My laboratory is interested understanding and exploiting the complex interplay of microbial pathogens with eukaryotic cells. To that end, we have investigated the key processes of microbial attachment and entry, intracellular survival, and host cell injury in the context of two important human pathogens, Pseudomonas aeruginosa and Chlamydia trachomatis . Each of these microorganisms has developed a unique strategy for successful survival that involves subverting and exploiting host cell pathways. Dissecting these processes will allow the development of new diagnostics, therapeutics, and vaccines and will provide a unique window into eukaryotic cell biology. Part of the lab focuses on how P. aeruginosa, an opportunistic pathogen of man, injures epithelial cells. The common element underlying these opportunistic infections is the ability of P. aeruginosa to colonize and further damage injured epithelium surfaces, leading to local tissue damage and dissemination to distant organs. We carried out a novel genetic screen to identify mutants that are deficient in injuring cells in vitro. This analysis has revealed that pili and products of a novel secretion system (type III secretion), are required for host cell injury by P. aeruginosa. In particular, we have identified three new type III-secreted virulence factors. These include a novel cytotoxin, ExoU, a bacterially-encoded apoptosis inducing factor, and a bacterially-encoded anti-internalization factor, ExoT, that acts as a GTPase activating protein (GAP) for Rho family GTPases. Current and future directions include further characterizing and determining the mechanism of action of these new virulence factors. For example, we have found that P. aeruginosa activates Rho upon entry and that the GAP activity of ExoT prevents bacterial internalization. Moreover, this entry pathway is downregulated as epithelial cells polarize and is upregulated during wound healing. These observations explain the relative resistance of intact epithelium to injury by P. aeruginosa and the susceptibility of injured epithelium to colonization and injury. These studies will expand our knowledge of bacterial pathogenesis, apoptosis, and will identify new targets for drug and vaccine development. C. trachomatis is the leading cause of venereal disease and preventable sterility in the United States and the most common cause of non-congenital blindness in third world countries. It replicates via a unique developmental cycle involving the serial alternation of two distinct forms sequestered within a membrane bound compartment (the "vacuole") in the cytoplasm of the infected epithelial cell. While this organism presents major experimental challenges, its importance as a human pathogen merits overcoming the difficulty in manipulating and growing the bacteria in the laboratory. Our work has encompassed several aspects of chlamydial pathogenesis that are unique to this obligate intracellular bacterium. Currently we are studying the mechanism of entry. and the role of the actin cytoskeleton in this process. As well, we have characterized the chlamydial vacuole in order to learn how it avoids fusion with the host cell lysosomes, a key feature of the ability of C. trachomatis to survive intracellularly. We have shown that sphingolipid precursors that are trafficked from the Trans Golgi Network to the C. trachomatis vacuole are required for intracellular growth. We are currently identifying the cellular pathways that Chlamydia subverts in order to acquire host cell sphingolipids. The latter is a prime example of how the study of microbial pathogens provides new tools and approaches to the study of eukaryotic cell biology.