Time-resolved transcriptomic profiling of Dictyostelium discoideum infection with Mycobacterium marinum reveals Atg9-dependent restriction via maintenace vacuolar integrity

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major global health challenge, highlighting the need to better understand host mechanisms that restrict mycobacterial infection. Mycobacterium marinum (Mm) shares key virulence mechanisms with Mtb and provides a suitable model for studying mycobacterial pathogenesis. Here, we used Dictyostelium discoideum (Dd), a genetically tractable phagocytic model with evolutionarily conserved pathways shared with mammalian macrophages, to investigate host responses to Mm infection. Time-resolved transcriptomic profiling across early, intermediate, and late stages of infection identified global infection-responsive genes, stage-specific pathways, and substantial conservation with transcriptional responses of human macrophages infected with Mtb. Autophagy and ESCRT pathways were dynamically regulated throughout infection, suggesting stage-specific roles in host defence. We focused on Atg9, an autophagy factor that was strongly induced during infection and recruited to damaged mycobacterium-containing vacuoles (MCVs). Atg9 promoted membrane damage control and maintained MCV integrity, thereby preventing premature bacterial escape into the cytosol of virulent Mm. Loss of Atg9 increased vacuolar damage, accelerated cytosolic escape, and enhanced intracellular bacterial growth, phenocopying the infection phenotype of atg1-deficient cells. Our findings further distinguish complementary host defence mechanisms acting at distinct stages of infection: Atg9-associated membrane repair, potentially involving ATG8ylation, limits vacuolar damage and bacterial escape during early infection, whereas Atg9-dependent xenophagic restriction contributes to bacterial clearance at later stages. Together, these findings establish Dd as a powerful model for dissecting conserved host responses to mycobacteria and identify Atg9-dependent membrane protection as a key host resistance mechanism with potential relevance to TB pathogenesis and therapeutic intervention.