Roles of putative type II secretion and type IV pilus systems in the virulence of uropathogenic Escherichia coli

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Abstract

Background: Type II secretion systems (T2SS) and the evolutionarily related type IV pili (T4P) are important virulence determinants in many Gram-negative bacterial pathogens. However, the roles of T2SS and T4P in the virulence of extraintestinal pathogenic Escherichia coli have not been determined. Methodology/Principal Findings: To investigate the functions of putative T2SS and T4P gene clusters present in the model uropathogenic E. coli (UPEC) strains UTI89 and CFT073, we deleted the secretin gene present in each cluster. The secretin forms a channel in the outer membrane that is essential for the function of T2S and T4P systems. We compared the secretin deletion mutants with their wild type counterparts using tissue culture assays and the CBA/J mouse model of ascending urinary tract infection. No deficiencies were observed with any of the mutants in adherence, invasion or replication in human bladder or kidney cell lines, but UTI89 ΔhofQ and UTI89 ΔgspD exhibited approximately 2-fold defects in fluxing out of bladder epithelial cells. In the mouse infection model, each of the knockout mutants was able to establish successful infections in the bladder and kidneys by day one post-infection. However, UTI89 ΔhofQ and a CFT073 ΔhofQ ΔyheF double mutant both exhibited defects in colonizing the kidneys by day seven post-infection. Conclusions/Significance: Based on our results, we propose that the putative T4P and T2S systems are virulence determinants of UPEC important for persistence in the urinary tract, particularly in renal tissues. © 2009 Kulkarni et al.

Figures

  • Figure 1. Schematic Representation of the T2SS and T4P gene clusters in UTI89 and CFT073. The E. coli K12 and ETEC homologous T2SS gene clusters are shown in black and red, respectively. T4P genes are shown in blue. For the K12-T2SS, the E. coli K12 gene names are listed above the arrows and the generic gsp nomenclature is given below. The OM secretin genes are indicated by the hatched arrows. The open arrows are genes that do not show homology to any of the known T2SS or T4P genes. doi:10.1371/journal.pone.0004752.g001
  • Table 1. Primers used in this study.
  • Figure 2. Efflux of UTI89 WT and secretin mutant strains from 5637 human bladder epithelial cells. A modified gentamicin protection assay (see Materials and Methods) was used to calculate efflux of WT UTI89 or the indicated deletion mutant from monolayers of 5637 human bladder epithelial cells. Cell lines were infected with bacteria the previous day and incubated overnight in the presence of gentamicin to kill extracellular bacteria. Cells were then incubated for 6 h in the absence of gentamicin. The efflux index was calculated by dividing the number of bacteria released into the medium after the 6 h incubation without gentamicin by the intracellular bacteria present after the overnight incubation. The efflux index for each strain was normalized to the WT strain. The UTI89 DgspD and DhofQ mutants consistently exhibited significantly reduced efflux index values compared to bladder cells infected with WT UTI89. Efflux index values for the UTI89 DyheF mutant were not always reduced compared to WT. Each deletion mutant was compared with the WT strain at least 3 times. Values are shown as a percentage of WT; error bars indicate SD; *, p,0.05; **, p,0.01. doi:10.1371/journal.pone.0004752.g002
  • Figure 3. Mouse infection experiments with UTI89. Eight weekold, female CBA/J mice were infected by trans-urethral catheterization with the indicated WT or secretin knockout strains of UTI89. Bladders and kidneys were harvested on day 7 post-infection, weighed, homogenized and CFU/g of organ weight determined. The UTI89 DhofQ mutant was consistently defective in colonizing kidneys (p = 0.03) (B), whereas no differences were observed in CFU recovered from the bladder (A). The kidney colonization defect of UTI89 DhofQ (p = 0.027) was rescued by reintroduction of hofQ in trans (p = 0.013) (C). The bars indicate median CFU values. The limit of detection is indicated by the dotted line. In panels (A) and (B), the data from two independent experiments were combined together. Panel (C) shows data from one experiment. P values were calculated by Mann-Whitney test for nonparametric data with one-tailed P value. doi:10.1371/journal.pone.0004752.g003
  • Figure 4. Mouse infection experiments with CFT073. Eight weekold, female CBA/J mice were infected by trans-urethral catheterization with the indicated WT or secretin knockout strains of CFT073. Bladders and kidneys were harvested on day 7 post-infection, weighed, homogenized and CFU/g of organ weight determined. The CFT073 DyheF DhofQ double mutant was significantly defective in colonizing the kidneys (p = 0.013) (B), while bladder CFU (A) were lowered as compared to WT, but this difference was not significant. The kidney colonization defect of the CFT073 DyheF DhofQ double mutant (p = 0.005) was restored to WT-like levels by plasmid-borne hofQ (p = 0.038) (C). Addition of yheF in trans also increased colonization levels, but this was not statistically significant (p = 0.393) (C). The bars indicate median CFU values. The limit of detection is indicated by the dotted line. In panels (A) and (B), the data from two independent experiments were combined together. Panel (C) shows data from one experiment. P values were calculated by Mann-Whitney test for nonparametric data with one-tailed P value. doi:10.1371/journal.pone.0004752.g004

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CITATION STYLE

APA

Kulkarni, R., Dhakal, B. K., Slechta, E. S., Kurtz, Z., Mulvey, M. A., & Thanassi, D. G. (2009). Roles of putative type II secretion and type IV pilus systems in the virulence of uropathogenic Escherichia coli. PLoS ONE, 4(3). https://doi.org/10.1371/journal.pone.0004752

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