PacL-organized membrane-associated effluxosomes coordinate multi-metal resistance in Mycobacterium tuberculosis

Metal ion homeostasis is crucial for bacterial pathogens to withstand metal-induced stress during infection. However, the mechanisms underlying bacterial resistance to metal stress remain incompletely understood, particularly how bacteria coordinate responses to simultaneous exposure to multiple metals. Here, we uncover a previously unrecognized mechanism by which Mycobacterium tuberculosis, the causative agent of tuberculosis, orchestrates a coordinated response to multi-metal stress. We demonstrate that M. tuberculosis assembles dynamic, membrane-associated platforms, organized by PacL proteins, that confer resistance to multiple metals simultaneously. PacL proteins function as scaffolds, clustering multiple P-type ATPase (P-ATPase) pumps, CtpC, CtpG, and CtpV, into functional complexes we term "effluxosomes". Our findings show that PacL proteins are critical for stabilizing CtpG within membrane-associated clusters, conferring cadmium tolerance, while CtpC serves as a backup, promoting cross-resistance to both zinc and cadmium. Using super-resolution microscopy and single-particle tracking, we elucidate the 3D structure and dynamics of effluxosomes in the mycobacterial membrane. We further demonstrate that conserved residues within the transmembrane domain of PacL proteins are crucial for the assembly of dynamic effluxosomes, which are essential for P-ATPase activity. Additionally, we reveal that PacL1 exhibits metallochaperone activity, binding zinc, cadmium, and copper via a conserved C-terminal motif. Proximity labeling further identifies an extensive PacL1 interaction network, encompassing multiple proteins involved in stress adaptation. Our findings introduce effluxosomes as dynamic, membrane-associated efflux machineries that mediate coordinated multi-metal resistance in M. tuberculosis, providing new insights into bacterial metal homeostasis and unveiling potential antimicrobial targets.