Pseudomonas aeruginosa is a leading cause of nosocomial infections and responsible for significant morbidity and mortality amongst hospitalized patients. Virulence of this bacterium in an animal model of acute pneumonia correlates with in vitro cytotoxicity towards polarized Madin- Darby canine kidney (MDCK) cells. Preliminary results suggest that the newly described type III secretion system of Pseudomonas is necessary for this cytotoxicity and that this system secretes three proteins in addition to exoenzymes S and T: a 73 kDa protein named PepA, a 40 kDa protein, and a 32 kDa protein. We hypothesize that these proteins are virulence factors and will further characterize them. The gene encoding PepA has already been sequenced, and the genes encoding the 40 kDa and 32 kDa proteins will likewise be cloned and sequenced. Mutagenesis studies will be performed to confirm that these proteins are indeed secreted via the type III pathway. Enzymatic assays and confocal immunofluorescence microscopy will be used to determine which, if any, of these proteins are translocated to the cytoplasmic compartment of MDCK cells. Mutants will be made using allelic replacement techniques and used to demonstrate which proteins are necessary for cytotoxicity and virulence in a mouse model of acute pneumonia. Examination of clinical isolates will be performed to determine which of these genes are variable traits and whether their presence correlates with virulence. Promoter fusions and immunoblots will be used to identify environmental conditions that induce expression of type III system genes and activate this secretion pathway. Finally, the role of pili in type III secretion will be examined. Together, these studies will aid in the understanding of the Pseudomonas type III secretion system and suggest targets for therapeutic interventions. Dr. Alan Hauser is in his 22nd month as a post-doctoral fellow in Dr. Joanne Engel's laboratory. He is dedicated to developing into as independent investigator in the field of bacterial pathogenesis. The experiments on Pseudomonas outlined in this proposal will complement his past work on Streptococcus and increase his exposure to numerous experimental techniques. Drs. Engel and Mostov have much experience in the fields of bacterial pathogenesis and cell biology. UCSF is one of the premier biological research centers in the country.