Mycobacterium tuberculosis phagosome Ca2+ leakage triggers multimembrane ATG8/LC3 lipidation to restrict damage in human macrophages.

The role of canonical autophagy in controlling Mycobacterium tuberculosis (Mtb), referred to as xenophagy, is understood to involve targeting Mtb to autophagosomes, which subsequently fuse with lysosomes for degradation. Here, we found that Ca2+ leakage after Mtb phagosome damage in human macrophages is the signal that triggers autophagy-related protein 8/microtubule-associated proteins 1A/1B light chain 3 (ATG8/LC3) lipidation. Unexpectedly, ATG8/LC3 lipidation did not target Mtb to lysosomes, excluding the canonical xenophagy. Upon Mtb phagosome damage, the Ca2+ leakage-dependent ATG8/LC3 lipidation occurred on multiple membranes instead of single or double membranes excluding the noncanonical autophagy pathways. Mechanistically, Ca2+ leakage from the phagosome triggered the recruitment of the V-ATPase-ATG16L1 complex independently of FIP200, ATG13, and proton gradient disruption. Furthermore, the Ca2+ leakage-dependent ATG8/LC3 lipidation limited Mtb phagosome damage and restricted Mtb replication. Together, we uncovered Ca2+ leakage as the key signal that triggers ATG8/LC3 lipidation on multiple membranes to mitigate Mtb phagosome damage.