MECANISMES DE VIRULENCE DES ESCHERICHIA COLI UROPATHOGENES PORTEURS DES ADHESINES DE LA FAMILLE AFA/DR

CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

The secreted autotransporter toxin, Sat, functions as a virulence factor in Afa/Dr diffusely adhering Escherichia coli by promoting lesions in tight junction of polarized epithelial cells.

International audienceAfa/Dr diffusely adhering Escherichia coli (DAEC) strains are responsible for urinary tract and intestinal infections. Both in intestine and kidney, the epithelial cells forming epithelium are sealed by junctional domains. We provide evidence that the Secreted autotransporter toxin, Sat, belonging to the subfamily of serine protease autotransporters of Enterobacteriaceae (SPATEs), acts as a virulence factor in Afa/Dr DAEC by promoting lesions in the tight junctions (TJs) of polarized epithelial Caco-2/TC7 cells. Southern blot analysis reveals that the prototype strains of the subclass-1 and subclass-2 typical Afa/Dr DAEC strains, hybridize with a sat probe. Using the wild-type IH11128 strain, the recombinant E. coli AAEC185 strain that expresses Sat, the recombinant E. coli that expresses both Dr adhesin and Sat, we report that Sat in monolayers of cultured enterocyte-like Caco-2/TC7 cells, induces rearrangements of the TJs-associated proteins ZO-1, ZO-3 and occludin, and increases the formation of domes as the result of an increase in the paracellular permeability without affecting the transepithelial electrical resistance of the cell monolayers. Moreover, we observe that Sat-induced disassembly of TJs-associated proteins is dependent on the serine protease motif. Finally, an analysis of the prevalence of the sat gene in three collections of Afa/Dr DAEC strains collected from the stools of children with and without diarrhoea, and from the urine of patients with urinary tract infection (UTI) shows that: (i) the sat gene is highly prevalent in UTI-associated Afa/Dr DAEC strains (88% positive), (ii) the sat gene is generally absent from Afa/Dr DAEC strains collected from the stools of children without diarrhoea (16% positive); whereas (iii) it is present in about half of the strains collected from the stools of children with diarrhoea (46% positive)

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

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

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

Perinatal hormones favor CC17 Group B Streptococcus intestinal translocation through M cells and hypervirulence in neonates

International audienceGroup B Streptococcus (GBS) is the leading cause of invasive bacterial neonatal infections. Late-onset diseases (LOD) occur between 7 and 89 days of life and are largely due to the CC17 GBS hypervirulent clone. We studied the impact of estradiol (E2) and progesterone (P4), which impregnate the fetus during pregnancy, on GBS neonatal infection in cellular and mouse models of hormonal exposure corresponding to concentrations found at birth (E2-P4 C 0) and over 7 days old (E2-P4 C 7). Using representative GBS isolates, we show that E2-P4 C 7 concentrations specifically favor CC17 GBS meningitis following mice oral infection. CC17 GBS crosses the intestinal barrier through M cells. This process mediated by the CC17-specific surface protein Srr2 is enhanced by E2-P4 C 7 concentrations which promote M cell differentiation and CC17 GBS invasiveness. Our findings provide an explanation for CC17 GBS responsibility in LOD in link with neonatal gastrointestinal tract maturation and hormonal imprint

Insights into Streptococcus agalactiae PI-2b pilus biosynthesis and role in adherence to host cells

International audienceThe core PI-2b pilus present in "hypervirulent" ST-17 Streptococcus agalactiae strains consists of three pilin subunits (Spb1, Ap1 and Ap2) assembled by sortase SrtC1 and cell-wall anchored by Srt2. Spb1 was shown to be the major pilin and Ap2 the anchor pilin. Ap1 is a putative adhesin. Two additional genes, orf and lep, are part of this operon. The contribution of Lep and Ap1 to the biogenesis of the PI-2b pilus was investigated. Concerning the role of PI-2b, we found that higher PI-2b expression resulted in higher adherence to human brain endothelial cells and higher phagocytosis by human THP1 macrophages