PROJECT SUMMARY/ABSTRACT The bacterial pathogen Pseudomonas aeruginosa is a frequent cause of nosocomial infections and life- threatening lung infections in people with the genetic disorder cystic fibrosis. P. aeruginosa infections are difficult and costly to treat due to inherent and acquired antibiotic resistance, underscoring the need for new treatments. A better understanding of how P. aeruginosa causes infections will be instrumental for this. P. aeruginosa survives in a human host and causes disease by producing multiple virulence factors. One such virulence factor, the serine protease PrpL, causes severe tissue damage and degrades components of the immune system. Expression of prpL is regulated by two environmental factors experienced by P. aeruginosa during an infection: low iron availability and temperature. Expression of prpL is upregulated by low iron availability through a known mechanism and downregulated at 37C compared to 25C through an unknown mechanism. The Goldberg Lab has found that the transcription factors MvaT/MvaU and LasR are required for this prpL thermoregulation, but the mechanism of prpL thermoregulation remains to be fully elucidated. How iron and temperature coregulate prpL and the importance of this for P. aeruginosa virulence is also unknown. Given that low iron availability upregulates prpL while 37C conditions downregulate it, iron and temperature may balance PrpL production and be important for P. aeruginosa virulence. Based on these findings, I hypothesize that thermoregulation of prpL occurs through temperature-dependent binding of MvaT/MvaU and LasR to the prpL promoter and that iron/temperature coregulation of prpL is important for P. aeruginosa virulence. I will test this hypothesis using genetic and biochemical approaches, and an animal model of infection. In Aim 1, I will define the prpL thermoregulatory mechanism by determining if MvaT/MvaU and LasR positively or negatively regulate prpL transcription at 25C and 37C, and by characterizing the impact of temperature on the binding of MvaT/MvaU and LasR to the prpL promoter. In Aim 2, I will determine how temperatures balances production and activity of PrpL by measuring prpL gene expression, the amount of PrpL secreted, and the total enzymatic activity of secreted PrpL across a 20C-42C range. In Aim 3, I will characterize the role of prpL iron/temperature coregulation in P. aeruginosa virulence by infecting larvae of the moth Galleria mellonella. Larvae will be infected with a P. aeruginosa strain in which prpL is regulated by low iron availability and a strain in which prpL is not, and larvae from both groups will be housed at 25C and 37C to measure how iron and temperature coregulation of prpL affects P. aeruginosa virulence. A mechanistic study of prpL thermoregulation will address a major gap in the knowledge of virulence factor thermoregulation in P. aeruginosa. Understanding how PrpL is regulated by temperature and iron to facilitate P. aeru...