PROJECT SUMMARY. Pathogenic microbes sense increased temperature as they transition from the external environment to the human host. Pseudomonas aeruginosa is an important opportunistic pathogen responsible for community- acquired and ventilator-associated pneumonia and infections after burns or corneal abrasion. In the hospital setting, it mainly infects immunocompromised individuals and is the leading cause of chronic life-threatening lung infections in people living with cystic fibrosis. P. aeruginosa is naturally antibiotic resistant and infections are notoriously difficult to treat. Preventing the establishment of infection necessitates a better understanding of how microbes switch from the external environment to the human host. P. aeruginosa can survive in a wide-range of environments and temperatures, but must go through transcriptional shifts when transitioning between these environments; global regulatory systems used during infection have been studied, but an emphasis has been on transcriptional differences between acute and chronic infections, planktonic lifestyle to the biofilm mode of growth, and regulatory pathways that respond to cell density (quorum-sensing) and nutrient availability. Modulation by temperature, our focus, has been described for a number of bacterial pathogens, in addition to P. aeruginosa, however the regulatory factors responsible have not been well studied. We propose to identify P. aeruginosa regulators that modulate gene expression in response to variations in temperature. To address this, we will focus on one known virulence factor, PrpL, a lysyl-endopeptidase (also known as protease IV, Piv, PIV, PA4175, or PA14_09900). In laboratory strains of P. aeruginosa, prpL is more highly expressed at 22C-28C compared to 37C. Preliminary Results we have generated indicate that the temperature regulation of prpL is at the level of transcription initiation and furthermore that the known regulators of prpL do not control the thermo-responsiveness of this gene. Here, we will identify the regulator that controls prpL expression at different temperatures and determine whether this regulator controls the expression of other genes. We will also address whether this temperature regulation of prpL is a conserved trait in other strains from varied sources. We suggest that understanding the temperature regulation of transcription of prpL will be informative not only for this important virulence factor, but may also potentially shed light on a novel type of regulatory pathway for other thermo-regulated genes important in the establishment of infection. These studies will lay the foundation for future R01 grant applications to further dissect the regulatory mechanism underlying how bacteria respond to temperature, how this promotes survival in various environments, and finally the role of this type of regulation in P. aeruginosa pathogenesis.