The T3SS translocator components EspA and EspD are proteolyzed by EspC.

<p>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 <i>espC</i> expression, respectively. (C, D, E) The EspB-D-A containing supernatant of mid-exponential DMEM-cultured-Δ<i>espC</i> 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.</p

The Serine Protease EspC from Enteropathogenic <i>Escherichia coli</i> Regulates Pore Formation and Cytotoxicity Mediated by the Type III Secretion System

<div><p>Type III secretion systems (T3SSs) are specialized macromolecular machines critical for bacterial virulence, and allowing the injection of bacterial effectors into host cells. The T3SS-dependent injection process requires the prior insertion of a protein complex, the translocon, into host cell membranes consisting of two-T3SS hydrophobic proteins, associated with pore-forming activity. In all described T3SS to date, a hydrophilic protein connects one hydrophobic component to the T3SS needle, presumably insuring the continuum between the hollow needle and the translocon. In the case of Enteropathogenic <i>Escherichia coli</i> (EPEC), the hydrophilic component EspA polymerizes into a filament connecting the T3SS needle to the translocon composed of the EspB and EspD hydrophobic proteins. Here, we identify EspA and EspD as targets of EspC, a serine protease autotransporter of Enterobacteriaceae (SPATE). We found that <i>in vitro</i>, EspC preferentially targets EspA associated with EspD, but was less efficient at proteolyzing EspA alone. Consistently, we found that EspC did not regulate EspA filaments at the surface of primed bacteria that was devoid of EspD, but controlled the levels of EspD and EspA secreted <i>in vitro</i> or upon cell contact. While still proficient for T3SS-mediated injection of bacterial effectors and cytoskeletal reorganization, an <i>espC</i> mutant showed increased levels of cell-associated EspA and EspD, as well as increased pore formation activity associated with cytotoxicity. EspP from enterohaemorrhagic <i>E</i>. <i>coli</i> (EHEC) also targeted translocator components and its activity was interchangeable with that of EspC, suggesting a common and important function of these SPATEs. These findings reveal a novel regulatory mechanism of T3SS-mediated pore formation and cytotoxicity control during EPEC/EHEC infection.</p></div

EspC preferentially targets EspA/EspD-containing structures.

<p>(A-C) Proteins from the supernatant of Δ<i>espC</i> 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.</p

EspC negatively regulates the amounts of EspA and EspD secreted upon cell contact.

<p>(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.</p

EspC does not regulate EspA filament structures at the surface of primed bacteria.

<p>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.</p

EspC controls pore formation mediated by the T3SS during cell infection.

<p>(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 Δ<i>escN</i> 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.</p

Additional file 4: Figure S1. of Draft genome sequence and characterization of commensal Escherichia coli strain BG1 isolated from bovine gastro-intestinal tract

Hierarchical clustering of E. coli strains according to adherence systems encoding genes. The dendrogram and associated heatmap are generated on the basis of gene presence/absence considering 78 genes involved in adherence, using binary distance and complete clustering method, R version 3.3.1. [43]. Blue color indicates gene presence, red gene absence. The origin of each strain is identified with B (Bovine) or H (Human). The color of the strain name corresponds to its phylogroup as in Fig. 2. (DOCX 67 kb

Additional file 2: Table S2. of Draft genome sequence and characterization of commensal Escherichia coli strain BG1 isolated from bovine gastro-intestinal tract

Genes encoding virulence factors in the E. coli BG1 genome (XLSX 31 kb

Additional file 6: Figure S2. of Draft genome sequence and characterization of commensal Escherichia coli strain BG1 isolated from bovine gastro-intestinal tract

Nucleotide sequence alignment of the eutT gene. The sequences of the eutT gene and the translated EutT polypeptide were aligned respectively from E. coli strains BG1 and EDL933 using Seaview version 4.6.1 [56]. (DOCX 15 kb

DataSheet1.XLSX

<p>Healthy cattle are the primary reservoir for O157:H7 Shiga toxin-producing E. coli responsible for human food-borne infections. Because farm environment acts as a source of cattle contamination, it is important to better understand the factors controlling the persistence of E. coli O157:H7 outside the bovine gut. The E. coli O157:H7 strain MC2, identified as a persistent strain in French farms, possessed the characteristics required to cause human infections and genetic markers associated with clinical O157:H7 isolates. Therefore, the capacity of E. coli MC2 to survive during its transit through the bovine gastro-intestinal tract (GIT) and to respond to stresses potentially encountered in extra-intestinal environments was analyzed. E. coli MC2 survived in rumen fluids, grew in the content of posterior digestive compartments and survived in bovine feces at 15°C predicting a successful transit of the bacteria along the bovine GIT and its persistence outside the bovine intestine. E. coli MC2 possessed the genetic information encoding 14 adherence systems including adhesins with properties related to colonization of the bovine intestine (F9 fimbriae, EhaA and EspP autotransporters, HCP pilus, FdeC adhesin) reflecting the capacity of the bacteria to colonize different segments of the bovine GIT. E. coli MC2 was also a strong biofilm producer when incubated in fecal samples at low temperature and had a greater ability to form biofilms than the bovine commensal E. coli strain BG1. Furthermore, in contrast to BG1, E. coli MC2 responded to temperature stresses by inducing the genes cspA and htrA during its survival in bovine feces at 15°C. E. coli MC2 also activated genes that are part of the GhoT/GhoS, HicA/HicB and EcnB/EcnA toxin/antitoxin systems involved in the response of E. coli to nutrient starvation and chemical stresses. In summary, the large number of colonization factors known to bind to intestinal epithelium and to biotic or abiotic surfaces, the capacity to produce biofilms and to activate stress fitness genes in bovine feces could explain the persistence of E. coli MC2 in the farm environment.</p