ATG16L1 Knockout Cell Line (A549)

() represents a major human bacterial pathogen leading to high morbidity and mortality in children and the elderly. Recent research emphasizes the role of extracellular vesicles (EVs) in bacterial pathogenicity. However, the contribution of EVs (pEVs) to host-microbe interactions has remained unclear. Here, we observed that infections in mice led to severe lung injuries and alveolar epithelial barrier (AEB) dysfunction. Infections of reduced the protein expression of tight junction protein OCLN (occludin) and activated macroautophagy/autophagy in lung tissues of mice and A549 cells. Mechanically, induced autophagosomal degradation of OCLN leading to AEB impairment in the A549 monolayer. released the pEVs that could be internalized by alveolar epithelial cells. Through proteomics, we profiled the cargo proteins inside pEVs and found that these pEVs contained many virulence factors, among which we identified a eukaryotic-like serine-threonine kinase protein StkP. The internalized StkP could induce the phosphorylation of BECN1 (beclin 1) at Ser93 and Ser96 sites, initiating autophagy and resulting in autophagy-dependent OCLN degradation and AEB dysfunction. Finally, the deletion of in completely protected infected mice from death, significantly alleviated OCLN degradation , and largely abolished the AEB disruption caused by pEVs . Overall, our results suggested that pEVs played a crucial role in the spread of virulence factors. The cargo protein StkP in pEVs could communicate with host target proteins and even hijack the BECN1 autophagy initiation pathway, contributing to AEB disruption and bacterial pathogenicity.: AEB: alveolarepithelial barrier; AECs: alveolar epithelial cells; ATG16L1: autophagy related 16 like 1; ATP:adenosine 5'-triphosphate; BafA: bafilomycin A; BBB: blood-brain barrier; CFU: colony-forming unit; co-IP: co-immunoprecipitation; CQ:chloroquine; CTRL: control; DiO: 3,3'-dioctadecylox-acarbocyanineperchlorate; DOX: doxycycline; DTT: dithiothreitol; ECIS: electricalcell-substrate impedance sensing; eGFP: enhanced green fluorescentprotein; erm: erythromycin-resistance expression cassette; Ery: erythromycin; eSTKs: eukaryotic-like serine-threoninekinases; EVs: extracellular vesicles; HA: hemagglutinin; H&E: hematoxylin and eosin; HsLC3B: human LC3B; hpi: hours post-infection; IP: immunoprecipitation; KD: knockdown; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LC/MS: liquid chromatography-mass spectrometry; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MVs: membranevesicles; NC:negative control; NETs:neutrophil extracellular traps; OD: optical density; OMVs: outer membrane vesicles; PBS: phosphate-buffered saline; pEVs: extracellular vesicles; protK: proteinase K; Rapa: rapamycin; RNAi: RNA interference; ; SNF:supernatant fluid; sgRNA: single guide RNA; ; TEER: trans-epithelium electrical resistance; moi: multiplicity ofinfection; TEM:transmission electron microscope; TJproteins: tight junction proteins; TJP1/ZO-1: tight junction protein1; TSA: tryptic soy agar; WB: western blot; WT: wild-type.