SummaryThe ability of Staphylococcus aureus to invade mammalian cells may explain its capacity to colonize mucosa and to persist in tissues after bacteraemia. To date, the underlying molecular mechanisms of cellular invasion by S. aureus are unknown, despite its high prevalence and dif®culties in treatment. Here, we show cellular invasion as a novel function for an S. aureus adhesin, previously implicated solely in attachment. S. aureus, but not S. epidermidis, invaded epithelial 293 cells in a temperature-and F-actindependent manner. Formaldehyde-®xed and live bacteria were equally invasive, suggesting that no active bacterial process was involved. All clinical S. aureus isolates analysed, but only a subset of laboratory strains, were invasive. Fibronectin-binding proteins (FnBPs) acted as S. aureus invasins, because: (i) FnBP deletion mutants of invasive laboratory strains lost invasiveness; (ii) expression of FnBPs in noninvasive strains conferred invasiveness; and (iii) the soluble isolated ®bronectin-binding domain of FnBP (D1±D4) completely blocked invasion. Integrin a 5 b 1 served as host cell receptor, which interacted with staphylococcal FnBPs through cellular or soluble ®bronectin. FnBP-de®cient mutants lost invasiveness for epithelial cells, endothelial cells and ®broblasts. Thus, ®bronectin-dependent bridging between S. aureus FnBPs and host cell integrin a 5 b 1 is a conserved mechanism for S. aureus invasion of human cells. This may prove useful in developing new therapeutic and vaccine strategies for S. aureus infections.

The importance of the fibrinogen-binding adhesin clumping factor A (ClfA) in the pathogenesis of Staphylococcus aureus septic arthritis was examined in an animal model. The protective effect of active and passive immunization with ClfA also was investigated in S. aureus infection models. The severity of arthritis was markedly reduced in mice challenged intravenously with a clfA mutant, compared with mice infected with the wild-type strain. Mice immunized with recombinant ClfA and challenged with S. aureus developed less-severe arthritis than did mice immunized with a control antigen. Passive immunization of mice with rat and rabbit anti-ClfA antibodies protected against S. aureus arthritis and sepsis-induced death, indicating that the protection by active immunization is antibody mediated. Taken together, these data strongly suggest that ClfA is a crucial virulence determinant for septic arthritis and an excellent target for the generation of immune therapies directed against S. aureus.

SummaryClumping factor of Staphylococcus aureus is a fibrinogen-binding protein that is located on the bacterial cell surface. The protein has an unusual repeat domain (region R) comprising mainly the dipeptide aspartate and serine. To determine if region R has a role in the surface display of the fibrinogen-binding region A domain, deletions lacking the region R encoding region of the clfA gene were generated. To determine the minimum length of region R required for wild-type levels of ClfA expression, variants with truncated region R domains were constructed. S. aureus cells expressing mutated clfA genes were tested for (i) proteins released by lysostaphin treatment that reacted with antisera specific for region A, (ii) clumping in soluble fibrinogen, (iii) adherence to immobilized fibrinogen and (iv) expression of the ClfA antigen on the cell surface by fluorescent activated cell sorting analysis. Each construct expressed three major immunoreactive proteins, two of which were putative Nterminal degradation products. Region R residues greater than 40 were required between region A and W (72 residues between region A and the LPDTG sorting signal) for wild-type levels of clumping in fibrinogen. A stepwise decrease in clumping titre was observed as the distance between region A and LPDTG was decreased from 72 to 4 residues. Similarly, a decrease in binding of anti-ClfA serum and in binding to fibrinogen-coated plastic surfaces was observed with cells expressing ClfA with 40 region R residues or less. Nevertheless, low levels of adherence to fibrinogen and binding to anti-ClfA serum occurred with ClfA derivatives that lacked region R altogether. This indicates that a small proportion of the ClfA molecules are linked to peptidoglycan very close to the cell surface but that residues greater than 72 are needed to allow sufficient ClfA molecules to span the entire cell wall and to display the biologically active A domain in a form that can participate fully in fibrinogen binding.

Staphylococcus epidermidis can express three different cell-surface-associated proteins, designated SdrF, SdrG and SdrH, that contain serine-aspartate dipeptide repeats. Proteins SdrF and SdrG are similar in sequence and structural organization to the Sdr proteins of Staphylococcus aureus and comprise unique 625-and 548-residue A regions at their N termini, respectively, followed by 110-119-residue B-repeat regions and SD-repeat regions. The C termini contain LPXTG motifs and hydrophobic amino acid segments characteristic of surface proteins covalently anchored to peptidoglycan. In contrast, SdrH has a short 60-residue A region at its N terminus followed by a SD-repeat region, a unique 277-residue C region and a C-terminal hydrophobic segment. SdrH lacks a LPXTG motif. Recombinant proteins representing the A regions of SdrF, SdrG and SdrH were expressed and purified from Escherichia coli. Antisera specific to these proteins were raised in rabbits and used to identify Sdr proteins expressed by S. epidermidis. Only SdrF was released from lysostaphin-generated protoplasts of cells grown to late-exponential phase. SdrG and SdrH remained associated with the protoplast fraction and thus appear to be ineffectively sorted along the conventional pathway used for cell-wall-anchored proteins. In Southern hybridization analyses, the sdrG and sdrH genes were present in all 16 strains tested, whilst sdrF was present in 12 strains. Antisera from 16 patients who had recovered from S. epidermidis infections contained antibodies that reacted with recombinant A regions of SdrG and SdrH, suggesting that these proteins can be expressed during infection.

Staphylococcus epidermidis strains HB and K28 express surface proteins called Fbe or SdrG, respectively, that have sequence similarity to the clumping factors ClfA and ClfB of Staphylococcus aureus. A mutation in the fbe gene of strain HB was isolated by directed plasmid integration using the broad-host-range temperature-sensitive plasmid pG M Host9 (pVE6155). An internal fragment of fbe was cloned into pG M Host9 and the chimaeric plasmid was mobilized from S. aureus RN4220 to S. epidermidis 9142 by conjugation promoted by plasmid pGO1. The plasmid was then transferred to S. epidermidis strain HB by phage-48-mediated transduction. The plasmid integrated into the chromosomal fbe gene at a frequency of 28i10 N4 . All the survivors tested had a copy of pG M Host9' fbe ' integrated into the chromosomal fbe gene either as a single copy or as a tandem array. Western immunoblotting showed that the wallassociated Fbe protein was absent in the mutant. Wild-type S. epidermidis HB adhered to immobilized fibrinogen in a dose-dependent and saturable fashion whereas the mutant did not bind. The Fbe proteins of HB and K28 were expressed at a high level in Lactococcus lactis MG1363 using the expression vector pKS80. These strains adhered strongly to immobilized fibrinogen. These results confirm that Fbe is a fibrinogen-binding adhesin.