Cell-surface polypeptides as determinants of hydrophobicity in Streptococcus gordonii and Streptococcus sanguis

COLLOIDS SURFACES E L 8 EV I E R

Colloids and Surfaces B: Biointerfaces 5(19951135 142

Cell-surface polypeptides as determinants of hydrophobicity in Streptococcus gordonii and Streptococcus sanguis Roderick McNab, Ann R. Holmes, Howard F. J e n k i n s o n * Department o[Oral Biology amt Oral Pathology, Unirersity o[Ota~o. PO Bo.\ 647, Dunedi,, New Zealamt Received 11 January 1995, accepted 5 April 1995

Abstract ('ell-surfi~ce hydrophobicity is well-established as a factor in oral streptococcal adherence. While proteins generally have been implicated in contributing to cell-surtime hydrophobicity of the more hydrophobic strains of Streptococcus gordonii and Streptococcus sanguis, the determinants of hydrophobicity have remained largely uncharacterized. Targeted gene inactivation experiments in Streptococcus mutans suggest that a wall-associated polypeptide, termed antigen 1/11, is a major determinant of cell-surface hydrophobicity. In S. gordonii, production of a high molecular mass wallassociated polypeptide denoted CshA is associated with hydrophobicity. Polypeptides anligenically related to CshA are produced by all wild-type strains of S. gordonii tested and by S. sanguis, but not by S. mutans or Streptococcus parasang,uis. The cell-surface hydrophobicity of wlrious strains correlated with the amount of cell-surface-exposed CshA-like polypeptide present in those strains. CshA polypeptide contains an extensive region of 13 repeat blocks of 101 amino acids, rich in glycine, proline, threonine and valine, and predicted to form an elastic structure. Cell-surface hydrophobicity in S. gordonii and S. sanguis is suggested to result from the exposure of hydrophobic amino acid residues present within the amino acid repeat blocks of CshA-like polypeptides.

Kerwords: Cell-surface proteins: Hydrophobicity; Oral streptococci: Proteins: Streptoco¢'c,s ~,,ordo,ii: Streptococcus san g,uis

1. Introduction: hydrophobicity and adherence in oral streptococci An i m p o r t a n t early step in the c o l o n i z a t i o n of the h u m a n b o d y by b a c t e r i a is the a d h e r e n c e of the m i c r o o r g a n i s m s to a suitable surface substratum. In the oral cavity, n u m e r o u s sites are available for bacterial adherence, a n d these include host epithelial cell surfaces, s a l i v a - c o a t e d h a r d surfaces such as teeth and prostheses (when present), a n d surfaces of o t h e r m i c r o b i a l cells present in biofilms. The bacterial cell surface acts as the forum for *Corresponding author. Tel: 164)-3-4797076. Fax: (641-34790673: cmail: howard.jenkinson(a!stoncbow.otago.ac.nz lt927-7765 95/$09.50 ,t: 1995 Elsevier Science B.V. All rights reserved SSDI 0c~27-7765(95)01213-3

a d h e r e n t interactions, and in the streptococci, which are m a j o r c o m p o n e n t s of the oral microbiota, adherence is m e d i a t e d t h r o u g h multiple surface c o m p o n e n t s , or adhesins [ 1 ] . B r o a d l y speaking, a d h e r e n c e of plaque streptococci such as Streptococcus gordonii and Streptococcus sanguis has been d e m o n s t r a t e d to result from two types of basic mechanisms. The first, various ligand receptor reactions, involve protein (lectin) c a r b o h y d r a t e or protein protein interactions, and are implicit in the e x p l a n a t i o n for site-specific c o l o n i z a t i o n that is observed a m o n g s t oral streptococci [ 2 ] . The second m e c h a n i s m involves physicochemical forces, and there is c o m p e l l i n g evidence that h y d r o p h o b i c and electrostatic interactions

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either promote or indeed are essential for streptococcal cell adherence [3]. The relative roles of these mechanisms in the cooperative binding of streptococci to various substrata, in particular to salivary pellicle, has been the focus of research for many laboratories for at least two decades. Yet today, the molecular mediators of oral streptococcal cell adherence are still largely uncharacterized. Cell-surface hydrophobicity has been implicated strongly in the ability of oral streptococci to adhere to oral surfaces, particularly to glycoproteins present in salivary pellicle [4]. This has come mainly from studies showing positive correlations between bacterial cell-surface hydrophobicity and the ability of cells to adhere. Thus, for those streptococcal strains that adhere to experimental salivary pellicles, it has been observed generally ( but not always) that the greater the cell-surface hydrophobicity, the greater the proportion of input cells that bind [ 5-9]. Furthermore, hydrophobic bond-disrupting agents such as tetramethyl urea abolish the binding of streptococcal cells to experimental salivary pellicle [10]. Despite the evidence that hydrophobic forces are influential in streptococcal adherence, the components of the bacterial cell surface conferring hydrophobicity are poorly defined. Clearly, surface proteins are major determinants since proteolytic treatments of bacterial cells have been shown to reduce both hydrophobicity and adherence [11 13]. Also, more hydrophilic mutant strains of Streptococcus mutans and Streptococcus sanguis were altered in their electrophoretic profiles of cell-surface proteins [ 14]. Lipoteichoic acid, an amphipathic lipoglycan, may also influence cellsurface hydrophobicity [15]. Thus the molecular complexity of the streptococcal cell wall layers may contribute multiple components not only for adherence but also for overall cell-surface hydrophobicity. In this article we will review some of the results, mainly from genetic experiments, that suggest that different polypeptides may influence cell-surface hydrophobicity in different species of oral streptococci. We will then provide evidence suggesting that hydrophobicity of Streptococcus gordonii and S. sanguis (previously to Ref. [16] both classified as S. sanguis) may be determined by the expression on the cell surface of a high molecular mass

polypeptide antigen that is present in all hydrophobic strains of the species examined.

2. Cell-surface proteins associated with hydrophobicity of S. gordonii Once hydrophobicity was firmly established as a factor in streptococcal adherence, the search was on for the "hydrophobins". It was evident from the outset, however, that the task of identifying the determinants of hydrophobicity would not be simple because surface-labelling experiments revealed a complex array of cell-surface polypeptides in S. gordonii [12,17]. Some of these surfaceexposed polypeptides could be extracted from the cell surface with detergents, and sodium lauroylsarcosinate extracts contained proportionally large amounts of a small hydrophobic polypeptide (apparent molecular mass, 16 kDa) which appeared to be a potential candidate for contributing to cellsurface hydrophobicity [12]. This polypeptide turned out to be a phosphocarrier protein (HPr) similar to HPrs of other streptococci and Grampositive bacteria [18] that are normally involved in the uptake of sugars via the phosphotransferase system [19]. A specific modified form of this polypeptide, lacking amino-terminal methionine, is also found associated with the cell surfaces of Streptococcus salivarius [20], S. mutans [21], and Streptococcus pyogenes [22], but the function of the modified form of HPr remains an enigma. Since, however, the presence of this polypeptide amongst these organisms does not appear to correlate with their cell-surface hydrophobicity, it is probably not a major determinant of this property. It cannot be excluded from consideration, though, because, as discussed later in this article, structurally closely related polypeptides in different species of oral streptococci have been shown to confer different cell-surface properties. The analysis of the cell-surface composition of mutant strains of S. gordonii selected for antibiotic resistance [23], for altered cell-surface hydrophobicity [24] or for altered autoaggregation [25] revealed additional polypeptides that might be associated with hydrophobic properties. The most convincing association of specific polypeptides with

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surface hydrophobicity was for mutant strains OB70R and OB74, derived from S. gordonii Challis (DL1), with reduced or increased hydrophobicity, respectively [24]. Hydrophobicity was positively correlated with the ability of the cells to adhere to experimental salivary pellicle and v~:ith the presence of two cell-surface-exposed cell-envelope polypeptides of molecular masses 43 and 45 kDa [24]. These polypeptides were underrepresented in the membrane of the less hydrophobic mutant strain OB70R, and were over-expressed in the more hydrophobic strain OB74. Subsequently these polypeptides were shown by radioactive labelling experiments with [3H]-palmitic acid to carry covalently associated lipid [26] and to be amongst an additional 13 lipoproteins expressed by S. ,vordonii DLI. However, the role that these lipoproteins play in adherence and hydrophobicity remains unknown, despite the identification now of a number of lipoproteins that may function as adhesins in streptococci including SarA [27], Fim A [28], SsaB [29] and ScaA [30] {reviewed in Ref.[31]). Lipid modification of the aminoterminal cysteine residue of these lipoproteins probably serves to anchor them at the outer surface of the cytoplasmic membrane. Streptococcal lipoproteins are on the cell surface and are accessible to both cell-labelling reagents [24,27] and to antibodies [32] and may be secreted into the extracellular medium [33]. However, it seems unlikely that the lipid moieties of these polypeptides are, in general, determinants of cell hydrophobicity since, For example, mutants of S. ,eordonii lacking SarA tipoprotein are unalTected in cell-surface hydrophobicily [27]. Indeed the increased cell-surface hydrophobicity of S. ,~ordonii OB74 cells does not appear now to be atttributable directly to overexpression of the 43 and 45 kDa lipoproteins, but to increased expression of a much larger wall-associated polypeptide as described in the following sections.

3, Gene inactivation studies reveal common themes for hydrophobicity

Comparative studies of different strains of streptococci and of wlrious mutant strains derived

B." BioimeUaces' 5 / 1995! 135 142

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thereof have been useful in identifying potential hydrophobins. Nevertheless a common theme fl'om those studies has not emerged; that is, hydrophobic properties of streptococci have not been attributed to the production of a specific type of protein or set of proteins. Targeted gene inactivation studies in streptococci have shed new light on the hydrophobicity problem. The characterization of mutant strains deficient in the production of specific celtsurface-associated polypeptides has enabled the determination of which of these are involved ill the hydrophobic effect. The first demonstration that a specific polypeptide contributed to cell-surface hydrophobicity came from gene inactivation experiments. In S. mulans NG5 {serotype c) disruption of the SpaP gene encoding a 166 kDa cell-surface polypeptide resulted in reduced cell-surface hydrophobicity [34]. In another serotype c strain MT8148, inaclivation of the pac gene encoding a polypeptide 96% identical in amino acid sequence to SpaP also caused a reduction in the cell-surface hydrophobicity [35] its well as a decrcased ability of the cells to adhere to experimental salivary pellicle [36]. This demonstrated that in X. muums the correlation previously observed between hydrophobicity and adherence wets likely to be the result of the major adhesin and hydrophobin being one and the same polypeptide. More recently, it was shown that loss of the SpaP-like polypeptide ~called AgB) from another S. mutans serotype c strain LTll led to reduced cell-surface hydrophobicily and to reduced amounts of cell-surtEce lipoteichoic acid [37]. However the reduction in hydrophobicity was not due directly to the loss of lipoteichoic acid since another class of LTI I mutants that had lost more than 9()9} lipoteichoic acid from their cell surfaces were unaffected with respect to cell-surface hydrophobicity 137]. SpaP, PAc and AgB are members of the antigen 1/11 family of polypeptides found m a variety of oral streptococci [31]. These proteins are thought to have lectin-like properties and may mediate the binding of cells to salivary glycoproteins [38 4(I]. Despite the evidence for a common binding function for these polypeptides, gene inactivation experiments reveal an alternative cell-surface function for the antigen I11 polypeptide SspA in

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R. McNab et al./Colloids Surj~tces B." Biointerjitces 5 (1995) 135 142

S. gordonii DL1. Mutants of S. gordonii DL1 deficient in SspA production were not affected with respect to hydrophobicity and nor were they altered in their ability to bind to salivary pellicle [-41]. Therefore components other than the antigen I/II polypeptide are involved in determination of these properties in S. gordonii. Possibly, changes in crucial amino acids within the antigen I/II polypeptide of S. gordonii may have rendered the polypeptide less hydrophobic than the S. mutans counterpart. Alternatively, the molecular environment of the cell surface of S. mutans might influence the hydrophobic nature of the polypeptide. These possibilities might be addressed by expressing the S. gordonii SspA polypeptide on cells of a S p a P - m u t a n t of S. mutans. The second demonstration by gene inactivation that a specific polypeptide contributed to cellsurface hydrophobicity came with the discovery of the CshA polypeptide in S, gordonii DL1. Mutants of S. gordonii that failed to retain this protein on the cell surface were reduced in hydrophobicity and were deficient in their ability to coaggregate with another oral bacterium, Actinomyces naeslundii [42]. The properties of this polypeptide, its structure, and how it might confer cell-surface hydrophobicity on S. gordonii and S. sanguis form the basis for the remainder of this article.

4. CshA polypeptide in S. gordonii CshA is a polypeptide of molecular mass 259 kDa that is anchored through a region at the carboxy-terminal end of the polypeptide to the cell wall of S. gordonii DL1 [42]. The gene encoding CshA has been cloned and sequenced in full (Gen/EMBL accession number X65164). The deduced amino acid sequence of CshA precursor (2508 amino acids) exhibits several features characteristic of Gram-positive bacterial wall-associated polypeptides [-43] including the presence of amino acid repeat blocks and a proline-rich domain at the carboxy terminus involved in cell-wall anchoring. The CshA precursor polypeptide sequence can be divided into four regions: (i) an amino-terminal signal sequence of 41 amino acids; (ii) amino acid residues 42-878 comprising a non-repetitive

domain predicted to be predominantly s-helical; (iii) an extensive region of amino acid repeat blocks representing approximately 60% of the mature polypeptide (amino acid residues 879 2417); and (iv) a wall-anchor domain (residues 2418 2508). Isogenic mutants lacking CshA showed a more than 50% reduction in cell-surface hydrophobicity [44]. During the course of these studies on CshA, a second gene was identified at a separate chromosomal locus encoding a highly similar polypeptide denoted CshB. Mutants were constructed that were deficient in CshB, and were found to be less affected with respect to cell-surface hydrophobicity than were CshA mutants [44].

5. CshA-like polypeptides and hydrophobicity in S. gordonii and S. sanguis To investigate the production of CshA-like polypeptides by various strains of oral streptococci, bacteria were grown in brain heart infusion broth (Difco) to the early stationary phase of growth, harvested, and cell-wall-associated proteins were extracted and separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE). Nitrocellulose blots of polypeptides were then incubated with antibodies raised to a recombinant polypeptide purified from Escherichia coli and consisting of four carboxy-terminal repeat blocks of amino acids of CshA fused to the carboxyterminus of /~-galactosidase [-42]. Antibodies to the fusion polypeptide, but not to/:~-galactosidase, reacted with polypeptide antigens produced by a variety of streptococci. In general, all wild-type strains of S. gordonii tested produced one or more CshA-like antigenic polypeptide bands (Fig. 1). Strain OB277 in which the cshA and cshB genes were inactivated [-44] produced no antibodyreactive material. Three strains of S. sanguis tested all produced antibody cross-reactive bands, although reactions were weaker for strains ATCC 10556 and 12 compared with strain 133.79 (Fig. 1). No antibody-reactive bands were detected on immunoblots of proteins from Streptococcus parasanguis EW213 (Fig. 1), or from S. mutans NCTC 10449 (serotype c) and S. salivarius HB (results not shown).

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Fig. 2. CshA-like antigen production and cell-surface hydrophobicity in various strains of S. ,gordomi and S..~an*,,uis. iA) ELISA of whole cells with antibodies to CshA amino acid repeat blocks (see caption to Fig. I). Streptococcal cells were immobilized onto microtitre plate wells, reacted with doubling dilutions of antiserum or pre-immune serum, and antibody binding was measured. following reaction with peroxidase-linked secondary antibody and enzyme substrate 1,2-pllenylenediamine, by absorbance at 492 nm. A serum dihition (1:800) was selected that gave absorbance values that fell on the linear portion of an antibody titration curve. Values for antiserum reactivities were corrected for absorbance values obtained with pre-immune serum. For ease of presentation, the highest absorbance value (0.321 for cells of strain OB74 was assigned a value of 10 ELISA units and the reactivities of all other stratus were expressed relative to this. (B) The hydrophobicity of the cells was measured by hexadecune partitioning [23]. Cell suspensions in 0.1 M NaH2POa/NaOH buffer, pH 6.8 (optical density (()D) lit 540 nm. 11.8 (1.5 ml)) werc mixed with hexadecane 10.04 ml). The phases were allowed to separate, and the percentage of input cells adhering to the hexadecune was determined [23]. The error burs are standard deviations of the means of quadruplicate samples from experiments repeated three times.

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R. McNab et aL/Colloids Sur[aces B." Biointel;/itces 5 (1995) 135 142

reacted less well with CshA antibodies although two of these strains (10556 and 133.79} were as hydrophobic as the wild-type S. gordonii strains. Clearly, ELISA titres would be lower for the S. sanguis strains if antibodies raised to CshA reacted less well with the heterologous antigens. In summary, therefore, (1) the amino acid repeat blocks of CshA and CshA-like polypeptides are exposed at the streptococcal cell surface; and (2) the expression of CshA and CshA-like polypeptides correlates with the cell-surface hydrophobicity.

6. Structure-function aspects of the amino acid repeat block present in CshA polypeptide Amino acid repeat blocks are present in most Gram-positive bacterial-wall polypeptides [43] and in some instances have been ascribed ligand-binding functions. The amino acid repeat blocks of the immunoglobulin-binding proteins of group A streptococci bind immunoglobulins, human serum albumin and factor H [45]. These repeat block regions are predicted to form an ~helical coil structure typical of fibrous eukaryotic proteins such as tropomyosin [46]. Within the carboxy-terminal domains of the streptococcal glucosyltransferase enzymes [47] and pneumococcal surface protein PspA [48], amino acid repeat blocks are present that are involved in carbohydrate polymer binding [47]. The amino acid repeat region of CshA comprises 13 blocks of a 101 amino acid residue repeat, flanked at the anaino-terminal end of the region by one incomplete repeat of 36 residues, and at the carboxy-terminal end by two blocks of 95 residues each [44]. The repeat blocks bear no strong resemblance to any other amino acid repeat block regions present in Gram-positive bacterial proteins except perhaps to sequences within a collagenbinding adhesion of Staphylococcus aureus [49]. The repeat blocks of CshA are dominated by the amino acids glycine, proline, threonine and valine, and the 101 amino acid residue sequence is remarkably well-conserved across the 13 complete blocks (six of the blocks contain more than 90% identical amino acid residues). A Garnier Osguthorpe

Robson [50] prediction of the secondary structure for a representative repeat block (number 11, residues 1824 1924) and a corresponding Kyte Doolittle [ 51 ] hydropathy plot are shown in Fig. 3. Each 101 amino acid residue repeat contains four regions of/:t-strand which coincide with predicted regions of hydrophobicity. Most of the remainder of the sequence comprises coil and turn with the exception of a central ~-helical region of six amino acid residues (Fig. 3). It is not possible yet to make an accurate prediction as to the conformation adopted by these repeat regions; however, several possibilities may be considered based on structural similarities with other proteins. The high glycine, proline and threonine content, together with the anticipated coil and sheet structure suggest an open confirmation with elastic properties. There are only four cysteine residues within CshA (two within the amino-terminal region and one each within repeat blocks 2 and 11 i; therefore, unlike the eukaryotic extracellular matrix protein elastin, which is also rich in glycine, proline and threonine, structural rigidity could not be maintained through extensive disulphide bond formation. The high glycine and proline content of the CshA repeat block sequence might suggest a structural similarity to collagen types I and XIV, and in fact the motif GXP (where X is any amino acid residue) which is important for formation of the collagen triple helix, is present four or five times in each repeat block of CshA [44]. How might therefore the CshA repeat blocks be involved in conferring cell-surface hydrophobicity? The CshA polypeptide is predicted to be hydrophilic overall: however, it could be envisaged that a protein that was extensively hydrophobic would not be a good candidate for conferring cell-surface hydrophobicity. This is because hydrophobic regions of polypeptides are likely to be sequestered within the non-polar regions of the polypeptides, or are likely to be associated with the cytoplasmic membrane, and therefore would not be exposed at the cell surface. An essentially hydrophilic protein with exposed hydrophobic residues, or groups of residues, is a more attractive proposition for a determinant of cell-surface hydrophobicity. In a repeat-block structure such as that within CshA, the exposure of one hydrophobic amino acid resi-

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= 13 Fig. 3. Secondary structure and hydropathy predictions of a repeat block of amino acids within CshA of S. g , rdo;fii DLI. Repeat block number 11 [44] comprising 101 amino acid residues was analyzed by the Gamier Osguthorpe Robson and Kyte Doolittle algorithms available on the GCG computer programme from the University of Wisconsin. The locations within the repeat exhibiting random coil, turn. sheet and >helix are designated. The positive values of the hydropathy index indicate more hydropbobic residues.

due in each of thirteen repeats might confer a cellsurface hydrophobicity that could not otherwise be achieved by a run of 13 hydrophobic residues. If this hypothesis is correct, then cell-surface hydrophobicity in S. gordonii might be modulated not only by varying the expression levels of the CshA polypeptide but also by altering the numbers of amino acid repeat blocks. This latter event might occur as a result of intragenic recombination events within the regions of the gene encoding the repeat blocks of amino acids. The molecular basis for the hydrophobic efl'ect that is correlated with the amino acid repeat block region of CshA is currently under investigation.

7. Conclusions Cell-surface hydrophobicity in different species of streptococci is likely to result from the expression of different cell-surface molecules. In S. gordonii DL1, CshA polypeptide appears to contribute to a greater extent to the hydrophobicity of the cell surface. The polypeptide is also involved in the coaggregation of S. gordonii cells with ,4. naeshmdii, but it is not necessary for the adherence of S. gordonii to salivary pellicle. CshAlike polypeptides are present in other strains of S. :mrdonii and in S. sanguis. Their presence and

amount at the cell surface correlates well with the degree of cell-surface hydrophobicity in strains of these species. CshA-like antigenic polypeptides were not detected on S. mtaans or S. parasangHis, organisms that are comparatively less hydrophobic than strains of S. gordoHii and S. sanguis.

Acknowledgements We thank M.C. Herzberg, P.E. Kolenbrander, R.J. Lamont, B.C. McBride and M.D.P. Willcox for their gifts of bacterial strains. The authors' research is supported by the Wetlcome Trust, U.K. and by the Health Research Council of New Zealand.

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