The segment of the U.S. population comprised of those 65 years and older is growing most rapidly. Many of these individuals will reside in nursing homes and require urinary catheterization for management of incontinence which leads inevitably to polymicrobial bacteriuria. Proteus mirabilis represents one of the most prevalent and persistent uropathogens in this patient population frequently causing acute pyelonephritis. This species is equipped with a number of putative virulence factors that may contribute to pathogenesis of upper urinary tract infection including urease, fimbriae, flagella, hemolysin, and the ability to invade kidney epithelium. Having demonstrated that the enzyme urease is a critical virulence determinant, the present work will be extended to the molecular mechanisms of pathogenesis by determining the contribution to virulence of fimbriae and flagella as well as understand the synergistic interaction with urease and the secreted hemolysin. To understand the role of putative virulence determinants of P. mirabilis in the development of acute pyelonephritis, the Project outlines methods to: 1) Isolate and sequence the genes encoding P. mirabilis MR/P fimbriae and flagellin determinants; 2) identify and characterize the polypeptides encoded by the MR/P and flagellin determinants; 3) construct separate isogenic strains of P. mirabilis containing specific deletions within MR/P and flagellin gene sequences; 4) determine the effect of specific mutations in MR/P fimbriae, flagellin, urease, and hemolysin on a) in vitro cytotoxicity for, b) adherence to, and c) internalization by cultured human renal epithelial cells; and 5) compare the infectivity and pathogenicity of the P. mirabilis parent strain with MR/P-fimbriae-deficient, flagella-deficient (non-motile), hemolysin-negative, and urease-negative mutants in CBA mouse models of ascending urinary tract infection.
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