Virulence plasmid harbored by uropathogenic Escherichia coli functions in acute stages of pathogenesis

Abstract

Urinary tract infections (UTIs), the majority of which are caused by uropathogenic Escherichia coli (UPEC), afflict nearly 60% of women within their lifetimes. Studies in mice and humans have revealed that UPEC strains undergo a complex pathogenesis cycle that involves both the formation of intracellular bacterial communities (IBC) and the colonization of extracellular niches. Despite the commonality of the UPEC pathogenesis cycle, no specific urovirulence genetic profile has been determined; this is likely due to the fluid nature of the UPEC genome as the result of horizontal gene transfer and numerous genes of unknown function. UTI89 has a large extrachromosomal element termed pUTI89 with many characteristics of UPEC pathogenicity islands and that likely arose due to horizontal gene transfer. The pUTI89 plasmid has characteristics of both F plasmids and other known virulence plasmids. We sought to determine whether pUTI89 is important for virulence. Both in vitro and in vivo assays were used to examine the function of pUTI89 using plasmid-cured UTI89. No differences were observed between UTI89 and plasmid-cured UTI89 based on growth, type 1 pilus expression, or biofilm formation. However, in a mouse model of UTI, a significant decrease in bacterial invasion, CFU and IBC formation of the pUTI89-cured strain was observed at early time points postinfection compared to the wild type. Through directed deletions of specific operons on pUTI89, the cjr operon was partially implicated in this observed defect. Our findings implicate pUTI89 in the early aspects of infection.

FIG. 1.

pUTI89 diagram. pUTI89 can be divided into two major portions. One half contains genes involved in conjugative DNA transfer (in blue), and the other half contains genes present on plasmids from pathogenic E. coli and other pathogenic bacteria, as well as genes encoded chromosomally in other bacteria (in red). Indicated next to each region is the gene name, organism it is also found in, and/or the operon function.

FIG. 2.

Confirmation of pUTI89 curing by PCR. pUTI89 was cured from UTI89 by ethidium bromide treatment. Plasmid purification, followed by PCR with primer pairs A to H, was used to identify plasmidless strains. UTI89 with intact pUTI89 (top panel) reveals a banding pattern as observed with very little genomic contamination, as evidenced by the ompA band. PCR of the plasmid-cured strain (bottom panel) revealed no positive PCR confirming the elimination of pUTI89.

FIG. 3.

In vitro characteristics of pUTI89. Plasmid-cured UTI89 behaved identically to UTI89. (A) Growth of UTI89 and ΔpUTI89 were equivalent in LB broth (solid line) and M9 plus 10-μg/ml niacin broth (dashed line). (B) Type 1 pili expression based on FimH and FimA immunoblots was equivalent in both strains. Densitometry analysis was used to quantify the bands observed (ImageJ software). (C) Biofilm formations under type 1-dependent and curli-dependent conditions were indistinguishable in UTI89 versus ΔpUTI89. (D) Both UTI89 and ΔpUTI89 exhibited equal mannose-sensitive HA titers, indicating equal expression levels and functionality of type 1 pili.

FIG. 4.

In vivo time course of UTI89 and ΔpUTI89. (A) ΔpUTI89 tested in our mouse model of UTI revealed a significant decrease in bladder colonization at 6 h postinfection compared to wild-type UIT89 (P < 0.0001 [Mann-Whitney]). However, by 24 h postinfection and beyond, colonization was equivalent in both strains. (B) Additional clones of ΔpUTI89 confirmed that the defect was due to the loss of pUTI89 and not additional mutations within the genome (P < 0.001 by Mann-Whitney).

FIG. 5.

Colonization and invasion of UTI89 versus ΔpUTI89. Gentamicin protection assays performed at 1 and 6 h revealed a significant decrease in both the extracellular (luminal) and intracellular population for ΔpUTI89. A more significant decrease in luminal colonization with ΔpUTI89 was observed at 6 h postinfection (P < 0.001 [Mann-Whitney]).

FIG. 6.

Confocal microscopy and LacZ staining for IBCs. Mice were infected with UTI89 or ΔpUTI89 and bladders were harvested at 6 h postinfection, splayed, and stained with WGA (bladder surface in green) and SYTO61 (bacteria in red). Confocal microscopy revealed IBC formation in bladders infected with either isolate. However, extensive filamentation was only observed in wild-type UTI89. IBCs were quantitated by LacZ staining revealing a significant defect (P < 0.001 [Mann-Whitney]) in the number of IBCs observed in ΔpUTI89-infected bladders.

FIG. 7.

Identification of virulence factors on pUTI89. Regions on pUTI89 were deleted by using the lambda red recombinase system. Deletions encompassing UP028-30 and UP062-63 (○) resulted in a significant reduction in bacterial load at 6 h postinfection (P < 0.05 and P < 0.0001 [Mann-Whitney], respectively). UP062-63 encodes the stability operon, and UP028-30 encodes the cjr operon.