Zn(2+) Intoxication of Mycobacterium marinum during Dictyostelium discoideum Infection Is Counteracted by Induction of the Pathogen Zn(2+) Exporter CtpC

Macrophages use diverse strategies to restrict intracellular pathogens, including either depriving the bacteria of (micro)nutrients such as transition metals or intoxicating them via metal accumulation. Little is known about the chemical warfare between Mycobacterium marinum, a close relative of Mycobacterium tuberculosis (Mtb), and its hosts. We use the professional phagocyte Dictyostelium discoideum to investigate the role of Zn(2+) during M. marinum infection. We show that M. marinum senses toxic levels of Zn(2+) and responds by upregulating one of its isoforms of the Zn(2+) efflux transporter CtpC. Deletion of ctpC (MMAR_1271) leads to growth inhibition in broth supplemented with Zn(2+) as well as reduced intracellular growth. Both phenotypes were fully rescued by constitutive ectopic expression of the Mtb CtpC orthologue demonstrating that MMAR_1271 is the functional CtpC Zn(2+) efflux transporter in M. marinum. Infection leads to the accumulation of Zn(2+) inside the Mycobacterium-containing vacuole (MCV), achieved by the induction and recruitment of the D. discoideum Zn(2+) efflux pumps ZntA and ZntB. In cells lacking ZntA, there is further attenuation of M. marinum growth, presumably due to a compensatory efflux of Zn(2+) into the MCV, carried out by ZntB, the main Zn(2+) transporter in endosomes and phagosomes. Counterintuitively, bacterial growth is also impaired in zntB KO cells, in which MCVs appear to accumulate less Zn(2+) than in wild-type cells, suggesting restriction by other Zn(2+)-mediated mechanisms. Absence of CtpC further epistatically attenuates the intracellular proliferation of M. marinum in zntA and zntB KO cells, confirming that mycobacteria face noxious levels of Zn(2+).