Fig. 5.
Analysis of T3SS-1- and T3SS-2-dependent secretion of AvrA in vitro by Western blot. wt, invG (T3SS-1 defective), ssaK (TTSS-2 defective) and invG ssaK (lacking both T3SSs) strains of S. Enteritidis containing AvrA, SipA and SseJ tagged with FLAG were grown under culture conditions that mimicked the intestinal niche and the intracellular environment as indicated in Methods. The secretion of the effectors was investigated in bacterial supernatants. SipA and SseJ were used as controls for T3SS-1- and TTSS-2-dependent secretion, respectively. C1 and C2, negative controls (culture medium used for SPI-1 and SPI-2 inducing conditions, respectively). Samples were subjected to SDS-PAGE and tagged proteins were detected by anti-FLAG antibodies. Each lane was loaded with material from approximately 106 c.f.u. bacteria. Data are representative from three independent experiments.