ISOGENIC P-FIMBRIAL DELETION MUTANTS OF PYELONEPHRITOGENIC ESCHERICHIA-COLI - THE ROLE OF ALPHA-GAL(1-4)BETA-GAL BINDING IN VIRULENCE OF A WILD-TYPE STRAIN

Escherichia coli strains causing acute pyelonephritis often express mu ltiple fimbrial types and haemolysin, which may contribute to their ab ility to adhere to, and interact with, kidney epithelial cells. Strain CFT073, a pap+, sfa+, pil+, hly+ pyelonephritis strain, previously es tablished as virulent in the CBA mouse model of ascending urinary trac t infection and cytotoxic for cultured human renal epithelial cells, w as 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 se lection suicide vector containing the sacB gene of Bacillus subtilis. Deletion mutations were introduced into each of the two constructs, wi thin papEFG of one operon and papDEFG of the other. Suicide vectors ca rrying pap deletions were mobilized from E. coli SM10 lambda pir into CFT073 (Nal(R)) and cointegrates were passaged on non-selective medium . The first pap mutation was identified by screening a Southern blot o f DNA from sucrose-resistant colonies using a papEFG probe. This mutan t retained the MRHA+ phenotype since a second functional copy of pap w as still present. A double pap-deletion mutant, UPEC76, confirmed by S outhern blotting, was unable to agglutinate human type 0 erythrocytes or alphaGal(1-4)betaGal-coated latex beads. CBA mice (N = 100) were ch allenged transurethrally with 10(5), 10(6), 10(7), or 10(9) cfu of str ains CFT073 or UPEC76. After one week, quantitative cultures of urine, bladder, and kidney were done and histologic changes were examined. N o substantive differences in organism concentration or histological fi ndings between parent and mutant were detected in urine, bladder, or k idney at any challenge concentration. We conclude that adherence by P fimbriae of uropathogenic E. coli strain CFT073 plays only a subtle ro le in the development of acute pyelonephritis in the CBA mouse model.