THE PROJECT'S BROAD, LONG TERM OBJECTIVES are to clarify the role in acute bacterial cystitis of four defined virulence factors (VFs) of Escherichia coli both, singly and in combination with one another, and to develop an intervention to prevent cystitis by interfering with the function of one of these VFs.
SPECIFIC AIMS are: 1) to refine a standard mouse model of UTI for the study of cystitis; 2) to use molecular methods for in vitro mutagenesis and allelic exchange to create isogenic VF-deficient mutants of a virulent wild-type E. coli urosepsis isolate; 3) to use these isogenic mutants in the mouse model to determine the contribution to cystitis of specific VFs singly and in combination with one another; 4) to purify from natural sources an inhibitor of one of the four VFs (P fimbriae); and 5) to evaluate the ability of this inhibitor to prevent adherence of E. coli to epithelial cells from women with frequent UTI's and to protect mice from experimental cystitis. EXPERIMENTAL DESIGN AND METHODS. Modified inoculation techniques will be used to avoid nonphysiological vesicoureteral reflux and renal trauma in the mouse model of cystitis. A suicide plasmid system will be used to introduce mutagenized VF determinants into a virulent wild-type E. coli strain in exchange for functional wild-type alleles. The virulence of the resulting isogenic mutants will be compared in the mouse model to reveal the importance of each VF and of combined VFs in the pathogenesis of cystitis. The P1-antigen-containing carbohydrate component of pigeon ovomucoid will be isolated using biochemical methods and tested for its ability 1) to block the adherence of P fimbriated E. coli to human urinary and vaginal epithelial cells from women with frequent UTIs and 2) to protect mice from cystitis due to P fimbriated E. coli. THE HEALTH RELATEDNESS OF THE PROJECT lies in the need for better ways to prevent acute bacterial cystitis, a major health problem of women that is responsible for tremendous morbidity and increased health care costs. Efforts to protect women from bacterial cystitis by interfering with E. coli VFs will be furthered by 1) identifying the specific VFs most responsible for cystitis (which would help guide the future development of anti-VF interventions), and 2) developing an affordable and effective anti-adhesin intervention for the prevention of cystitis.
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