| Bacteria have evolved many systems to secrete proteins that are involved in pathogenesis. By evolving targeted delivery systems, it is thought that these pathogens have the advantage of direct (and undiluted) delivery of effectors to a specific cellular location where they can intersect and influence host mechanisms. Several plant and animal Gram-negative bacterial pathogens have a specialized secretion system, called type III (T3SS), to deliver effectors directly from their cytoplasm into host cells, meaning that those effectors cross several biochemically distinct barriers, including the bacterial inner membrane, peptidoglycan layer, and outer membrane as well as the host plasma membrane and even potentially intracellular host membranes. These type III bacterial effector proteins are injected into the host cell where they affect a wide variety of events from promoting phagocytosis, to preventing phagocytosis, to subverting other host signalling pathways. We explored the mechanism by which enteropathogenic Escherichia coli (EPEC) inserts its receptor Tir into host cell membranes, a process which triggers intimate attachment of the bacterium to the host through Tir's outer membrane ligand intimin. We used a mechanical method of cell lysis and ultracentrifugation to fractionate infected HeLa cells to investigate the biology and biochemistry of Tir delivery and translocation, and found that the T3SS was essential for Tir delivery. This method demonstrated that the translocation of Tir into the host cell membrane requires its transmembrane domains, but not tyrosine-phosphorylation or binding to Tir's ligand, intimin. Bacterial fractionation revealed that the type III effectors Tir and EspB required a complete type III apparatus for any degree of export by EPEC indicating a continuous channel. Three type III components were studied and localized to the cytoplasm/associated with the inner membrane (EscN), inner membrane (EscV) and outer membrane (EscC). Remarkably, localization of the EscC secretin to the outer membrane was altered in escV and escN mutants but not in the escF needle protein mutant, suggesting that correct insertion and function of EscC secretin in the outer membrane depends not only on the sec-dependent export pathway but also other type III apparatus components. As effectors like Tir exit the bacteria through the T3SS, they should interact with components of the apparatus. To examine this hypothesis, we performed experiments to determine if there were any interactions between Tir and its chaperone CesT, and EPEC's type III apparatus. By affinity chromatography, gel overlay and immunoprecipitation, we have found that Tir and CesT interact with the type III ATPase EscN. Tir is not necessary for CesT and EscN interactions, and conversely EscN binds Tir specifically without its chaperone CesT. (Abstract shortened by UMI.)... |
