Zinc intoxication of Mycobacterium marinum during Dictyostelium discoideum infection is counteracted by induction of the pathogen Zn2+ exporter CtpC

Macrophages use diverse strategies to kill or restrict intracellular pathogens. Some of these strategies involve the deprivation of bacteria from (micro)nutrients such as transition metals, and the bacteria intoxication through metal accumulation. Little is known about the chemical warfare between Mycobacterium marinum , a close relative of the human pathogen M. tuberculosis , and its hosts. Here we use the professional phagocyte Dictyostelium discoideum to investigate the role of free zinc during M. marinum infection. We show that M. marinum infection induces the accumulation of free zinc inside the Mycobacterium-containing vacuole (MCV), achieved by the induction and recruitment of the D. discoideum zinc efflux pumps ZntA and ZntB. In cells lacking the ZntA detoxifying transporter there is further attenuation of M. marinum growth, possibly due to a compensatory efflux of free zinc into the MCV. This efflux is presumably carried out by ZntB, the main zinc transporter in endosomes and phagosomes. Counterintuitively, M. marinum growth is also impaired in zntB KO cells, where MCVs accumulate less zinc. We also demonstrate that M. marinum senses toxic levels of zinc and responds by upregulating its zinc exporter CtpC, which supports bacteria survival under these restrictive conditions. Attenuation of M. marinum intracellular proliferation in zntA and zntB KO cells is accentuated in the absence of CtpC, confirming that mycobacteria face noxious levels of free zinc. Altogether, we show for the first time that M. marinum infection induces a deleterious zinc elevation in D. discoideum , which is counteracted by the bacteria with the induction of its zinc exporter CtpC.