Characterization of the Atl-mediated staphylococcal internalization mechanism.

Staphylococcus aureus internalization by non-professional phagocytes is considered a main pathogenicity mechanism leading to chronic infections. The well-established mechanism of Staphylococcus aureus internalization is mediated by fibronectin (Fn)-binding proteins (FnBPs), Fn as a bridging molecule and the host cell α5ß1 integrin. We previously identified a novel alternative internalization mechanism in Staphylococcus aureus, which involves the major autolysin Atl and the host cell heat shock cognate protein 70 (Hsc70). Atl-dependent internalization is also employed by the coagulase-negative Staphylococcus epidermidis, where it might represent the major or even sole internalization mechanism, because of the lack of FnBP-homologous proteins. In this study, we aimed to further characterize the Atl-dependent staphylococcal internalization mechanism. We performed biomolecular interaction analysis (BIA) to quantify the adhesive properties of Atl and found multivalent and high affinity interactions of Atl with Fn and Hsc70. Confocal laser scanning microscopy (CLSM) and a flow-cytometric internalization assay in combination with different pharmacological inhibitors suggested an involvement of the α5ß1 integrin, Fn and Hsc70 and subsequent signaling events mediated by Src and phosphoinositide 3 (PI3) kinases in the Atl-dependent staphylococcal uptake by EA.hy 926 cells. Further characterization of the endocytic machinery implicated a role for clathrin-dependent receptor-mediated endocytosis involving actin cytoskeletal rearrangements and microtubules. In conclusion, Atl ubiquitous among staphylococcal species may substitute for the FnBPs ensuring low-level internalization via a mechanism that seems to share important features with the FnBP-mediated staphylococcal uptake potentially being the prerequisite for the development of therapy-resistant chronic infections by staphylococcal strains that lack FnBPs.

Characterization of the modular design of the autolysin/adhesin Aaa from Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is a frequent cause of serious and life-threatening infections, such as endocarditis, osteomyelitis, pneumonia, and sepsis. Its adherence to various host structures is crucial for the establishment of diseases. Adherence may be mediated by a variety of adhesins, among them the autolysin/adhesins Atl and Aaa. Aaa is composed of three N-terminal repeated sequences homologous to a lysin motif (LysM) that can confer cell wall attachment and a C-terminally located cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain having bacteriolytic activity in many proteins. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show by surface plasmon resonance that the LysM domain binds to fibrinogen, fibronectin, and vitronectin respresenting a novel adhesive function for this domain. Moreover, we demonstrated that the CHAP domain not only mediates the bacteriolytic activity, but also adherence to fibrinogen, fibronectin, and vitronectin, thus demonstrating for the first time an adhesive function for this domain. Adherence of an S. aureus aaa mutant and the complemented aaa mutant is slightly decreased and increased, respectively, to vitronectin, but not to fibrinogen and fibronectin, which might at least in part result from an increased expression of atl in the aaa mutant. Furthermore, an S. aureus atl mutant that showed enhanced adherence to fibrinogen, fibronectin, and endothelial cells also demonstrated increased aaa expression and production of Aaa. Thus, the redundant functions of Aaa and Atl might at least in part be interchangeable. Lastly, RT-PCR and zymographic analysis revealed that aaa is negatively regulated by the global virulence gene regulators agr and SarA. CONCLUSIONS/SIGNIFICANCE: We identified novel functions for two widely distributed protein domains, LysM and CHAP, i.e. the adherence to the extracellular matrix proteins fibrinogen, fibronectin, and vitronectin. The adhesive properties of Aaa might promote S. aureus colonization of host extracellular matrix and tissue, suggesting a role for Aaa in the pathogenesis of S. aureus infections.

A novel staphylococcal internalization mechanism involves the major autolysin Atl and heat shock cognate protein Hsc70 as host cell receptor.

Staphylococcus aureus and Staphylococcus epidermidis can cause serious chronic and recurrent infections that are difficult to eradicate. An important pathogenicity factor in these infections caused by S. aureus is its ability to be internalized by non-professional phagocytes thereby evading the host immune system and antibiotic treatment. Here, we report a novel mechanism involved in staphylococcal internalization by host cells, which is mediated by the major autolysin/adhesins Atl and AtlE from S. aureus and S. epidermidis respectively. In a flow cytometric internalization assay, atl and atlE mutants are significantly reduced in their capacities to be internalized by endothelial cells. Moreover, pre-incubation of endothelial cells with recombinant Atl dose-dependently inhibited internalization. As putative Atl-host cell receptor, the heat shock cognate protein Hsc70 was identified by mass spectrometry. The importance of Hsc70 in internalization was demonstrated by the inhibition of S. aureus internalization with anti-Hsc70 antibodies. In conclusion, this novel Atl- or AtlE-mediated internalization mechanism may represent a 'back-up' mechanism in S. aureus internalization, while it may represent the major or even sole mechanism involved in the internalization of coagulase-negative staphylococci and thus may play an important role in the pathogenesis of chronic and relapsing infections with these serious pathogens.