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OP002. The GCN2/eIF2α/ATF4 signaling pathway is necessary for autophagy response to infection with Crohn's disease-associated adherent-invasive Escherichia coli

H. Nguyen1, J. Carriere1, G. Dalmasso1, A.-C. Maurin2, A. Bruhat2, A. Darfeuille-Michaud1, 1University of Auvergne, UMR 1071/Inserm, Clermont-Ferrand, France, 2INRA, Human nutrition unit (UNH), Theix, France


A high prevalence of the adherent-invasive E. coli (AIEC) in the intestinal mucosa of Crohn's disease patients has been shown. Upon AIEC infection, autophagy is induced in host cells to restrain AIEC intracellular replication. The mechanism underlying such autophagy induction, however, remains largely unknown. Recently we showed that the GCN2/eIF2α/ATF4 pathway is essential for stress-induced autophagy gene expression. Activation of GCN2 leads to eIF2α phosphorylation, suppressing global mRNA translation while increasing translation of specific mRNAs, such as the transcription factor ATF4. Here, we investigated the role of this pathway in autophagy response to AIEC infection.


Autophagic activity was assessed by Western blot and immunofluorescent labelling of LC3. Intracellular bacterial number was determined by invasion assay and confocal microscopy. Binding of ATF4 to autophagy gene promoter was assessed by Chromatin immunoprecipitation (ChIP) assay. Wild type (WT) and GCN2 knockout (KO) mice were infected with an AIEC reference strain LF82 by gavage.


LF82 infection of human intestinal epithelial T84 cells activated the GCN2/eIF2α/ATF4 pathway as shown by increased phospho-GCN2 and phospho-eIF2α levels, enhanced ATF4 protein expression, and upregulated mRNA expression levels of ATF4 target genes. To explore the role of this pathway in host response to AIEC infection, we used GCN2-deficient mouse embryonic fibroblasts (GCN2−/− MEF). GCN2 depletion suppressed eIF2α activation and inhibited the increase in ATF4 protein level induced by LF82 infection. mRNA expression levels of the autophagy genes p62, MAP1lc3, Beclin1, atg3 and atg7 were increased in WT MEF upon LF82 infection, and this was blocked in GCN2−/− MEF. ChIP assay showed that GCN2 depletion inhibited the LF82-induced binding of ATF4 to the promoters of these autophagy genes. Autophagy induction upon LF82 infection was suppressed in GCN2−/− MEF, leading to increased LF82 intracellular replication and enhanced pro-inflammatory cytokine production. In vivo study consistently showed that LF82 infection activated the GCN2/eIF2α/ATF4 pathway in enterocytes from WT mice, but not those from GCN2 KO mice. Upon AIEC infection, autophagy was induced in WT mouse-derived enterocytes, which was not observed in KO mice. LF82 persistence in the gut was increased in KO mice, shown by higher fecal count of LF82, compared to WT mice. This was accompanied with aggravated intestinal inflammation in infected KO mice.


The GCN2/eIF2α/ATF4 pathway is activated in host cells upon AIEC infection, which is served as a defense mechanism to induce a functional autophagy to control the intracellular replication of AIEC.