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. A fundamental challenge in developing antibiotics is that many pathogens replicate inside host cells rendering them inaccessible to antimicrobial agents. Critical processes for pathogen growth in host cells represent the most promising new targets for therapeutic intervention. Most bacterial pathogens that grow inside host cells do so in a specialized compartment called the replication vacuole. The formation and maintenance of a replication vacuole are paramount to bacterial survival and growth as it provides a source of nutrients and protection against host surveillance systems that detect and eliminate pathogens. Disrupting this process would thus limit bacterial burden and enabling pathogen killing by the host. Despite the crucial role of replication vacuole biogenesis, the mechanisms responsible are poorly understood. A major obstacle in determining how a pathogen generates and sustains a replication vacuole is redundancy. Bacteria form a replication vacuole by secreting bacterial proteins into the host cell to modulate a variety of host biological processes. While the secretion machinery itself is essential for disease, individual secreted proteins are dispensable. This often occurs because the activity of one secreted protein can compensate for the loss of another. However, many pathogens also employ multiple strategies for generating a replication vacuole. Redundancy makes it very difficult to define the roles of individual secreted proteins in disease and thus, identify promising targets for new antibiotics. We have developed a genetic screening technique to resolve redundancy amongst secreted proteins of the bacterial pathogen Legionella, the cause of a life-threatening pneumonia. We have defined sets of secreted proteins that contribute to the same strategy in replication vacuole formation and separate but redundant strategies used to accomplish this task. The goal of this research is to determine how individual components that constitute a single strategy promote replication vacuole formation, how independent strategies contribute to this process and how they compensate for one another. This work will provide unprecedented insight into a critical event determining the outcome of an infection and a means to develop new strategies to prevent and treat disease.