Figure 3

Disruption of lipid rafts and microtubules, but not microfilaments, inhibits the activation of the Rho GTPase Rac1 in C. jejuni -infected HeLa cells. Assays were performed to assess the delivery of the C. jejuni CiaC protein to the cytosol of HeLa cells (Panel A) and to determine the level of Rac1 activity in C. jejuni-infected HeLa cells (Panel B). HeLa cells were pre-treated methyl-β-cyclodextrin (MβCD) (5 mM), nocodazole (20 mM), cytochalasin D (1 mM), and TAE 226 (5 μM) and infected with C. jejuni as outlined in ‘the ‘Methods’ section. Panels: A) Delivery of the CiaC-ACD fusion protein from C. jejuni to the cytosol of HeLa cells was assayed using the adenylate cyclase domain (ACD) reporter delivery assay. The concentration of cAMP within HeLa cells was assayed 30 min post-infection. The error bars represent mean ± standard deviation (SD) from five independent experiments. The level of cAMP for cells infected with C. jejuni in the absence of an inhibitor was significantly greater than for the negative control MetK-ACD, as judged by one-way ANOVA followed by post-hoc Tukey’s analysis. None of the treatments resulted in a value significantly (P < 0.05) different from the control (HeLa cells infected with C. jejuni in the absence of a host cell inhibitor). B) Rac1 activation in host cells infected with C. jejuni. Whole cell lysates were analyzed for activated Rac1 by G-LISA™. The mean ± SD of total active Rac1 is indicated in Relative Optical Density. The data shown represent analysis of four samples. The asterisks indicate a significant difference (P < 0.01) in the level of Rac1 activation in cells infected with C. jejuni in the presence versus absence of a host cell inhibitor, as judged by one-way ANOVA followed by post-hoc Tukey’s analysis.