Among the aged, E.coli is the most common organism causing acute pyelonephritis (pyelo) and bacteremia in the community, hospital, and nursing home. Fifty percent of pyelo E. coli are hemolytic versus 5-10% of fecal E. coli. The pathogenesis of hemolytic E. coli infection is obscure. This project's overall hypothesis is that E. coli fimbriae bind to the target cell cytoplasmic membrane, thus allowing effective delivery of secreted hemolysin to the membrane with subsequent pore formation, ion and molecular flux, and cell death. Resources used to test this hypothesis will include: 1) a prototypic hemolytic E. coli, strain CFT073, virulent in humans and mice; and 2) a putative target cell for pyelo, the human renal proximal tubular epithelial cell (HRPTEC). CFT073 binds in large numbers to HRPTEC by fimbriae and is cytotoxic. By transposon mutagenesis, the cytotoxicity was demonstrated to be mediated by hemolysin. This Project will test the following hypotheses: 1) That the HRPTEC-binding fimbriae enhance the cytotoxicity of hemolysin. Deletion for transposon mutagenesis of CFT073 to the four permutations of +/- hemolysin and +/- HRPTEC-binding fimbriae will be performed. The ability of CFT073 and these three mutants to cause cytotoxicity to HRPTEC and pyelo in the mouse model will be compared. 2) That the cytotoxicity of hemolytic e. coli varies among human proximal tubular, collecting duct, and pelvic epithelial cells from the same human donors. A target cell for pyelo E. coli must be an epithelial cell behind which is the renal interstitium. These three are such cells and will be collected from each of 10 donors and exposed to CFT073, its hemolysin- negative mutant, and a fecal strain; adherence and cytotoxicity will be compared. 3) That the cytotoxicity of HRPTEC by hemolytic E. coli is greater in cells from aged than from younger humans. Membrane alterations are among the changes of aging. Preliminary findings suggest an increased susceptibility of cells from older than younger donors. This project will examine CFT073 adherence and cytotoxicity to HRPTEC of more than 100 donors spanning nine decades of life. Detection of change as little as one percentage point per decade and of the degree of contribution of adherence to cytotoxicity by age will be possible. 4) That hemolysin kills HRPTEC by formation of transmembrane pores with subsequent fluxes of ions and small molecules. Ion fluxes in HRPTEC as a result of hemolysin-induced pore formation will be measured radiometrically and spectrophotometrically with fluorescent probes sensitive to nanomolar concentrations of ions to determine if fluxes occur and precede cell death.
Showing the most recent 10 out of 59 publications
