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. 2001 Jun;69(6):3954-64.
doi: 10.1128/IAI.69.6.3954-3964.2001.

Mutation of the gene encoding cytotoxic necrotizing factor type 1 (cnf(1)) attenuates the virulence of uropathogenic Escherichia coli

K E Rippere-Lampe  1 , A D O'Brien, R Conran, H A Lockman
Affiliations

Mutation of the gene encoding cytotoxic necrotizing factor type 1 (cnf(1)) attenuates the virulence of uropathogenic Escherichia coli

K E Rippere-Lampe (VSports) et al. Infect Immun. 2001 Jun.

"VSports在线直播" Abstract

Cytotoxic necrotizing factor type 1 (CNF1) is a 115-kDa toxin that activates Rho GTPases and is produced by uropathogenic Escherichia coli (UPEC). While both epidemiological studies that link CNF1 production by E. coli with urinary tract disease and the cytopathic effects of CNF1 on cultured urinary tract cells are suggestive of a role for the toxin as a UPEC virulence factor, few in vivo studies to test this possibility have been reported. Therefore, in this investigation, we evaluated the importance of CNF1 in a murine model of urinary tract infection (UTI) by comparing the degree of colonization and damage induced by three different CNF1-producing E. coli strains with isogenic CNF1-deficient derivatives. The data from single-strain challenge experiments with C3H/HeOuJ mice indicated a trend toward higher counts of the wild-type strains in the urine and bladders of these animals up to 3 days after challenge in two of three strain pairs. Furthermore, this difference was statistically significant at day 2 of infection with one strain pair, C189 and C189cnf(1). To control for the animal-to-animal variability inherent in this model, we infected C3H/HeOuJ mice with a mixture of CNF1-positive and -negative isogenic derivatives of CP9 VSports手机版. The CNF1-positive strain was recovered in higher numbers than the CNF1-negative strain in the urine, bladders, and kidneys of the mice up to 9 days postinfection. These striking coinfection findings, taken with the trends observed in single-strain infections, led us to conclude that CNF1-negative strains were generally attenuated compared to the wild type in the C3H/HeOuJ mouse model of UTI. Furthermore, histopathological examination of bladder specimens from mice infected with CNF1-positive strains consistently showed deeper, more extensive inflammation than in those infected with the isogenic mutants. Lastly, we found that CNF1-positive strain CP9 was better able to resist killing by fresh human neutrophils than were CP9cnf(1) bacteria. From these data in aggregate, we propose that CNF1 production increases the capacity of UPEC strains to resist killing by neutrophils, which in turn permits these bacteria to gain access to deeper tissue and persist better in the lower urinary tract. .

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"VSports" Figures

FIG. 1
FIG. 1
Comparison of different CNF1-positive UPEC strains in the C3H/HeOuJ mouse model of ascending UTI. Ten female C3H/HeOuJ mice per bacterial strain were inoculated with 1.0 × 108 CFU and sacrificed 3 days later. Urine, bladders, and kidneys were collected and homogenized, and serial dilutions were plated for colony counts. (A) Bacterial counts in urine. (B) Bacterial counts in bladders. (C) Bacterial counts in kidneys. Each point represents one sample. The height of the bar represents the mean for those samples. In all cases except C189 in the bladder, the counts for J96 were significantly lower than those of CP9, C85, and C189 (analysis of variance, P < 0.0001, followed by Student's t test, P < 0.05).
FIG. 2
FIG. 2
Kinetics of UTI of C3H/HeOuJ mice with CP9 and the cnf1 isogenic mutant CP9cnf1. Thirty female C3H/HeOuJ mice were inoculated transurethrally with 1.9 × 107 CFU of either CP9 or CP9cnf1 bacteria and sacrificed 1, 3, and 5 days later. Urine, bladder, and kidney samples were recovered, homogenized, and plated for colony counts. Urine samples were not obtained from every mouse, and mice that did not become infected were not included in the analysis. (A) Bacterial counts 1 day after infection. (B) Bacterial counts 3 days after infection. (C) Bacterial counts 5 days after infection. Each point represents one sample. The height of the bar represents the mean for those samples. Symbols: ▵, CP9; ▴, CP9cnf1.
FIG. 3
FIG. 3
Comparison of CP9 and the cnf1 isogenic mutant CP9cnf1 at 1 to 3 days after inoculation of C3H/HeOuJ or BALB/c mice. (A) Twenty female C3H/HeOuJ mice were inoculated with 1.9 × 107 CFU and sacrificed 1 day later. Organ samples were collected, homogenized, serially diluted, and plated on LB agar. Symbols: ▵, CP9; ▴, CP9cnf1. (B) Fifteen female BALB/c mice were infected with 1.9 × 107 CFU and sacrificed for colony counts 1 day later. Symbols: ▵, CP9; ▴, CP9cnf1. (C) Fifteen female C3H/HeOuJ mice were infected with 1.9 × 107 CFU and sacrificed 2 days later. Symbols: ▵, CP9; ▴, CP9cnf1. (D) Ten female C3H/HeOuJ mice were infected with 1 × 108 CFU and euthanatized 3 days later. Counts of CP9 bacteria were significantly higher than those of CP9cnf1 in the urine and bladders (Student's t test, P < 0.05). Symbols: ▵, CP9; ▴, CP9cnf1. Each point represents one sample. The height of the bar represents the mean for those samples. Urine samples were not obtained from every mouse, and mice that did not become infected were not included in the analysis.
FIG. 4
FIG. 4
Single-strain infection comparing C189 with C189cnf1 2 days after inoculation. Seven female C3H/HeOuJ mice were inoculated with 1.0 × 108 CFU and sacrificed 2 days after challenge. Points represent individual samples, and the height of each bar represents the geometric mean of those samples. Counts of C189 were significantly higher than those of C189cnf1 in the urine and bladders (Student's t test, P < 0.05). Symbols: ▵, C189, ▴, C189 cnf1.
FIG. 5
FIG. 5
Mixed infections of C3H/HeOuJ mice. Two strains were mixed in equal numbers and inoculated into female mice. Mice received 2.0 × 108 CFU and were sacrificed 2 days after infection. (A) Mixed infections comparing CP9 (CNF1+ Lac+) with CP9lacZ (CNF1+ Lac−). Eleven female C3H/HeOuJ mice were inoculated. Symbols: ▵, CP9; ▴, CP9lacZ. (B) Mixed infections comparing CP9lacZ with CP9cnf1. Twenty female C3H/HeOuJ mice were inoculated. Counts of CP9lacZ were significantly higher than those of CP9cnf1 in the urine, bladders, and kidneys (Student's t test, P < 0.05). Symbols: ▵, CP9lacZ, ▴, CP9cnf1. Points represent bacterial numbers in individual samples, and bars represent the means of those samples.
FIG. 6
FIG. 6
CNF1 contributes to bacterial survival in a mixed infection. Time courses of mixed infections with CP9lacZ and CP9cnf1 are shown. The bacterial strains were mixed in equal numbers, and C3H/HeOuJ mice received 2.0 × 108 CFU. Mice were sacrificed 6 h, 2 days, 4 days, 7 days, and 9 days after infection, and colony counts were performed on collected samples. (A) Mean bacterial numbers in the urine of infected mice. (B) Mean bacterial numbers in the bladders of infected mice. (C) Mean bacterial numbers in the kidneys of infected mice. ——, CP9lacZ; –––, CP9cnf1. Each point represents the mean number of CFU per sample for three mice.
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