PROJECT SUMMARY Infectious disease is a major threat to human health worldwide. The emergence of antibiotic resistance pathogens necessitates the development of new drugs to treat infection. Virulence factors that pathogens employ to promote their survival and growth in host cells represent promising targets for therapeutic intervention. Most bacterial pathogens employ sophisticated secretion systems to translocate bacterial proteins called effectors into the host cell to modulate host cell processes for their own benefit. The bacterial pathogen Legionella, which causes life-threatening pneumonia in humans, has one of the largest repertoires of effector proteins described to date. Defects in secretion have pleiotropic effects on Legionella pathogenesis as they prevent both the formation of a replication permissive vacuole and bacterial escape from a degradative lysosomal compartment. This limits bacterial burden by preventing their proliferation while enabling pathogen killing by the host cell. Despite the crucial role of the secretion system itself, the contributions of individual effectors and the critical events responsible for Legionella pathogenesis remain poorly understood. A major obstacle in defining how Legionella causes disease is the lack virulence defects associated with loss of individual effectors. This can result from redundancy between effectors or the analysis of a limited set of host cell types and/or virulence traits. Moreover, effectors are typically studied in isolation despite the coordinated activities of many effectors during infection, with some modulating parallel or complementary host pathways, some functioning in pairs as on/off switches and some regulating the activity of other effectors. Defining critical events responsible for disease and the effectors governing these processes requires a high throughput strategy to simultaneously analyze the entire collection of effectors under a variety of conditions. The goal of the proposed work is to generate a library L. pneumophila mutants representing all 384 effectors and, in the process, a set of genetic tools to systematically interrogate effector regulation and function. Collectively, these reagents will provide a foundation for numerous key avenues of investigation and an invaluable resource to the research community. Such a systems level approach to studying effectors is not only unprecedented but paramount to characterizing a critical set of virulence factors in Legionella pathogenesis and thus the development of new strategies to prevent and treat disease.