PefA, an ALG-2-like Ca2+ sensor, regulates ESCRT and autophagy responses to mycobacterial vacuole damage

Calcium influx is a universal early signal triggering membrane repair pathways, yet how Ca2+ sensors coordinate the balance between ESCRT-mediated sealing and autophagy-based responses at damaged endolysosomes and pathogen-containing vacuoles remains unclear. Here we use the Dictyostelium discoideum-Mycobacterium marinum infection model, a surrogate to study intracellular pathogenesis of Mycobacterium tuberculosis, combined with genetic, imaging, and proteomic analyses to identify the penta-EF-hand protein PefA, an ALG-2-like Ca2+ sensor, as a Ca2+-responsive regulator that orchestrates recruitment of the E3 ubiquitin ligase TrafE, ESCRT components, and the autophagy machinery to damaged membranes. PefA is transcriptionally upregulated and accumulates at the mycobacterial vacuole, promoting timely repair that preserves vacuolar integrity and supports bacterial replication. Loss of PefA impairs ESCRT and autophagy engagement, leading to premature bacterial escape into the cytosol and altered infection outcomes. These findings uncover a conserved Ca2+-dependent mechanism linking membrane damage sensing to coordinated repair pathways and shapes host-pathogen interactions, with direct relevance to tuberculosis pathogenesis and host resilience to infection. TeaserThe PefA calcium sensor times membrane repair and autophagy to control vacuole integrity during mycobacterial infection.