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 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 Zn2+ during M. marinum infection. We show that M. marinum senses toxic levels of Zn2+ and responds by upregulating one of its isoforms of the Zn2+ efflux transporter CtpC. Deletion of ctpC (MMAR_1271) leads to growth inhibition in broth supplemented with Zn2+ 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 Zn2+ efflux transporter in M. marinum. Infection leads to 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 ZntA, there is further attenuation of M. marinum growth, presumably due to a compensatory efflux of Zn2+ into the MCV, carried out by ZntB, the main Zn2+ transporter in endosomes and phagosomes. Counterintuitively, bacterial growth is also impaired in zntB KO cells, in which MCVs appear to accumulate less Zn2+ than in wild-type cells, suggesting restriction by other Zn2+-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 Zn2+. IMPORTANCE Microelements are essential for the function of the innate immune system. A deficiency in zinc or copper results in an increased susceptibility to bacterial infections. Zn2+ serves as an important catalytic and structural cofactor for a variety of enzymes including transcription factors and enzymes involved in cell signaling. But Zn2+ is toxic at high concentrations and represents a cell-autonomous immunity strategy that ensures killing of intracellular bacteria in a process called zinc poisoning. The cytosolic and lumenal Zn2+ concentrations result from the balance of import into the cytosol via ZIP influx transporters and efflux via ZnT transporters. Here, we show that Zn2+ poisoning is involved in restricting Mycobacterium marinum infections. Our study extends observations during Mycobacterium tuberculosis infection and explores for the first time how the interplay of ZnT transporters affects mycobacterial infection by impacting Zn2+ homeostasis.