? Urinary tract infections (UTIs) are the second most common infectious disease in the United States and result in medical expenditures exceeding one billion dollars annually. Despite the prevalence of the UTIs current medical regimens are unable to eradicate infections or prevent their occurrence. E. coli cause 85% of UTIs and are able to thrive within host cells, but the mechanisms allowing for internalization and survival are unclear. Recently, we have shown that during acute infections uropathogenic E. coli (UPEC) form intracellular bacterial communities (IBCs), which provide a survival advantage and are a source for ongoing disease. Moreover, the IBCs are present in the cytoplasm of the superficial umbrella cells, not membrane bound vacuoles as seen in cancer cell monolayers. We wish to understand how UPEC enter superficial umbrella cells, localize to the cytoplasm to form IBCs and adapt to survive long term within the bladder. This proposal will use a novel in vitro model of IBCs in conjunction with our well defined murine cystitis model to study bacterial entry in differentiated umbrella cells and identify processes, such as uroplakin recycling, cytoskeletal rearrangement, membrane trafficking, protein synthesis and cell differentiation required for IBC formation. Routine microbiology and invasion assays will be used along with immunohistochemistry and fluorescent confocal microscopic techniques to track bacteria internalization, and their localization will be confirmed by electron microscopy. The FimH adhesin, vital for binding and internalization, will specifically be assessed for its ability to target bacterial delivery to the umbrella cell cytoplasm. We will use co-infection of UPEC and K-12 bacteria to determine how UPEC alter the urothelium to improve bacterial survival. Finally, we will use targeted inactivation of a quorum sensing signaling pathway to determine its role in IBC maturation and dispersal. The specific knowledge gained from these studies will enhance our understanding of bacterial pathogenesis, intracellular trafficking in umbrella cells and communication within bacterial communities. Using the in vivo mouse model, I have already made contributions to the understanding of UTI pathogenesis, and I have developed an in vitro model of IBC formation that will greatly enhance our ability to dissect the early stages of UPEC infection. My ongoing relationship with Dr. Scott Hultgren, a recognized leader in this field, will provide the guidance and support needed for continued success. I am currently an instructor in Pediatric Gastroenterology with greater than75% effort committed to research. Over the next 5 years, I will develop a research course focusing on the host-pathogen interactions of intestinal flora in the urinary and gastrointestinal tracts that will be the basis of an NIH RO1 application and future independent investigations. The services and infrastructure at Washington University are of the highest caliber and together with the leadership of senior faculty, will provide an ideal environment for a young investigator to establish a solid foundation in academic science. ? ?
