Role of the vpe carbohydrate permease in Escherichia coli urovirulence and fitness in vivo

Abstract

Uropathogenic Escherichia coli (UPEC) strains are a leading cause of infections in humans, but the mechanisms governing host colonization by this bacterium remain poorly understood. Previous studies have identified numerous gene clusters encoding proteins involved in sugar transport, in pathogen-specific islands. We investigated the role in fitness and virulence of the vpe operon encoding an EII complex of the phosphotransferase (PTS) system, which is found more frequently in human strains from infected urine and blood (45%) than in E. coli isolated from healthy humans (15%). We studied the role of this locus in vivo, using the UPEC E. coli strain AL511, mutants, and complemented derivatives in two experimental mouse models of infection. Mutant strains displayed attenuated virulence in a mouse model of sepsis. A role in kidney colonization was also demonstrated by coinfection experiments in a mouse model of pyelonephritis. Electron microscopy examinations showed that the vpeBC mutant produced much smaller amounts of a capsule-like surface material than the wild type, particularly when growing in human urine. Complementation of the vpeBC mutation led to an increase in the amount of exopolysaccharide, resistance to serum killing, and virulence. It was therefore clear that the loss of vpe genes was responsible for all the observed phenotypes. We also demonstrated the involvement of the vpe locus in gut colonization in the streptomycin-treated mouse model of intestinal colonization. These findings confirm that carbohydrate transport and metabolism underlie the ability of UPEC strains to colonize the host intestine and to infect various host sites.

Fig 1

Genomic organization of the vpe gene cluster, which is frequently linked to the deoK gene cluster, in a conserved 9-kb region.

Fig 2

Effect of growth phase and urine on the transcription of the vpe genes from AL511. Transcription of the vpeA, -B, -C, and -R genes was analyzed by qRT-PCR after growth in either LB broth or urine. (A) A growth phase-dependent effect of urine on gene expression was demonstrated. Fold change indicates the ratio of growth in stationary phase to that in exponential phase. (B) The induction of transcription by urine is illustrated for bacteria in the stationary growth phase. Fold change refers to the ratio of growth in urine to that in LB broth.

Fig 3

Regulatory effect of vpeR on transcription of the vpeABC operon. Relative expression of the genes in the AL511 vpeR and AL511 strains was analyzed by qRT-PCR after growth in either LB broth or urine. Downregulation was observed for cells grown in LB broth (A and B) and in urine (C and D), in the exponential growth and stationary phases.

Fig 4

Survival of independent AL511 vpeBC mutants in coculture experiments. The parental AL511 strain and the AL511 vpeBC mutant were cocultured in both LB broth (A) and human urine (B) for 2 weeks. The mutant was outcompeted by the wild-type strain only in urine. Similar results were obtained when AL511 was cocultured with an independent AL511 vpeBC derivative (vpeBC [Km-FRT]) in urine (C). No competition was observed in urine cocultures of two independent AL511 derivatives (vpeBC [Km-FRT] and vpeBC [FRT]) (D). Similar results were obtained in at least two independent experiments.

Fig 5

Competitive colonization of the urinary tract (UT) by AL511 and AL511 vpeBC. Two independent colonization experiments were performed on a total of 24 mice displaying UT colonization in the bladder 4 days after the simultaneous administration of the two strains (1:1 ratio). (A) The results are reported as log CFU/g of organ for AL511 (◆) and AL511 vpeBC (♢). The horizontal bars represent the mean values. The asterisks indicate significant differences, with P values of <0.05 in a paired one-tailed t test. (B) The results are reported as log CI values. Each point (△) corresponds to a single mouse, and the horizontal bars represent the mean values. The asterisk indicates mean values of less than −0.3, indicating significant attenuation of the mutant.

Fig 6

Transmission electron micrographs of exopolysaccharide production by the wild type and vpe mutants of the AL511 strain grown in urine. Surface-expressed polysaccharides are visible as electron-dense material on the surface of bacteria stained with ferritin and examined by transmission electron microscopy. Representative images of wild-type AL511 (A), AL511 vpeBC (B), and complemented AL511 vpeBC (C) are shown. Thick, dark, circumferential staining (solid arrow) was observed at the surface of most (89%) AL511 cells. In contrast, only a thin layer of irregular staining was identified at the surface of AL511 vpeBC cells. Labeling was discrete, with detached material visible (dashed arrow). The expression of pZEZeovpeBC restored capsule production to wild-type levels, in terms of the number of stained bacteria and the thickness of the ferritin-stained layer. This thickness was estimated by measurements taken at five different points on seven bacteria. The mean scores were 129, 27, and 84 nm for AL511, AL511 vpeBC, and complemented strains, respectively. Bars, 1 μm.

Fig 7

Transcription of genes involved in exopolysaccharide production in AL511. Transcription of genes specific for the assembly and export of group 4 capsule at the cell surface (gfcC and etk) and of genes specific for the ABC-dependent mechanism of O9-antigen biosynthesis (wbdB, wzt, and wzm) was analyzed by qRT-PCR after growth to stationary phase in either LB broth or urine. (A) An effect of urine on the expression of genes involved in O9-antigen synthesis is illustrated. The indicated fold change is the ratio of growth in urine to that in LB broth. (B) The relative expression of the genes in the AL511 vpeBC and AL511 strains was analyzed after growth in urine. The expression of all the genes was induced. The relative expression of the genes in the AL511 vpeR and AL511 strains was set to 1 in urine (data not shown).

Fig 8

Kinetics of the killing of AL511 wild type and vpe mutants by human serum. Changes in cell viability were estimated after exposing bacteria to 20% normal (A) or heat-treated (B) human serum. All points are the means of at least three independent determinations in two independent experiments. Similar data were obtained with serum samples from two other healthy people.

Fig 9

Competitive colonization of the mouse intestine by AL511 and AL511 vpeBC. Two independent colonization experiments were performed. Eleven mice had intestinal colonization 1 day after the simultaneous administration of the two strains (1:1 ratio) by oral force-feeding. At the times indicated, fecal samples were plated on LB agar with and without kanamycin. (A) The results are reported as log CFU/g of feces for AL511 (◆) and AL511 vpeBC (♢). We arbitrarily attributed a value of 10² CFU/g of feces to a strain if no bacteria were recovered on plates. The horizontal bars represent the mean values. The asterisks indicate significant differences, with P values of <0.05 in a paired one-tailed t test. (B) The results of the experiments are reported as log CI values. The CI was calculated as the ratio of colony counts for the mutant to those for the wild type recovered from feces at the various times divided by the initial ratio of mutant to wild-type CFU. Each point (○) corresponds to a single mouse, and the horizontal bars represent the mean values. The asterisks indicate mean values of less than −0.3, reflecting significant attenuation of the mutant.