Molecular characterization of the Escherichia coli asymptomatic bacteriuria strain 83972: the taming of a pathogen

Abstract

Escherichia coli 83972 is a clinical asymptomatia bacteriuric isolate that is able to colonize the human urinary bladder without inducing an immune response. Here we demonstrate that one of the mechanisms by which this strain has become attenuated is through the mutation of its genes encoding type 1 and P fimbriae.

FIG. 1.

(A) Physical map of the fim gene cluster from E. coli MG1655 indicating the arrangement of the genes, the position of relevant restriction enzyme sites (E, EcoRI; EV, EcoRV), and the region covered by each of the probes. (B) Southern blot analysis of total genomic DNA from E. coli MG1655 and 83972 digested to completion with either EcoRI or EcoRV and probed with the fimE gene, the fimA, fimB, and fimC genes, and the fimH gene (as indicated). The sizes of the hybridizing fragments are indicated in kilobase pairs. (C) Structure of the fim locus in E. coli 83972 indicating the precise start and end points of the deletion between fimB and fimD.

FIG. 2.

(A) Transmission electron micrographs of E. coli 83972 grown (i) in LB broth and (ii) on LB agar. Almost all cells harvested after growth on agar produced P fimbriae, while less than 5% of cells grown as liquid cultures produced any fimbriae. This may explain previous observations that E. coli 83972 does not produce fimbriae. (B) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified fimbrial proteins from E. coli 83972 (lane 2). The N-terminal amino acid sequence of the PapA fimbrial protein is indicated in brackets. Molecular size markers (lane 1) are 250, 98, 64, 50, 36, 30, 16, and 6 kDa. (C) Corresponding Western blot analysis of the same purified fimbrial preparation using a PapG-specific polyclonal antiserum indicating the expression of the PapG adhesin. (D) Physical map of the pap gene cluster from E. coli 83972 indicating the arrangement of the genes and the amino acid identity of each of the predicted proteins with the respective protein from E. coli CFT073. The predicted translational product of the papA gene is identical to the first 10 residues identified by N-terminal sequencing. PapA belongs to the F14 allele group, while PapG belongs to the class III allele group (nucleotide accession number, DQ010312).

FIG. 3.

Agglutination of human type A RBC (A) and binding to human uroepithelial cells (B) by (i) E. coli 83972, (ii) E. coli 83972 (pDD3; contains all pap genes from UPEC strain J96 except papG), and (iii) E. coli 83972 (pDD4; papG gene from J96). Functional activity was observed only in the presence of the plasmid containing the papG gene from E. coli J96. Shown are the binding phenotypes under low (10×) (A) and high (63×) (B) magnifications.

FIG. 4.

Alignment of the amino acid sequence deduced from the nucleotide sequence of the E. coli 83972 papG gene with the closely related sequence of the uropathogenic E. coli 536 prfG gene. Residues identical to those of the E. coli 83972 papG gene are indicated by dots; gaps introduced into the alignment are indicated by dashed lines. The residues conserved between PapG I, II, and III alleles are shaded.