<i>Mycobacterium tuberculosis</i> phagosome Ca ²⁺ leakage triggers multimembrane ATG8/LC3 lipidation to restrict damage in human macrophages

The role of canonical autophagy in controlling <i>Mycobacterium tuberculosis</i> (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.