PROJECT SUMMARY Understanding bacterial physiology during host infection is critical for designing effective prevention and treatment strategies. Traditionally our knowledge of bacterial pathogenesis has been derived from experiments conducted in standard laboratory models. Due to the limitations of these models in mimicking human infection and the fact that bacterial physiology is largely shaped by the environment, important features associated with bacterial pathogenesis can be overlooked in these studies. In this proposal, we aim to understand a novel gene expression signature of Pseudomonas aeruginosa (Pa) during human infection. Pa is a prominent causative agent of a wide variety of acute and chronic infections in humans. Through comparative transcriptomic analysis, we recently identified an uncharacterized gene (PA1414) that is often the most highly expressed Pa gene during human infection. However, no function has yet been attributed to this gene. Here we provide multiple lines of evidence demonstrating that: (1) PA1414 can be induced by host-associated environmental signals such as oxygen deprivation, which in turn promotes Pa biofilm formation. (2) PA1414 encodes a novel small RNA (sRNA). (3) induction of PA1414 promotes a localized non-disseminating chronic infection in a mouse wound model. Combined with the fact that PA1414 is restricted to the taxonomic group of Pa, we hypothesize that this sRNA is a key player in governing the acute-to-chronic infection switch in Pa. Our hypothesis will be addressed by experiments proposed in two specific aims. First, we will define how PA1414 induction impacts on Pa gene expression to alter its lifestyle during infection. Second, we will elucidate the mechanism of action of the PA1414- encoded sRNA and uncover its molecular targets. The information generated from this study will fill a critical void in our understanding of Pa physiology during human infection. In addition, considering that PA1414 is a well- conserved Pa-specific gene and the encoded sRNA is highly abundant in humans, this work will lay the foundation for future development of novel therapeutic strategies targeting Pa infection.