, 2008). In our current studies, the HEp-2 cells were cocultured with the wild-type or the isogenic scl1-inactivated mutant GAS that were either treated or untreated with cFn or Lm. Following internalization, the numbers of surviving intracellular bacteria were determined. The Scl1-deficient GAS cells were internalized significantly less than click here the wild-type strain in ECM-free medium (Fig. 3). Following preincubation with cFn and Lm, the wild-type strain exhibited about a 4- and 6.5-fold

increase in internalization, respectively, compared with ECM-untreated cells. The scl1-inactivated strain preincubated with cFn and Lm also showed about a 2.2- and a 2.8-fold increase in internalization compared with the ECM-untreated mutant cells; however, the overall levels of mutant internalization were lower compared with the wild-type strain under each corresponding experimental condition, emphasizing the contribution of Scl1 to cell invasion by GAS. It should be noted that the in vivo relevance of GAS internalization by human cells mediated by ECM binding Pictilisib has been debated in recent years. In spite of this, recent investigations using nuclear magnetic resonance spectroscopy, circular dichroism analyses, and experiments with monoclonal antibodies identified structural changes caused by fibronectin upon binding to bacterial

proteins that result in an enhanced Fn recognition by integrins (Bingham et al., 2008; Margarit et al., 2009). It is tempting to speculate that Scl1 binding to cFn and Lm may exert similar biological effects. It was shown previously by our group

that Scl1 from M41-type GAS binds the human collagen integrin receptors, which mediates GAS internalization by host cells (Caswell et al., 2007, 2008a). Integrins bind the GLPGER sequence directly within the Scl1-CL region. Here, we show the V-region of the same Scl1.41 protein binds to cFn and Lm, which also increases GAS internalization by HEp-2 cells. We think it is unlikely that cFn and Lm binding to the globular V domain affects Scl1-CL region binding to α2β1 and α11β1; Nintedanib (BIBF 1120) however, we cannot fully exclude such a possibility. The HEp-2 cells express the α2, α3, α5, and β1 integrin subunits (Caswell et al., 2007), and are thus capable of producing the α2β1, α3β1, and α5β1 heterodimers with the ability to bind collagen, laminin, and fibronectin, respectively (Watt, 2002). The α11β1 integrin expression is restricted to fibroblasts (Popova et al., 2007) and, thus, may not be present on the surface of HEp-2 cells. Therefore, Scl1 may be contributing to internalization of M41-type GAS by HEp-2 cells by two mechanisms: direct binding to the α2β1 integrin and ECM-bridging mechanism via integrins α3β1 and α5β1.