Fig 1.
Deletion of effector genes in EPEC E2348/69.
A. Representation of the LEE pathogenicity island. Effector genes are labeled in red. The order of deletion is numbered: deletion 1, 2, 3, 4 are for map, espG, espF and espH respectively. The espZ and tir genes were deleted after the non-LEE effectors, with order 12 and 13 respectively. Scale of 5 kb is indicated at the bottom. B. Effector genes located outside the LEE are localized in integrative elements (IE) and prophages (PP). Effector genes are labeled in red. Pseudogenes are specified with asterisk. The red bars indicate the deletions carried out. The order of deletion is numbered. Scale of 5 kb is indicated at the bottom.
Table 1.
EPEC strains used in this work.
Fig 2.
Functionality of T3SS of the EPEC effector mutants.
A. Analysis of secreted and bacterial proteins in the indicated EPEC strains after 4 h of growth in DMEM at 37°C. Top panel: Coomassie staining of proteins found in the extracellular medium labeling the translocators EspABD and the autotransporter EspC. Molecular standards are shown in kDa. Bottom panels: Western blots of bacterial whole-cell protein extracts incubated with polyclonal antibodies to detect EscC, EscJ, EscD injectisome proteins and EspB translocator protein. Detection of cytoplasmic GroEL was used as a loading control. Discontinuous lines indicate borders of independent gels. B. Immunofluorescence confocal microscopy of HeLa cells infected for 90 min with WT EPEC, EPEC2, EPEC1 and EPEC0 strains. EPEC bacteria are labeled with anti-intimin-280 polyclonal (green), actin is labeled with TRITC-phalloidin (red) and cell nuclei are labeled with DAPI (gray). Actin polymerization beneath the adherent bacteria is observed in EPECwt, EPEC2 and EPEC1 strains. Scale bar 5 μm. C. Quantification of the number of HeLa cells with actin pedestals after infection with the indicated strains WT, EPEC2, EPEC1 and EPEC0. The data shown are the mean of two independent experiments with standard deviation (SD). In each experiment, one hundred cells per infection sample were counted. D. Protein translocation into HeLa cells of β-lactamase (Bla) fusion by EPEC effector mutants. HeLa cells were infected for 90 min with the indicated EPEC strains (WT EPEC, EPECΔescN, EPEC2, EPEC1 EPEC0), expressing EspF1-20-Bla fusion (WT EPEC, EPECΔescN, EPEC2, EPEC1, EPEC0) or the control vector pCX340, and then incubated with the BLA substrate CCF2/AM for additional 1 h. Bla activity was quantified measuring the emission ratio of fluorescence at 450/520 nm. Results are the mean of three independent experiments with standard deviation (SD). One way ANOVA Tukey's Multiple Comparison Test. **p<0.01 and ***p <0.001.
Fig 3.
Induction of filopodia by EPEC effector mutants with integrated map.
Immunofluorescence microscopy of Swiss 3T3 cells infected for 10 min with EPEC2, EPEC1 and EPEC0 and isogenic strains with map integrated in the chromosome. EPEC was detected with rabbit polyclonal anti-O127 (red) and actin was stained with Oregon-green phalloidin (green). Filopodia spikes are labelled with arrowheads. Actin accumulation beneath bacteria is indicated with arrows. Scale bar 2 μm.
Fig 4.
Translocation of NleC by EPEC effectors mutants induces p65 degradation.
A. Western blot detecting NF-κB p65 protein in HeLa cells infected for 4 h (1 h+3 h gentamicin) with EPEC2, EPEC1 and EPEC0 and isogenic strains with nleC integrated in the chromosome. Uninfected (UI) cells and cells infected with WT EPEC are used as controls. Detection of α-tubulin was used as a loading control. B. Quantification of p65 in Hela cells infected with the indicated strains. Protein loading was normalized with α-tubulin. Results are the mean of three independent experiments with standard deviation (SD). One way ANOVA Tukey's Multiple Comparison Test. **p <0.01; ***p <0.001.
Fig 5.
A/E lesion formation in human intestinal biopsies infected with EPEC effector mutant strains.
Representative scanning electron micrographs of human duodenal biopsies infected with the indicated EPEC strains. WT EPEC and EPEC9 strains induce the characteristic A/E lesions in the intestinal mucosal surface, forming bacterial microcolonies (asterisk) and elongation of microvilli at the periphery of the microcolony (arrowheads). Biopsies infected with EPEC2, EPEC1, EPEC0 show a smooth epithelial surface without any adherent bacteria. Most biopsies infected by EPEC2LEE (10/11) also showed an intact mucosal surface without bacteria. Scale bar 2 μm.
Table 2.
A/E lesions in human intestinal biopsies infected with EPEC effector mutants.
Table 3.
A/E lesions formation by EPEC deletion mutants in IE effectors.