PROJECT SUMMARY
Bacterial pathogens and their infectious sequelae remain a huge burden on the health care system, sickening
millions in developed countries and killing tens of millions (often children) in developing countries. Many
clinically relevant bacterial pathogens establish an intracellular niche in order to replicate, survive and/or
persist within the host. These intracellular bacteria either occupy a membrane-bound vacuole or lyse their
nascent phagosome to live freely within the cytosol. The fundamental processes governing intracellular niche
selection are poorly understood. Here we propose to fill this knowledge gap and investigate whether a key
virulence determinant to Gram-negative bacteria, the type III secretion system (T3SS), directs the intracellular
lifestyle of pathogenic bacteria. The T3SS or injectisome, is a complex needle-like nanomachine anchored in
the 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 bacterial
proteins, known as translocators, oligomerize to form this pore. We have found that replacing the gene
encoding 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, allows
STm to proficiently lyse its nascent vacuole and colonize the cytosol. Based upon these findings, we
hypothesize that intrinsic properties of the translocator proteins define the efficiency of bacteria-containing
vacuole lysis, and therefore the intracellular niche occupied by Gram-negative pathogens. We will test our
hypothesis by pursuing two Specific Aims. First, we will determine whether translocator proteins from cytosolic
bacteria have greater intrinsic membrane-destabilizing activity than those from vacuolar bacteria. Here we will
genetically replace translocator proteins in a vacuolar (STm) and cytosolic (C. violaceum) pathogen with
orthologs from other members of the Inv/Mxi-Spa T3SS family and measure the effect on bacteria-containing
vacuole lysis. We will also construct translocator chimeras to identify functional regions that define their
phagolytic properties. Second, we will identify whether the translocon itself or type III effector activities of the
translocator proteins account for differential bacteria-containing vacuole lysis. Our proposed studies will
specifically address the role of type III translocators in phagosomal membrane lysis. This will improve our
understanding of the pathogenic mechanisms utilized by bacteria to colonize host cells, and could aid in the
rational design of therapeutics against the T3SS injectisome, which is shared by many clinically relevant Gram-
negative pathogens.