A Novel Salmonella Effector Reveals The Mechanism Of Bacterial Autophagy Pathway

Selective autophagy in response to pathogen infection,known as bacterial autophagy(xenophagy),plays a critical role in host defense.The Holy Grail of xenophagy study is to understand how the cells sense intracellular bacteria and signal the ATG machinery for autophagic induction.Extensive efforts from many leading laboratories have been invested into this topic,but contradictory results are recorded in the literature.In this study,we for the first-time exploited genome-wide transposon screen to probe bacterial autophagy and identified a novel T3SS effector SebX in S.Typhimurium with an extremely potent autophagy-inhibiting activity.Removing sebX from S.Typhimurium increases the percentage of autophagy-targeted bacteria to 80%,explaining why only a small fraction of wild-type S.Typhimurium is targeted by host autophagy.We further found that SebX by itself can inhibit xenophagy regardless of the bacterial species but does not affect other autophagy pathways including canonical autophagy.Identification of SebX promotes us to design and successfully perform a sophisticated FACS-based CRISPR/Cas9 screen for host genes essential for bacterial autophagy.The screen reveals a novel V-ATPase-ATG16L1 signaling axis that responds specifically to bacterial infections and mediates initiation of bacterial autophagy.The V-ATPase-ATG16L1 association requires the WD40 domain of ATG16L1 and is sensitive to inhibition by SebX.We also show that SebX inhibition of V-ATPase-ATG16L1 association promotes S.Typhimurium proliferation in infected mice.Next,we obtain the crystal structure of SebX which reveals it is a novel ADP-ribosyltransferase(like cholera toxin,it is activated by ARF binding).After enrichment of the modified substrate and mass spectrometry analysis,we identified that ATP6V0C,a subunit of the V-ATPase complex,is the host target and substrate of SebX and SebX specifically ADP-ribosylates Q124 of ATP6V0C during bacterial infection.Finally,just like the effect of SebX action,ATP6V0C Q124 mutation determines the occurrence of bacterial autophagy but without affecting canonical autophagy pathway.The identification of SebX effector is unexpected,provides the hard evidence for recognition mechanism of bacterial autophagy.Our discovery of the V-ATPase-ATG16L1 signaling axis answers the longstanding question about autophagic recognition of bacterial infection,which will transform the study of bacterial autophagy and impact the field of innate immunity.