Vacuole Lysis By Type III Translocator Proteins

<p>PROJECT SUMMARYBacterial pathogens and their infectious sequelae remain a huge burden on the health care system, sickeningmillions in developed countries and killing tens of millions (often children) in developing countries. Manyclinically relevant bacterial pathogens establish an intracellular niche in order to replicate, survive and/orpersist within the host. These intracellular bacteria either occupy a membrane-bound vacuole or lyse theirnascent phagosome to live freely within the cytosol. The fundamental processes governing intracellular nicheselection are poorly understood. Here we propose to fill this knowledge gap and investigate whether a keyvirulence determinant to Gram-negative bacteria, the type III secretion system (T3SS), directs the intracellularlifestyle of pathogenic bacteria. The T3SS or injectisome, is a complex needle-like nanomachine anchored inthe bacterial membrane that acts as a conduit for the passage of bacterial effectors directly into the host cell.Contact of the needle tip with host cell membranes, specifically the plasma membrane and endocytic bacteria-containing vacuole membrane, triggers the formation of a membrane-spanning translocon pore. Two bacterialproteins, known as translocators, oligomerize to form this pore. We have found that replacing the geneencoding a translocator protein in the vacuolar bacterium, Salmonella enterica serovar Typhimurium (STm),with its ortholog from a cytosolic bacterium, either Shigella flexneri or Chromobacterium violaceum, allowsSTm to proficiently lyse its nascent vacuole and colonize the cytosol. Based upon these findings, wehypothesize that intrinsic properties of the translocator proteins define the efficiency of bacteria-containingvacuole lysis, and therefore the intracellular niche occupied by Gram-negative pathogens. We will test ourhypothesis by pursuing two Specific Aims. First, we will determine whether translocator proteins from cytosolicbacteria have greater intrinsic membrane-destabilizing activity than those from vacuolar bacteria. Here we willgenetically replace translocator proteins in a vacuolar (STm) and cytosolic (C. violaceum) pathogen withorthologs from other members of the Inv/Mxi-Spa T3SS family and measure the effect on bacteria-containingvacuole lysis. We will also construct translocator chimeras to identify functional regions that define theirphagolytic properties. Second, we will identify whether the translocon itself or type III effector activities of thetranslocator proteins account for differential bacteria-containing vacuole lysis. Our proposed studies willspecifically address the role of type III translocators in phagosomal membrane lysis. This will improve ourunderstanding of the pathogenic mechanisms utilized by bacteria to colonize host cells, and could aid in therational design of therapeutics against the T3SS injectisome, which is shared by many clinically relevant Gram-negative pathogens.</p>