Department of Oral and Dental Science, University of Bristol, Lower Maudlin St, Bristol BS1 2LY, UK1
Department of Microbiology, Eastman Dental Institute, University College London, 256 Grays Inn Rd, London WC1X 8LD, UK2
Author for correspondence: Roderick McNab. Tel: +44 1932 822000. Fax: +44 1932 822100. e-mail: scunner_99{at}yahoo.com
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ABSTRACT |
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Keywords: oral streptococcus, chloramphenicol O-acetyltransferase reporter, gene expression
Abbreviations: CAT, chloramphenicol O-acetyltransferase
The GenBank accession number for the sequence reported in this paper is X65164.
a Present address: GlaxoSmithKline Consumer Healthcare, St Georges Avenue, Weybridge, Surrey KT13 0DE, UK.
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INTRODUCTION |
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Fibronectin is a dimeric glycoprotein (approx. 440 kDa) that is present in a soluble form in plasma and extracellular fluids; it is also present in a fibrillar form on cell surfaces. Both the soluble and cellular forms of fibronectin may be incorporated into the extracellular tissue matrix (reviewed by Romberger, 1997 ). While fibronectin has critical roles in eukaryotic cellular processes, such as adhesion, migration and differentiation, it is also a substrate for the attachment of bacteria. The binding of pathogenic Streptococcus pyogenes and Staphylococcus aureus to epithelial cells via fibronectin facilitates their internalization (Joh et al., 1999
) and systemic spread within the host.
The best-characterized fibronectin-binding proteins from streptococci and staphylococci share a similar mosaic architecture (Joh et al., 1994 , 1999
). These cell-surface proteins possess an N-terminal signal peptide for sec-dependent secretion and an LPXTG C-terminal motif for covalent anchorage to cell-wall peptidoglycan (Navarre & Schneewind, 1999
). A consensus sequence [ED(T/S)(9 or 10 X)GG(3 or 4 X)(I/V)DF, where X is any amino acid] that occurs in multiple copies within the carboxy-terminal amino acid repeat regions of these proteins is important for their fibronectin-binding activities (Joh et al., 1994
, 1999
). Streptococci also express several atypical fibronectin-binding proteins. These proteins do not contain conventional secretion, anchorage or fibronectin-binding sequences; examples include FBP54 (Courtney et al., 1994
) and SDH (Pancholi & Fischetti, 1992
) in Streptococcus pyogenes, and PavA in Streptococcus pneumoniae (Holmes et al., 2001
).
Fibronectin is present on oral epithelial cells and in submandibular/sublingual and parotid saliva, and is deposited within the acquired pellicle that coats the tooth surface (Zetter et al., 1979 ; Babu et al., 1983
; Babu & Dabbous, 1986
; Ericson & Tynelius-Bratthall, 1986
). A range of oral streptococci have been shown to bind fibronectin (Babu et al., 1983
; Babu & Dabbous, 1986
; Ericson & Tynelius-Bratthall, 1986
; van der Flier et al., 1995
; Willcox et al., 1995
) but, in general, little is known about the adhesins involved in this binding. Most molecular studies have been carried out on S. gordonii, which binds to conformationally specific determinants of immobilized human fibronectin that are not presented by soluble plasma fibronectin (Lowrance et al., 1988
). Fibronectin binding is mediated by a 259 kDa cell-wall-anchored protein, CshA, which also confers cell-surface hydrophobicity and co-aggregation properties with Actinomyces spp., Candida albicans and S. oralis (Holmes et al., 1996
; McNab et al., 1996
). CshA is the structural and functional component of the 60 nm long peritrichous fibrils that are present on the S. gordonii cell surface (McNab et al., 1999
). Isogenic cshA mutants of S. gordonii show approximately 50% reduction in their ability to bind to immobilized human fibronectin when compared to the wild-type (McNab et al., 1996
), and are unable to colonize the murine oral cavity. There are no significant amino acid sequence similarities between CshA and other wall-anchored staphylococcal and streptococcal fibronectin-binding proteins, perhaps reflecting the binding specificity of CshA for immobilized fibronectin.
Sequencing upstream and downstream of the cshA locus in S. gordonii revealed a gene, downstream of cshA and convergently transcribed, encoding a polypeptide with high sequence similarity to FBP54, a fibronectin-binding protein in S. pyogenes (Courtney et al., 1994 ). This raised the possibility that there was a host-association locus on the S. gordonii chromosome that encoded a second adhesin (designated FbpA) mediating attachment of S. gordonii to fibronectin. In this paper, we demonstrate that while FbpA binds to immobilized human fibronectin, the effects observed on the ability of S. gordonii to bind fibronectin upon inactivation of the fbpA gene probably arise through modulation of the expression of CshA.
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METHODS |
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DNA sequence information 3' to the TGA stop codon of cshA was obtained in two stages, as outlined below. Plasmid pPH1704 contained a 2·2 kb S. gordonii DNA fragment, 1·5 kb of which has been shown to encode the carboxy terminus of CshA (McNab & Jenkinson, 1992 ). The sequence of the remaining 0·7 kb of the S. gordonii DNA fragment was determined and used to design primers for inverse PCR amplification of flanking DNA (McNab et al., 1994
). Briefly, S. gordonii DL1 genomic DNA was digested with SspI, self-ligated at 3·5 ng µl-1 and then used as a template for PCR (Expand High Fidelity System; Boehringer Mannheim) with the primers CTERM (5'-TTATACAGGACAGAAAACCC-3'; complementary to nucleotides 82988317, cshA locus; GenBank accession no. X65164) and IFBPA (5'-GCTCCACCTGATCTTGGTCG-3'; complementary to nucleotides 89078926) (30 cycles, 60 °C annealing temperature, 3 min extension at 72 °C). The PCR product (3·63 kb) was purified and subcloned into pUC19 for sequencing.
Total RNA was prepared from S. gordonii cells as described previously (McNab & Jenkinson, 1998 ). RNA samples (10 µg lane-1) were prepared for electrophoresis and separated on 1·2% (w/v) agarose gels. The RNA was then transferred to a nylon membrane, as described by Kroczek & Siebert (1990)
. Hybridization of the blots with a 32P-labelled fbpA gene fragment was performed as described by Church & Gilbert (1984)
, with solutions containing 8% (w/v) dextran sulphate. The blots were washed three times each for 20 min with 2xSSC containing 0·1% SDS at 65 °C; the radiolabelled bands were detected by autoradiography.
Preparation of antiserum to FbpA.
Antibodies were raised against FbpA that had been produced initially as a glutathione S-transferase fusion protein. Primers HJ30 (5'-ACGGATCCGCAGCAATCGCAAAAGCC-3'; BamHI site shown in bold type; complementary to nucleotides 97659794) and HJ31 (5'-TCAATCATGTCGACTTGGACAAG-3'; SalI site shown in bold type; complementary to nucleotides 83668388) were used in PCR to amplify an internal fragment of fbpA encoding amino acid residues 39511. The PCR product was digested with a combination of BamHI and SalI and cloned into pGEX-5X-3 (Amersham Pharmacia Biotech) that had been similarly digested. FbpA from E. coli lysates was purified on a glutathione-Sepharose column, as described by McNab et al. (1996) . Antibodies against recombinant FbpA were raised in New Zealand White rabbits by intramuscular injection of 80 µg of protein; booster injections (30 µg each) were administered after 2 and 3 months.
Purification of the recombinant FbpA polypeptide.
To produce N-terminal His-tagged FbpA for use in protein-binding assays, the entire coding region of FbpA was amplified by PCR from S. gordonii DL1 genomic DNA using the primers HisF1 (5'-TTCGACGGATCCTTTTTACATCAC-3'; BamHI site shown in bold type) and HisR1 (5'-AAGAGAGAGTCGACCCTATTTATA-3'; SalI site shown in bold type). The PCR product was digested with BamHI and SalI and ligated into pQE30 that had been similarly digested. The plasmid construct was then transformed into E. coli M15. Expression of the His-tagged FbpA protein was induced by the addition of 0·01 mM IPTG to cultures in the exponential phase of growth. After a further 4 h incubation at 30 °C, cells were harvested by centrifugation, suspended in lysis buffer [0·05 M NaH2PO4, 250 mM NaCl, pH 8·0, containing 1 mM PMSF and 0·1 % (v/v) protease inhibitor cocktail (Sigma) for use with bacterial cell extracts] and then lysed by sonication. Proteins were extracted from the lysates with 8 M urea, and FbpA was purified on a nickel-NTA resin column, according to the manufacturers guidelines. Eluted proteins were dialysed extensively against de-ionized water at 4 °C.
Construction of the fbpA mutant strain.
A portion of the fbpA gene (encoding amino acids 1175 of FbpA) was amplified by PCR using primers FLPID3 (5'-GTCTTTCTAGAGATTTTTTTTAC-3'; XbaI site shown in bold type) and FLPID4 (5'-CAGTGAAAGGATCCAGACTG-3'; BamHI site shown in bold type). The 525 bp product was purified, digested with a combination of XbaI and BamHI, and then ligated with the integration vector pSF143 (Tao et al., 1992 ), which had been similarly digested. The resultant plasmid, pSFBP1, was transformed into competent S. gordonii DL1 cells with selection for tetracycline resistance to generate recombinant strain UB1060. Southern-blot analysis (using the ECL DNA Labelling and Detection System; Amersham Life Sciences) was performed to confirm that the plasmid had been integrated into the S. gordonii chromosome at the predicted locus.
S. gordonii strain OB507 carries a chromosomal cshA promotercat (p-cshAcat) fusion (McNab & Jenkinson, 1998 ). To generate a fbpA knockout mutant in this reporter strain, the PCR product amplified using primers FLPID3 and FLPID4 was digested with XbaI and BamHI, and ligated with integration vector pFW5 (Podbielski et al., 1996
), which had been similarly digested. The resulting plasmid, pFWFLP1, was inserted onto the S. gordonii OB507 chromosome by transformation and selection for tetracycline and spectinomycin resistance, to generate strain UB1245.
Construction of the strain carrying p-flpAcat.
The fbpA promoter region was amplified by PCR using primers PROMF (5'-TTCTTCTAGATTTATCAGTAAATTACC-3'; XbaI site shown in bold type; complementary to nucleotides 1018510211, cshA locus) and FUSION2 (5'-GCCTCCGGATTTCGAGCTCCTTGAAAGTTACTATAGAA-3'; complementary to nucleotides 99089928). To enable ligation-PCR, FUSION2 carried a 5' extension of 13 bp which was complementary to the 3' end of FUSION1 (see below). A promoterless chloramphenicol O-acetyltransferase (CAT) gene (cat) was amplified from pMH109 (Hudson & Stewart, 1986 ) using primers FUSION1 (5'-TTCTATAGTAACTTTCAAGGAGCTCGAAATCCGGAGGC-3') and Tet/Cat (5'-GGATCCGGGCCACCTCGACC-3'; BamHI site shown in bold type). FUSION1 carried a 5' extension of 19 bp which was complementary to the 3' end of FUSION2. PCR products of the correct size were purified, mixed and used in PCR amplification with primers PROMF and Tet/Cat. The resulting fbpA promotercat (p-fbpAcat) fusion product (1·4 kb) was digested with XbaI and BamHI, and then ligated with pFW5 that had been similarly digested, to generate pFWFUSION1. Plasmid pFWFUSION1 was introduced onto the S. gordonii chromosome by transformation and selection for spectinomycin resistance, to generate strain UB1300. The insertion also conferred resistance to S. gordonii against 10 µg chloramphenicol ml-1.
Binding of recombinant FbpA to immobilized fibronectin.
Human fibronectin (Boehringer Mannheim) was applied to microtitre plate wells and additional sites were blocked with BSA, as described previously (McNab et al., 1996 ). Recombinant N-terminal His6-tagged FbpA (1·25 µg) was incubated in fibronectin-coated or BSA-coated (control) wells for 2 h at 37 °C. Wells were washed with TNMC buffer (1 mM Tris/HCl, 150 mM NaCl, 0·1 mM MgCl2, 0·1 mM CaCl2, pH 8·0) and incubated with His5 antibody (Qiagen; 1:1000) for 1 h at room temperature. After rinsing the wells with TNMC, bound antibody was detected with horseradish-peroxidase-conjugated anti-mouse IgG (Dako; 1:1000) and o-phenylenediamine. ELISA (A492) values for protein bound to fibronectin were corrected by subtracting the background value detected for the binding of protein to the BSA-blocked (control) wells. To measure the effects of gelatin on FbpA binding to fibronectin, FbpA was added to fibronectin-coated wells that had been pre-incubated with gelatin (1 mg ml-1) for 1 h at 37 °C.
Hydrophobicity, adhesion and aggregation assays.
Cell-surface hydrophobicity was estimated as the percentage of total cells partitioning with hexadecane (Jenkinson, 1987 ). The adhesion of S. gordonii cells to fibronectin was performed as described previously (McNab et al., 1996
). Briefly, S. gordonii cells were radiolabelled by growth in TY-glucose medium containing [methyl-3H]thymidine [6 µCi ml-1 (78 Ci mmol-1, 2·89 TBq mmol-1); Amersham]. They were then washed thoroughly and added to microtitre plate wells that had been fibronectin-coated and BSA-blocked as before. Plates were incubated at 37 °C for 2 h with gentle shaking, and the numbers of bound cells were then determined by scintillation counting (Jenkinson et al., 1993
). The saliva-mediated agglutination of bacterial cells was determined spectrophotometrically using the method described previously by Jenkinson et al. (1993)
, with the decrease in the OD600 value being measured over time.
Analysis of bacterial proteins.
Bacteria from a late-exponential phase culture (30 ml) of S. gordonii DL1, OB235 cshA3 or UB1060 fbpA were harvested by centrifugation (4000 g, 10 min, 4 °C), washed once in TP buffer [50 mM Tris/HCl, pH 7·8, containing 5% (v/v) protease inhibitor cocktail; 5 ml] and suspended in 0·2 ml spheroplasting buffer [20 mM Tris/HCl, pH 6·8, 10 mM MgCl2, 26% (w/v) raffinose; Jenkinson et al., 1993 ]. Mutanolysin (500 U ml-1; Sigma) and lysozyme (0·2 mg ml-1) were added, and the suspension was incubated at 37 °C for 20 min. TP buffer (0·2 ml) was then added to the suspension, and the cells were disrupted by sonication (4x15 s at 280 W cm-2, with cooling on ice between each pulse) using an Ikasonic U50 hand-held sonicator fitted with a 3-mm-diameter probe (IKA-Werke GMBH, Staufen, Germany). The suspension was fractionated by centrifugation (12000 g, 10 min, 4 °C) and the supernatant (soluble fraction) was carefully removed. The cell-envelope pellet was washed twice by suspension and centrifugation in TP buffer, and the proteins were solubilized from the fractions with 1% (w/v) SDS (final concentration) by heating for 10 min at 90 °C. The solutions were then mixed with loading dye (Jenkinson et al., 1993
). Proteins were separated by SDS-PAGE on 8% (w/v) acrylamide gels and then transferred to Hybond-C nitrocellulose membranes (Amersham) by electroblotting (McNab & Jenkinson, 1992
). Antibodies against FbpA were affinity-purified by incubating FbpA antiserum with a nitrocellulose strip containing recombinant FbpA. Bound antibodies were eluted with 0·2 M glycine/HCl, pH 2·5, as described previously (Jenkinson & Easingwood, 1990
). Antibody binding on immunoblots was detected by using peroxidase-conjugated swine immunoglobulins to rabbit IgG and enhanced chemiluminescence (ECL; Amersham), according to manufacturers instructions.
The immunoreactivity of CshA on intact cells was determined by ELISA, as described previously (Holmes et al., 1996 ), using antibodies raised against a recombinant fragment of the CshA polypeptide comprising the N-terminal 844 aa residues of the mature CshA polypeptide (N-CshA; McNab et al., 1996
).
Preparation of cell-free extracts and CAT assay.
Cell-free extracts were prepared and assayed for CAT activities as described previously (McNab & Jenkinson, 1998 ). Briefly, streptococcal cells in BHY medium (10 ml) were harvested by centrifugation (4000 g, 10 min, 4 °C) and washed once in TPE buffer (100 mM Tris/HCl, pH 7·8, containing 1 mM PMSF and 1 mM EDTA). The bacteria were then resuspended in spheroplasting buffer. Mutanolysin (final concentration 500 U ml-1) was added and the suspension was incubated for 30 min at 37 °C. Cells were disrupted by vortexing with glass beads. The suspensions were then centrifuged (12000 g, 20 min, 4 °C) to remove the glass beads and cell debris; the supernatants were removed for enzyme assays. Protein concentrations were determined by using a Bio-Rad protein assay kit, with bovine
-globulin as the standard. CAT activity was determined by the spectrophotometric method of Shaw (1975)
, utilizing a Shimadzu UV-1201 recording spectrophotometer. The reaction rate was determined from the linear portion of the graph and corrected for the background change in A412. The corrected value was divided by 0·0136 to yield CAT activity expressed as nmol chloramphenicol acetylated min-1 (mg protein)-1 at 37 °C.
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RESULTS |
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S. gordonii FbpA was encoded on the opposite strand to CshA, with the cshA and fbpA stop codons separated by 75 bp (Fig. 1a). Within this region was a single potential stemloop structure previously identified as a
-independent transcriptional terminator for cshA (overscored in Fig. 1a
). At the 5' end of fbpA there was a potential ribosome-binding site (RBS) (GGAGA) and an extended -10 promoter sequence (underscored in Fig. 1b
), comprising a -16 region (TTTG; Voskuil & Chambliss, 1998
) upstream of a canonical -10 region (TATAAT), positioned appropriately to the fbpA start codon (Fig. 1b
). No recognizable -35 sequence was present in the promoter region of fbpA. However, the extended -10 sequence can function naturally in the absence of a -35 site (Sabelnikov et al., 1995
), and it was predicted that fbpA was expressed from its own promoter. The deduced amino acid sequence of FbpA contained no conventional N-terminal hydrophobic leader or signal peptidase cleavage site to direct export via the general secretory pathway. Also, there was no Gram-positive bacterial cell-wall anchorage motif (LPXTG, where X is any amino acid; Navarre & Schneewind, 1999
) at the C-terminal end of FbpA. The sequence was relatively leucine rich (61 residues out of 550), a feature previously noted for FBP54 of S. pyogenes (Courtney et al., 1994
).
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The ability of wild-type and UB1060 fbpA cells to adhere to immobilized human fibronectin was measured using a microtitre-plate-based assay (see Methods). The adhesion of mid-exponential phase cells of UB1060 to fibronectin was reduced by approximately 25%, when compared with wild-type cells harvested at the same phase of growth (Table 2). However, stationary phase cells of UB1060 and DL1-Challis adhered to fibronectin to approximately the same extent as each other (Table 2
). The binding of wild-type or UB1060 cells to fibronectin was inhibited by gelatin to a similar degree (45·2%±3·0%) (data not shown). The adhesion of S. gordonii OB235 cshA3 cells to fibronectin was reduced by approximately 45 %, irrespective of whether the cells were harvested in the exponential or stationary phases of growth (Table 2
). Inactivation of fbpA also resulted in significantly reduced surface hydrophobicity of UB1060 cells harvested in the mid-exponential phase of growth, but this reduction was not seen in cells harvested during the stationary phase of growth (Table 2
). The hydrophobicity of OB235 (cshA3) was greatly reduced irrespective of the growth phase; this was in accordance with previous results correlating CshA with surface hydrophobicity (McNab & Jenkinson, 1992
). Saliva-mediated aggregation of exponential or stationary phase cells was unaffected in UB1060, when compared with the wild-type (data not shown).
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By contrast to cshA expression, which increases in the late-exponential phase of growth, fbpA promoter activity remained at a relatively constant level throughout growth and into the stationary phase (Fig. 4). However, fbpA promoter activity was consistently lower (five- to eightfold) than that of the cshA promoter. An fbpA mRNA transcript of approximately 1850 nt was visualized in Northern-blot analysis of RNA isolated from cells of S. gordonii DL1. In support of the CAT reporter data, the levels of fbpA mRNA with respect to total RNA did not alter significantly throughout growth and into the stationary phase (Fig. 5
).
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DISCUSSION |
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FbpA of S. gordonii exhibits several features that distinguish it from the other fibronectin-binding proteins of Gram-positive bacteria that have been characterized to date. FbpA does not contain sequences that closely match recognized motifs involved in binding fibronectin, suggesting that binding may be mediated by a novel mechanism. In support of this, streptococcal and staphylococcal proteins that contain consensus fibronectin-binding motifs are able to bind soluble and immobilized fibronectin, generally interacting with the N-terminal fibrin- and heparin-binding domain of fibronectin (Joh et al., 1999 ). Like CshA, FbpA binds to only the immobilized form of fibronectin and, since these two proteins show no amino acid similarity, their mechanisms of fibronectin binding may be different or may involve protein configurations that are not easily defined through linear sequence motifs.
The inactivation of fbpA was associated with a significant reduction in the fibronectin binding and cell-surface hydrophobicity of S. gordonii cells harvested at the exponential phase of growth. However, our results suggest that these phenotypic effects resulted from a reduction in the expression of CshA. Although we cannot rule out that, under some growth conditions, FbpA may mediate adhesion of S. gordonii to fibronectin, it would appear that the CshA-independent mechanism of binding of stationary phase cells is not due to FbpA. In support of this, we have not been able to conclusively demonstrate cell-surface localization of FbpA by immunogold electron microscopy using affinity-purified FbpA antibodies (data not shown). By contrast, the orthologous PavA protein of S. pneumoniae, which partly mediates pneumococcal cell adhesion to fibronectin (Holmes et al., 2001 ), has been shown to be cell-surface exposed. PavA and FbpA both appear to partition between the soluble (cytoplasmic) and envelope (containing membrane and wall-associated proteins) fractions of cells. Neither of these proteins carries conventional secretory (signal peptide) nor cell-surface anchorage sequences, so the mechanism by which PavA is secreted and becomes cell-surface-associated is currently unknown.
The ability of bacteria to sense and respond to environmental influences is integral to their survival and adaptation within the host (Finlay & Falkow, 1997 ). The level of O2 varies at host sites and bacteria respond to this. Fibronectin-binding protein PrtF1 of S. pyogenes is maximally expressed under aerobic conditions (VanHeyningen et al., 1993
), and this is thought to enhance colonization at superficial body sites. We have also shown that O2 levels may directly regulate the expression of FbpA, but that this does not significantly impact on CshA production. The cshA promoter is apparently under complex regulatory control, since it has been demonstrated that maximal cshA promoter activity in the early-stationary phase of growth requires a functional Hpp oligopeptide transport system (McNab & Jenkinson, 1998
) as well as the co-production of CshB (McNab & Jenkinson, 2000
). HppA, which is an oligopeptide-binding protein (Jenkinson et al., 1996
), is thought to be involved in sensing an extracellular signal that modulates the expression of cshA in a cell-density-dependent manner. The data presented here suggest that FbpA provides another layer of control on CshA expression during the exponential phase of growth. It is unclear how this operates at present, and we have been unable to show by gel-shift assay that purified FbpA binds to the cshA promoter (data not shown). It is possible that FbpA may act as an accessory factor involved in HppA-dependent peptide signal detection, or as an independent regulator which would allow the integration of multiple sensory signals at the level of adhesin gene transcription. In S. pyogenes, Mga positively regulates the expression of several virulence factors, including SOF and Fba, at the transcriptional level (McLandsborough & Cleary, 1995
; Terao et al., 2001
). The expression and activity of Mga is, in turn, affected by various environmental stimuli (Okada et al., 1993
; Podbielski et al., 1992
), and an additional level of control occurs through Nra, a negative regulator of Mga transcription whose expression is cell-density-dependent (Podbielski et al., 1999
). Nra also directly regulates the expression of the fibronectin-binding protein PrtF2 (Podbielski et al., 1999
). PrtF, another fibronectin-binding protein of group A streptococci, is positively regulated by the transcriptional factor RofA, whose expression is elevated under anaerobic conditions (Fogg et al., 1994
, 1997
). These multiple layers of regulation controlling the expression of fibronectin-binding proteins in streptococci highlight the importance of detecting and integrating spatial and temporal information for colonization and survival within the host.
Orthologues of FbpA are present in a wide range of streptococci, in Lactococcus lactis, in Bacillus subtilis and in some Gram-negative species of bacteria. The PavA protein of S. pneumoniae is essential for pneumococcal virulence (Holmes et al., 2001 ; Lau et al., 2001
), while a promoter trap screening system has demonstrated that an FbpA-like protein in Streptococcus suis is expressed during experimental infection of piglets (Smith et al., 2001
). It has also been shown that mice immunized with recombinant FBP54 were protected against S. pyogenes infection (Kawabata et al., 2001
). Thus, while the role of these FbpA-like proteins in streptococcal infections are not yet understood, they are important for bacterial growth and survival within the host and they may provide novel targets for disease intervention.
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ACKNOWLEDGEMENTS |
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Received 25 October 2001;
revised 14 February 2002;
accepted 19 February 2002.
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