Vacuolar-ATPase inhibitors are antimicrobial agents active against intracellular mycobacteria

<i>Mycobacterium tuberculosis</i> (Mtb) evades host defenses by inhibiting phagosome acidification in part through the secreted phosphatase PtpA, binding to the vacuolar ATPase (v-ATPase), and disrupting downstream cellular events. We investigated the antimicrobial effects of three v-ATPase inhibitors, Bafilomycin A1 (BafA1), Bafilomycin D, and Cladoniamide B (ClaB), on the growth of Mtb, <i>M. abscessus</i> (Mabs), and <i>M. bovis</i> BCG in THP-1 and murine infection models. We found potent inhibition of intracellular growth with MIC₅₀ in the nanomolar range, with compounds showing a bacteriostatic inhibition of Mtb growth in THP-1 macrophages. Axenic bacteria were not affected by 2 µM compound in broth, although lysate from macrophages incubated with ClaB resulted in a 50% reduction in bacterial growth in broth, which was further enhanced by the addition of zinc. We further discovered that BafA1 amplifies the cytotoxic effects of Mtb infection and limits Mtb's ability to delay apoptosis in host cells. BafA1 antimicrobial activity was abolished in Mtb PtpA knockout mutant, and BafA1 binding to PtpA was shown via an <i>in vitro</i> thermal shift assay. Our findings reveal a complex interplay between v-ATPase inhibition, host cell responses, and bacterial survival, challenging traditional views on phagosome acidification in pathogenesis and suggesting novel avenues for host-directed therapies against intracellular mycobacterial infections.