Localization of all four ZnT zinc transporters in Dictyostelium and impact of ZntA and ZntB knockout on bacteria killing.

Professional phagocytes have developed an extensive repertoire of autonomous immunity strategies to ensure killing of bacteria. Besides phagosome acidification and the generation of reactive oxygen species, deprivation of nutrients and the lumenal accumulation of toxic metals are essential to kill ingested bacteria or inhibit the growth of intracellular pathogens. Here, we used the soil amoeba Dictyostelium discoideum, a professional phagocyte that digests bacteria for nutritional purposes, to decipher the role of zinc poisoning during phagocytosis of nonpathogenic bacteria and visualize the temporal and spatial dynamics of compartmentalized, free zinc using fluorescent probes. Immediately after particle uptake, zinc is delivered to phagosomes by fusion with 'zincosomes' of endosomal origin, and also by the action of one or more zinc transporters. We localized the four Dictyostelium ZnT transporters to endosomes, the contractile vacuole and the Golgi complex, and studied the impact of znt knockouts on zinc homeostasis. We show that zinc is delivered into the lumen of Mycobacterium smegmatis-containing vacuoles, and that Escherichia coli deficient in the zinc efflux P1B-type ATPase ZntA are killed faster than wild-type bacteria.