Genomic islands of uropathogenic Escherichia coli contribute to virulence

Abstract

Uropathogenic Escherichia coli (UPEC) strain CFT073 contains 13 large genomic islands ranging in size from 32 kb to 123 kb. Eleven of these genomic islands were individually deleted from the genome, and nine isogenic mutants were tested for their ability to colonize the CBA/J mouse model of ascending urinary tract infection. Three genomic island mutants (Delta PAI-aspV, Delta PAI-metV, and Delta PAI-asnT) were significantly outcompeted by wild-type CFT073 in the bladders and/or kidneys following transurethral cochallenge (P <or= 0.0139). The PAI-metV mutant also showed significant attenuation in the ability to independently colonize the kidneys (P = 0.0011). Specific genes within these islands contributed to the observed phenotype, including a previously uncharacterized iron acquisition cluster, fbpABCD (c0294 to c0297 [c0294-97]), autotransporter, picU (c0350), and RTX family exoprotein, tosA (c0363) in the PAI-aspV island. The double deletion mutant with deletions in both copies of the fbp iron acquisition operon (Deltac0294-97 Delta c2518-15) was significantly outcompeted by wild-type CFT073 in cochallenge. Strains with mutations in a type VI secretion system within the PAI-metV island did not show attenuation. The attenuation of the PAI-metV island was localized to genes c3405-10, encoding a putative phosphotransferase transport system, which is common to UPEC and avian pathogenic E. coli strains but absent from E. coli K-12. We have shown that, in addition to encoding virulence genes, genomic islands contribute to the overall fitness of UPEC strain CFT073 in vivo.

FIG. 1.

(A) Thirteen genomic islands of >30 kb in E. coli CFT073. Isogenic mutants were constructed in 11 GIs (shaded) using the lambda red recombinase system. Deletion mutants of nine of these mutants (shaded blue and red) were tested in cochallenge with wild-type strain CFT073 in the CBA/J mouse model of ascending UTI. Deletion mutants of six islands shown in blue did not show levels of colonization that were statistically different from that of CFT073. Mutants with deletion of genes in the three islands shown in red (PAI-aspV, PAI-metV, and PAI-asnT) were significantly outcompeted by wild-type strain CFT073 in the bladders and/or kidneys (P < 0.05). Smaller deletion mutants spanning PAI-aspV (PAI-aspV1, PAI-aspV2, PAI-aspV3, and PAI-aspV4) and PAI-metV (PAI-metV1 and PAI-metV2) were also constructed and tested in cochallenge with strain CFT073. Isogenic mutants in GIs PAI-icdA and PAI-smpB were not created (white). (B) Confirmation of homologous recombination and subsequent replacement of each GI with a kanamycin resistance cassette. PCR amplification of isogenic mutants with primers flanking the targeted GIs (32 to 123 kb) resulted in PCR products with the predicted sizes: ΔPAI-pheV (2,204 bp), ΔPAI-pheU (3,820 bp), ΔPAI-aspV (1,883 bp), φΔ-b0847 (1,938 bp), ΔPAI-serX (2,061 bp), φΔ-potB (2,290 bp), ΔGI-asnW (2,113 bp), ΔGI-cobU (2,122 bp), ΔPAI-metV (1,746 bp), ΔGI-selC (1,850 bp), and ΔPAI-asnT (1,943 bp). The estimated sizes of PCR products were consistent with the predicted sizes.

FIG. 2.

Colonization levels in the bladder and kidneys of CBA/J mice at 48 hpi. Cochallenge of wild-type CFT073 with mutant strains or ΔPAI-aspV (n = 20), ΔPAI-metV (n = 14), and ΔPAI-asnT (n = 10) in the bladder (A) and kidneys (B). Independent challenges of wild-type CFT073 (n = 13) with mutant strains ΔPAI-aspV (n = 11), ΔPAI-metV (n = 10), and ΔPAI-asnT (n = 10) in the bladder (C) and kidneys (D). Bars indicate the median level of colonization (CFU/g tissue). A P value of <0.05 was considered statistically significant. P values for cochallenge infections were determined using the Wilcoxon matched-pair test. P values for independent challenges were determined using the Mann-Whitney test.

FIG. 3.

Colonization levels in the bladder (A) and kidneys (B) of CBA/J mice at 48 hpi during cochallenge of wild-type CFT073 with mutant strains ΔPAI-aspV1 (n = 10), ΔPAI-aspV2 (n = 10), ΔPAI-aspV3 (n = 9), and ΔPAI-aspV4 (n = 10).

FIG. 4.

Colonization levels in the bladder (A) and kidneys (B) of CBA/J mice at 48 hpi during cochallenge of strains with mutations in individual genes of known function against the entire PAI mutant in which the gene is located. Cochallenge of the ΔcdiA mutant with the ΔPAI-aspV mutant (n = 9), ΔpicU mutant with ΔPAI-aspV mutant (n = 10), and Δc0294-97 mutant with ΔPAI-aspV mutant (n = 9).

FIG. 5.

Colonization levels in the bladder and kidneys of CBA/J mice at 48 hpi during cochallenge of wild-type CFT073 with the Δc0363 (tosA) RTX toxin mutant (n = 14). The colonization levels in bladders (squares) and kidneys (circles) are indicated.

FIG. 6.

(A) Colonization levels in the bladder and kidneys of CBA/J mice at 48 hpi during cochallenge of wild-type CFT073 with the double iron system Δc0294-97 Δc2518-15 (Δfbp) mutant (n = 16). The colonization levels in bladders (squares) and kidneys (circles) are indicated. (B) Growth of wild-type CFT073, Δc0294-97 Δc2518-15 (Δfbp) mutant, and an enterobactin/aerobactin-negative (entF::kan iucB::cam) strain of CFT073 on CAS siderophore agar. Siderophore production is indicated by orange halos around the bacterial growth. (C) Expression of the cloned fbp locus under the control of a constitutive em7 promoter (E. coli TOP10/pGENfbp) and the vector-only negative control (pGEN-MCS). The predicted molecular mass of the c0294 protein is 78.4 kDa.

FIG. 7.

Colonization levels in the bladder (A) and kidneys (B) of CBA/J mice at 48 hpi during cochallenge of wild-type CFT073 with the ΔPAI-metV1 mutant (n = 9) and ΔPAI-metV2 mutant (n = 9).

FIG. 8.

Colonization levels in the bladder and kidneys of CBA/J mice at 48 hpi during cochallenge of wild-type CFT073 with the Δc3405-10 mutant (n = 9). The colonization levels in bladders (squares) and kidneys (circles) are indicated.