Based on these findings, we concluded that the focal complex is usually a central player in epithelial cells detecting and responding to bacterial pathogens

Based on these findings, we concluded that the focal complex is usually a central player in epithelial cells detecting and responding to bacterial pathogens. Open in a separate window Fig. response. Panels: (A) The invasiveness of the mutant was determined by the gentamicin protection assay. INT 407 cells were infected with the wild-type strain and mutant. Host cell association was assessed 30 min following contamination. Gentamicin was added to infected cells 3 hr after contamination and incubated for 3 additional hr. (B) INT 407 cells were infected with a wild-type strain and mutant. Uninfected cells served as a negative control. IL-8 secretion was quantified by ELISA. A significant difference was not detected in the amount of IL-8 secreted in response to the mutant as compared to the wild-type strain as judged by ELISA of IL-8 in supernatants. (C) The expression of the Rac1 activating guanine exchange factor Dock180 was reduced by siRNA treatment of INT 407 cells. Scrambled siRNA (Scrm) treated cells served as a transfection control and uninfected cells served as a negative control. The cells were infected with Vehicle treated cells infected with served as a positive control and uninfected cells served as a negative control. Lysates were prepared after a 30 min Rabbit Polyclonal to COX5A contamination and probed for activation of Akt by immunoblot with antibodies against phospho-Akt (pAkt). The blot was reprobed with antibodies against actin as a loading control. (B) INT 407 cells were treated with FAK inhibitor TAE 226 or with PI3 kinase inhibitor LY294002 and infected with contamination was measured with antibodies against phospho-NF-B (pNF-B). The blot was re-probed with antibodies against actin as a loading control. (C) INT 407 cells were treated with LY294002 and infected with served as a positive control and uninfected cells served as a negative control. IL-8 secretion was quantified by ELISA. Fig. S6. and require paxillin to trigger maximal IL-8 secretion from epithelial cells. INT 407 cells were treated with siRNA specific to paxillin or scrambled siRNA (Scrm). The amount of IL-8 in supernatants collected from INT 407 cells infected with and was quantified by ELISA. The IL-8 values were normalized to untreated cells infected with wild-type bacteria, and the value generated for the uninfected cells was subtracted from all values. to Digoxigenin determine if the FC is required for induction of chemokine signaling in response to bacterial pathogens. Our data indicate that Digoxigenin secretion of IL-8 is usually brought on by and serovar Typhimurium in response to engagement of 1 1 integrins. Additionally, we found that the secretion of IL-8 from infected epithelial cells requires FAK, Src, and paxillin, which in turn are necessary for Erk 1/2 recruitment and activation. Targeting the FC component paxillin with siRNA prevented IL-8 secretion from cells infected with several bacterial pathogens, including serovar TyphimuriumRaf)]. The activated triple kinase then phosphorylates its target MAP kinase-kinase (MEK 1/2), Digoxigenin leading to phosphorylation of the MAP kinase (Erk 1/2) (Schaeffer possesses two fibronectin binding proteins termed CadF and FlpA (Konkel invasion antigens (Cia proteins) to host cells (Konkel are co-cultured with epithelial cells (Konkel adhesins and secreted proteins act cooperatively to subvert components of the host cell focal adhesion complex to facilitate internalization, and that these virulence proteins also contribute to IL-8 secretion. The recurring theme of bacterial conversation with components of the FC system, including the ECM components and the integrins, led us to hypothesize that these proteins are serving a critical role in bacterial pathogenesis. The goal of this work was to identify membrane associated and cytosolic signaling components required for Erk 1/2 activation as a prerequisite for IL-8 secretion in response to bacterial pathogens. We hypothesized that bacterial activation of the FC directly contributes to the activation of the MAP kinase signaling pathway in epithelial cells. We demonstrate that 1 integrins, FAK, Src, and paxillin are required for Erk 1/2 activation and IL-8 secretion in response to serovar.