Cytotoxic necrotizing factor type 1 (CNF1) is a member of a family of bacterial toxins that target the Rho family of small GTP-binding proteins in mammalian cells. CNF1 deamidates a single glutamine residue in RhoA, Cdc42, and Rac1 but not in Ras. This deamidation results in the constitutive activation of these GTPases which can trigger actin stress fiber formation, multinucleation, or cell death, depending on the target cell and dose of toxin. CNF1 is frequently produced by Escherichia coli strains that cause urinary tract infections (UTIs), such as cystitis, prostatitis, and pyelonephritis. In support of this epidemiological connection, we recently showed that CNF1 not only induces apoptosis in 5637 human uroepithelial cells but also provides a growth advantage to uropathogenic E. coli (UPEC) in a mouse model of ascending UTI when compared to CNF1-negative isogenic mutants. Additionally, we found that CNF1 enhances the degree of inflammation and resulting tissue damage in bladders of infected mice and in prostates of rats challenged intraurethrally with CNF1-producing UPEC. Finally, we discovered that CNF1- producing UPEC survive better than CNF1-negative isogenic mutants in the presence of human polymorphonuclear leukocytes (PMNs). Taken together, these findings have led us to propose the following hypothesis. CNF1 enhances the pathogenicity of UPEC by: i.) promoting uroepithelial cell shedding; ii.) evoking a large influx of PMNs while providing toxin-producing E. coli protection against PMN-mediated killing, and; iii.) facilitating deeper invasion of the bladder or prostate by the infecting strain.
The specific aims designed to test this theory are to: 1.) further define the role that CNF1 plays in the pathogenesis of UPEC-mediated cystitis in the mouse and prostatitis in the rat by analyzing the interaction of CNF1 or CNF1-expressing UPEC with PMNs from these animals and by defining the CNF1-mediated cytokine response that evokes PMN influx; 2) investigate the cellular and cytokine responses of a human bladder organoid to CNF1 or a CNF1-producing UPEC strain; 3.) identify the functional receptor for CNF1 by sequential biochemical and molecular approaches, and; 4.) continue to evaluate CNF1 structure and function by characterizing the CNF1 epitopes recognized by neutralizing monoclonal antibodies and by analyzing chimeric molecules comprised of portions of CNF1 and the related toxins CNF2, Pasteurella multocida toxin, and the Bordetella dermonecrotic toxin.
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