Escherichia coli is the most common cause of urinary tract infections (UTIs) in otherwise healthy individuals. The uropathogenic E. coli isolates that cause these infections are characteristically of certain serotypes and express P fimbriae, alpha- hemolysin, and aerobactin. Recent findings from Europe indicate that the cytotoxic necrotizing factor type 1 (CNF1), a toxin little studied in the U.S., is also frequently produced by uropathogenic E. coli. CNF1 and the immunologically related CNF2 are 110-115 kDa polypeptides that induce multinucleation and actin polymerization in eukaryotic cells and necrosis and death of some animals. We recently cloned and sequenced cnf2 and found that the N-terminal half of CNF2 is homologous to the dermonecrotic toxin of Pasteurella multocida, a toxin that mediates the pathology of progressive rhinitis in pigs. We also observed that CNF2 modifies a small GTP-binding protein designated Rho that is involved in stress fiber assembly. The long term goals of this proposal are to examine the role that CNF1 plays in the virulence of uropathogenic E. coli and to determine the precise mechanism by which CNF1 modifies Rho.
The specific aims are designed to achieve these goals are to: 1) analyze the role of CNF1 in the pathogenicity of E. coli strain with J96 by constructing a cnf1-negative derivative of that strain and comparing the mutant with the wild-type for virulence in a mouse model of ascending UTI and in human kidney and bladder cells; 2) investigate the nature of the chemical modification of Rho by CNF1 and clarify how that modification leads to actin polymerization; 3) dissect the relationship between the structure of CNF1 and its function by generating a set of mutant CNF1s through deletion, regionally-directed, and site-specific mutagenesis and preparing monoclonal and monospecific antibodies as probes for toxin structural integrity; 4) attempt to identify the functional receptor for CNF1.
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