Mycobacterial HBHA drives mitochondrial damage-mediated apoptosis via inhibiting mitophagy in macrophages.

Heparin-binding hemagglutinin adhesin (HBHA), an important adhesion protein located on the surface of Mycobacterium tuberculosis (Mtb), plays a critical role in the pathogen infection process. Macrophages serve as the primary effector cells that modulate the host immune response. It has been established that HBHA can regulate macrophage autophagy and apoptosis; however, the precise mechanism underlying HBHA's effect on macrophage apoptosis remains to be fully elucidated. In this study, HBHA was employed to stimulate mouse macrophage RAW 264.7 cells. Subsequently, apoptosis (including DNA fragmentation, the rate of apoptosis, and apoptosis-related proteins), mitochondrial damage (including mitochondrial morphology, membrane potential, and permeability transition), reactive oxygen species (ROS) generation, and mitophagy (including mitochondrial-lysosome formation and mitophagy-related pathway proteins) were systematically evaluated. The ROS scavenger N-acetyl cysteine (NAC) and the mitophagy inducer Carbonylcyanide 3-chlorophenylhydrazone (CCCP) were utilized to investigate the specific mechanism by which HBHA regulates macrophage apoptosis. The results demonstrated that HBHA stimulation significantly promoted apoptosis and ROS production in RAW 264.7 cells, leading to mitochondrial structural damage, decreased membrane potential, and increased permeability. Additionally, HBHA inhibited the formation of mitophagy-lysosome complexes and the activation of mitophagy-related pathways. NAC intervention partially reversed the effects of HBHA on ROS production, mitochondrial dysfunction, and apoptosis in RAW 264.7 cells. However, CCCP intervention effectively suppressed HBHA-induced apoptosis in RAW 264.7 cells by activating mitophagy. HBHA induces mitochondrial damage through the promotion of ROS production and the inhibition of mitophagy, ultimately leading to macrophage apoptosis. Our findings offer a novel perspective on the molecular mechanism by which Mtb evades macrophage immune clearance.