Integrin-mediated Invasion of Staphylococcus aureus into Human Cells Requires Src Family Protein-tyrosine Kinases

Abstract

Staphylococcus aureus, a common cause of nosocomial infections, is able to invade eukaryotic cells by indirectly engaging ␤ 1 integrin-containing host receptors, whereas non-pathogenic Staphylococcus carnosus is not invasive. Here, we identify intracellular signals involved in integrin-initiated internalization of S. aureus. In particular, the host cell actin cytoskeleton and Src family protein-tyrosine kinases (PTKs) are essential to mediate S. aureus invasion. Src PTKs are activated in response to pathogenic S. aureus, but not S. carnosus. In addition, pharmacological and genetic interference with Src PTK function reduces bacterial internalization. Importantly, Src PTK-deficient cells are resistant to S. aureus invasion, demonstrating the essentiality of host Src PTKs in integrin-mediated uptake of this pathogen.The Gram-positive microorganism Staphylococcus aureus is a common cause of nosocomial infections (1, 2). Introduction of staphylococci is facilitated by indwelling medical devices that provide a surface for colonization and a platform for further dissemination. Due to widespread and often multiple antibiotic resistance of S. aureus, infections with this microbe are increasingly difficult to treat.A prominent characteristic of different S. aureus isolates associated with disease is the expression of extracellular matrix (ECM) 1 -binding proteins that have been collectively termed MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) (3, 4). In particular, the cell wall-attached fibronectin-binding proteins A and B (FnBPA and FnBPB) confer a tight association of the bacteria with the ECM protein fibronectin (Fn) (5, 6). The interaction of FnBPs with fibronectin is mediated by direct protein-protein interactions between multiple Fn-binding domains of the bacterial proteins connecting as extended antiparallel ␤-strands to the fibronectin type 1 modules in the amino-terminal domain of Fn (7). As Fn is a common constituent of the extracellular matrix in different tissues and is also abundant in serum, it is thought that Fn deposition on introduced materials such as bone-implanted metals promotes the attachment of S. aureus on medical devices (8).Eukaryotic cells also possess specific surface receptors that bind to Fn. Most prominently, the integrin ␣ 5 ␤ 1 serves as a Fn receptor on multiple cell types. Integrin ␣ 5 ␤ 1 recognizes a short peptide motif, the Arg-Gly-Asp (RGD) sequence, found within one of the type III repeats of Fn. As staphylococcal FnBP associates with the N terminus of Fn independently of the RGD sequence (7, 9), simultaneous association of both S. aureus and human cells with Fn should be possible. Indeed, it has been shown recently that Fn acts as a molecular bridge, linking FnBP-expressing S. aureus with integrin ␣ 5 ␤ 1 on the surface of human cells (for review see Ref. 10). This interaction not only tightly anchors S. aureus to its eukaryotic host cell, but also promotes the internalization of the microorganisms by human epithelial and endothelial cells (11-13) ...

Microscopic quantification of bacterial invasion by a novel antibody-independent staining method

“…3B). A similar pattern of invasiveness has been observed before using antibiotic protection assays and antibody-dependent staining procedures (Agerer et al, 2003), indicating that the covalent modification of S. aureus by FITC and biotin does not impair the integrin-mediated internalisation process. Taken together, these results demonstrate that the novel antibody-independent FBA staining procedure is a convenient and reliable technique for distinguishing between extracellular and intracellular bacteria.…”

“…Previously, we and others have demonstrated that S. aureus is able to exploit fibronectin and host cell h1 integrins to gain access to an intracellular compartment, whereas the related nonpathogenic species S. carnosus does not invade eukaryotic cells (Sinha et al, 1999;Fowler et al, 2000;Agerer et al, 2003). Therefore, we labelled both species of staphylococci with FITC and biotin and then infected 293 cells for 2 h. Again, processing of the fixed samples according to the FBA staining procedure resulted in a clear discrimination between extracellular and intracellular bacteria (Fig.…”

“…In the case of S. aureus, invasion is triggered by fibronectin recruitment to the surface of these Gram-positive microbes. Fibronectin then serves as a molecular bridge to allow bacterial attachment to host h1 integrins (Sinha et al, 1999;Fowler et al, 2000;Agerer et al, 2003). To analyse bacterial invasion in vitro and to quantify the invasive potential of pathogens, two experimental approaches have been used extensively.…”

Targeting of αv integrins interferes with FAK activation and smooth muscle cell migration and invasion

“…Cell lysis immunoprecipitation, Western blotting, and gelatine zymography. Cells were solubilized in a modified RIPA lysis buffer containing 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS [20]. Immunoprecipitations, SDS-PAGE, Western blotting, and gelatine zymography were performed as described [19,21].…”

Quantification of bacterial invasion into adherent cells by flow cytometry

“…Fn then serves as a molecular link connecting the bacteria with the principal host cell fibronectin receptor, the integrin a 5 h 1 (Fowler et al, 2000). Previously, we and others have observed that FnBP-initiated uptake via integrin a 5 h 1 is the major route of S. aureus internalization in vitro (Sinha et al, 1999;Sinha et al, 2000;Agerer et al, 2003). Therefore, S. aureus and the nonFn-binding, non-pathogenic S. carnosus were labelled with CFSE and used to infect fibroblasts isolated from integrin h1-deficient mouse embryos (GD25 cells) as well as GD25 cells stably expressing human integrin h1 (Fig.…”