The densitometry values are averages from three independent experiments and are expressed as a ratio of CesT/EscJ signals as assayed by Quantity One software. A dependent, match paired student’s t test was used to assess statistical significance between values (denoted by an asterisk). A representative immunoblot from the experiments is shown. (B) Sucrose density gradient fractionation of membrane preparations from the indicated strains. EscJ and intimin are known inner and outer membrane proteins and their immune-detection served to indicate fractions enriched for inner and outer membranes separated upon ultracentrifugation. Note the altered distribution of CesT in the

presence Selleck 4SC-202 of EscU(N262A) and EscU(P263A). Figure 6 EscU or EscU variants from EPEC lysates do not co-purify with immunoprecipitated CesT. Cell lysates were generated from the indicated bacterial strains and exposed to anti-CesT antibodies in a co-immunoprecipitation experiment. The lysate inputs were probed with the indicated antibodies (top panel). Anti-RNA polymerase antibodies were used to detect RNA polymerase amounts within the lysates which are expected to be equivalent. The elution fractions were probed with the indicated antibodies.

tir and cesT null mutants were included as control strains in the experiment. Note that Tir is unstable in the absence of CesT and therefore was not detected in the elution APR-246 fraction. The lane designations apply to all the panels. Taken together, these data indicate that total CesT membrane levels were not statistically different for EscU variant expressing strains, although the nature of CesT association with the inner membrane was altered in the absence ID-8 or with limited EscU auto-cleavage. CesT retained normal effector binding function in the absence of EscU auto-cleavage and EscU did not co-immunoprecipitate with CesT. Discussion The T3SS is one of the most complex secretory systems in prokaryotic biology,

being composed of at least 10 conserved protein components [17]. The YscU/FlhB proteins have been studied in considerable detail, although the phenotypes Akt inhibitor associated with secretion are highly variable among bacteria and even within the same species [24, 30–32, 49, 50]. The intein-like auto-cleavage mechanism of EscU was previously elucidated through protein crystallography studies. It was proposed that EscU auto-cleavage likely results in an interface for important protein interactions for type III secretion. In this study, we provide evidence to suggest that EscU auto-cleavage supports efficient type III effector translocation. We also observed that the multicargo type III chaperone CesT was less efficiently associated with the inner membrane (Figure 6), which may partly explain the deficiency in type III effector translocation.