Relationship of CellSurface Morphology and Composition of ...

Vol. 55, No. 2

INFECTION AND IMMUNITY, Feb. 1987, p. 438-445 0019-9567/87/020438-08$02.00/0 Copyright © 1987, American Society for Microbiology

Relationship of Cell Surface Morphology and Composition of Streptococcus salivarius K+ to Adherence and Hydrophobicity ANTON H. WEERKAMP,1* HENNY C. VAN DER MEI,1 AND JAN W. SLOT2 Department of Oral Biology, Dental School, University of Groningen, 9713 AV Groningen,l and Department of Electron Microscopy, Medical School, University of Utrecht, Utrecht,2 The Netherlands Received 16 April 1986/Accepted 9 October 1986

The cell surfaces of a range of variants of Streptococcus salivarius HB, altered in cell wail antigen composition, were compared with those of the parent with respect to adherence, ability to adsorb to hexadecane, morphology, and exposure of lipoteichoic acid (LTA). Adherence to host surfaces was measured by using both saliva-coated hydroxyapatite beads and tissue-cultured HeLa cells, and interbacterial adherence was measured by using Veillonella alcalescens Vl cells. Progressive loss of the protease-sensitive fibril classes was generally associated with decreasing ability to adsorb to hexadecane. However, increased exposure of protein antigen C (AgC) increased the apparent hydrophobicity of the cell. This correlated with the finding that AgC was the most hydrophobic of the solubilized fibrillar cell wall antigens. Collectively, this demonstrates that adsorption to hydrophobic ligands is directly related to the density of the fibrillar layer on the cells and the properties and surface exposure of specific fibril classes. The involvement of hydrophobic interactions in AgC-associated attachment was suggested by its sensitivity to low levels of the hydrophobic bond-breaking agent tetramethyl urea, although the reduction was not to the level of adherence observed with strains lacking AgC. However, hydrophobicity was less essential to other adherence reactions. Circumstantial evidence, including (i) immunoelectron microscopy, showing that LTA was virtually absent from the fibrillar layer, (ii) whole-cell enzyme-linked immunosorbent assay, suggesting that surface exposure of LTA related inversely to the density of the fibrillar layer, and (iii) agarose gel electrophoresis, showing that LTA was not specifically associated with protein fibrillar antigens, strongly suggested that LTA does not confer hydrophobic properties to these cells and is not involved in adherence reactions associated with the cell wall protein antigens.

According to one recent model (4), hydrophobic bonds adjacent to stereospecific surface molecules stabilize interactions mediated by these compounds. According to macroscopic models of bacterial adhesion (2, 34) the bacterial surface hydrophobicity (surface free energy) is an important factor in the interaction with other surfaces by virtue of its effect on the interfacial free energy of adhesion. Despite the apparent involvement of hydrophobic domains in adherence reactions, little is known about the nature of the molecules which confer hydrophobicity to the cell surface of oral and other streptococci. Several authors have demonstrated a relationship between the presence of surface appendages on these bacteria and hydrophobicity (8, 10, 18, 26), similar to observations made with gram-negative bacteria (7). Recently, McBride and co-workers (14, 18) showed that the loss of hydrophobicity of Streptococcus sanguis and Streptococcus mutans strains coincided with the loss of several high-molecular-weight proteins from the cell wall. However, no conclusive evidence was given that one or more of these proteins were directly responsible for hydrophobicity. Similarly, a relationship between the M protein of group A streptococci and surface hydrophobicity has been suggested (26), but later studies have presented evidence that lipoteichoic acid (LTA) is the major hydrophobicity-conferring component in these bacteria (15, 16). Both M protein and LTA have been implicated in the adherence of group A streptococci to epithelial surfaces (1, 5). We previously identified two high-molecular-weight proteinaceous cell wall-associated antigens in Streptococcus salivarius (29), respectively suggested to be involved in adherence to host tissues, including pellicle-coated tooth surfaces, and to mediate coaggregation with Veillonella cells and other gram-negative bacteria. The latter antigen is

Microbial adherence to tissues and to other microorganisms is thought to be an important factor in the formation of the specific microfloras found in association with a host. In the human oral cavity, distinct bacterial ecosystems thus form at specific sites, partly owing to interactions between bacterial surface components and the oral surfaces. A wide variety of interactions, both long and short range, have been implicated in the complex adhesion process, including van der Waals dispersion, hydrogen bonding, and electrostatic and hydrophobic interactions (25). Several studies have suggested an important role for hydrophobic interactions in microbial adherence (24). Considering their ability to adhere to hydrophobic ligands (3, 9, 20-22, 36), many freshly isolated oral bacteria indeed appear to have hydrophobic domains on their surface. However, contact angle measurements on layers of oral streptococcal strains suggested that most strains possess a relatively low overall surface hydrophobicity, particularly after being coated with saliva (34). Nonhydrophobic variants of oral streptococci (8, 10, 18, 34, 37) and cells grown under various growth conditions (12) have been used to relate hydrophobic cell surface properties with the ability to adhere to oral surfaces. However, these experiments could not conclusively demonstrate a direct relationship, since a variety of surface components is usually lost from these cells simultaneously. Recently (3), evidence was supplied by use of oral actinomycetes which showed that although hydrophobicity and adherence to saliva-coated hydroxyapatite (SHA) were statistically correlated traits, hydrophobic interactions appeared not to be of major importance for the adherence reaction itself. *

Corresponding author. 438

SURFACE FIBRILS AND HYDROPHOBICITY OF S. SALIVARIUS

VOL. 55, 1987

TABLE 1. Antigenic composition of S. salivarius strains Strain

Cell wall antigensa

Reference

HB HB-7 HB-V5 HB-V51 HB-C12

B+ C+ D+ E+ F+ B+ C-b D+ E- F+ B- C+ D+ E+ F+ B- C-b D+ E- F+ B- C- D-c E- F+

29 28, 29 29, 30 26 This study

All strains are Lancefield group K+. Accumulated intracellularly. c Excreted in increased amounts compared to parent strain.

a

b

thought to involve a galactose-specific lectin (32, 33). S. salivarius (serogroup K) cells possess a typical fibrillar surface layer (28), which consists of fibrils of at least three different lengths (28, 35). Recently, we demonstrated that two specific fibril classes could be identified with the two antigens involved in adhesion (35), and we described a series of mutant strains which lack specific fibrils and particular adhesive properties (28). This knowledge of the complex surface topology of these bacteria could be used to locate and identify the molecules or structures which confer hydrophobicity to the cell surface. In the present paper, we demonstrate that hydrophobic properties of the cell surface are associated with specific fibrillar protein antigens on the surface of S. salivarius cells. It is proposed that the hydrophobicity of these compounds is an intrinsic property rather than the result of association with LTA. MATERIALS AND METHODS

Bacterial strains and growth conditions. S. salivarius HB and mutant strains HB-7, HB-V5, and HB-VS1 have been described previously (28, 29, 31). Strain HB-C12 was a spontaneous stable variant obtained after prolonged cultivation of strain HB in a chemostat at high specific growth rate. It lacked the ability to attach to buccal epithelial cells and saliva-coated SHA and was impaired in coaggregation with veillonellae. The antigenic structures of the strains are shown in Table 1. For each experiment, the bacteria were grown from a frozen stock in batch culture in Todd-Hewitt broth for 16 h at 37°C. Cells were harvested by centrifugation for 5 min at 8,000 x g, washed twice with phosphate-buffered saline (PBS; pH 7.0), and resuspended in PBS. Preparation of cell wall digests. Procedures for the preparation of cell walls by shaking them with glass beads in a Braun cell disintegrator and subsequently washing them with Triton X-100 were described previously (29). For some purposes, the Triton X-100 treatment was omitted and the walls were washed only with PBS. Mutanolysin digests were made of freeze-dried walls (20 mg [dry weight]) per ml, containing 100 U of mutanolysin; Sigma Chemical Co.) by previously described methods (29). Immunological procedures. Polyvalent monospecific antisera against wall antigens B (AgB), C (AgC), and D (AgD) were prepared by immunoaffinity chromatography of whole antiserum against S. salivarius HB walls on columns containing the immobilized purified antigens, as previously described (28, 29). Specific antiserum against the polyglycerolphosphate backbone of Lactobacillus teichoic acid (lot 644; 2.07 mg of antibody per ml), was kindly donated by K. W. Knox,

439

School of Dental Medicine, University of Sydney, Sydney, Australia. Standard crossed-immunoelectrophoretic techniques were performed with 1% agarose gels made in Tris-Veronal (Winthrop Laboratories) buffer (pH 8.6), as described previously (29). Immunoblotting on nitrocellulose filter paper (Bio-Rad Laboratories), washing, blocking, and immunodetection procedures with colloidal gold-labeled protein A followed by silver amplification were carried out by the method of Moeremans et al. (17). Protein blotting from agarose gels was done simply by overlaying the gel with nitrocellulose paper, several layers of filter paper, and a light weight, thus allowing fluid to be removed from the gel by suction. Hexadecane adsorption test. A modification of the method described by Rosenberg et al. (23) was used for the hexadecane assays carried out on modified cells. An initial cell concentration of approximately 2 x 109 cells in a total volume of 1.2 ml of PBS and 75 ,u1 of hexadecane was used. The agitation time was 60 s. For comparison of the mutant strains, the more accurate assay recently described by Lichtenberg et al. (13), which is based on a kinetic analysis of the adsorption to hexadecane, was used. The data were expressed as the removal coefficient (k), which is the slope of a linear function between the hexadecane/water ratio and the rate constant of removal of the bacteria from the bulk aqueous phase to the hexadecane interface. Although the lines did not pass through the origin as stated in the original publication (13), the linear correlation coefficients were always better than 0.96. Hydrophobic interaction chromatography. To determine the relative hydrophobicity of cell wall-associated antigens, mutanolysin digests of crude cell wall preparations (not treated with Triton X-100) were applied to a column of Octyl-Sepharose 4B (7 by 1 cm; Pharmacia, Inc.) preequilibrated in 50 mM potassium phosphate buffer (pH 7.3) containing 25% saturated ammonium sulfate. The sample, previously brought to the same ammonium sulfate concentration, was eluted with a linear gradient of decreasing concentration of ammonium sulfate (25 to 0% saturation) and simultaneously increasing concentration of ethylene glycol (0 to 50%, vol/vol). Subsequently, the column was eluted with 1 M tetramethyl urea in phosphatee buffer (one column volume) and finally with buffer containing 1% (vol/vol) Triton X-100. Fractions were collected, dialyzed extensively against distilled water, and freeze-dried. Quantitation of LTA. LTA in culture media and cell wall digests was determined in a semiquantitative manner by using an immunodot-blotting test. Twofold serial dilutions of the test solution (2 IlI) were spotted on nitrocellulose filter and allowed to dry. The nitrocellulose was quenched in Tris-buffered saline (pH 7.4) containing 1% bovine serum albumin for 30 min. Subsequently, the filters were incubated with LTA antiserum (diluted 1:200) in TBS containing 0.1% bovine serum albumin or control preimmune serum for 2 h at room temperature. The filters were than treated as described above. The relative concentration of cell surface-located LTA was determined by a modification of the procedure described by Miorner et al. (16). A 50-,u aliquot of a washed-cell suspension containing approximately 5 x 109 cells in Trisbuffered saline-0.4% bovine serum albumin, was incubated for 60 min at 30°C with 100 RId of LTA antiserum (diluted 1:100) or control preimmune serum. The cells were centrifuged in an Eppendorf type 5414S centrifuge for 3 min, washed twice with Tris-buffered saline-bovine serum albu-

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TABLE 2. Adherence properties and surface hydrophobicity of S. salivarius strains Strain

Adsorption hexadecane (k [min-'])to(% of HB)

HB HB-7 HB-V5 HB-V51 HB-C12

52 (100) 44 (84.6) 76 (146.1) 27 (51.9) 2 (3.8)

aExpressed as the ratio of bound over unbound cells. b Average of three different cultures; standard deviation ±

Adherence to SHA HB) (B/U)a (% of

1.01 0.08 0.92 0.11 0.06

(100) (7.9) (91.1) (10.9) (5.9)

Adherence to confluent monolayers of tissue-cultured HeLa cells was measured as follows. HeLa cells were grown in RPMI 1640 medium (GIBCO Laboratories) containing 10% fetal calf serum in tissue culture polystyrene petri dishes (25 cm2). After aspiration of the medium, the cell layers were incubated for 90 min at 37°C with 2 ml of the bacterial suspensions containing 108 cells per ml. During incubation, the dishes were slightly agitated on a reciprocal shaking platform (20 strokes per min). The cells were washed four times with PBS, fixed with 2% glutaraldehyde, and stained with crystal violet. The number of bacteria attached per cell was estimated under a light microscope by counting at least 50 cells randomly distributed over the surface of the petri dish. The average standard deviation in the counts was 10%. Coaggregation of the streptococci with Veillonella alcalescens Vl was estimated in a spectrophotometric assay by the method of Ericson and Rundegren (6). To 5 ml of a cell suspension in PBS (optical density at 660 nm, 0.60) was added 400 p.1 of a V. alcalescens suspension (optical density, 2.5), and the extinction was monitored continuously. The data were linearized according to the formula (6) ln [(Ao A)IA] = mt + b, where Ao and A are the optical densities at times zero and t, respectively. The value of m was used as a measure of the aggregating activity. Replicate experiments with the same culture were reproducible within 5% standard

deviation.

12.3 2.0 12.6 2.4 0.4

(100) (16.3) (102.4) (19.5) (3.3)

Coaggregation cellswith Veillonella (m) 0.132 (100) NT 0.010 (7.5) 0.009 (6.7) 0.065 (49.2)

10%.

min, and subsequently incubated with alkaline phosphataselabeled goat anti-rabbit immunoglobulin G antiserum (diluted 1:1,000). After another 60 min, the cells were washed twice and incubated with 200 p.1 of enzyme substrate. The reaction was terminated after 20 min by the addition of 50 ,u1 of 3 N NaOH. The A405 of the supernatant minus that of the control serum was taken as a measure of the surface LTA concentration. Since with many strains the nonspecific binding of rabbit immunoglobulin G could not be completely blocked, care was taken to apply the same amount of total immunoglobulin G in both the experimental and control incubations to obtain more realistic base-line values. Immunoelectron microscopy. The subcellular localization of LTA and cell wall-associated protein antigens was determined by using immunoelectron microscopy on ultrathin cryosections. The sandwich labeling technique involving the use of colloidal gold-complexed protein A was described previously (28). Adherence and aggregation experiments. Adherence of bacteria to SHA was determined essentially as described previously (30). All experiments were performed in triplicate. Since the adherence of S. salivarius HB to SHA involves a single high-affinity type of interaction, the indirect method of calculating the number of bound cells was used

(19).

Adherence to HeLa cellsb (no. of bacteria/cell) (% of HB)

RESULTS Cell surface properties of the bacterial strains. Table 1 shows the antigenic composition of the cell walls of S. salivarius HB and the mutant strains derived. At least five antigens, labeled B to F, could be observed by crossed immunoelectrophoresis in the mutanolysin digest of strain HB. The major antigenic components AgB, AgC, and AgD were identified previously (29). The identity and possible functions of AgE and AgF have not yet been established. All strains possessed similar amounts of AgF and also possessed the carbohydrate group K antigen. Mutant HB-7 lacked the host attachment factor AgC and, in addition, appeared to lack AgE. In strain HB-V5, the veillonella-binding protein AgB was absent, but the walls were otherwise similar to those of the parent strain HB. Strain HB-V51 lacked AgB, AgC, and AgE, whereas strain HB-C12 was missing virtually all cell wall antigens that react with anti-HB antiserum, except AgF. None of the mutant strains excreted the antigens in higher amounts than did the parent strain during growth, with the exception of HB-C12, which excreted increased amounts of AgD. The cell surface hydrophobicity of the strains as probed by the adherence to hexadecane is shown in Table 2. The hydrophobicity of the cells ranges on a relative scale from very strong to weak, correlating with the progressive loss of cell wall antigenic compounds in these strains. Mutant HB-7 adsorbed significantly less well to hexadecane than did the parent strain HB but still retained a relatively high level of hydrophobicity compared with those of many other streptococci. A further decrease was observed with mutant HB-V51, and HB-C12 bound only very weakly to hexadecane. In contrast, mutant HB-V5, which lacks only AgB, appeared significantly more hydrophobic than parent strain HB and in fact was the most hydrophobic in this test of about 20 different oral streptococci tested (unpublished observation). Comparison of the biological adherence properties of the strains with the adsorption to hexadecane showed an incomplete correlation with respect to both adherence to SHA and HeLa cells and showed no correlation in the case of Veillonella coaggregation (Table 2). Microscopic observation of the adherence of the various strains to human buccal epithelial cells yielded results very similar to those obtained for HeLa cells, ranging from buccal epithelial cells carrying many bacteria (HB and HB-V5) to those nearly free of any attached cells (HB-7, HB-V51, and HB-C12) (results not shown). Surface properties of modified cells. Whole cells of the strains were subjected to various treatments and subsequently tested for the ability to bind to hexadecane (Table 3). Only treatment with proteases and extraction with sodium


VOL. 55, 1987

TABLE 3. Effect of cell surface modifications on the hydrophobicity of S. salivarius HB % Adsorption to hexadecane (% of untreated value)

Treatment

Cells None ................................ ..... 100°C, 20 min ................................... 3% SDS, 2 h, room temp ....................... 45% aqueous phenol, 1 h, room temp ...... .... 10% trichloroacetic acid, 2 h, 60°C ...... ....... Sonication, 6x, 30 s each .......... ............ Pronase (1 mg/ml), 3 h, 37°C ........ ........... Pronase (heat-inactivated) .......... ............ Trypsin (1 mg/ml), 3 h, 37°C .................... Trypsin + trypsin inhibitor (each 1 mg/ml)

89 (100) 94 (105.6) 28 (31.5) 71(79.8) 78 (87.6) 75 (84.3) 7 (7.9) 85 (95.5) 45 (50.6) 83 (93.3)

Cell walls None ..................................... 2% Triton X-100 extracted ......................

58 67

.....

ANTIGEN CONC. (% of total)

8OF 60w

4O* B

20O

L1. 0-15

D

16-25

26-35

% ETHYLENEGLYCOL

dodecyl sulfate (SDS) followed by extensive washing significantly reduced surface hydrophobicity. In contrast, all other treatments had only a limited effect. Methods designed to extract LTA from the cells, such as aqueous phenol and trichloracetic acid extraction, appeared to impair adsorption to hexadecane only slightly, whereas boiling resulted in a slightly increased adsorption. Isolated cell walls, both before and after treatment with Triton X-100, retained a surface hydrophobicity which was relatively high but which was significantly reduced compared with that of whole cells. Relative hydrophobicity of solubilized cell wall antigens. Mutanolysin digests of strain HB cell walls which had not been treated with Triton X-100 were subjected to hydrophobic interaction chromatography on a column of OctylSepharose 4B to determine the relative hydrophobicity of the wall antigens. When the digest was applied directly to the column in 50 mM phosphate buffer, all of the AgC but only part of AgB was bound. Therefore, samples were subsequently applied in a 25% saturated ammonium sulfate solution, under which conditions all antigens completely bound to the column. Elution of the column with decreasing ammonium sulfate and simultaneously increasing ethylene glycol concentrations to increase the polarity of the solvent selectively desorbed some of the antigens (Fig. 1). AgB was the most readily released and hence the least hydrophobic compound, followed by AgD. AgC could be only partly released in this way but was largely recovered upon elution of the column with tetramethyl urea, an agent known to break hydrophobic bonds. Quantitative recovery of this antigen required elution with Triton X-100, indicating that the compound has strongly hydrophobic properties. Effect of tetramethyl urea on adherence. To assess the role of hydrophobic bonds in adherence, we studied the influence of the presence of the hydrophobic bond-breaking agent tetramethyl urea (20). Adherence to SHA, in which AgC is involved, and coaggregation with Veillonella spp., mediated by AgB, were chosen because of the ability to quantitate these reactions accurately. Concentrations of TMU as low as 50 mM reduce the adherence of strain HB to SHA (Fig. 2). Complete inhibition was not achieved even at concentrations 50-fold higher. The half-maximal effect was reached at approximately 0.2 M. In contrast to this, the effect of urea

441

36-50 .

TRITON

TMU

FIG. 1. Hydrophobic interaction chromatography on OctylSepharose 4B of mutanolysin-solubilized cell walls of S. salivarius HB. A digest of crude cell walls, not treated with Triton X-100, was applied to the column and sequentially eluted with a linear 0 to 50% (vol/vol) gradient of ethylene glycol, 0.5 M tetramethyl urea, and 1% Triton X-100. Concentrations of antigens in pooled fractions were estimated by crossed immunoelectrophoresis and expressed as the percentage of the totally eluted amount of the antigens. Symbols: EZJ, AgB; cm, AgC; _, AgD.

was negligible at these concentrations. Approximately 4 M urea was required to obtain a 50% reduction in adherence to SHA. As a control, a similar experiment was done with mutant strain HB-7, which shows only a very weak adher-

B/u 1.0

0.5

0.01

A

A

\

OQ05 0.1

0.5

1

5

CONCENTRATION (M)

FIG. 2. Effect of urea (A, L) and tetramethyl urea (0, 0) on the adherence of S. salivarius to SHA. The results are expressed as the ratio of bound (B) over unbound (U) cells. Solid symbols, strain HB; open symbols, strain HB-7.

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ence to SHA. At all inhibitor concentrations tested, this strain yielded similar low adherence values. The effects of both tetramethyl urea and urea were negligible up to concentrations of 2 M, when the interbacterial coaggregation was studied. Hence, this reaction seems not to involve essential hydrophobic bonds, in contrast to adherence to SHA. Association of protein antigens with LTA. To determine whether, in analogy with group A streptococci (14, 15), LTA may be responsible for the surface hydrophobicity of S. salivarius, several tests were applied. The presence of LTA at the cell surface of various mutant strains and modified cells were measured by using a modified whole-cell enzymelinked immunosorbent assay technique involving antiserum against the polyglycerolphosphate backbone of LTA (16). As a control, the technique was applied to specific antisera against cell wall antigens of strain HB and was shown to accurately detect the presence of these antigens in appropriate strains (Table 4). The results with anti-LTA show an inverse relationship between the antigenic complexity of the cell wall and the relative amount of LTA detectable at the cell surface. Comparison of the range of mutants indicated that the progressive loss of protein antigens correlates with increased binding of anti-LTA antibodies. Concomitantly, a decrease in the binding of nonspecific rabbit immunoglobulin G was observed in the mutants (not shown). These results suggest that the protein surface antigens shield LTA from the external environment. This is further supported by the observation that pronase treatment of cells resulted in exposure of LTA (Table 4). In a second experiment, the relative amount of LTA in cell wall digests of the strains was measured. Similar titers (1:512) were obtained with all strains, except that digests of strain HB-V5 seemed to contain a slightly higher amount. To see whether LTA in the digest of HB cell walls was associated with any of the cell wall antigens, we subjected the preparation to electrophoresis in an agarose gel, followed by blotting to nitrocellulose and immunodetection of LTA. LTA was not specifically associated with the wall protein antigens but migrated close to the electrophoresis front (Fig. 3). Aqueous phenol-extracted LTA from the same strain that was partially purified by Sepharose CL-2B gel filtration and extraction with chloroform-methanol (11) also migrated close to the electrophoresis front. The somewhat different electrophoretic behavior of the two preparations may reflect differences in association with other compounds. TABLE 4. Surface exposure of LTA and protein antigens in S. salivarius strains LTA

AgB

AgC

Strain

A405' (%

A405 (% Control value)

A405 (% Control value)

HB HB-7 HB-V5 HB-V51 HB-C12 HB (pronase) HB-7 (pronase)Y

0.42 (100) 0.69 (164) 0.48 (115) 0.78 (206) 1.14 (273) 0.88 (210) 1.28 (186)

1.48 (100) 1.60 (116) 0.02 (1) 0.08 (5) 0.24 (16)

1.68 (100) 0.78 (46.4) NTb NT 0.20 (12.5)

Control value)

a Whole-cell enzyme-linked immunosorbent assay; expressed as the net increase in A405 compared with preimmune serum. Average standard deviation in all tests was 7%. bNT, Not tested. c Treated with pronase (1 mg/ml) for 3 h at 370C.

1

2

3

4

D~I~

c99 B

FIG. 3. Agarose gel electrophoresis of a mutanolysin digest of S. salivarius HB cell walls (lanes 1 to 3). Antigens were detected after immunoblotting to nitrocellulose with protein A-colloidal gold and total antiserum against HB walls (lane 1), serum specific for AgC (lane 2), and serum specific for LTA (lane 3). B, C, and D refer to the positions of the antigens in the gel. Lane 4 shows the electrophoresis of partially purified LTA after immunodetection with anti-LTA. The origin of the electrophoresis is at the bottom.

In a further test, immunoelectron microscopy was done to localize LTA on cryosections of HB cells. The results show that LTA is clearly associated with the cytoplasmic membrane, but very little or none is found in the periferal fibrillar layer of the cell envelope (Fig. 4). For comparison, it is shown that AgC is almost exclusively associated with the fibrillar layer. DISCUSSION The availability of mutants of S. salivarius HB which were originally selected on the basis of the absence of specific adhesive properties and were subsequently found to lack distinct fibrillar surface antigens (28, 29, 35) created the opportunity to investigate the role of these structures in hydrophobic properties of the cell surface. The results of this study demonstrate that hydrophobicity, assessed from the ability to adsorb to hydrophobic ligands, is a function of both the fibrillar density on the cell surface and the properties and exposure of specific fibril classes. An association of hydrophobic properties with cell surface proteins and fibrillar structures of streptococci and oral actinomycetes has been suggested in previous studies (8, 10, 14, 15, 18, 26, 38), but no conclusive evidence was supplied, or conflicting findings were reported (8, 16, 26). At least three classes of fibrils on the surface of S. salivarius HB cells have been recognized (28, 35). The longest (168 nm) fibrils are apparently not proteinaceous in nature since they cannot be removed by protease treatment of the cells, in contrast to the other fibril classes. Therefore, the long fibrils do not seem to contribute significantly to surface hydrophobicity, since treatment with protease removes most or all hydrophobic sites from the cell surface. The function and nature of this fibril class is still unknown. A class of 72-nm, protease-sensitive fibrils corresponds to the host attachment factor AgC (29) and is absent from the cell surface of mutant HB-7. Mutants of this type had a lower ability to adsorb to hexadecane than did the parent strain;


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-M

m

FIG. 4. Ultrathin cryosections of S. salivarius HB indirectly immunolabeled with a protein A-colloidal gold (6 nm) probe and anti-LTA antiserum. The insert shows immunolabeling with anti-AgC antiserum. Bar, 0.5 ,um.

this corresponds to the finding that solubilized AgC was the most hydrophobic of the fibrillar antigens. It corresponds also to the finding that mutant HB-V5, which lacks the medium-sized (91 nm) fibrils, is more hydrophobic than the parent strain, since an increased surface exposure of AgC would be expected in this mutant. The 91-nm fibrils, corresponding to AgB, the veillonella-binding protein (33), have only weak hydrophobic properties as indicated by hydrophobic interaction chromatography. However, they apparently still contribute significantly to hydrophobic properties of the cell surface, since mutant cells having predominantly AgB at the cell surface (i.e., HB-7) retain a relatively high surface hydrophobicity. This is further supported by the observation that the simultaneous absence of both AgB and AgC in mutant HB-V51 results in a strong decrease in hydrophobicity, although not its complete loss. Mutant HB-V51 was previously shown to carry very sparsely distributed short (63 nm) fibrils (28), which may have been responsible for the residual hydrophobicity. Indeed, the supplementary reduction in hydrophobicity observed for strain HB-C12 corresponds with the virtual absence of all fibrils from this strain (P. Handley, personal communication). Further evidence for the association of hydrophobic properties with surface fibrils is provided by the cell modification studies, which suggested that this property is associated with proteinaceous compounds. Circumstantial evidence for the noninvolvement of LTA in surface hydrophobicity was provided by our experiments, which showed that (i) surface exposure of LTA is inversely correlated with cell surface hydrophobicity, (ii) LTA is not specifically associated with isolated fibrillar proteins, and (iii) LTA localizes predominantly in the cytoplasmic membrane region of the cell

envelope but is virtually absent from the fibrillar layer in immunoelectronmicroscopy. These results contrast with the model accounting for surface hydrophobicity of S. pyogenes (15, 16). Surprisingly, treatment of S. salivarius with SDS at room temperature strongly reduced the ability of the organism to adsorb to hexadecane but did not extract significant amounts of the antigens (not shown). This suggests that SDS is bound to and masks hydrophobic sites on the cell surface. In contrast to our findings, Morris et al. (18) did not observe a significant reduction in hydrophobicity of S. sanguis, even after it was boiled in the presence of SDS. Boiling did not destroy hydrophobicity nor extract antigens from the S. salivarius cells; this finding is similar to results for S. sanguis (18) but contrasts with findings for S. mutans and S. pyogenes (14, 21). However, it was noted that boiling removed cell surface proteins from S. mutans, which suggests that in S. salivarius and S. sanguis, the proteins are incorporated into the cell wall in a quite different way. Another contrasting finding with respect to S. mutans was that in nonhydrophobic variants of S. salivarius, the missing wall protein antigens were not excreted into the culture medium during growth, with the exception of AgD, which was excreted by strain HB-C12. On the basis of all evidence, it appears that hydrophobic properties of the cell surface are associated with the presence of a variety of specific (fibrillar) proteins, which exhibit various degrees of intrinsic hydrophobicity. In a previous report on the subcellular localization of cell wall proteins in parent and mutant strains (28), we suggested that synthesis, translocation, and incorporation of these compounds are independent processes. This may explain our failure to select stable, nonhydrophobic mutants of S.

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salivarius by repeated adsorption of mutagenized cells to hexadecane or Octyl-Sepharose. It should be noted in this respect that prolonged growth at high specific growth rate apparently induces phenotypes which have lost multiple surface components (27) and finally leads to the isolation of mutant HB-C12. In a similar way, this may also explain why successful selection of nonhydrophobic mutants with other oral streptococci generally resulted in strains which had simultaneously lost multiple surface components and a variety of adhesive functions (10, 14, 18). A recent experiment in which S. sanguis mutants were selected for specific adhesive functions yielded a range of six phenotypes differing in surface properties; this was more similar to our findings (8). The reactions mediated by the fibrillar antigens of S. salivarius are highly specific and, at least in the case of AgB, involve lectinlike interactions (32, 33). Therefore, the hydrophobicity in itself cannot be the sole factor determining specificity. McBride et al. (18) recently suggested that hydrophobic sites in S. sanguis are not localized close to the adhesin involved in adsorption to SHA. For oral actinomycetes, it was demonstrated that although hydrophobicity and adsorption to SHA are statistically correlated, hydrophobic interactions appeared not to be of major importance to the reaction itself (3). However, hydrophobic interactions may function to stabilize the specific interactions of the cell with the adhesion substratum by forming additional bonds (4, 9). Indeed, provided that tetramethyl urea disrupts the bacterium-receptor interaction and does not act by inducing conformational changes in either of the surfaces which may prevent their proper interaction, its reductive effect on the adherence of S. salivarius HB to SHA may support such a model. This reduction was not down to the level of adhesion of mutant HB-7, which lacks AgC but retains a fairly high surface hydrophobicity. A reductive effect was not noticed on the coaggregation of strain HB with veillonellae, a reaction which is mediated by the less hydrophobic AgB. Such findings are indicative of a mosaic pattern of surface hydrophobicity accommodating the specific requirements of various surface functions. In addition to creating a suitable microenvironment supporting the formation or stabilization of specific bonds, the overall hydrophobicity of the cell surface may also be of importance to adherence, since it affects the interfacial free energy between the bacterial cell and the substratum (2, 34).

INFECT. IMMUN.

6. 7.

8. 9. 10. 11. 12.

13. 14.

15,

16.

17.

18.

19.

ACKNOWLEDGMENT We are indebted to Caroline Tijhof for excellent technical assist-

20.

ance.

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