HIF1A regulates xenophagic degradation of adherent and invasive <i>Escherichia coli</i> (AIEC)

<p>The hypoxia inducible transcription factor HIF1 activates autophagy, a general catabolic pathway involved in the maintenance of cellular homeostasis. Dysfunction in both autophagy and HIF1 has been implicated in an increasing number of human diseases, including inflammatory bowel disease (IBD), such as Crohn disease (CD). Adherent invasive <i>E. coli</i> (AIEC) colonize ileal mucosa of CD patients and strongly promote gastrointestinal inflammatory disorders by activation of HIF-dependent responses. Here, we aim to characterize the contribution of HIF1 in xenophagy, a specialized form of autophagy involved in the degradation of intracellular bacteria. Our results showed that endogenous HIF1A knockdown increased AIEC survival in intestinal epithelial cells. We demonstrate that the increase in survival rate correlates with a dramatic impairment of the autophagic flux at the autolysosomal maturation step. Furthermore, we show that AIEC remained within single-membrane LC3-II-positive vesicles and that they were unable to induce the phosphorylation of ULK1. These results suggested that, in the absence of HIF1A, AIEC were found within LC3-associated phagosomes. Using blocking antibodies against TLR5 and CEACAM6, the 2 well-known AIEC-bound receptors, we showed that downstream receptor signaling was necessary to mediate ULK1 phosphorylation. Finally, we provide evidence that HIF1 mediates CEACAM6 expression and that CEACAM6 is necessary to recruit ULK1 in a bacteria-containing signaling hub. Collectively, these results identify a new function for HIF1 in AIEC-dedicated xenophagy, and suggest that coactivation of autophagy and HIF1A expression may be a potential new therapy to resolve AIEC infection in CD patients.</p

DNA damage was detected by the comet assay in HeLa cells exposed to <i>E. coli</i> for 3 h.

<p>DNA damage was not detected in HeLa cells infected with the <i>E. coli</i> strain IHE3034 Δ<i>clbP</i> harboring a defective <i>pks</i> island (A). Comet assay was positive in HeLa cells infected with the <i>E. coli</i> strain IHE3034 harboring <i>pks</i> island (B) or with <i>E. coli</i> devoid of known cyclomodulin-encoding genes (C).</p

Cytopathic effect induced by <i>E. coli</i> live bacteria or protein extracts on epithelial cells at three-day post-infection.

<p>For CNF and CDT, the cytopathic effect is only observable with bacterial lysates. In contrast, for colibactin and CIF, a contact between bacteria and host cells is required. Colibactin, CDT and CIF induced cytopathic effects as seen by enlarged nuclei and cell distension (megalocytosis), while CNF induced multinucleation and enlargement of HeLa cells.</p

Distribution of <i>E. coli</i> strains producing various cyclomodulins according to phylogroups and specimen origins.

<p>A, <i>E. coli</i> strains (n = 70) isolated from CRC samples (n = 38), and B, <i>E. coli</i> strains (n = 46) isolated from diverticulosis samples (n = 31).</p

Colonization of diverticulosis and CRC samples by <i>E. coli</i> phylogroups (A, B1, B2 and D).

<p>Colonization of diverticulosis and CRC samples by <i>E. coli</i> phylogroups (A, B1, B2 and D).</p

Adhesion ability of <i>E. coli</i> strains isolated from diverticulosis and CRC (proximal and distal) samples to Int-407 intestinal epithelial cells.

<p>A, adhesion ability according to the specimen origin and E. coli phylogroup. B, Adhesion ability of <i>E. coli</i> strains isolated from diverticulosis and CRC (proximal and distal) samples and belonging to A and D phylogroups according to their ability to produce or not a cyclomodulin/genotoxin. The results are expressed as number of adherent bacteria per cell after 3 h infection period. Data are means+/−SEM for at least 3 independent experiments.</p

Proteolytic activity of meprins α and β on AIEC LF82 outer membrane proteins and flagellin.

<p>Total protein extracts from untreated or meprin-treated (100 µg/mL) whole bacteria were immunoblotted for OmpA and OmpC/F (A) or Flagellin (B). The inner membrane protein Lep was used as internal control. Amounts of proteins were quantified by using <i>Image J</i> software. Results are expressed as protein amount relative to Lep. Data are mean ± SEM for at least three independent experiments. Student's <i>t</i>-test, * <i>P</i><0.05.</p

Meprin treatment affect mannose residue recognition by AIEC and AIEC-induced IL-8 secretion by T84 cells.

<p>A, ability of type 1 pili to bind D-mannose residues as determined by a yeast aggregation test. AIEC LF82 bacteria were treated with 100 µg/ml of meprin α or β at 37°C for 120 min. A fixed amount of inactivated yeast cells (<i>Saccharomyces cerevisiae</i>) suspension and decreasing concentrations of treated and untreated bacteria were mixed, and the loss of the ability to form homogenous aggregation was used as the read-out for impaired type 1 pili-yeast interaction. B, Amount of IL-8 secreted by uninfected or AIEC LF82- or type 1 pili negative mutant LF82-Δ<i>fimA</i>-infected T84 cells, at 24 h post-infection. AIEC LF82 and LF82-Δ<i>fimA</i> bacteria were treated with 100 µg/ml of meprins. Il-8 secretion was determined by ELISA. Data are expressed as fold increase in the amount of secreted IL-8 ± SEM by T84 cells infected with untreated or treated bacteria relative to non infected cells. Student's <i>t</i>-test, * <i>P</i><0.05 for comparison between IL-8 secretion induced by untreated versus meprin-treated AIEC LF82 or LF82-Δ<i>fimA</i> bacteria. C, LF82-Δ<i>fimA</i> bacteria were pretreated with exogenous meprin α or meprin β at 100 µg/ml and undifferentiated T84 cells were infected at a MOI of 10. The number of associated bacteria was determined. Results are expressed as the percentage of cell-associated bacteria pretreated with exogenous meprins relative to untreated bacteria, defined as 100%.D, effect of meprins on recombinant human IL-8. Recombinant human IL-8 (110 ng/ml) was treated with meprin α or β (100 µg/ml), electroblotted and detected with mouse anti-human IL-8.</p

Effect of meprins on the ability of AIEC strains and <i>Salmonella</i> Typhimurium strain LT2 to adhere to and to invade intestinal epithelial cells.

<p>Bacteria were pretreated with exogenous meprin α or meprin β at 10 µg/ml. A and B, undifferentiated Intestine-407 (I407), Caco-2 and T84 cells infected with AIEC LF82 at a MOI of 10. The number of associated (A) and internalized (B) bacteria was determined. Results are expressed as the percentage of cell-associated (A) or intracellular bacteria (B) relative to initial inoculum. C and D, undifferentiated T84 cells were infected at a MOI of 10 with <i>Salmonella</i> Typhimurium and AIEC strains LF82, LF9, LF15 and LF31. The number of associated (C) and internalized (D) bacteria was determined. Results are expressed as the percentage of cell-associated (C) or intracellular bacteria (D) relative to untreated bacteria, defined as 100% (bar, C and D). Data are mean ± SEM for at least three independent experiments. Student's <i>t</i>-test, * <i>P</i><0.05.</p

Intestinal meprin α and meprin β mRNA.

<p>A and B, Mep1A (A) and Mep1B (B) mRNA levels were determined by TaqMan quantitative real time PCR and are displayed as amounts relative to the intestinal epithelial marker villin-1. Healthy controls (hc) were compared with ulcerative colitis (UC) and Crohn's disease (CD) patients. CD patient biopsies were separated into groups with normal appearance or with macroscopic inflammation, as determined by an experienced endoscopist. Statistics were performed using GraphPad Prism 5.0 Software, and <i>P</i> values were calculated with the non-parametric Mann-Whitney test. C and D, Mep1A (C) and Mep1B (D) mRNA levels in C57Bl/6J mouse ileum and colon uninfected or infected with AIEC LF82 bacteria. The effect of AIEC LF82 infection on meprin expression was determined in mouse ileum and colon by quantitative real time PCR. Data are displayed as meprin amounts relative to the housekeeping TATA box binding protein (TBP) gene. Statistics were performed using GraphPad Prism 5.0 Software, and <i>P</i> values were calculated with the one-way ANOVA test. Dot plots show individual samples with relative mRNA (cDNA) amounts on a linear scale. Horizontal bars represent the median.</p