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Escherichia coli  strains are responsible for a majority of human extra-intestinal infections, resulting in huge direct medical and social costs. We had previously shown that HlyF encoded by a large virulence plasmid harbored by pathogenic  E. coli  is not a hemolysin but a cytoplasmic enzyme leading to the overproduction of outer membrane vesicles (OMVs). Here, we showed that these specific OMVs inhibit the macroautophagic/autophagic flux by impairing the autophagosome-lysosome fusion, thus preventing the formation of acidic autolysosomes and autophagosome clearance. Furthermore, HlyF-associated OMVs were more prone to activate the non-canonical inflammasome pathway. Because autophagy and inflammation are crucial in the host’s response to infection especially during sepsis, our findings revealed an unsuspected role of OMVs in the crosstalk between bacteria and their host, highlighting the fact that these extracellular vesicles have exacerbated pathogenic properties. Abbreviations:  AIEC: adherent-invasive  E. coli BDI: bright detail intensityBMDM: bone marrow‐derived macrophagesCASP: caspase E. coli: Escherichia coli EHEC: enterohemorrhagic  E. coli ExPEC: extra-intestinal pathogenic  E. coli GSDMD: gasdermin DGFP: green fluorescent proteinHBSS: Hanks’ balanced salt solutionHlyF: hemolysin FIL1B/IL‐1B: interleukin 1 betaISX: ImageStreamX systemLPS: lipopolysaccharideMut: mutatedOMV: outer membrane vesicleRFP: red fluorescent proteinTEM: transmission electron microscopyWT: wild-type

Outer membrane vesicles produced by pathogenic strains of Escherichia coli block autophagic flux and exacerbate inflammasome activation