Figure 1
CiaD is delivered to the cytosol of human epithelial cells. (A) CiaD is secreted from C. jejuni. A C. jejuni wild-type strain transformed with the pRY111 vector harboring CiaD fused to the adenylate cyclase domain (CiaD-ACD, + designation) was analyzed by immunoblot analysis. CysM is a cytoplasmic protein (O-acetylserine sulfhydrylase B) involved in cysteine biosynthesis (Mr = 32.4 kDa). Also included was a C. jejuni wild-type strain without the pRY111 shuttle vector as a negative control (- designation) and a C. jejuni flgBC mutant expressing CiaD-ACD and a pRY111 shuttle vector. (B) CiaD is delivered to host cells. A C. jejuni wild-type strain, ciaD mutant, and flgBC mutant were transformed with pRY111 vector harboring the ciaD-ACD, ciaC-ACD, and metK-ACD inserts. C. jejuni harboring the metK-ACD vector was used as a negative control. The CiaC-ACD, a known delivered protein, was included as a positive control. The delivery of the CiaD-ACD, CiaC-ACD, and MetK-ACD fusion proteins to the cytosol of human INT 407 cells was determined using the ACD delivery assay described in Methods. The asterisk indicates that the amount of cAMP produced in the wild-type strain was significantly greater than the value obtained from the flgBC mutant, as judged by one-way ANOVA followed by post-hoc Tukey’s analysis (P < 0.05). Each error bar represents ± the standard error of the mean (SEM) of N = 9 samples from 3 independent experiments.