Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, Ireland1
Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK2
Author for correspondence: Timothy J. Foster. Tel: +353 1 6082014. Fax: +353 1 6799294. e-mail: tfoster{at}tcd.ie
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ABSTRACT |
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Keywords: surface protein, Lactococcus lactis, adhesion, MSCRAMM
Abbreviations: MSCRAMM, microbial surface component recognizing adhesive matrix molecule
a These authors made an equal contribution.
b Present address: Plant Bioscience Limited, Norwich Research Park, Colney Lane, Norwich NR4 7UH, UK.
c Present address: Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK.
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INTRODUCTION |
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Shortly after a plastic or metal device is introduced into the body, the surface of the device becomes coated with a conditioning layer of host proteins (Vaudaux et al., 1994 , 1995
; Francois et al., 2000
). Foreign-body infection caused by Staphylococcus aureus is attributed to its ability to attach to host proteins such as fibronectin and fibrinogen that are in the coating layer (Vaudaux et al., 1989
, 1994
). Adherence is mediated by surface protein adhesins of the MSCRAMM (microbial surface component recognizing adhesive matrix molecule) family (Patti et al., 1994
; Foster & Höök, 1998
). The fibrinogen-binding proteins ClfA and ClfB have been shown to be important MSCRAMMs in foreign-body infections caused by S. aureus (Ní Eidhin et al., 1998
; McDevitt et al., 1994
).
Some strains of S. epidermidis can attach to fibrinogen immobilized on a plastic surface in vitro (Baldassarri et al., 1997 ; Herrmann et al., 1988
; Nilsson et al., 1998
) so it is possible that this organism can also initiate device-related infections by attaching to the conditioned surface of an implant within the body. A fibrinogen-binding protein, Fbe, has been identified in S. epidermidis strain HB (Nilsson et al., 1998
; Pei et al., 1999
). S. epidermidis K28 expresses a closely related protein which we called SdrG (McCrea et al., 2000
). Fbe and SdrG are members of the ClfSdr multigene family of staphylococcal surface proteins (Fig. 1
; Josefsson et al., 1998
). They have a 50-residue signal sequence, a 548-residue fibrinogen-binding A domain (96% identical) followed by two B repeats of 113 and 111 residues. The proteins have repeats of the dipeptide Ser-Asp which are characteristic of the ClfSdr family. The SdrG protein from strain K28 has 56 residues in the dipeptide repeat region, while Fbe from strain HB has 216 residues. At the C-terminus the proteins have an LPXTG motif, a hydrophobic domain and positively charged residues associated with anchoring the protein to the cell surface (Navarre & Schneewind, 1999
).
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METHODS |
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L. lactis strain MG1363 (Wells et al., 1993 ) was used to express Fbe proteins from the expression vector pKS80, a derivative of pTREX1 (Wells & Schofield, 1996
) which provides a strong lactococcal promoter and a translational coupling mechanism to ensure efficient transcription and translation of genes cloned into the BclI site (Fig. 2
).
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Construction of pG+Host9fbe.
An 855 bp fragment was amplified from within the region of fbe (sdrG) encoding the unique A domain of the Fbe (SdrG) protein from S. epidermidis K28. The primer F1-GGCGAATTCAAAAGAACCCTCATCAGTCG annealed at position 435454 and included an EcoRI site at the 5' end (in italics) and primer R1-GGCAAGCTTGGTTTTTGATATTCAACCG annealed at position 12621280 and included a HindIII site at the 5' end (in italics). The product was purified using Wizard PCR Prep (Promega), cut for 16 h with 20 units EcoRI and HindIII and ligated with pG+Host9 cut with the same enzymes. The ligated DNA was transformed into E. coli T61 and transformants were selected for by growth on erythromycin (100 µg ml-1). DNA was isolated and transformed by electroporation (Oskouian & Stewart, 1990 ) into S. aureus RN4220(pGO1) selecting on gentamicin (10 µg ml-1) and erythromycin (100 µg ml-1).
Transfer of pG+Host9fbe into S. epidermidis.
Stationary-phase cultures of S. aureus RN4220(pGO1, pG+Host9fbe) and S. epidermidis 9142 grown in BHI broth were lawned on the surface of BHI agar and grown for 16 h at 30 °C. The lawns were emulsified in PBS and resuspended at an OD600 of 1·0. Portions of 1 ml of each were mixed, centrifuged and resuspended in 100 µl PBS and pipetted onto a 2 cm2 Protran nitrocellulose membrane (Schleicher & Schuell) on the surface of a BHI agar plate and incubated for 24 h at 30 °C. Cells were harvested in PBS and spread onto BHI agar containing 5 µg norfloxacin ml-1 and 10 µg erythromycin ml-1 and incubated for 34 d at 30 °C. Transconjugants were screened for those that were sensitive to gentamicin. Phage 48 (Nedelmann et al., 1998 ) was propagated on one such transconjugant and pG+Host9fbe was transduced into strain HB selecting for resistance to erythromycin at 30 °C.
Isolation of fbe mutants.
S. epidermidis carrying pG+Host9fbe was grown in TSB containing erythromycin (100 µg ml-1) at 28 °C. Cultures were diluted and spread on TSA containing erythromycin and incubated at 43 °C.
Cloning of fbe in L. lactis.
The fbe genes of S. epidermidis strains HB and K28 were amplified by PCR using Pfu polymerase from the putative translational initiation codon to the TAA stop codon and cloned into the L. lactis expression vector pKS80, which provides a lactococcal promoter, ribosome-binding sites and a translational coupling mechanism to ensure efficient initiation of translation of the cloned gene (Fig. 2). The primers used were LsdrGF1-CGCGGATCCTGATTAATAAAAAAAATAATTTACTAAC and LsdrGR1-CGCGGATCCTTAATTTTTATTTTTTCTATTTTTGCGAC. Both primers introduce a BamHI site (italicized) to facilitate cloning of the product into the BclI site of the vector. The ligation mixture was transformed into L. lactis MG1363 cells made competent by washing and suspension in 0·5 M sucrose and 10% glycerol as previously described (Wells et al., 1993
). The cells had been grown for 6 h in 300 ml M17 broth (Difco) containing 0·5% glucose at 30 °C without shaking. Transformants were selected on M17 agar incorporating 0·5% glucose and erythromycin (5 µg ml-1) at 30 °C. Transformants expressing Fbe were identified by whole-cell immunoblotting. Cultures were grown to stationary phase in 2 ml M17 broth with 0·5% glucose and 5 µg erythromycin ml-1 and 10 µl was spotted onto a Protran nitrocellulose membrane. After blocking with 10% skimmed milk for 1 h, membranes were incubated with anti-Fbe (SdrG) region A antibodies (1:1000; McCrea et al., 2000
), washed three times with TBS, incubated with protein A coupled to horseradish peroxidase (1:500; Sigma) and detected by LumiGLO chemiluminescence substrate (New England Biolabs).
Detection of Fbe expression by Western immunoblotting.
L. lactis transformants were grown to late-exponential phase in M17 broth containing 5 µg erythromycin ml-1, washed twice in 5 mM EDTA, pH 8·0, and concentrated to an OD600 of 40 in 1 ml 26% raffinose (Sigma) in 20 mM Tris/HCl, pH 8·0, 10 mM MgCl2. Cell-wall-associated proteins were released by incubation at 37 °C with occasional shaking with 500 U mutanolysin ml-1 and 200 µg lysozyme ml-1 in the presence of protease inhibitors (Complete Mini; Roche) and 10 mM PMSF. Protoplasts were removed by centrifugation at 12000 g for 10 min and 200 µl of the supernatant was added to the final sample (Laemmli, 1970 ), boiled for 10 min and subjected to PAGE (4·5% acrylamide stacking gel, 7·5% acrylamide separating gel). Gels were electroblotted onto membranes as described previously (McCrea et al., 2000
) and were incubated with antibodies and developed as described above.
S. epidermidis was grown for 3·5 h in 50 ml TSB, without shaking, at 37 °C to an OD600 of 0·8. Cells were collected by centrifugation, washed in PBS, resuspended to an OD600 of 10 in 250 µl raffinose buffer (see above) containing 80 µg lysostaphin ml-1 (AMBI, New York) and incubated for 20 min at 37 °C. Samples were prepared for electrophoresis and immunoblotting as described above.
Whole-cell dot immunoblot titrations used to compare the level of Fbe expression were performed as described by Hartford et al. (2001) .
Southern hybridization.
Genomic DNA (5 µg) was cleaved with 20 units HindIII and separated on a 0·8% agarose gel. Transfer of DNA to nitrocellulose membranes was performed as described previously (McCrea et al., 2000 ). The same PCR primers used in the construction of pG+Host9fbe were used to amplify a fragment in the presence of DIG-dUTP (Roche). This was used as a probe in hybridization experiments. Hybridization was detected by chemiluminescence (Roche).
Adherence to immobilized fibrinogen.
Adherence assays were performed in 96-well ELISA dishes as described previously (Hartford et al., 1997 , 2001
) with several modifications. L. lactis and S. epidermidis cells were grown under the same conditions used to prepare cells for releasing wall-associated proteins for Western immunoblotting. Cells were washed in PBS and resuspended to an OD600 of 4 in PBS. Wells in ELISA dishes were coated for 16 h at 4 °C with human fibrinogen (Calbiochem; 0·0120 µg ml-1). Following blocking for 1 h at 37 °C with 2 mg BSA ml-1 (Sigma), wells were washed three times with PBS and 100 µl of a cell suspension was added and incubated for 2 h at 37 °C. Wells were washed three times with PBS and adherent cells were fixed by adding 100 µl 25% formaldehyde and incubating at room temperature for 30 min. After washing three times with PBS, cells were stained with 5% crystal violet for 1 min, washed three times with PBS and 100 µl 5% acetic acid was added to each well to dissolve the cell-associated dye. The A570 was measured in an ELISA plate reader.
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RESULTS |
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Isolation and characterization of pG+Host9 insertions in the fbe gene of S. epidermidis HB
S. epidermidis HB bearing the ts plasmid pG+Host9fbe was grown at 43 °C in order to isolate variants where the ts plasmid had integrated into the fbe gene to survive the restrictive temperature for plasmid replication. The efficiency of plating was 2·8x10-4. Colonies that grew were putative integrants and fbe mutants where the plasmid had integrated into and disrupted the chromosomal fbe gene. Several were picked and genomic DNA was analysed by Southern blot hybridization of HindIII-cleaved genomic DNA using a probe for the A region of fbe. The wild-type fbe (sdrG) gene is present on an approximately 12 kb fragment (Fig. 3; McCrea et al., 2000
). The integrants fell into two classes: (i) those that lacked the wild-type fragment and possessed two smaller HindIII fragments of 10 kb and 6·2 kb that reacted with the probe (the additional HindIII site is contained within the plasmid vector); (ii) those that lacked the wild-type fragment and possessed three reactive fragments, two of which were the same as in the previous case and one that corresponded to the size of the pG+Host9fbe plasmid (4·6 kb). From our previous studies we predicted that the first type was caused by a single-copy integration event and the second by an integration and tandem duplication event (McDevitt et al., 1993
; Supersac et al., 1998
). The proportion of single-copy integration events to tandem duplications was 1:8.
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Expression of Fbe
Cell-wall-associated proteins were released from S. epidermidis strain HB and the fbe insertion mutant by digestion with lysostaphin in the presence of 30% raffinose. Western immunoblots were probed with anti-Fbe (SdrG) region A antibodies. A single immunoreactive protein of 190 kDa was expressed by strain HB wild-type but not by the fbe mutant (Fig. 4).
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DISCUSSION |
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The major conclusion to be drawn from this paper is that the Fbe (SdrG) proteins of S. epidermidis HB and K28 are surface proteins that promote adherence of bacteria to immobilized fibrinogen substrates. They could thus contribute to the initiation of foreign-body infection by allowing bacteria to adhere to biomaterial surfaces that have become coated with host proteins after implantation. This mechanism could complement the previously described adherence of S. epidermidis to naked plastic surfaces, which presumably occurs prior to implantation.
Fbe was expressed at a sufficiently high level to allow detectable adherence only when S. epidermidis HB was grown to early exponential phase in broth without shaking. The failure of the fbe mutant of strain HB to adhere indicated that binding was due to the Fbe protein. However, even under optimum conditions adherence was poor compared to L. lactis expressing Fbe. We estimated that there was at least 500-fold more Fbe protein present on the surface of L. lactis compared to S. epidermidis. Enhanced expression by L. lactis was reflected by the higher level of bacterial adherence.
This paper thus demonstrates the merits of using a surrogate Gram-positive host to express a single protein to complement studies with null mutants of the natural host. L. lactis MG1363 has been used as a host for expression of large LPXTG-anchored surface proteins from Streptococcus gordonii (Holmes et al., 1998 ) and S. aureus (Que et al., 2000
). We have used this system to express several surface proteins from S. aureus with equal success (L. OBrien & T. J. Foster, unpublished data).
One problem that we have identified with L. lactis MG1363 as a surrogate host is that solubilization of the cell-wall-associated surface protein by mutanolysin and lysozyme treatment resulted in extensive degradation. We partially circumvented this by shortening the solubilization time from 60 min to 15 min. It is our belief that there is relatively little degradation of the protein on the surface of intact exponential phase L. lactis cells because of the high levels detected by quantitative whole-cell immunoblotting and because of the high dose-dependent and saturable adherence to fibrinogen. Proteases that are impervious to inhibition by the protease inhibitor cocktail must be activated during protoplast formation.
The apparent molecular mass of the Fbe protein of S. epidermidis HB (190 kDa) is higher than that observed for SdrG expressed by strain K28 (170 kDa; McCrea et al., 2000 ). This is most likely due to the K28 protein having an R region of 56 residues compared to that of the HB protein, which comprises 216 residues.
The pG+Host vectors have been used for isolating allele replacement mutations and for mutagenesis by directed plasmid integration in several different Gram-positive bacteria including L. lactis (Biswas et al., 1993 ), Streptococcus pyogenes (Perez-Casal et al., 1994
) and S. aureus (Supersac et al., 1998
). This is the first report of mutagenesis with this vector in S. epidermidis. Success was possible because of our ability to mobilize the plasmid from S. aureus to strain 9142 by pGO1-promoted conjugation. One advantage of using pG+Host9 to create insertion mutations is the stability of the integrated plasmid after prolonged growth at 37 °C in the absence of drug selection. This offers a considerable advantage over some other Ts plasmids whose mutant Rep proteins become active at that temperature. It is possible that pG+Host integrants would be stable during bacterial growth in experimental infections and mutations caused by plasmid integration could be used for analysing virulence.
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ACKNOWLEDGEMENTS |
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Received 5 February 2001;
revised 22 May 2001;
accepted 23 May 2001.