Uropathogenic Escherichia coli
Fimbriae and Motility StudiesSecreted Autotransporter Studies (Sat, Pic, and Tsh)|
Genomic Studies on Uropathogenic E. coliReferences
Secreted Autotransporter Studies (Sat, Pic, and Tsh)
7. Autotransporter genes pic and tsh are associated with E. coli strains that cause acute pyelonephritis and are expressed during UTI. We identified two chromosomal open reading frames in UPEC strain CFT073 which are highly homologous to serine protease autotransporters Pic and Tsh. Both cloned determinants were correlated with the presence of 105- to 110-kDa proteins in the culture supernatants. Furthermore, in cellular fractionation experiments, 30-kDa ß-barrel portions of the autotransporter homologues were identified in the outer membrane. Furthermore, Pic-containing culture supernatants have serine protease activity. In reverse transcription-PCR analyses, pic and tsh were expressed by bacteria isolated from urine of transurethrally infected mice. The tsh determinant was identified in 63% of our clinical UPEC strain isolates (n = 87) and in 33% of fecal strains (n = 27), whereas pic was present in 31% of the pyelonephritis (n = 67) and 7% of the fecal strains. There was no significant correlation between cystitis strains (n = 20) and the pic determinant.
Heimer, Susan R., David A. Rasko, C. Virginia Lockatell, David E. Johnson, and Harry L.T. Mobley. 2004. Autotransporter genes pic and tsh are associated with E. coli strains causing acute pyelonephritis and expressed during UTI. Infect. Immun. 72:593-597.
8. Sat, the secreted autotransporter toxin of uropathogenic E. coli, is a vacuolating cytotoxin for bladder and kidney epithelial cells. The secreted autotransporter toxin (Sat) of UPEC exhibits cytopathic activity upon incubation with bladder and kidney epithelial cells. Sat elicited elongation of cells and apparent loosening of cellular junctions upon incubation with Vero kidney cells. Additionally, incubation with Sat triggered significant vacuolation within the cytoplasm of both human bladder (UM-UC-3) and kidney (HEK293) cell lines. Following transurethral infection of CBA mice with a sat mutant, no reduction of CFU in urine, bladder, or kidney tissue was seen compared to that in mice infected with wild-type E. coli CFT073. However, significant histological changes were observed within the kidneys of mice infected with wild-type E. coli CFT073, including dissolution of the glomerular membrane and vacuolation of proximal tubule cells. Such damage was not observed in kidney sections of mice infected with a Sat-deficient mutant. These results indicate that Sat elicits defined damage to kidney epithelium during upper UTI and thus contributes to pathogenesis of UTI.
Guyer, D.M., Radulovic S., Jones, F.E., and H.L.T. Mobley. 2002. Sat, the Secreted Autotransporter Toxin of Uropathogenic E. coli, is a Vacuolating Cytotoxin for Bladder and Kidney Epithelial Cells. Infect. Immun. 70:4539-4546.
9. Protease activity, secretion, cell entry, cytotoxicity, and cellular targets of secreted autotransporter toxin (Sat) of uropathogenic E. coli. Sat is found predominantly in uropathogenic E. coli, is a member of the SPATE (Serine Protease Autotransporters of Enterobacteriaceae) family and exhibits serine protease activity and cytopathic activity on various cell-types. To assess the contribution of the serine protease active site to the mechanism of action of Sat, mutations were made in the first (Ser256Ile), the second (Ser258Ala), or both serine residues (Ser256Ile/Ser258Ala) within this motif by site-directed mutagenesis. Nomutation affected secretion of the mature passenger domain or release into the supernatant. Mutations in the first or both serines reduced protease activity to background levels (p < 0.001 ); a single mutation in the second serine reduced activity by 60% compared to wild-type (p < 0.001 ). After reverting the mutation Ser256Ile back to wild-type (Ile256Ser), we confirmed Ser256 as the catalytically active serine. Indeed, the Ser256Ile mutation abrogates the cytotoxicity of Sat on human bladder (UM-UC-3) and kidney (HEK293) epithelial cells, characterized by rounding, elongation, and high level of cell detachment. Moreover, Sat, but not its protease-negative mutant, mediates contraction of the cytoskeleton and loss of actin as well as degradation of specific membrane (fodrin and leukocyte function-associated molecule-1) and nuclear (microtubule-associated proteins, LIM domain only protein 7, Rap GTPase-activating protein, and Poly(ADP-ribose) polymerase) proteins in vitro. Sat was internalized by relevant host cells and localized to the cytoskeletal fraction where specific cleavable target proteins reside.
Maroncle, Nathalie M., Kelsey E. Sivick, Rebecca Brady, Faye-Ellen Stokes, and Harry L.T. Mobley. 2006. Protease Activity, Secretion, Cell Entry, Cytotoxicity, and Cellular Targets of Secreted Autotransporter Toxin of uropathogenic Escherichia coli. Infect. Immun. 74:6124-6134 .