Abstract
<title>Abstract</title> <p>Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of global mortality from a single infectious agent. The mitochondrial fission protein dynamin-related protein 1 (Drp1) is known to modulate host immunity, but its role in anti-mycobacterial defense is unclear. Here, we found that Mtb infection triggers Drp1 phosphorylation at Ser616 in macrophages and TB patient tissues. Genetic or pharmacological inhibition of Drp1 enhanced intracellular Mtb survival, whereas Drp1 overexpression restricted bacterial loads. Myeloid-specific Drp1 knockout mice exhibited exacerbated pulmonary infection and inflammation. Mechanistically, Drp1 promoted xenophagic clearance of Mtb, as evidenced by increased LC3-II lipidation, p62 recruitment to Mtb, lysosomal-associated membrane protein 1 (LAMP1) colocalization with bacteria, and chloroquine-sensitive autophagic flux. Mechanistically, Drp1 promoted xenophagic clearance of Mtb, as evidenced by increased LC3-II lipidation, p62 recruitment to Mtb, LAMP1 colocalization with bacteria, and chloroquine-sensitive autophagic flux. These effects were independent of AMP-activated protein kinase (AMPK)/mechanistic target of rapamycin (mTOR) and Parkin pathways but required ROS production and hypoxia-inducible factor-1α (HIF-1α) stabilization, as ROS scavenging abolished, while HIF-1α activation restored Drp1-mediated autophagy and bacterial control. Carbonyl cyanide m-chlorophenyl hydrazine (CCCP) treatment rescued autophagic flux and bacterial killing in Drp1-silenced cells. Importantly, a phosphorylation-deficient S616A mutant failed to promote ROS production, HIF-1α stabilization, autophagic flux, lysosomal delivery, and Mtb clearance, identifying Ser616 phosphorylation as the critical switch. Together, these findings identify phosphorylated Drp1 (S616) as a key upstream regulatory node in macrophage anti-tuberculosis immunity and provide a mechanistic basis for targeting Drp1 S616 phosphorylation as a potential host-directed therapeutic strategy against TB.</p>