Roles of the Tol-Pal system in the Type III secretion system and flagella-mediated virulence in enterohemorrhagic Escherichia coli

Abstract

The Tol-Pal system is a protein complex that is highly conserved in many gram-negative bacteria. We show here that the Tol-Pal system is associated with the enteric pathogenesis of enterohemorrhagic E. coli (EHEC). Deletion of tolB, which is required for the Tol-Pal system decreased motility, secretion of the Type III secretion system proteins EspA/B, and the ability of bacteria to adhere to and to form attaching and effacing (A/E) lesions in host cells, but the expression level of LEE genes, including espA/B that encode Type III secretion system proteins were not affected. The Citrobacter rodentium, tolB mutant, that is traditionally used to estimate Type III secretion system associated virulence in mice did not cause lethality in mice while it induced anti-bacterial immunity. We also found that the pal mutant, which lacks activity of the Tol-Pal system, exhibited lower motility and EspA/B secretion than the wild-type parent. These combined results indicate that the Tol-Pal system contributes to the virulence of EHEC associated with the Type III secretion system and flagellar activity for infection at enteric sites. This finding provides evidence that the Tol-Pal system may be an effective target for the treatment of infectious diseases caused by pathogenic E. coli.

Figure 1

Motilities and flagellar production of the wild-type parent, tolB mutant and pal mutant, or wild-type parent and tolB mutant carrying pTH18kr (empty vector) or pTH18krtolB (tolB expression plasmid). (a) and (d) Bacterial migrations on LB medium containing 0.3% agar were pictured. (b) and (e) Flagella and bacteria cells stained with Victoria blue/tannic acid were pictured on the microscopy using 100 × objective. (c) Cell lysates and secreted proteins from bacteria containing a VSVG-tagged FliC expression plasmid. The proteins including VSVG-tagged FliC were separated by SDS/PAGE, and VSVG-tagged FliC was visualized by western-blotting with VSVG antibody. The full-length blot/gel is presented in Supplementary Fig. 1.

Figure 2

Cell Morphology and growth of the wild-type parent and tolB mutant. All strains were grown in LB medium at 37 °C. (a) Phase-contrast images of bacteria were pictured on the microscopy using 100 × objective. Bacterial growth were monitored by measuring CFU (b) and OD₆₀₀ (c).

Figure 3

Determination of EspB and EspA levels in culture supernatant and whole cell extract and transcript levels of LEE genes in the wild-type parent, tolB mutant and pal mutant, or wild-type parent and tolB mutant carrying pTH18kr (empty vector) or pTH18krtolB (tolB expression plasmid). (a, b) The wild-type, tolB mutant and pal mutant were grown in LB medium or DMEM. For complementation test, we grew the wild-type parent and tolB mutant carrying pTH18kr or pTH18krtolB in DMEM. The proteins including EspB and EspA were separated by SDS/PAGE, and EspB and EspA were visualized by western-blotting with EspB and EspA antiserums, respectively. Full-length blots/gels are presented in Supplementary Fig. 2 and 3. (c) Transcript levels were described as relative values to that of rpoD (housekeeping gene). Data plotted are the means of two biological replicates, error bars indicate the ranges.

Figure 4

Shiga toxin production of the wild-type parent, tolB mutant and pal mutant. (a) Stx1 and Stx2 latex agglutination titers of culture supernatant from each indicated strain were determined. (b) Transcript levels of stx1 and stx2. Transcript levels were described as relative values to that of rpoD (housekeeping gene). Data plotted are the means of two biological replicates, error bars indicate the ranges.

Figure 5

Adhesion to and formation of A/E lesions in HeLa cells for the wild-type parent and tolB mutant. (a) Y-axis on the graphs shows percent (%) of CFU values of adhered bacteria relative to total bacterial cell numbers. Data plotted are the means; error bars indicate the standard deviations. Asterisks denote significance for values relative to the wild-type control (P < 0.05). (b) Bacteria and nuclei of HeLa cells stained with Hoechst33342 and actins strained with rhodamine-phalloidin were imaged, respectively, as green and red colors on the microscopy using 100 × objective. Arrows on picture indicate A/E lesions.

Figure 6

Virulence of the wild-type and tolB mutant strains of C. rodentium in C3H/HeJ mice. (a) Survival of the C3H/HeJ mice infected with the wild-type parent and tolB mutant. The mice (N = 5 mice per strain for DBS100 parent and the tolB mutant, and N = 2 mice for non-infection control) were daily monitored. (b) Change in body weight of the C3H/HeJ mice infected with the wild-type parent and tolB mutant. The connecting lines denote the mean and error bars denote range for the data of control mice and standard deviation for the data of mice infected with the parent and tolB mutant. Asterisks denote significance for values of survival rate and body weight of mice infected with tolB mutant relative to those infected with the parent strain (P < 0.05). (c, d) Length of colons from mice infected with the wild-type parent, tolB mutant and non-pathogenic K-12 strain, and control mice (without infection). Colons were isolated at 7 days post-infection. Asterisks denote significance for values of colon length of mice infected with tolB mutant relative to those infected with the parent strain, and control mice (P < 0.05). (e) Survival of the wild-type parent and tolB mutant after challenged at an acidic condition (pH3.0). Y-axis on the graphs shows percent (%) of CFU values of cells after incubation in the acidified LB medium relative to CFU values of cells after incubation in the regular LB medium. Data plotted are the means; error bars indicate the standard deviations.

Figure 7

Immune response in mice infected with the wild-type parent, tolB mutant and non-pathogenic K-12 strain, and control mice (without infection). (a–c) Levels of IgG1 and IgG2a from serum and IgA from feces in mice at 7 days post-infection. Each data point represents a sample from an individual mouse. Horizontal bars indicate the mean values. (d) Levels of IFN-γ and IL-17 in spleen cells of mice, re-stimulated with an extract from C. rodentium DBS100. Data plotted are the means; error bars indicate the standard deviations. Asterisks denote significance for levels of antibodies and cytokines of mice infected with the wild-type or tolB mutant relative to those of control mice (P < 0.05).