The goal of this study was to characterize the Yersinia pestis KIM OmpX protein. Yersinia spp. provide a model for studying several virulence processes including attachment to, and internalization by, host cells. For Yersinia enterocolitica and Yersinia pseudotuberculosis, Ail, YadA and Inv, have been implicated in these processes. In Y. pestis, YadA and Inv are inactivated. Genomic analysis of two Y. pestis strains revealed four loci with sequence homology to Ail. One of these genes, designated y1324 in the Y. pestis KIM database, encodes a protein designated OmpX. The mature protein has a predicted molecular mass of 17.47 kDa, shares approximately 70 % sequence identity with Y. enterocolitica Ail, and has an identical homologue, designated Ail, in the Y. pestis CO92 database. The present study compared the Y. pestis KIM6 + parental strain with a mutant derivative having an engineered disruption of the OmpX structural gene. The parental strain (and a merodiploid control strain) expressed OmpX at 28 and 37 6C, and the protein was detectable throughout all phases of growth. OmpX was required for efficient adherence to, and internalization by, cultured HEp-2 cell monolayers and conferred resistance to the bactericidal effect of human serum. Deletion of ompX resulted in a significantly reduced autoaggregation phenotype and loss of pellicle formation in vitro. These results suggest that Y. pestis OmpX shares functional homology with Y. enterocolitica Ail in adherence, internalization into epithelial cells and serum resistance.

Staphylococcus aureus expresses several surface proteins that promote adherence to host cell extracellular matrix proteins, including fibronectin (Fn). Since this organism has recently been shown to be internalized by nonprofessional phagocytes, a process that typically requires high-affinity binding to host cell receptors, we investigated the role of its Fn binding proteins (FnBPs) and other surface proteins in internalization by the bovine mammary gland epithelial cell line (MAC-T). Efficient internalization of S. aureus 8325-4 required expression of FnBPs; an isogenic mutant (DU5883), not expressing FnBPs, was reduced by more than 95% in its ability to invade MAC-T cells. Moreover, D3, a synthetic peptide derived from the ligand binding domain of FnBP, inhibited the internalization of the 8325-4 strain in a dose-dependent fashion and the efficiency of staphylococcal internalization was partially correlated with Fn binding ability. Interestingly, Fn also inhibited the internalization and adherence of S. aureus 8325-4 in a dose-dependent manner. In contrast to internalization, adherence of DU5883 to MAC-T was reduced by only approximately 40%, suggesting that surface binding proteins, other than FnBPs, can mediate bacterial adherence to cells. Adherence via these proteins, however, does not necessarily result in internalization of the staphylococci. An inhibitor of protein tyrosine kinase, genistein, reduced MAC-T internalization of S. aureus by 95%, indicating a requirement for a host signal transduction system in this process. Taken together, these results indicate that S. aureusinvades nonprofessional phagocytes by a mechanism requiring interaction between FnBP and the host cell, leading to signal transduction and subsequent rearrangement of the host cell cytoskeleton.

In recent years several new staphylococcal enterotoxins (SEs) have been described, which currently have largely unknown frequencies of occurrence and roles in human or animal disease. One hundred and ninety-one Staphylococcus aureus isolates from cows (99), goats (39), sheep (23), rabbits (15), chickens (15) and a cat (1) were screened for SE genes sea-see, seg-seo and seq and for the tst gene encoding staphylococcal toxic shock syndrome toxin-1 using multiplex PCRs and individual PCRs for the seb and sek genes. One hundred and ten isolates tested positive for at least one of these 16 superantigen (SAg)-encoding genes. There were statistically significant differences in the frequencies of some of these SAg genes between isolates from different animals. No strain possessed either the sea or see gene. The sec gene was present in 51 isolates, the sed gene in eight and the seb gene in one. The seh gene was found in four strains and the sek and seq genes together in one isolate. The most common combinations of genes were the egc cluster, bearing the seg, sei, sem, sen and seo genes, in 47 isolates, the sec, sel and tst gene combination typical of the SaPIbov pathogenicity island in 44 isolates, the egc cluster lacking the seg gene in 11 isolates, the sed and sej genes in nine isolates, and the sec and tst genes without the sel gene in seven isolates. The higher frequencies of the sec and tst genes together and the lower frequencies of the egc gene cluster among the SAg gene-positive sheep or goat isolates compared to bovine isolates were statistically significant. Of 36 bovine isolates that were mitogenic for human T lymphocytes, four were negative for the 16 SAg genes tested for, while a further 14 gave borderline results in the mitogenicity assay, 12 of which were SAg gene-negative. Twenty-nine strains lacking all the SAg genes did not induce T-cell proliferation. This survey indicates that novel SE genes seg, sei, sel, sem, sen and seo along with the sec and tst genes predominate in S. aureus from animal hosts. The mitogenicity assays indicate that further uncharacterized SAgs may be present in bovine isolates. INTRODUCTIONStaphylococcus aureus is a major human pathogen that causes a wide variety of diseases ranging in severity from food poisoning (McCormick et al., 2001;Le Loir et al., 2003) and life-threatening toxic shock syndrome (Llewelyn & Cohen, 2002;Proft & Fraser, 2003) to less serious infections, e.g. boils (Stulberg et al., 2002). S. aureus can also cause a number of infections in animals, such as tick-associated pyaemia in lambs (Webster & Mitchell, 1989), staphylococcosis in rabbits (Hermans et al., 2003), oedematous and necrotic dermatitis, septicaemia, abcesses and chondronecrosis in chickens McNamee et al., 1998;Takeuchi et al., 2002) S. aureus is the most frequent cause of bovine mastitis, a disease that is of economic importance worldwide (Beck et al., 1992;Miles et al., 1992). Typically staphylococcal mastitis is chronic in nature, with subclinical mastitis being the most common form (Gruet et a...

Bacteria have developed various mechanisms for inducing internalization into nonprofessional phagocytes (8,9). A shared requirement is that a molecular interaction must occur between a bacterial surface adhesin and a host ligand in the cytoplasmic membrane. This interaction must be of sufficiently high affinity to induce signal transduction through the membrane, resulting in cytoskeletal rearrangements and uptake of the organism (14, 37). For some organisms, a simple model involving the binding of a bacterial adhesin to its host receptor is sufficient to induce uptake. Yersinia species and Listeria monocytogenes are examples of organisms with this pattern of uptake. The adhesins on these facultative intracellular pathogens bind directly to integrins or E-cadherin, respectively, with an affinity that is sufficient to induce internalization (15,24).A number of reports have described bacterial surface adhesins, which adhere to host extracellular matrix (ECM) proteins. The ECM binding proteins are termed MSCRAMMS, and Staphylococcus aureus expresses several of these proteins with different ligand specificities (17,18,27). We demonstrated previously that the S. aureus surface adhesin responsible for stimulating signal transduction upon uptake by nonprofessional phagocytes is one of its MSCRAMMs, fibronectin (Fn) binding protein (FnBP) (6). Our data were confirmed in two additional publications (22,28). This finding raised several questions regarding the nature of the molecular interactions at the host cell surface.Fn is the ECM protein commonly associated with integrins. It is known that Fn is bivalent and can serve as a bridging molecule between FnBP and the host cell integrins (17,26, 39). Although other bacteria use Fn as a link to adhere to host tissues, the mechanisms by which this linkage could induce internalization are less clear. For example, Tran Van Nhieu and Isberg showed that coating of S. aureus with Fn did not lead to efficient internalization (37). It was proposed that the binding affinity between Fn and integrins was not sufficient to induce uptake. Interestingly, at least one organism, Neisseria gonorrhoeae has overcome this limitation by employing a heparin-containing accessory coreceptor, which is necessary to induce maximal internalization by HEp-2 cells (39).The present study was initiated to identify potential cellular ligands and other molecular requirements for uptake of S. aureus by nonprofessional phagocytes. Using a variety of methods, we found that FnBP binds directly to heat shock protein 60 (Hsp60) on the membranes of human and bovine epithelial cells. Fn and ␤ 1 integrins are also required for maximal uptake. Based on these combined results, a potential model to explain the molecular interactions leading to uptake of S. aureus is proposed.