Isogenic P‐fimbrial deletion mutants of pyelonephritogenic Escherichia coli: the role of α Gal(1–4)β Gal binding in virulence of a wild‐type strain

Abstract

Escherichia coli strains causing acute pyelonephritis often express multiple fimbrial types and haemolysin, which may contribute to their ability to adhere to, and interact with, kidney epithelial cells. Strain CFT073, a pap+, sfa+, pil+, hly+ pyelonephritis strain, previously established as virulent in the CBA mouse model of ascending urinary tract infection and cytotoxic for cultured human renal epithelial cells, was selected for construction of isogenic strains. From a gene bank of this strain, two distinct copies of the pap operon were isolated. The two P-fimbrial determinants were subcloned into pCVD442, a positive selection suicide vector containing the sacB gene of Bacillus subtilis. Deletion mutations were introduced into each of the two constructs, within papEFG of one operon and papDEFG of the other. Suicide vectors carrying pap deletions were mobilized from E. coli SM10 lambda pir into CFT073 (NalR) and cointegrates were passaged on non-selective medium. The first pap mutation was identified by screening a Southern blot of DNA from sucrose-resistant colonies using a papEFG probe. This mutant retained the MRHA+ phenotype since a second functional copy of pap was still present. A double pap-deletion mutant, UPEC76, confirmed by Southern blotting, was unable to agglutinate human type O erythrocytes or alpha Gal(1-4)beta Gal-coated latex beads. CBA mice (N = 100) were challenged transurethrally with 10(5), 10(6), 10(7), or 10(9) cfu of strains CFT073 or UPEC76. After one week, quantitative cultures of urine, bladder, and kidney were done and histologic changes were examined. No substantive differences in organism concentration or histological findings between parent and mutant were detected in urine, bladder, or kidney at any challenge concentration. We conclude that adherence by P fimbriae of uropathogenic E. coli strain CFT073 plays only a subtle role in the development of acute pyelonephritis in the CBA mouse model.

The osmotic stress response and virulence in pyelonephritis isolates of Escherichia coli: contributions of RpoS, ProP, ProU and other systems The GenBank accession numbers for the DNA sequences of the rpoS loci in E. coli strains HU734 and CFT073 are AF275947 and AF270497, respectively.

“…Introduction of genetic lesions affecting P pili to E. coli HU734 (Hagberg et al, 1983b) and E. coli DS17 (Roberts et al, 1994) reduced their virulence as indicated by the murine and primate models, respectively. Although these and other data clearly establish an important role for P pili as virulence determinants in pyelonephritis, the elimination of P pili did not reduce the virulence of pyelonephritis isolate CFT073 as determined by the murine model (Mobley et al, 1993). These and other observations suggest that E. coli CFT073 is more virulent than E. coli HU734 (Mobley et al, 1993).…”

ibeA, a virulence factor of avian pathogenic Escherichia coli

“…Among the functional similarities, the finding that type 1 pili are expressed in the early steps of avian colibacillosis and that P pili are expressed in the later steps is reminiscent of the finding that the UPEC strain J96 expresses either type 1 pili or P-pili, but not both at the same time, the expression of type 1 pili being inhibited by the PapB protein (Mobley et al, 1993;Pourbakhsh et al, 1997).…”

“…Virulence determinants common to APEC and ExPEC have been identified. They include the aerobactin iron transport system, the K1 capsule and type 1 and P fimbriae (Bahrani-Mougeot et al, 2002;Dho-Moulin & Fairbrother, 1999;Gunther et al, 2002;Hoffman et al, 1999;Mobley et al, 1993;Pourbakhsh et al, 1997;Torres et al, 2001). Vandemaele and colleagues also found a high level of homology between papG sequences, the P pilus adhesin gene from APEC isolates and papG sequences from mammalian E. coli isolates, including human isolates (Vandemaele et al, 2003;Johnson et al, 1997).…”

Salmonella typhimurium Coordinately Regulates FliC Location and Reduces Dendritic Cell Activation and Antigen Presentation to CD4+ T cells

“…Motility was measured 18 h after inoculation to Luria-Bertani motility (0.4% agar) plates. Suicide vector pCVD442 (pir-dependent replicon, Amp R , sacB gene conferring sucrose sensitivity) was used to replace the WT fliC allele on the chromosome as described previously (45). Briefly, plasmid pCVD442 carrying a mutant fliC allele was transferred to FljB-null SL1344 (BC687) by conjugation, and the resulting Amp R Salmonella were then grown in sucrose-containing medium to select for loss of sacB gene and plasmid DNA.…”