0~~~ 0 A A

0~~~

I\Ix

,1* 0~~~~

.\\Y

\A'\xas

-\ =A 0

F

FA--, ~- - --A

0.1 0.2 0.3 0.4 0.5HEXADECANE ADDED(mL)

FIG. 1. Partitioning of S. sanguis. 0, S. sanguis;0, S. salivarius; A, B. subtilis; A, S. mutans 6715.

Morristown, N.J. 2-(N-morpholino)ethanesulfonicacid was a product of Calbiochem, San Diego, Calif.

RESULTSHydrophobicity of S. sanguis. The assumption

that hydrophobic-hydrophobic interactionscould stabilize the cell-pellicle complex wasderived from theoretical considerations of posi-tive cooperativity (4, 20; Doyle et al., in press).Experimental verification for the premise that S.sanguis possesses a hydrophobic surface is pro-vided below. The assay described by Rosenberget al. (26) was used in these studies. Whenhexadecane or some other water-immiscible sol-vent is mixed with an aqueous suspension ofcells, the cells will exhibit an affinity for one ofthe two phases upon partitioning. S. sanguistended to bind to the hexadecane phase (Fig. 1).When the results were quantitated by measuringthe remaining opacity of the aqueous phase, itwas seen that the extent of the adherence of thecells for the hexadecane layer is a function of thehexadecane concentration (Fig. 1). Because sat-uration appeared to occur at approximately 0.2ml of hexadecane per ml of cell suspension, wechose this hydrocarbon concentration for subse-quent studies. In contrast to the hydrophobicitydemonstrated by S. sanguis, we noted that S.salivarius and B. subtilis had little or no tenden-cy to partition into the nonaqueous phase (Fig.1). Furthermore, when S. sanguis MJM 19 (20)and several other clinical isolates of S. sanguiswere subjected to the assay, results similar tothose for S. sanguis Challis were obtained (datanot shown). Results for S. mutans 6715 are alsoshown in Fig. 1. The data reveal that this orga-nism is at least as hydrophobic as S. sanguis

Challis. This result for S. mutans 6715 is incontrast to our previous claim (Doyle et al., inpress), based on the partitioning of clinical iso-lates of S. mutans in hexadecane-water, that S.mutans was hydrophilic. Heterogeneity in thehydrophobicity of streptococci has also beenobserved by Miorner et al. (18). They haveobserved that the binding of proteins by strepto-cocci can markedly change the isoionic pointsand surface hydrophobicities of the bacteria.To confirm the results described above, tolu-

ene was substituted for hexadecane in the assayprocedures. The data obtained showed that S.sanguis, but not S. salivarius or B. subtilis,could be partitioned into the toluene phase oftoluene-water mixtures. Finally, to rule out arti-facts caused by possible membrane disruption,S. sanguis Challis was radiolabeled with[3H]thymidine and mixed with either hexadec-ane or toluene (0.2 ml of hydrocarbon per 1.0 mlof cell suspension). The suspension was centri-fuged to sediment the cells, and samples wereremoved from the hydrocarbon and aqueouslayers. No radioactivity was found in eitherphase, indicating that organic solvents did notdisrupt the cells.Enhancement of the hydrophobicity of S. san-

guis by salt. According to theory, hydrophobicgroups can be readily "salted out" with variouskinds of salts (16, 24, 29). In fact, one conve-nient means of determining the hydrophobicityof bacteria is to measure the extent of clumpinginduced by ammonium sulfate (16). S. sanguiswas mixed with dilute salt solutions (sodiumchloride); hexadecane was added; and the sus-pensions were blended in a Vortex mixer. Afterattainment of phase separation, it was found thatthe sodium chloride enhanced the affinity of S.sanguis for the hexadecane phase (Fig. 2). Onlysmall amounts of salt (16) were required to

0.4O

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0.3

02

0.1

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0.05 0.1 0.25 '0.5

[No Cl]FIG. 2. Effect of salt on phase partitioning of S.

sanguis with hexadecane. Sodium chloride was addedto cells at the indicated concentrations before theassay.

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640 NESBITT, DOYLE, AND TAYLOR

60

50

- 40

zL.0$ 30-.

lmIr 20

10

pHFIG. 3. Effect of pH on ph;

sanguis with hexadecane. Dataaverage offour determinations (:0, distilled-deionized water. Allto a 500 mM concentration. BupH 3.0, citric acid; pH 4.0, pot5.0, sodium acetate; pH 6.0, 2-(sulfonic acid; pH 7.0, imidazole;sodium carbonate; pH 10.0, sod

achieve maximal expressionon the cells. The results stsurface of S. sanguis may ]

that are readily salted out in cEffect of hydrogen ion conc

phobicity of S. Sanguis. It hmented that the hydrophobiteins and other amphipathicmodified by hydrogen ionswould be expected that thspecies would be the mostVarious buffers were prepartrations to circumvent thstrength (Fig. 2). Cells wawater were then suspendedthe suspensions were subjecassay procedures. It was obsenvironment was optimal I

cells into the hexadecane X

TABLE 1. Partitioning of S. sanguis by ahexadecane-water mixture at various temperaturea

Temperature Turbidity(°C) (% of initial)'23 70.5 + 8.837 55.8 ± 9.760 48.5 ± 8.3

a Cells were grown in Todd-Hewitt broth, harvestedby centrifugation, and washed twice in cold distilled-deionized water. The cells were then suspended inwater to an absorbance of 0.5.

b Values given are averages (t standard deviation)of six separate determinations.

results shown (Fig. 3) suggest, but do not statis-tically prove, that hydrogen ion concentrationsrepresenting pH values between 3 and 4 wererequired to elicit the maximal expression ofhydrophobicity. A distilled water-hexadecane-cell control is also shown (Fig. 3). In all cases,the buffer enhanced the hydrophobic nature ofthe cell surface of S. sanguis.Adherence and hydrophobicity of S. sanguis as

a function of temperature. Increased tempera-tures are used to strengthen hydrophobic bonds.It could be predicted that elevated temperatures

' ' ' would result in an enhancement of the hydro-7 8 9 lO phobic characteristics of S. sanguis. In turn, if

hydrophobic forces alone are responsible forase partitioning of S. adherence of the bacteria to SHA, then anpoints represent the increase in the number of cells bound to the

t standard deviation). hydroxylapatite should accompany an increasebuffers were prepared in temperature. When the assays were conduct-iffers were as follows: ed at temperatures above ambient (22 to 23°C),tassium phthalate; pH more cells were attracted to the hexadecaneN-%morpholino)ethane- (Table 1). It was not practical to conduct thepH 8.0, Tris; pH 9.0, assays at temperatures lower than 22'C becauselium carbonate. the hexadecane tends to solidify.

Adherence assays were conducted with SHAbeads by standard procedures (3, 20). The cell

l of hydrophobicity density was adjusted so that the amount boundiggest that the cell was not near saturation (20). Adherence washave apolar groups measured at 4, 23, and 37°C. There was nodilute salt solutions. significant change in the bound/unbound ratiocentration on hydro- over the temperature range studied (Fig. 4).Las been well docu- Adherence, therefore, appears to be insensitiveic character of pro- to temperature changes, whereas the surfacec molecules can be hydrophobicity of S. sanguis is increased by an(16, 19, 24, 29). It increase in temperature..e most protonated Inhibition of adherence by hydrophobic bond-hydrophobic form. disrupting agents. In assessing how moleculesed at 0.5 M concen- interact to form stable complexes, it is expediente effects of ionic to use inhibiting agents of defined specificity.shed with distilled We reasoned that if agents such as urea could bein the buffers, and used to inhibit adherence, then substitutedAted to the standard ureas, such as TMU, should be even more.erved that a low-pH effective inhibitors if hydrophobic bonds con-for partitioning the tribute to the binding between cell and pellicle.phase (Fig. 3). The When assays were performed in the presence of

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ADHERENCE AND HYDROPHOBICITY 641

0.4

0.3

D 0.2c.

0.11

4 23TEMPERATURE (°C)

37

FIG. 4. Effect of temperature on binding of S.sanguis to SHA. A cell density corresponding to anoptical density of 0.4 was added to the beads, and theadherence assay was performed as described in thetext. The number of adherent cells at 23°C represents3,910 cpm of 13,915 cpm added to the beads. B/U,Bound/unbound ratio.

urea or TMU, it was found that TMU was amuch more potent inhibitor of adherence (Fig.5). For example, at 0.125 M, urea reduced thebound/unbound ratio from 0.43 to approximately0.30, whereas TMU reduced the ratio to 0.21(Fig. 5). When higher concentrations of urea orTMU (1 to 2 M) were used, no additional inhibi-tion of adherence was observed.Some ions, such as Li' and SCN-, are known

breakers of hydrophobic interactions, whereasother ions, such as K+, do not greatly influencehydrophobic bonding (24, 29). Several salts weretested for their ability to prevent the adherenceof S. sanguis to SHA beads (Table 2). When 1.0M K+ ion was used, adherence was actually

0.5

0

: 0.40oUREA

0 *TETRAMETHYL;k 8 UREA

, 0.3 00

z.0.

w

\.IZ 0.1 \

0

0.1 0.5 1.0 2.0[INHIBITOR]

FIG. 5. Effect of denaturants on the adherence ofS. sanguis to SHA. The amount of the denaturantrepresents the final concentration used in the adher-ence assay. The amount bound at a denaturant con-centration of zero was 5,718 cpm of 18,815 cpm addedto the beads.

TABLE 2. Effect of salts and chaotropes on theadherence of S. sanguis to SHAa

Inhibitor B/U % ofcontrol

None (control) 0.48b 100KCI 0.64 133NaCl 0.32 72LiCl 0.09 19NaSCN 0.03 6Urea 0.27 56TMU 0.06 13SDS (1%, wt/vol) 0.02 4

a Mixtures of cells suspended to a density of 236 ,ug/ml in buffer and inhibitor were added to 40 mg of SHA.The final concentration of the inhibitors, with theexception of SDS, was 1.0 M. Controls consisted ofcells suspended in buffer without inhibitor. B/U,Bound/unbound ratio.

b The control value represents 4,249 cpm bound of13,261 total cpm added to SHA.

enhanced. In contrast, the Na+ cation partiallyinhibited adherence, whereas the Li' ion was ahighly effective inhibitor. In addition, both SDSand the SCN- anion were potent inhibitors ofadherence. Data are also included for urea andTMU to show their relative effectiveness ofinhibition compared with the other chaotropes.There was no evidence to suggest that the inhibi-tors aggregated the bacteria. The inhibition ofadherence is therefore probably not a result ofcellular aggregation. We conclude that adher-ence is highly sensitive to agents capable ofdisrupting hydrophobic bonding. It must be con-sidered also that other forces, such as ionic orhydrogen bonding, may be destroyed by highconcentrations of ions or denaturants.Amino acid composition of cell walls of S.

sanguis Challis. Although the foregoing resultsshow that S. sanguis possesses hydrophobicproperties associated with its cell surface, theorigin of the hydrophobic groups remains ob-scure. In other work, we have observed that cellwalls of S. mutans contain non-peptidoglycanamino acids (21). The presence of hydrophobicamino acids in cell walls does not necessarilyconfer a hydrophobic character to a bacterium,but their presence constitutes a natural andconvenient parameter for more detailed studies.Suspensions of washed S. sanguis Challis wereburst in a French press, and the walls wereisolated by conventional methods (21). The wallpreparations were subjected to multiple treat-ments with boiling SDS, which were followed bydialysis against 8.0 M urea, to remove adventi-tiously bound proteins and other molecules. The"'cleaned" walls were then subjected to HClhydrolysis, and the hydrolysate was examinedfor amino acids. The results (Table 3) reveal thatseveral amino acids not generally regarded as

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642 NESBITT, DOYLE, AND TAYLOR

TABLE 3. Amino acid composition of cell walls ofS. sanguis Challisa

Amino acid Amt(nmollmg)Aspartic acid .......................... 80Threonine ............................. 40Serine ................................ 35Glutamic acid ......................... 475Glycine ............................... 50Alanine ............................... 1,530Cysteine .............................. 25Valine ............................... 53Methionine ............................ 5Isoleucine ............................. 25Leucine ............................... 20Tyrosine .............................. 20Phenylalanine ......................... 13Lysine ............................... 490Histidine .............................. 5Arginine .............................. 15

a Cells were grown in brain heart infusion broth,harvested, and washed twice in cold distilled-deion-ized water. The walls were extracted sequentially withSDS, LiCl, and urea as described previously (21).

components of peptidoglycan (14, 21), such asaspartic acid, leucine, isoleucine, tyrosine,phenylalanine, valine, histidine, arginine, ser-ine, threonine, aiid methionine, were present inthe wall preparation. Amino acids such as ala-nine, lysine, and glutamic acid were present inhigh quantities and probably originate from thepeptidoglycan (8, 14, 21). The results provideevidence to suggest that a polypeptide(s) whichcontains amino acids possessing hydrophobicside chains is tightly bound to the cell walls of S.sanguis.

If the wall-associated and hydrophobic aminoacids are involved in the affinity that S. sanguisdisplays for hexadecane, then the isolated wallsshould partition into the nonaqueous layer. Wallpreparations were suspended to 1.0 mg/ml indistilled water and stirred overnight to achieve ahomogeneous suspension (5). The walls (opticaldensity of 0.90 at a 500-nm wavelength and a 1-cm path length) were diluted, and hexadecanewas added (0.2 ml of hexadecane per ml ofaqueous suspension). The suspensions wereblended in a Vortex mixer for 2 min, and thephases were allowed to separate. Absorbancemeasurements were then made on the aqueouslayers to quantitate the extent of partitioning ofwalls between the two solvent phases. The wallsdisplayed an affinity for the hexadecane layer(Table 4). The distribution of walls between thesolvents was independent of the amount of wallassayed. When B. subtilis 168 walls were sub-jected to the same partitioning procedures, theirdistribution was not modified by the hydrocar-

bon. We conclude that the walls of S. sanguishave an affinity for hexadecane.

DISCUSSIONThe results from this study support the view

that hydrophobic interactions contribute to theadherence of S. sanguis to SHA. To account forthe observation that adherence involves positivecooperativity (20), it was necessary to predictthat hydrophobic interactions were providing a

stable environment for other weak interactions,such as ionic or hydrogen bonds (Doyle et al., inpress). As a corollary to this reasoning, we

predicted that the cell surface of S. sanguisshould exhibit characteristics of hydrophobicity.The present studies verified that S. sanguis has a

hydrophobic surface.The observations that low pH (Fig. 3), elevat-

ed temperatures (Table 1), and dilute sodiumchloride (Fig. 2) increased the tendency of S.sanguis to adhere to hexadecane are consistentwith the suggestion that the bacteria assumed an

increased hydrophobicity. In a study of thesurface hydrophobic characteristics of Esche-richia coli, it has been noted that low concentra-tions of the salt ammonium sulfate are effectivein eluting only those cells with the most hydro-phobic surfaces from hydrophobic gels (16).Conversely, high concentrations of salt are nec-essary in eluting cells with less hydrophobicsurfaces (16). When sodium chloride was addedto aqueous cell suspensions of S. sanguis, theirpartitioning in hexadecane was enhanced (Fig.2). 5. sanguis partitioned maximally under con-ditions of low pH (Fig. 3). Stinson et al. (27)have examined the adherence of a number oforal streptococci to glass under conditions ofvarying pH and have found that maximal adhe-

TABLE 4. Adherence of cell walls to hexadecaneaAmt % Decrease

Organism assayed in turbidity(4Lg)

S. sanguis 1,000 55500 50250 55

B. subtilis 168 1,000 0500 0250 0

a Cell walls were prepared according to methodsdescribed previously (20, 21). The assay mixturesconsisted of 2.0 ml of wall suspension in water and 400,ul of hexadecane. In the absence of hexadecane, S.sanguis walls gave an absorbance of 0.9 for 500 ,ug ofwall per ml, whereas B. subtilis walls gave an absor-bance reading of 0.83. After being blended in a Vortexmixer in the presence of hexadecane, the suspensionswere again assayed for turbidity.

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ADHERENCE AND HYDROPHOBICITY 643

sion occurs in a low-pH environment for certainS. mutans strains. Because carboxyl groups ofamino acids such as aspartic acid and glutamicacid are readily protonated within the pH rangeat which significant adhesion was observed,Stinson et al. concluded that the involvement ofproteins in the adhesion of these organisms wasprobable (27). It must also be borne in mind thatthe negative forces present on both bacterial andhost surfaces may be minimized by low pH (19),a condition most likely to favor hydrophobicinteractions. It is quite probable, however, thatprotonation of carboxyl groups is responsible forenhancing the hydrophobicity of the surface ofS. sanguis, thereby allowing an even greaterdegree of interaction with hexadecane than thatobserved at higher pH values.The adherence of S. sanguis to SHA must

involve several kinds of interactions. If theadherence were purely ionic in nature, then itwould be expected that salts should inhibit thebinding. This was clearly not the case, as 1.0 MK+ ion actually enhanced the adherence (Table2). The K+ ion, however, has little effect onhydrophobic interactions (24, 29). Other ions,such as Na+, Li', and SCN-, were inhibitory,although the inhibition induced by Na+ wasmarginal. The possibility of lectin-like interac-tions contributing to the adherence must beconsidered (9, 10). Salts may or may not inhibitlectin interactions with carbohydrate-containingmolecules. Concentrated sodium chloride has noeffect on concanavalin A-glycogen complex for-mation, but dilute salts readily inhibit concanav-alin A-teichoic acid interactions (6). This is dueto the fact that salts can change the solutionstructure of the teichoic acids (6). We believethat when interactions involving ionic, dipole-ion, or hydrogen bonds occur between the celland the pellicle, the interactions will be stable ifhydrophobic bonds can also occur (Doyle et al.,in press). This view of adherence explains whyK+ ion did not inhibit, whereas Li' and SCN-were effective inhibitors. The effects of urea,TMU, and SDS are also predictable. All of theseagents can disrupt macromolecular structures.Urea is less hydrophobic than TMU, and itrecently has been reported that the former is thebetter denaturant of proteins (23), although earli-er reports have suggested that TMU is thesuperior denaturant (7, 12). The results of Orsta-vik (22), who has shown that the adherence of astreptococcus (designated as OI5) to saliva-coat-ed enamel slabs is sensitive to 2.0 M Na+,concentrated urea, and Tween 80, may be ex-plained on the basis of the disruption of hydro-phobic bonds.The observation that an increase in tempera-

ture increased the hydrophobic tendency of S.sanguis yet had little effect on adherence (Table

1 and Fig. 4) also needs explanation. Nonhydro-phobic bonds would be weakened at elevatedtemperatures, whereas hydrophobic bondswould be strengthened. If multiple interactionsare required for a productive union between celland pellicle, then the enhanced hydrophobiceffect at higher temperatures may be diminishedby the disruption of other bonds.The origin of the hydrophobicity of S. sanguis

probably resides in the cell wall of the organism.Hydrophobic amino acids, possibly in covalent-ly bound polypeptide (21), were present in deter-gent-denaturant-extracted and dialyzed cellwalls (Table 3). The walls also displayed anaffinity for hexadecane, a quality not inherent inall bacterial walls (Table 4). If cell wall polypep-tide is responsible for the affinity of walls forapolar solvents, then the polypeptide must con-tain a sequence of amino acids which make itshydrophobic properties refractory to the effectsof the extraction procedures. The role of "fim-briae," or surface structures emanating from thesurfaces, of S. sanguis in contributing to thehydrophobicity of the bacterium is not clear.The fimbriae are removed by sudden pressurechanges and by chemical reagents, such as urea(25), used in the preparation of the walls. It ispossible, however, that both walls and fimbriaecontribute to the hydrophobicity of S. sanguis.Our present view of streptococcal adherence

is summarized below. Salivary proteins are ad-sorbed on the hydroxyapatite surface. The pro-teins may consist of lectin-like molecules or ofmolecules which may form ionic or ion-dipolecomplexes with streptococcal surfaces, and theymust contain molecules capable of interactingwith cells via the hydrophobic effect. The cellsmust contain molecules on their surfaces com-plementary to those on the pellicle, but must inaddition possess hydrophobic residues. Cellswhich do not fulfill all of these criteria wouldinteract weakly with the pellicle. Presumably, S.mutans falls into this category, even thoughcertain strains possess hydrophobic characteris-tics. Cells, such as S. sanguis, which possessboth hydrophobic sites and sites complementaryto the pellicle would adhere strongly and proba-bly demonstrate positive cooperativity (20;Doyle et al., in press) in in vitro assays.

ACKNOWLEDGMENT

This work was supported in part by Public Health Servicegrant NIDR-DE 05102 from the National Institutes of Health.

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