Studies from Dr. Feng Shao's laboratory reveal the molecular mechanism of V-ATPase-ATG16L1-mediated xenophagy pathway using a Salmonella effector

On July 18, 2019 ---- Studies from Dr. Feng Shao's laboratory reveal the molecular mechanism of V-ATPase-ATG16L1-mediated xenophagy pathway using the Salmonella effector SopF. The work entitled “A Bacterial Effector Reveals the V-ATPase-ATG16L1 Axis that Initiates Xenophagy” is published in the journal Cell.

The process of eukaryotic cells recognizing intracellular pathogens through selective autophagy is named xenophagy. Xenophagy plays an important role in host innate immune defense against bacteria. The Holy Grail of xenophagy study is to understand how the cells sense intracellular bacteria and initiate the autophagy pathway. Extensive efforts from many leading laboratories have been invested in this topic, but contradictory results are recorded in literature.

Salmonella is often used as the bacterial model for studying the mechanism of xenophagy. However, it has long been noted that only a small portion of Salmonella that invade host cells are recognized by autophagy. In addition to studying the innate immune pathways in host cells, Shao’s laboratory has also long been devoted to the study of bacterial effectors that interfere with the host immune system. They hypothesized that there might be effector(s) in Salmonella that inhibits xenophagy. Through transposon screening of Salmonella, the researchers identified a novel Salmonella type III secretion system (T3SS) effector, SopF, that inhibits xenophagy. Deletion of sopF in Salmonella increased the autophagic recognition of bacteria to 80%. The researchers also found that ectopic expression of SopF in eukaryotic cells could inhibit xenophagy triggered by different kinds of bacteria without affecting the canonical autophagy pathway. Therefore, the discovery of SopF provides an excellent tool for studying the mechanism of xenophagy.

After an elaborate experimental design, the researchers used a combination of CRISPR and flow cytometry techniques to identify that the V-ATPase complex may be involved in xenophagy. Three sets of parallel experiments were used to demonstrate that the V-ATPase complex mediates xenophagy pathway. Under the condition of bacterial infection, the researchers detected that the V-ATPase complex recruited the autophagy protein ATG16L1 to the bacteria-containing vacuoles and initiated LC3 lipidation. They also demonstrated that that bacteria-induced vacuolar membrane damage is sensed by V-ATPase. The interaction between V-ATPase and ATG16L1 is dependent on the WD40 domain of ATG16L1 (this domain does not participate in the canonical autophagy and does not exist in yeast). And SopF promotes the spread and proliferation of Salmonella in vivo by hijacking the V-ATPase-ATG16L1 association.

By solving the crystal structure of SopF, the researchers found that it belongs to the ADP-ribosyltransferase superfamily. After enrichment of the modified substrates and mass spectrometry analysis, the researchers demonstrated that ATP6V0C, a subunit of the V-ATPase complex, is the host target of SopF. SopF catalyzes ADP-ribosylation of ATP6V0C on Gln124. After substituting the Gln124 with Ala, V-ATPase can no longer bind to ATG16L1, and the xenophagy process is completely inhibited. Thus, Gln124 in ATP6V0C is essential for infection-induced recruitment of ATG16L1 to the V-ATPase and bacterial autophagosome formation.

Dr. Yue Xu, a Ph.D. student at Shao’s laboratory (joint graduate program with China Agricultural University) is the first author of this article. Dr. Ping Zhou (a postdoc at Shao’s laboratory) and Dr. Sen Cheng (a Ph.D. student at Xioayun Liu’s laboratory at Peking University) have made great contributions to this work. Other authors of the paper include Dr. Qiuhe Lu, Dr. Xuyan Shi, Dr. Zhiwei Zhou, Dr. Wenqing Gao, Da Li, Dr. Huabin He, Dr. Jingjin Ding at Shao’s laboratory, and Li Lin at protein center, and Kathrin Nowak, Ann-Katrin Hopp, Dr. Michael O Hottiger at University of Zurich, Switzerland. Dr. Feng Shao is the corresponding author of this article. The work was supported by the Basic Science Center Project of NSFC, the National Key Research and Development Program of China, and the Strategic Priority Research Program of the Chinese Academy of Sciences. Research in the laboratory of M.O.H. is funded by the Kanton of Zurich and the Swiss National Science Foundation. This work was carried out at National Institute of Biological Sciences, Beijing.