INTRODUCTION

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The pathogenesis of infective endocarditis is a complex phenomenon, involving numerous host-pathogen interactions. Infection of the endocardium is initiated by the attachment of blood-borne organisms to platelets, fibrin, and extracellular matrix proteins on the damaged valve surface (10, 19). The relative importance of these host binding factors to colonization is uncertain. However, since a variety of endocarditis-associated organisms, including streptococci and staphylococci, can bind platelets directly in vitro (16, 36, 42, 43), it is likely that direct binding to platelets in vivo contributes to the initiation of endocardial infection.

The subsequent development of mature, macroscopic vegetations may also be mediated in part by the direct binding of platelets to bacteria. Binding of circulating platelets to organisms on the valve surface may result in the further accumulation of platelets at the site of endocardial infection. In addition, such binding may be a mechanism for the reattachment of bacteria shed into the circulation back onto the valve surface (33). Evidence for these processes in vivo came initially from histologic studies of animals with experimental endocarditis, in which the progressive accumulation of platelets and bacteria was observed at the outer margins of maturing vegetations (9). More recently, the induction of selective thrombocytopenia in rabbits with early endocarditis has resulted in vegetations of significantly reduced mass, indicating that platelets continue to be deposited on the infected endocardium and are a major structural component of vegetations (35). In addition, diminished platelet binding in vitro by Staphylococcus aureus has been associated with reduced virulence in an animal model of endocarditis, as manifested by decreased concentrations of bacteria within vegetations and a reduced incidence of peripheral embolization and hematogenous dissemination of infection (34).

Among the viridans group streptococci, Streptococcus mitis is a leading cause of infective endocarditis (8, 27). In addition to its long-recognized association with endocardial infection, this organism has recently emerged as a major cause of bacteremia in the immunocompromised host (4, 15, 26, 38). Therapy of S. mitis infections has become problematic, due to a high prevalence of multidrug resistance reported in this organism (6, 25). Despite the clinical importance of S. mitis, few studies have addressed its virulence determinants, particularly with regard to endocarditis and the role of platelets in pathogenesis (23).

In view of the role of platelet binding by endocarditis-associated organisms and the importance of S. mitis as an endocardial pathogen, we sought to identify bacterial components that contribute to platelet binding by strain SF100. These studies indicate that platelet binding by this organism is a complex interaction that involves at least two distinct loci. As shown below, one is likely to encode a small molecule transmembrane transporter and the other encodes cell surface proteins resembling structural components of streptococcal phages.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and reagents. The bacterial strains and plasmids used in this study are listed in Table 1. Strain SF100 is a streptomycin-resistant variant of S. mitis 12021, which was isolated from the blood of a patient with infective endocarditis. The species of this strain was confirmed by biochemical testing at the Centers for Disease Control (Atlanta, Ga.) and by 16S rRNA sequencing (MIDI Labs, Newark, Del.). Enterococcus faecalis RH110 carries Tn916E, a derivative of Tn916 in which the tet gene has been replaced by erm (28). These strains and their variants were grown in Todd-Hewitt broth (THB; Difco Laboratories, Detroit, Mich.) or on sheep blood agar (Remel, Lenexa, Kans.) at 37°C in a 5% CO₂ environment. When indicated, antibiotics were added to the media at the following concentrations: 15 µg of erythromycin per ml, 750 µg of streptomycin per ml, and 5 µg of chloramphenicol per ml. Escherichia coli strains were grown in Luria-Bertani broth containing 100 µg of ampicillin per ml or 15 µg of chloramphenicol per ml when appropriate. Tyrode's salts, trypsin, sodium lauroyl sarcosine (SLS), and Dulbecco's phosphate-buffered saline (DPBS) were purchased from Sigma (St. Louis, Mo.).

Quantitative assay for binding to immobilized platelets. The binding of streptococci to human platelets was assessed quantitatively as described previously (34). In brief, washed, fixed human platelets were immobilized in poly-L-lysine-coated 22-mm-diameter tissue culture wells, producing monolayers of 75 to 90% confluence. To reduce nonspecific adherence, the wells were then treated with a casein solution (1× blocking reagent [Roche, Indianapolis, Ind.] in DPBS) for 1 h at room temperature. After the blocking solution was removed by aspiration, the wells were inoculated with approximately 5 × 10⁶ CFU of streptococci suspended in 0.5 ml of DPBS and incubated at 37°C for 2 h, with gentle rocking to enhance mixing. Unbound bacteria were then removed by washing, and the platelet-bound organisms were recovered by treating the platelet monolayers with 1 mg of trypsin per ml in DPBS. The number of organisms bound was determined by plating serial dilutions of the suspension onto blood agar, and binding was expressed as a percentage of the inoculum. In control studies, trypsinization of SF100 had no effect on viability and was found to allow complete recovery of bound organisms. For experiments in which SF100 was treated with trypsin before being tested in the binding assay, bacteria were incubated for 60 min at room temperature in DPBS containing 1 mg of trypsin per ml and then washed repeatedly. All studies were done in triplicate, using platelets from multiple human donors.

Transposon mutagenesis and selection of low-binding variants. Transposon mutagenesis of SF100 was by done by filter mating, as described previously (29). In brief, 1 ml of an exponential-phase culture of SF100 (~5 × 10⁸ CFU) was combined with 10 ml of an exponential-phase culture of E. faecalis strain RH110. Cells were collected on a sterile 0.2-µm-pore-size filter (Millipore, Bedford, Mass.), placed on a blood agar plate, and incubated overnight at 37°C. The filter was then transferred to 10 ml of THB containing streptomycin and erythomycin and incubated for 3 h at 37°C to select for SF100 transconjugants.

Southern blot analysis. Chromosomal DNA was isolated from streptococci by adding 6.5 ml of fresh THB and 0.1 g of solid glycine to 3.5 ml of an overnight culture and incubating it for 90 min at 37°C. The bacteria were centrifuged, resuspended in 1 ml of distilled H₂O, and transferred to a microcentrifuge tube. Cell pellets were suspended in 100 µl of TE 50:5 (50 mM Tris, 5 mM EDTA [pH 8.0]), 50 µl of a lysis solution (50 mg of lysozyme per ml and 200 U of mutanolysin per ml in TE 50:5) was added, and the mixture was incubated for 1 h at 37°C. After addition of 340 µl of TE 50:5, 7.5 µl of 20% sodium dodecyl sulfate (SDS), and 2 µl of proteinase K (25 mg/ml), the suspensions were gently mixed and then incubated for 1 h at 37°C. An additional 200 µl of TE 50:5 was added, and the mixtures were extracted with 700 µl of phenol. After centrifugation, the aqueous phase was transferred to a Phase Lock Gel tube (Eppendorf, Westbury, N.Y.) and extracted twice with 2.5 ml of phenol-chloroform (1:1). The aqueous phase was then extracted with an equal volume of chloroform-isoamyl alcohol (24:1), and nucleic acid was precipitated from 400 µl of the aqueous phase by adding 40 µl of 3 M sodium acetate (pH 5.2) and 1 ml of ethanol. Pellets were rinsed with 70% ethanol in dH₂O, resuspended in 100 µl of 10 mM Tris-1 mM EDTA (pH 8) containing 0.5 µg of DNase-free RNase (Roche), and incubated at 37°C until completely dissolved. Following treatment with restriction enzymes and electrophoresis, digested chromosomal DNA was transferred to positively charged nylon membranes (Roche) using a Trans-Blot semidry transfer apparatus (Bio-Rad, Hercules, Calif.). The membranes were hybridized with digoxigenin-labeled probes and developed with the CDP-Star chemiluminescent substrate as recommended by the supplier (Roche).

DNA sequence analysis. To identify the sites of Tn916E insertion in the low-binding mutants, chromosomal regions flanking the transposon were isolated by cloning EcoRI fragments in the cosmid vector pWE15 as described previously (3). Following Tn916E excision from the cosmid clones, the EcoRI fragments (or portions thereof) were subcloned in pBluescript KS() or pBluescript SK() (Stratagene, La Jolla, Calif.) for sequence analysis by primer walking. Sequences upstream and downstream from the EcoRI fragment of PS101 (Fig. 1) were obtained by marker rescue of pVA891 from SacI- or ClaI-digested chromosomal DNA from strain PS163, which has pVA891 integrated in pblT. For PS116, a full-length EcoRI fragment (later estimated to be 20 kb by Southern blot analysis of SF100 chromosomal DNA) was not maintained in DH5 but, rather, tended to undergo deletion or rearrangement. However, a 2.3-kb HindIII fragment (Fig. 2, segment d) was maintained in one spontaneously deleted cosmid construct and was subcloned for sequence analysis. A chromosomal segment flanking the left end of Tn916E, which includes the erythromycin resistance marker (Fig. 2, segment b), was obtained by direct cloning of HindIII-digested PS116 chromosomal DNA in pBluescript. Since these constructs were also unstable, the cloned fragment was amplified by PCR, using the M13 40 universal primer and a primer complementary to bases 85 to 104 of the left end of the transposon (5'-CGAAAGCACATAGAATAAGG-3'), and the 2.0-kb PCR product was sequenced directly. The location of this fragment relative to segment d was confirmed by PCR amplification of SF100 chromosomal DNA, using a forward primer upstream from the PstI site in segment b and a reverse primer downstream from the XbaI site in segment d. The PstI-XbaI fragment (segment c) was then cloned in pBluescript and sequenced. Additional sequence was obtained after cloning products derived by inverse PCR of XbaI- or SacI-digested chromosomal DNA, using primers reading outward from the known sequence (segment e or segments a, f, and g, respectively).

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Map of the PS101 locus, and site of insertion of Tn916E. The broken arrow indicates an incomplete ORF. Bold letters correspond to the ribosome binding site of pblR. The underlined segment corresponds to the probable 10 region of the promoter element. The 35 region is within an inverted repeat, underscored with arrows. The site of insertion of Tn916E was determined by cloning the left flank of the transposon (containing the erythromycin resistance gene) and then sequencing with a primer complementary to the left end of the transposon. E, EcoRI; H, HincII; C, ClaI.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   Map of the PS116 locus. (A) Location of selected restriction sites, and fragments used for DNA sequence analysis. Segment d corresponds to the initially sequenced HindIII fragment. The sequence of segment b was obtained from a PCR product that was amplified from a metastable clone of the left flank of Tn916E. Segment c was cloned after PCR amplification of the region from SF100 chromosomal DNA. Segment e was cloned from a PCR product derived by inverse PCR of XbaI-digested chromosomal DNA. Segments a, f, and g were cloned from products derived by inverse PCR of SacI-digested chromosomal DNA. E, EcoRI; H, HindIII; Hp, HpaI; N, NheI; P, PstI; RV, EcoRV; S, SacI; X, XbaI. (B) Genes and gene products. The speckled region of pblB encodes a protein sequence showing 47% similarity to PspA of S. pneumoniae and 42% similarity to M protein of S. pyogenes. cc, coiled-coil domain.

Directed mutations. Selected genes were mutated by insertion-duplication or by gene replacement, using pVA891 (18). For insertion-duplication mutagenesis, internal portions of the target genes were cloned in pVA891 and the resulting plasmids were propagated in Escherichia coli strain DH5 prior to introduction to SF100 by natural transformation. To create strain PS344, in which open reading frame 3 (ORF3), pblA, ORF4, and pblB were deleted by gene replacement, the HindIII-SacI fragment (Fig. 2, segment g) was cloned adjacent to the SacI-BglII fragment (from segment a [Fig. 2]) in the BamHI site of pVA891. This plasmid was linearized with SacI and then used to transform SF100. Recombination at the expected site was confirmed by Southern blot analysis of chromosomal DNA isolated from the transformants.

Complementation. For trans-complementation of wild-type or mutated chromosomal genes, pblA and pblB were each cloned in the streptococcal expression vector pDC123 (7). The ribosome binding site and entire coding sequence of pblA or pblB was amplified by PCR, using the following BamHI-linked primers: 5'-AAGGATCCAATAGGAGGTGAGGATTAATGGCTACAG-3' and 5'-AAGGATCCATTAGATTCCCTCCCTTGC-3' for pblA or 5'-AAGGATCCTTGGAGGTATAAAATATGATTTACTT-3' and 5'-AAGGATCCTTTGTTTGTCCTGTTCGTTCATGC-3' for pblB. PCR products were then cloned in the BamHI site of pDC123 downstream from the constitutive cat/tet promoter. Plasmids were propagated in E. coli strain MC1061, and those with pblA or pblB in the proper orientation were used to transform S. mitis by electroporation.

Transformation of S. mitis. Introduction of pVA891 derivatives into SF100 was accomplished by natural transformation. In pilot studies, the frequency of transformation of SF100 was found to be very low (<10⁸ transformant per µg of DNA). To enhance the efficiency of transformation, a competence-stimulating peptide (CSP) specific for this S. mitis strain was identified, using the strategy of Håvarstein et al. (13). The comCDE locus of SF100 was amplified by PCR and sequenced. Analysis of comC indicated that the amino acid sequence of the CSP was DWRISETIRNLIFPRRK. This peptide was synthesized (Biomolecular Resource Center, University of California, San Francisco, Calif.) and was subsequently found to increase the transformation frequencies to approximately 10⁵ transformant per µg of DNA. To transform SF100 with pVA891 derivatives, overnight cultures were diluted 100-fold in fresh THB supplemented with 20% heat-inactivated horse serum, 200 ng of CSP per ml, and 1 µg of DNA per ml. Transformation mixtures were incubated 8 h at 37°C and then plated on blood agar containing erythromycin.

Production of polyclonal antisera. Since overexpression of full-length PblA or PblB was found to be toxic to E. coli host strains, two subdomains of each protein were used to generate antisera. For PblA, one domain was generated by in-frame fusion of the HindIII-BglII fragment of segment b (Fig. 2) to the glutathione S-transferase (GST) moiety of pGEX-3X (Amersham Pharmacia Biotech, Piscataway, N.J.). The second domain was created by fusing the entire pblA coding sequence with that of GST and then deleting the internal HindIII fragment of pblA, producing an in-frame fusion of the N- and C-terminal regions of PblA to GST. For PblB, the EcoRV-EcoRV or HpaI-EcoRV fragment of pblB (Fig. 2) was fused in frame with GST in pGEX-3X. These plasmids were introduced into E. coli strain BL21(DE3), and protein expression was induced as recommended (Amersham Pharmacia Biotech). Expressed proteins were separated by SDS-polyacrylamide gel electrophoresis and then stained with zinc (Pierce, Rockford, Ill.). Regions of the gel containing the overexpressed proteins were excised and used to immunize goats (Caltag Laboratories, South San Francisco, Calif.). To enhance the specificity of the antisera for PblA or PblB, each antiserum was adsorbed repeatedly with S. mitis strain PS344, a deletion mutant (described above) that does not express PblA or PblB.

Western blot analysis. Cell surface proteins were extracted from S. mitis strains using SLS or mutanolysin as described by Jenkinson (14). The extracted proteins were separated by electrophoresis through SDS-6.8% polyacrylamide gels under reducing conditions, transferred to Biotrace NT nitrocellulose membranes (Pall Corp., Ann Arbor, Mich.), and incubated with antisera. Antibody binding was detected with horseradish peroxidase-conjugated anti-goat immunoglobulin G IgG (Sigma) and developed with the Super Signal chemiluminescent detection system (Pierce).

RNA isolation and blotting. Total RNA was extracted from S. mitis strains using the RNeasy kit as recommended by the manufacturer (Qiagen, Valencia, Calif.), except that 500 U of mutanolysin per ml was included in the cell resuspension buffer. For dot blot analysis of pblR transcripts, 2 µg of RNA was spotted onto nylon membranes, hybridized with a digoxigenin-labeled DNA fragment of pblR, and developed with the CDP-Star chemiluminescent substrate as specified in the Genius System protocol (Roche). For Northern blot analysis of pblB transcripts, approximately 5 µg of RNA was denatured, loaded into wells of a 1% agarose gel, electrophoresed as described previously (2), and then transferred to a nylon membrane using the TurboBlotter system (Schleicher & Schuell, Keene, N.H.). The membrane was probed with a digoxigenin-labeled DNA fragment spanning the pblB coding region and developed as described above.

Statistical methods. Differences in platelet binding were compared by the unpaired t test, using the Welch modification when appropriate.

Nucleotide sequence accession numbers. The sequences generated from strains PS101 and PS116 have been deposited in GenBank under accession numbers AY007504 and AY007505, respectively. The sequence of the SF100 comC has been deposited under accession number AY007503.

	RESULTS

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Platelet binding by SF100 and isolation of low-binding mutants. Strain SF100 readily bound to human platelets immobilized in tissue culture wells. Initial characterization of platelet binding by this strain indicated that after 2 h of incubation with platelet monolayers, binding ranged from 1.4 to 3.7% of the applied inoculum, with a mean ± standard deviation of 3.3% ± 1.9%. In contrast, only 0.33% ± 0.24% of the inoculum bound to polylysine-coated plastic wells (P = 0.0002 compared with platelet binding; n = 12). Trypsinization of bacteria prior to testing reduced platelet binding by 88.7% ± 8.8% compared with that of untreated organisms (P < 0.0001; n = 4). These results indicated that the binding of SF100 to platelets was relatively selective and was mediated predominantly by one or more surface proteins.

DNA sequence analysis of the PS101 locus. To characterize the sites of Tn916E insertion in the low-binding mutants, chromosomal regions flanking the transposon were cloned and sequenced as described in Materials and Methods. The initial sequence of the PS101 locus was obtained from a 1.2-kb EcoRI fragment of chromosomal DNA. A 0.6-kb EcoRI-HincII fragment upstream from the site of the Tn916E insertion (Fig. 1) was then cloned in the suicide vector pVA891 and used to generate strain PS163 by insertion-duplication mutagenesis of strain SF100. Additional sequences downstream and upstream of the 1.2-kb EcoRI fragment were obtained by recovery of pVA891 and flanking DNA from PS163.

PblT contributes to platelet binding. To confirm that the reduced platelet binding observed with PS101 was due to transposon insertion within pblT, strain PS163 was tested for platelet binding. Compared with the parental strain, platelet binding by PS163 was reduced by 42.0% ± 10.1% (P < 0.0001) (Fig. 3). To assess whether the low-binding phenotype was due to a polar effect on downstream gene expression, strain PS361 was generated by insertion-duplication mutagenesis of pblR. Platelet binding by PS361 was not significantly different from that by SF100 (P = 0.0794) (Fig. 3). In addition, dot blot analysis of RNA from PS101 showed no difference in pblR transcript levels compared with RNA from SF100 (data not shown). These results indicated that platelet binding by SF100 is in part mediated by pblT and that the loss of binding seen with pblT disruption was not due to polar effects on the expression of pblR.

View larger version (53K): [in this window] [in a new window]   FIG. 3.   Platelet binding by insertion-duplication and deletion mutants. Values presented are percent of wild-type binding (mean ± standard deviation). Values that are significantly different from those of SF100 (P < 0.05) are indicated by asterisks. Strain genotypes: SF100, wild type; PS163, pblT::pVA891; PS361, pblR::pVA891; PS344, ORF1-pblB; PS301, pblA::pVA891; PS345, pblB::pVA891.

DNA sequence analysis of the PS116 locus. The chromosomal region of PS116 flanking Tn916E was sequenced in several stages, as diagrammed in Fig. 2. A total of 8.5 kb of sequence was compiled for this locus, including 3.9 kb upstream and 4.6 kb downstream from the point of the Tn916E insertion. Analysis of the combined sequences indicated that the entire region was likely to be part of a polycistronic operon, since there are just a few nucleotides between adjacent ORFs.

Role of PblA and PblB in platelet binding. To confirm the role of this second locus in platelet binding, the platelet binding phenotype of a mutant (PS344) carrying a deletion of ORF1 through pblB was assessed. Compared with the parental strain, platelet binding by PS344 was reduced by 36.1% ± 16.4% (P < 0.0001) (Fig. 3), confirming that disruption of the PS116 locus was linked to loss of binding.

Disruption of pblA is not transcriptionally polar. To determine whether disruption of pblA caused a polar mutation, RNA from strain PS301 was compared with RNA from strain SF100 by using a pblB probe. Northern blot analysis indicated that the pblB mRNA is part of a very large (much greater than 6.9 kb) polycistronic transcript in SF100 (Fig. 4, lane 1). Although the level of transcription of pblB in PS301 (lane 2) was somewhat reduced compared with the level in SF100, it was not abolished. Thus, integration of pVA891 into pblA in strain PS301 was not transcriptionally polar. However, the pblB transcript in PS301 was smaller. The difference in transcript size was probably due to either differential processing within pVA891 sequences or initiation of transcription from a promoter within pVA891.

View larger version (53K): [in this window] [in a new window]   FIG. 4.   Northern blot of RNA isolated from S. mitis strains and hybridized with a pblB probe. Lanes: 1, RNA from the parental strain SF100; 2, RNA from the pblA mutant strain PS301. Arrows indicate the predominant prgB transcripts. The bands seen at 2.5 kb correspond to nonspecific hybridization to the 23S rRNA.

Expression of PblB may be linked to that of PblA. To compare the expression of PblA and PblB in the wild-type and mutant strains, polyclonal antisera were generated to each of these proteins and expression was assessed by Western blotting. Preliminary experiments showed that both PblA and PblB could be extracted with SLS but not with a combination of mutanolysin and lysozyme (data not shown), indicating that they were associated with the cell wall but not covalently linked to the peptidoglycan (20). The anti-PblA serum reacted with proteins of 120, 110, and 80 kDa that were absent from the PS344 deletion mutant (Fig. 5A, lanes 1 and 2). The anti-PblB serum reacted with two protein bands: the predominant form had an apparent molecular mass of 110 kDa, and a minor form was apparent near the predicted molecular mass of 121 kDa (Fig. 5B, lane 1).

View larger version (38K): [in this window] [in a new window]   FIG. 5.   Western blot of SLS-extracted proteins from strain SF100 and from pblA and pblB mutant strains. Lanes: 1, SF100; 2, PS344 (PblA PblB); 3, PS301 (PblA); 4, PS345 (PblB). (A) The blot was reacted with anti-PblA serum. (B) The blot was reacted with anti-PblB serum.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

These results demonstrate that at least two distinct loci contribute to platelet binding by S. mitis strain SF100. DNA sequence analysis of the first region indicated that it contains an assortment of divergent and convergent ORFs. The gene responsible for platelet binding, pblT, is likely to encode a transmembrane transporter that resembles members of the major facilitator superfamily of small-molecule transporters. Solute binding proteins of the ATP binding cassette transporter family have been shown to facilitate adhesion by other microbes, such as Streptococcus parasanguis (11). Thus, it is possible that PblT affects platelet binding directly. Alternatively, it could play a secondary role, such as transport of a signal molecule or transport of a substrate required for adhesin assembly that cannot be produced de novo.

Binding is also mediated by one or more surface proteins encoded by the PS116 locus. Our data indicate that PblB is a likely adhesin, since the surface expression of this protein correlated with platelet binding, and PblB has several features typical of binding proteins. Of note, the N-terminal half of PblB shows similarity to two well-characterized streptococcal adhesins, PspA of S. pneumoniae and M protein of S. pyogenes. PblB is also predicted to form a coiled coil, which is characteristic of fibrillar proteins (17). Moreover, PblB is similar to receptor recognition proteins of various phages. Alternatively, it may serve as a scaffolding protein for another adhesin or may be part of a larger binding complex that includes PblA.

PblA has features that are more characteristic of a surface structure. It is predicted to have a signal peptide and has five regular repeats of a tryptophan-rich pentapeptide motif. Although the significance of these repeats for PblA is unknown, tryptophan-rich repeats have been shown to allow the association of proteins with choline residues in the cell wall of pneumococci and listeriae (5, 44) and the cell wall polysaccharides of S. mitis strains do contain choline (31). PblA may be associated with PblB on the cell surface, which could either stabilize PblB or affect its conformation. The possibility of such a multicomponent adhesin is suggested by the relatedness of the gene products to components of phage capsids.

The similarity of PblA and PblB to phage proteins also raises the question whether the locus can transfer between strains. A search for sequences similar to PblA and PblB within the unfinished genome sequences available from the University of Oklahoma Advanced Center for Genome Technology (http://www.genome.ou.edu) and The Institute for Genomic Research (http://www.tigr.org) indicated that homologs are present in S. pyogenes and E. faecalis but not in the sequences reported to date for S. mutans or S. pneumoniae. Interestingly, if present, the homologs again reside in a phage or cryptic phage locus (Fig. 6). That is, S. pyogenes has an arrangement of genes identical to that of pblA-ORF4-pblB, which are flanked by phage r1t-like sequences. E. faecalis has two sets of homologs: one set lies within a region of sequences similar to phage r1t sequences, and the other lies within a region of sequences similar to phage 01205 sequences. The sequence of the corresponding locus of SF100, by comparison, appears to be a mosaic that may have evolved from reassortment of the two streptococcal phage genomes. It is interesting to consider that, as with other virulence properties, they may be encoded within a mobile genetic element (40). Although it is unknown whether pblA and pblB reside within a functional phage, we have found that expression is greatly induced by mitomycin C and UV light (unpublished results), agents commonly used to induce the lytic cycle of prophages.

View larger version (15K): [in this window] [in a new window]   FIG. 6.   Homologs of PblA and PblB in other streptococcal species. Black arrows indicate similarity to Lactococcus lactis phage r1t sequences, and checkered arrows indicate similarity to S. thermophilus phage 01205 sequences. The gray arrow corresponds to ORF4, which was not similar to any sequences reported in the standard databases. In each of the loci diagrammed here, the pblA and pblB homologs are flanked by at least 6 kb of sequences corresponding to the structural protein region of the indicated phage. S. pyogenes has a cryptic or functional phage similar to r1t. E. faecalis has sequences similar to those of both r1t and 01205. The pblAB locus of S. mitis strain SF100, by comparison, appears to be a mosaic of the two streptococcal phage genomes.

These results indicate that platelet binding by S. mitis is a complex process, involving multiple bacterial factors. In part, binding appears to be mediated by PblA, PblB, and PblT. The precise mechanisms by which these proteins mediate binding are as yet unknown. These proteins may serve as direct adhesins or may be part of more complicated binding structures. It is also possible that other, as yet unidentified streptococcal adhesins contribute to platelet binding by S. mitis. Thus, additional studies are needed to address these issues. Further work is also required to determine whether platelet binding mediated by PblA, PblB, or PblT is important in the pathogenesis of infective endocarditis.