The host cell che mokine IL 8 is transcribed, translated, and secreted from host cells in response to a number of bacterial pathogens, in cluding C. jejuni, Pretreatment of INT 407 cells with DRB resulted in a reduction within the amount of IL 8 in supernatants from C. jejuni contaminated cells, suggesting that DRB correctly blocks the transcription of IL 8. Nevertheless, pretreatment of INT 407 cells with DRB re sulted in typical C. jejuni invasion of host cells, These effects propose that Erk 1 2 mediated transcriptional regulation is simply not involved in host cell actin cytoskeleton re arrangement needed for C. jejuni host cell invasion. Offered that Erk 1 two mediated transcriptional regulation is simply not demanded for cytoskeleton rearrangement, we per formed experiments to find out if cytosolic signaling mediated by Erk one two was altered or impaired.
We chose to investigate the Erk one two mediated phosphorylation from the cytosolic actin binding protein cortactin, a acknowledged target of Erk one 2 in addition to a part of your actin polymerization and nucleation complex, In contrast to infection of INT 407 cells that has a C. jejuni wild sort strain, the C. jejuni ciaD mutant was deficient in maximal phosphoryl ation of cortactin in the Erk 1 find more info two phosphorylation websites S405 and S418, as judged by immunoblot analysis together with the S418 and S405 phospho specific antibodies to cortactin, INT 407 cells infected together with the C. jejuni ciaD complemented isolate restored the phosphorylation of cortactin to amounts indistinguishable from infection that has a C. jejuni wild form strain, This discovering indi cates that CiaD mediated activation of Erk 1 two leads to the phosphorylation of cortactin on serine residues.
Consist ent with all the fact that CiaD mediates Erk one two activation and Erk 1 two mediates the phosphorylation of cortactin on S405 and S418, hop over to these guys we noticed that pretreatment of INT 407 cells together with the MEK 1 2 inhibitor PD98059 lowered phos phorylation of cortactin on S418 in response to C. jejuni infection, comparable for the degree observed in uninfected cells, The inhibition of cortactin serine phosphoryl ation by therapy of cells with PD98059 is in agreement with published information, However, this is the initial report displaying that cortactin turns into activated in response to C. jejuni infection. Given that CiaD is needed for max imal cortactin activation, we assessed the function of cortactin phosphorylation in C. jejuni invasion of host cells. Cortactin serine phosphorylation is needed for maximal invasion To determine if cortactin is required for C. jejuni invasion of host cells, we made use of minor interfering RNA to knockdown cortactin and siRNA to knockdown the down stream complicated protein N WASP.