1 Microbiology Department, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, Ireland
2 INSERM E0230, Faculté Laennec, IFR 62, rue G. Paradin, 69008 Lyon, France
Correspondence
Timothy J. Foster
tfoster{at}tcd.ie
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ABSTRACT |
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INTRODUCTION |
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S. lugdunensis expresses several potential virulence factors, including the SLUSH synergistic toxins, haemolysins, extracellular enzymes and a glycocalyx (Donvito et al., 1997; Leung et al., 1998
). This paper reports the properties of the fibrinogen-binding protein of S. lugdunensis which is closely related to clumping factor A, a fibrinogen-binding protein of S. aureus.
The clumping factor A protein (ClfA) is an important virulence factor of S. aureus. It contributes to the pathogenesis of experimental septic arthritis and endocarditis in rabbits (Josefsson et al., 2001; Moreillon et al., 1995
). It binds to the extreme C-terminus of the
-chain of fibrinogen at the same site as the platelet integrin GPIIbIIIa (Medved et al., 1997
; McDevitt et al., 1997
). ClfA is the archetype of a family of surface-associated proteins with similar structural organization. The primary translation product comprises a secretory signal sequence followed by a 520 residue ligand-binding domain, then repeats of the dipeptide DS, a short cell-wall-spanning region, and a C-terminal domain that comprises a sortase signal LPXTG, a hydrophobic membrane-spanning domain and a series of positive-charged residues (McDevitt et al., 1994
; Fig. 1
). The DS repeats act as a flexible stalk to extend the ligand-binding A domain from the cell surface (Hartford et al., 1997
). The signal sequence is removed during protein secretion, the sortase cleaves LPXTG between the T and G, and in a transpeptidation reaction it joins the protein to uncross-linked nascent peptidoglycan precursor, which is then polymerized into new cell wall (Perry et al., 2002
; Mazmanian et al., 2001
).
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This paper describes the fibrinogen-binding protein Fbl of S. lugdunensis and compares it with ClfA of S. aureus. It studies both purified recombinant protein and the native protein displayed on the surface of the surrogate Gram-positive host Lactococcus lactis.
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METHODS |
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Cloning of fbl in L. lactis.
The fbl gene of S. lugdunensis strain N920143 was amplified from the putative translational initiation codon to the TAA stop codon by using Pfu polymerase. It was then cloned into the L. lactis expression vector pKS80 (Hartford et al., 2001), which provides a lactococcal promoter, initiation codon, ribosome-binding site and a translational coupling mechanism to ensure efficient initiation of translation of the cloned gene. The primers used are listed in Table 2
.
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Purification of rFbl40534.
L. lactis pKS80 : fblA was inoculated into GM17 supplemented with 5 µg erythromycin ml1 and grown for 16 h at 30 °C. Cells were pelleted by centrifugation at 7000 r.p.m. (Sorvall GS3 rotor, 5320 g) and 445 g ammonium sulphate l1 was added to the supernatant and mixed well. The precipitated protein was removed, dissolved in 50 ml H2O and dialysed against 10 mM Tris/HCl, 0·9 % NaCl, pH 7·4 at 4 °C. This was concentrated tenfold in a stirred-cell ultrafiltration chamber (Amicon) with a 50 kDa cut-off membrane. Proteins were separated by size fractionation through Sephadex G-50. Peak fractions were applied to a 5 ml HiTrap Q Sepharose column (Amersham Pharmacia Biotech). Bound protein was eluted with a continuous linear gradient of NaCl (50500 mM; total volume 100 ml) in 20 mM Tris, 2 mM EDTA (pH 7·9). Peak fractions were analysed by SDS-PAGE, then pooled and concentrated.
Antibodies.
Rabbit antibodies to the A region of ClfA were kindly provided by Judy Higgins (Microbiology Department, Trinity College, Dublin). Antibodies to rFbl40534 were raised in a young New Zealand White rabbit whose pre-immune sera showed no reaction with S. lugdunensis wall-associated antigens in Western blots. Protein (25 µg in 10mM Tris/HCl, 0·9 % NaCl, pH 7·4) was emulsified with an equal volume of Freund's complete adjuvant (500 µl) and injected subcutaneously. Three subsequent injections given at 2 week intervals contained Freund's incomplete adjuvant. The rabbits were bled, the serum was recovered and IgG was purified.
SDS-PAGE and Western immunoblotting.
Bacterial cells were suspended to an OD600 of 60 in 30 % raffinose and 20 mM MgCl2 in a final volume of 500 µl. A 10 µl volume of lysostaphin (10 mg ml1) and 8 µl of protease inhibitors (Complete mixture, Roche Molecular Biochemicals) were added and the suspension was incubated at 37 °C for 20 min. Protoplasts were removed by centrifugation at 6000 g for 15 min. Supernatants containing wall-associated proteins and protoplasts in 30 % raffinose were prepared for electrophoresis by boiling for 5 min in final sample buffer (0·125 M Tris/HCl, 4 %, w/v, SDS, 20 % glycerol, 10 %, v/v, 2-mercaptoethanol, 0·002 %, w/v, bromphenol blue) and analysed in 10 % (w/v) acrylamide gels. Gels were stained with Coomassie blue or electrophoretically transferred to PVDF Western-blotting membranes (Boehringer) by the semi-dry system (Bio-Rad). Membranes were blocked for 15 h at 4 °C with 10 % (w/v) blocking reagent (Marvel milk powder).
Anti-ClfA antibodies were used because they allowed detection of ClfA and Fbl in the same blot, whereas anti-Fbl antibodies cross-reacted too weakly to allow detection of ClfA in the same sample. They were used at a 1 : 1000 dilution followed by use of protein-A-conjugated horseradish peroxidase (Sigma; a 1 mg ml1 stock diluted 1 : 500) or goat anti-rabbit IgG at a 1 : 1000 dilution (Dako) to detect bound antibody. Membranes were developed using LumiGLO chemiluminescent substrate (New England Biolabs) according to the manufacturer's instructions and exposed to X-ray film.
Adherence assays.
Adherence of bacterial cells to immobilized fibrinogen was performed as described by Hartford et al. (1997). Briefly, microtitre plates were coated with 0·5 µg ml1 of the protein in PBS and incubated overnight at 4 °C. Bovine serum albumin (2 %, w/v, in PBS) was added and incubated for 1 h at 37 °C. The plates were washed three times with PBS, and 100 µl bacterial cell suspension (1x108 c.f.u. ml1) was added. The plates were incubated at 37 °C for 2 h and then washed three times with PBS. Bound cells were fixed with formaldehyde (25 %, v/v) for 30 min and then stained with crystal violet (0·5 % v/v) for 1 min. Absorbance was measured at 570 nm in an ELISA plate reader (Labsystems Multiskan Plus).
Inhibition of adherence.
The ability of recombinant Fbl region A and recombinant ClfA region A to inhibit the adherence of L. lactis strains to immobilized fibrinogen was determined as described by Hartford et al. (1997), with modifications. Microtitre plates were coated with 10 µg fibrinogen ml1 in PBS and incubated overnight at 4 °C. Bovine serum albumin (2 %, w/v, in PBS) was added and the plates were incubated for 1 h at 37 °C. A 100 µl volume of rFbl was added in doubling dilutions and incubated with gentle agitation for 1 h at room temperature. After this, 100 µl bacterial cell suspension (1x108 c.f.u. ml1) was added, the plates were incubated with gentle agitation at room temperature for 1·5 h, and then washed three times with PBS. Bound cells were fixed with formaldehyde (25 %, v/v) for 30 min and stained with crystal violet (0·5 %, v/v) for 1 min. Absorbance was measured at 570 nm in an ELISA plate reader (Labsystems Multiskan Plus).
The same procedure was used to study anti-Fbl antibody inhibition of adherence, except that bacteria were incubated with 35 mg ml1 anti-Fbl antibodies or pre-immune serum for 30 min prior to measuring adherence.
Fibrinogen binding by Fbl region A.
The ability of recombinant proteins to bind to immobilized fibrinogen was analysed using an ELISA-type assay. Microtitre plates were coated with fibrinogen (10 µg ml1 in PBS) at 4 °C. The plates were washed three times with PBS containing 0·5 % Tween 20, and blocked with 2 % bovine serum albumin for 2 h at 37 °C. After an additional three washes with PBS containing 0·5 % Tween 20, rFbl40534 was added and the plates were incubated at 37 °C for 2 h. The wells were washed again and incubated with anti-ClfA region A antibodies at 37 °C for 1 h. After further washing, horseradish-peroxidase-labelled goat anti-rabbit IgG (Sigma) was added at a 1 : 1000 dilution. Following incubation at 37 °C for 1 h and washing with PBS, 100 µl chromogenic substrate (580 µg tetramethylbenzidine ml1 and 0·0001 % H2O2 in 0·1 M sodium acetate buffer, pH 5·2) was added per well and developed for 10 min in the dark. The reaction was stopped by the addition of 2 M H2SO4 (50 µl per well). Plates were read at 450 nm. The apparent dissociation constant (KD) of each protein for fibrinogen was the protein concentration that resulted in 50 % binding (Davis et al., 2001).
Measurement of cell clumping.
S. aureus and S. lugdunensis were grown to stationary phase with aeration, harvested by centrifugation at 7000 r.p.m. (Sorvall GS3 rotor, 5320 g) for 10 min, and washed in PBS. L. lactis strains were grown statically at 30 °C for 16 h and harvested in the same way. A suspension of 4x108 c.f.u. in a 20 µl volume was added to fibrinogen (Calbiochem, 1 mg ml1 and doubling dilutions thereof) in a 96-well microtitre dish. The highest dilution of fibrinogen that produced clumping after 5 min agitation was defined as the clumping titre (Hartford et al., 1997).
Whole-cell blotting.
Cells from stationary-phase cultures were harvested by centrifugation, washed in PBS, and resuspended at an OD600 of 100. Volumes of 10 µl were spotted onto a nitrocellulose membrane (Protran) and dried. The membrane was blocked with 10 % (w/v) skim milk in PBS for 1 h, and probed with anti-ClfA region A antibodies at a 1 : 1000 dilution. Thereafter the membranes were treated as for Western immunoblotting.
Whole cells were tested for fibrinogen-binding activity by probing duplicate membranes with 10 ml of a 1 mg ml1 solution of fibrinogen in 10 % (w/v) skim milk in PBS for 1 h. Membranes were subsequently washed and probed with anti-fibrinogen antibodies conjugated to horseradish peroxidase (Dako) at a 1 : 1000 dilution. Thereafter the membranes were treated as for Western immunoblotting.
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RESULTS |
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Southern hybridization analysis using a probe corresponding to bases 14003001 of the clfA gene was performed with eight S. lugdunensis strains. The probe encoded the C-terminal end of the A-domain, the SD repeat region and the wall-spanning region of ClfA, including the LPDTG motif. A single hybridizing band of 9 kb was revealed in each strain when the genomic DNA was cut with HindIII (Fig. 2 shows one representative strain), suggesting that the fbl gene is ubiquitous in S. lugdunensis. The clfA probe hybridized to fbl because of identical bases in codons 1, 2, 3 and 5 of the 18 bp repeats.
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The eight S. lugdunensis strains were tested for their ability to adhere to immobilized fibrinogen. Each strain adhered to fibrinogen although the level of binding varied (Fig. 3). Adherence of each was completely inhibited by anti-Fbl antibodies, whereas pre-immune serum had no effect. This demonstrates that adherence of S. lugdunensis to immobilized fibrinogen is promoted exclusively by Fbl.
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Binding of anti-Fbl antibodies to ClfA and of anti-ClfA antibodies to Fbl
The interaction of rabbit anti-Fbl and anti-ClfA A domain antibodies with recombinant bacterial proteins was analysed by ELISA (data not shown). The anti-Fbl antibodies had a higher affinity for the cognate antigen rFbl40534 than for the ClfA A domain rClfA40534. Similarly, rabbit anti-rClfA A domain antibodies bound more strongly to the cognate antigen rClfA40559 than to rFbl40534.
Anti-Fbl antibodies potently inhibited adherence of S. lugdunensis N920143 to immobilized fibrinogen, confirming data in Fig. 3. Higher amounts were required to inhibit adherence of L. lactis Fbl+, reflecting a higher level of Fbl expression by this strain (Fig. 6a
). Complete inhibition of S. aureus Newman was only achieved at the highest concentration of antibody tested, whereas L. lactis ClfA+ was only inhibited by 50 %. Once again this reflects a different level of protein expression, and also shows that the anti-Fbl antibodies contain molecules that neutralize the fibrinogen-binding activity of ClfA.
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Pooled human IgG enriched for anti-ClfA antibodies (Veronate; Inhibitex) was tested for its ability to block ClfA- and Fbl-mediated fibrinogen binding. Veronate inhibited both ClfA- and Fbl-expressing bacteria from adhering to immobilized fibrinogen in a concentration-dependent manner (Fig. 6). Veronate blocked S. lugdunensis more efficiently than L. lactis Fbl+. This is presumably because there is less Fbl protein expressed on the surface of S. lugdunensis than L. lactis Fbl+. Similarly Veronate blocked the adherence of S. aureus Newman to immobilized fibrinogen more efficiently than it blocked L. lactis ClfA+.
Western immunoblotting analysis
S. lugdunensis N920143 was digested with lysostaphin in the presence of raffinose in order to stabilize protoplasts and solubilize cell-wall-associated proteins. Analysis by Western immunoblotting with anti-ClfA antibodies detected no immunoreactive proteins in the cell wall fraction (Fig. 7). Proteins of
175 and
60 kDa were detected in the protoplast fraction. No immunoreactive proteins were detected with pre-immune serum. The larger protein probably represents the native form of Fbl, and the smaller is probably a breakdown product. The predicted molecular mass of Fbl is 90 kDa, but like ClfA it appears to migrate aberrantly in SDS-PAGE at close to twice the predicted size (Hartford et al., 1997
).
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Recombinant Fbl40534 binding to fibrinogen
Recombinant Fbl40534 bound to immobilized fibrinogen in ELISA-type ligand-binding assays in a dose-dependent and saturable fashion, indicating a specific interaction. This formally demonstrates that the ligand-binding region of Fbl is in the A domain between residues 40 and 534. rFbl had an apparent KD of 1·5 µM compared with 150 nM for rClfA when bound proteins were detected by anti-Fbl antibodies (Fig. 8a) and anti-ClfA antibodies (Fig. 8b
). The higher affinity of the antibody for the cognate antigen did not affect the apparent KD.
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DISCUSSION |
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The clonality of S. lugdunensis was suggested by PCR amplification of the DNA encoding the R region of Fbl, which yielded two distinct sizes that differed by 100 bp. When similar analysis was performed with clfA, many different sizes of R region were observed, a feature that reflects the more diverse population structure of S. aureus shown by PFGE (McDevitt & Foster, 1995
; van der Mee-Marquet et al., 2003
).
When expressed on the surface of L. lactis, Fbl behaved in a similar manner to ClfA. It promoted adherence to immobilized fibrinogen and cell clumping in a fibrinogen solution. Recombinant ClfA region A inhibited the adherence of L. lactis Fbl+ to immobilized fibrinogen, indicating that Fbl interacts with the -chain of fibrinogen. Thus it can be concluded that Fbl binds to the same region of the
-chain of fibrinogen as ClfA, but whether precisely the same residues are recognized is not known.
S. lugdunensis cells did not clump in soluble fibrinogen. However when Fbl was expressed in L. lactis, Fbl-promoted clumping did occur. This suggests that the level of expression of the fibrinogen-binding protein determines the ability of cells to form clumps. Western immunoblotting and whole-cell dot immunoblotting blotting showed that there is 64-fold more Fbl on the surface of L. lactis Fbl+ than on S. lugdunensis N920143.
The predicted molecular mass of full-length Fbl is 95 kDa. However, when protoplasts of S. lugdunensis and L. lactis Fbl+ were analysed by SDS-PAGE and Western immunoblotting, the highest immunoreactive band had an apparent molecular mass of 175 kDa. Aberrant migration at approximately twice the predicted molecular mass is a property of other Clf/Sdr proteins and is attributed to the presence of the multiple S residues in region R (Hartford et al., 1997). When Fbl was isolated from L. lactis Fbl+, the protein appeared to have undergone proteolytic degradation. This is likely to happen during cell wall digestion and probably does not affect the functionality of surface-exposed Fbl on intact cells. Studies reported by other authors concluded that the majority of molecules of heterologously expressed protein were intact and fully functional on the surface of L. lactis cells, and that degradation only occurred during solubilization of the proteins when they became exposed to a membrane-associated HtrA-like protease (O'Brien et al., 2002
; Poquet et al., 2000
; Miyoshi et al., 2002
).
It is interesting to observe that while ClfA is covalently sorted to the cell wall peptidoglycan in S. aureus and L. lactis, Fbl does not appear to be sorted to the cell wall in either host. A sortase signal LPKTG is present in Fbl. However, other factors must interfere with its ability to become anchored to the cell wall. It has been shown that changes to a consensus sequence SIRK-G/S in the signal sequence of some staphylococcal surface proteins can impede efficient sorting (Bae & Schneewind, 2003). Differences in the signal sequence of Fbl at residues F9, H14 and K15 compared to ClfA might provide an explanation as to why Fbl sorting is inefficient.
A major focus of this study was to determine whether antibodies recognizing S. aureus ClfA would also bind to S. lugdunensis Fbl. S. lugdunensis is often misdiagnosed as S. aureus, and is a frequent cause of invasive endocarditis. Polyclonal anti-ClfA antibodies inhibited S. aureus infections in animals, but it is not clear whether antibodies that block ClfA binding to fibrinogen would have the same effect on S. lugdunensis. Anti-ClfA region A antibodies inhibited Fbl-mediated bacterial adherence to immobilized fibrinogen in a concentration-dependent manner, and anti-Fbl antibodies inhibited ClfA-mediated adherence. This indicates that Fbl and ClfA are sufficiently similar in amino acid sequence and in three-dimensional structure that they share epitopes that enable antibodies raised against one protein to inhibit the other.
Fbl has a tenfold lower affinity for fibrinogen than ClfA. It is possible that this is due in part to the absence of residues equivalent to residues 550559 in ClfA. ClfA220559 had a higher affinity for fibrinogen than ClfA220550, suggesting functional importance for residues 550559 (McDevitt et al., 1997). Nevertheless ClfA220550 was still able to bind to fibrinogen in a dose-dependent and saturable manner. It is possible that residues 550559 play a role in stabilizing the interaction between ClfA and fibrinogen, while not actually contributing directly to the structure of the binding domain (Deivanayagam et al., 2002
).
The crystal structure of rClfA220559 was solved without the bound ligand in place (apo-ClfA). Solving the structure of the fibrinogen -chain peptide co-crystallized with the minimum binding domain of ClfA region A would promote understanding of how exactly the terminal residues of the
-chain of fibrinogen interact with the residues of ClfA. Ponnuraj et al. (2003)
successfully co-crystallized the minimum binding domain of SdrG, the fibrinogen-binding protein of Staphylococcus epidermidis, with a modified fibrinogen
-chain peptide. They solved the structure of SdrG bound to its ligand and compared it with the structure of the unbound apo-SdrG. This led to the proposal of a dock, lock and latch model for binding, whereby an open form of SdrG first binds to the fibrinogen
-chain peptide, a loop at the end of SdrG N3 then encloses the peptide within the binding groove and the peptide is locked into position. This could be a universal mechanism for Clf-Sdr proteins binding to peptide ligands.
The residues in ClfA that have been shown to interact directly with the extreme C-terminal residues of the -chain peptide of fibrinogen (AGDV) are conserved in Fbl. They are located in the hydrophobic trench between N2 and N3. The majority of amino acid residue differences between Fbl and ClfA occur away from the fibrinogen-binding site and are proposed to contribute to antigenic differences. However Fbl has a tenfold lower affinity for fibrinogen than ClfA. It is not clear if the dock, lock and latch mechanism applies to Fbl and ClfA because the three-dimensional structure of ClfA has the putative latching peptide wrapped around N3 in the apo form. Using SdrG as a model, the putative latching peptide of ClfA would comprise residues at the extreme C-terminus of domain N3 (532537). Only one of these residues varies in Fbl (Fig. 1
). However, it is possible that the amino acids in
-strand E, in domain N2 lining the putative latching trough, which vary between Fbl and ClfA, affect the affinity of Fbl for fibrinogen. This region has diverged considerably between ClfA and Fbl and there is a greater proportion of charged residues in the former (Fig. 1
). In order to test this hypothesis, residues in ClfA could be changed to those of Fbl, and vice versa, particularly in the critical region of ClfA277DDVK280 bearing charged residues which are absent in the corresponding Fbl sequence.
Fbl and ClfA bind to a similar region of the -chain of fibrinogen. However, differences may occur in the precise interaction between residues in the binding trough and the
-chain. Comparison of the interaction of the variant fibrinogen
-chain peptides with rFbl40534 and rClfA40559 may shed some light on this.
Considering that Fbl exhibits many similar characteristics to ClfA, it is reasonable to assume that it is one of the main virulence factors of S. lugdunensis. It is encouraging to note that antibodies generated against bothFbl A domain and ClfA A domain recognize the heterologous proteins, thus indicating that common antigenic epitopes must be present on each protein allow antibodies (including those in pooled human IgG) to inhibit fibrinogen binding by Fbl. This suggests that polyclonal human IVIG Veronate (Vernachio et al., 2003) is likely to protect against S. lugdunensis as well as S. aureus infections.
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ACKNOWLEDGEMENTS |
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Received 18 May 2004;
revised 26 July 2004;
accepted 6 August 2004.
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