Seeinstructions): Pneumonic plague is highly lethal in humans, and the agent of this disease, Y. pestis, is considered a major threat as a biological weapon. Current strategies to prevent pneumonic plague, such as the use of vaccines or antibiotics, are non-effective or could be bypassed by genetic manipulation of the pathogen. A better understanding of Y. peso's pathogenesis is needed to guide the development of novel countermeasures. Y. pestis is a facultative intracellular pathogen that can survive and replicate in macrophages. Survival of Y. pestis in macrophages plays an important role in pathogenesis, most likely during the early stages of infection. Although much remains to be learned about the mechanism of Y. peso's survival in macrophages, we recently identified several bacterial genes important for intracellular survival, have determined that Y. pestis inhabits a novel phagosomal compartment in macrophages, called the Yers/n/a-containing vacuole (YCV), and shown the ability of Y. pestis to survive in activated macrophages. The YCV acquires markers of late endosomes and autophagosomes, but fails to acidify to the normal levels of a phagolysosome. We hypothesize 1) that Y. pestis actively prevents acidification of the YCV, possibly by direct inactivation of the vacuolar ATPase (vATPase); and 2) that blocking acidification is essential for YCV formation and survival of Y. pestis in macrophages. To further elucidate the mechanism of Y. pestis survival in macrophages, the following aims are proposed. 1) Genetic approaches will be used to identify Y. pestis genes that are required for intracellular proliferation (rip)and for blocking YCV acidification in macrophages. The proteins encoded by rip genes will be studied using molecular, biochemical and immunological techniques to reveal the underlying bacterial mechanism for YCV formation. 2) The cellular basis for YCV formation will be investigated using immunological or chemical probes on infected macrophages or purified YCVs, to better characterize the environment of YCV, and to determine how the activity of the vATPase is modulated. The contribution of host proteins to YCV formation will be explored by the use of gene deficient macrophages. 3) mice will be infected by the pulmonary route with rip mutants generated in Aim 1. Mouse survival and bacterial growth in organs will be measured to determine how intracellular survival contributes to virulence. Conditional expression of rip genes will be used to test the hypothesis that survival of Y. pestis in macrophages is critical for virulence at early stages of pneumonic plague. The studies proposed here will foster the development of new strategies to prevent or treat pneumonic plague in humans. RELEVANCE (See instructions): Plague is a highly virulent disease in humans and is considered a major threat as a biological weapon. Current strategies to prevent pneumonic plague, such as the use of vaccines or antibiotics, are non-effective or could be bypassed by intentional genetic manipulation of the pathogen. The studies proposed here will foster the development of new strategies to prevent or treat pneumonic plague in the human population.