Fig 1.
The T3SS translocator components EspA and EspD are proteolyzed by EspC.
EPEC strains were grown for 16 h in DMEM to induce T3S. (A, B) Protein contents in supernatant of the indicated bacterial strain were analyzed by Coomassie blue staining (A), or Western blot using the antibodies indicated on the left (B). pEspC+ and pEspC- indicate growth in inducing condition (arabinose) or repressing condition (glucose), for espC expression, respectively. (C, D, E) The EspB-D-A containing supernatant of mid-exponential DMEM-cultured-ΔespC strain was incubated for 16 h with recombinant EspC at the indicated concentrations and analysed by Coomassie blue staining (C), or Western blot using the antibodies indicated on the left (D and E). EspC- indicates incubation with extract in the absence of induction. (F, G) Incubation was performed with 25 nM EspC in the presence or absence of PMSF, or with EspC-S256I. (E, G) Quantification of the protein band integrated density was performed in at least 3 independent experiments as shown in (D) and (F), respectively, using the image J software. Results are expressed as the average ± SEM (E, G). *: p ≤ 0.05; **: p ≤ 0,01.
Fig 2.
EspC preferentially targets EspA/EspD-containing structures.
(A-C) Proteins from the supernatant of ΔespC strain were fractionated by anion exchange chromatography, using a linear NaCl gradient. (A) Fractions were analysed by Coomassie blue staining. (B) quantification of the EspA and EspD Coomassie stained bands integrated density in (A). (C) anti-EspA and anti-EspD Western blot analysis. Dashed lines indicate editing between lanes from the same gel. EspB eluted in the flow-through. The first peak containing EspA and EspD eluted at 175nM NaCl (peak A/D), the second peak containing EspA eluted at 290 mM NaCl (peak A). (D, E) Fractions corresponding to peak A or peak A/D were incubated with 40 nM of purified EspC for the indicated time. (D) Western blot analysis using the antibodies indicated on the left. Arrows indicate proteolytic degradation products observed for EspA. (E) quantification of the protein band integrated density. Results are expressed as the mean integrated density ± SEM from at least 3 independent experiments.
Fig 3.
EspC does not regulate EspA filament structures at the surface of primed bacteria.
EPEC strains were grown for 5 hrs in DMEM to induce T3S. (A) Epifluorescent micrographs showing EspA staining associated with bacteria primed for 30 min or 5 h in DMEM. (B) Average percentage of bacteria associated with EspA staining ± SEM, scored for at least 2900 bacteria for each sample in 3 independent experiments. The total number of analysed bacteria (n) is indicated. Scale bar = 5 μm. (C) Samples were analyzed by Coomassie blue staining. (D, E) Western blot using the antibodies indicated on the left. (C,D) bacterial supernatants; (E) bacterial pellets.
Fig 4.
EspC negatively regulates the amounts of EspA and EspD secreted upon cell contact.
(A) Confocal micrographs of cells infected for 45 min with the indicated bacteria and processed for fluorescence staining. Scale bar: 10 μm. Middle panels: magnification of insets in the corresponding left panels. Green: EspA; red: EspD staining; grey: DAPI staining. (B) Quantification of the average fluorescence intensity for EspA and EspD in microcolonies. (C) Total extracts of HeLa cells containing T3S effector proteins were subjected to anti-EspD Western Blot analysis. Anti-actin and anti-OmpA Western blotting were used as controls for cellular and bacterial loads, respectively.
Fig 5.
EspC controls pore formation mediated by the T3SS during cell infection.
(A-C) Cells were challenged with EPEC strains for 45 min in presence of the fluorescent membrane-impermeant dye LY and analyzed by epifluorescence microscopy. (A) Representative micrographs of fixed TC7 cells showing DAPI staining (right) or LY fluorescence (middle panels). Binary images were generated by thresholding images corresponding to the LY fluorescence (right panels). LY positive cells were scored from binary images and the average percentage of LY cells / total cells ± SEM is indicated for each samples in TC7 cells (B) or HeLa cells (C). (D-E) Cells were loaded with the fluorescent dye calcein prior to bacterial challenge for 45 min. (D) Representative micrographs of pseudocolored fluorescence images of cells challenged with the indicated bacteria. Dashed lines indicate contours delineated from phase contrast images of cells with fluorescence intensity below the applied threshold. (E) The relative percentage of calcein leakage was calculated after normalization to cells challenged with the T3SS-deficient ΔescN strain (Experimental Procedures). The total number of analysed cells (n) and number of experiments (N) is indicated. *: p ≤ 0.05; **: p ≤ 0,01. Scale bar: 20 μm.
Fig 6.
An espC mutant induces increased cytotoxicity.
HeLa cells were challenged with EPEC strains for 45 min and infection was carried on for 18 h in presence of gentamicin (Experimental Procedures). (A) Samples were fixed and processed for hematoxylin-eosin staining. Scale bar: 15 μm. (B, E) Cells were trypsinized and scored under the microscope. The results are expressed as the average percentage ± SEM of cells relative to uninfected cells (N = 6). (C) Samples were processed for DAPI staining to visualize the cell nuclei. Scale bar: 15 μm. (D) The average surface area of nuclei ± SEM is indicated (N = 3). *: p ≤ 0.05; **: p ≤ 0,01. A T3SS-dependent cytotoxicity is observed, the espC mutant being significantly more cytotoxic than WT.