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

ABSTRACT

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 Zn²⁺ during M. marinum infection. We show that M. marinum infection induces the accumulation of Zn²⁺ inside the Mycobacterium-containing vacuole (MCV), achieved by the induction and recruitment of the D. discoideum Zn²⁺ 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 Zn²⁺ into the MCV. This efflux is presumably carried out by ZntB, the main Zn²⁺ transporter in endosomes and phagosomes. Counterintuitively, M. marinum growth is also impaired in zntB KO cells, where MCVs accumulate less Zn²⁺. We also demonstrate that M. marinum senses toxic levels of Zn²⁺ and responds by upregulating its Zn²⁺ 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 Zn²⁺. Altogether, we show for the first time that M. marinum infection induces a deleterious Zn²⁺ elevation in D. discoideum, which is counteracted by the bacteria with the induction of its Zn²⁺ exporter CtpC.