Cellular invasion byStaphylococcus aureusreveals a functional link between focal adhesion kinase and cortactin in integrin-mediated internalisation

Abstract

Nosocomial infections by Staphylococcus aureus, a Gram-positive pathogen colonising human skin and mucosal surfaces, are an increasing health care problem. Clinical isolates almost invariably express fibronectin-binding proteins that, by indirectly linking the bacteria with host integrin α5β1, can promote uptake of the microorganisms by eukaryotic cells. Integrin engagement by pathogenic fibronectin-binding S. aureus, but not by non-pathogenic S. carnosus, triggered the recruitment of focal contact-associated proteins vinculin, tensin, zyxin and FAK to the sites of bacterial attachment. Moreover, dominant-negative versions of FAK-blocked integrin-mediated internalisation and FAK-deficient cells were severely impaired in their ability to internalise S. aureus. Pathogen binding induced tyrosine phosphorylation of several host proteins associated with bacterial attachment sites, including FAK and the Src substrate cortactin. In FAK-deficient cells, local recruitment of cortactin still occurred, whereas the integrin- and Src-dependent tyrosine phosphorylation of cortactin was abolished. As siRNA-mediated gene silencing of cortactin or mutation of critical amino acid residues within cortactin interfered with uptake of S. aureus, our results reveal a novel functional connection between integrin engagement, FAK activation and Src-mediated cortactin phosphorylation. Cooperation between FAK, Src and cortactin in integrin-mediated internalisation of bacteria also suggests a molecular scenario of how engagement of integrins could be coupled to membrane endocytosis.

Invasion of Host Cells bySalmonella typhimuriumRequires Focal Adhesion Kinase and p130Cas

“…Because it has been shown that c-Src is the major tyrosine kinase that phosphorylates Cas (Astier et al, 1997;Ruest et al, 2001;Agerer et al, 2005), we assayed Salmonella internalization in cells treated cells with a Src inhibitor, PP2. Although PP2 treatment efficiently inhibited tyrosine phosphorylation of Cas during Salmonella invasion (see Supplementary Material, Figure S3), bacterial internalization remained unaffected under these conditions ( Figure 6B).…”

“…Similarly, uptake of Yersinia upon invasin-mediated engagement of ␤ 1 integrin and Streptococcus via fibronectin-binding integrins is dependent on FAK (Alrutz and Isberg, 1998;Ozeri et al, 2001;Bruce-Staskal et al, 2002). More recently, Agerer et al (2005) showed that FAK plays an essential role in integrin ␣ 1 ␤ 5 -mediated invasion of Staphylococcus. However, each of these processes is accompanied by increased tyrosine phosphorylation of FAK, indicative of its activation during particle internalization.…”

“…However, each of these processes is accompanied by increased tyrosine phosphorylation of FAK, indicative of its activation during particle internalization. Moreover, the autophophorylation site Y397 has been shown to be necessary for internalization of both Yersinia and Staphylococcus, as expression of FAK Y397F impairs bacterial uptake (Alrutz and Isberg, 1998;Agerer et al, 2005). In each of these cases, it is likely that FAK activation results from integrin clustering.…”

“…The Shigella effector protein IpaA binds to vinculin, inducing local actin depolymerization that is essential for formation of the phagocytic apparatus (Finlay et al, 1991;Tran Van Nhieu et al, 1997;Bourdet-Sicard et al, 1999). Similarly, FAK has been shown to promote invasion of host cells by Yersinia and Staphylococcus (Alrutz and Isberg, 1998;Bruce-Staskal et al, 2002;Agerer et al, 2005). Interestingly, a number of focal adhesion proteins, such as talin and vinculin, have been observed to accumulate at Salmonella-induced membrane ruffles despite a lack of evidence showing involvement of integrins (Finlay et al, 1991).…”

Quantification of bacterial invasion into adherent cells by flow cytometry

“…In other cases, the bacteria use an indirect route by engaging cell surface receptors coupled to the cytoskeleton including integrins, cadherins or immunoglobulin-related cell adhesion molecules (Hauck, 2002). For example, S. flexneri as well as S. aureus are able to connect to cell surface integrins to trigger actin-dependent internalization (Watarai et al, 1996;Sinha et al, 1999;Agerer et al, 2005). Listeria monocytogenes targets host E-cadherin by means of the InlA protein to enter epithelial cells (Mengaud et al, 1996;Lecuit et al, 1999).…”