Think zinc: Role of zinc poisoning in the intraphagosomal killing of bacteria by the amoeba Dictyostelium

Abstract

31Professional phagocytes have developed an extensive repertoire of autonomous immunity strategies 32 to ensure killing of bacteria. Besides phagosome acidification and the generation of reactive oxygen species, 33 deprivation of nutrients and the lumenal accumulation of toxic metals are essential to kill ingested bacteria or 34 inhibit growth of intracellular pathogens. We use the soil amoeba Dictyostelium discoideum, a professional 35 phagocyte that digests bacteria for nutritional purposes, to decipher the role of zinc poisoning during 36 phagocytosis of non-pathogenic bacteria and visualize the temporal and spatial dynamics of 37 compartmentalized, free zinc using fluorescent probes. Immediately after particle uptake, zinc is delivered to 38 phagosomes by fusion with "zincosomes" of endosomal origin, but also by the action of one or more zinc 39 transporters. We localize the four Dictyostelium ZnT transporters to endosomes, the contractile vacuole and 40 the Golgi apparatus, and study the impact of znt knockouts on zinc homeostasis. Finally, we show that zinc is 41 delivered into the lumen of Mycobacterium smegmatis-containing vacuoles, and that Escherichia coli 42 deficient in the zinc efflux P 1B -type ATPase ZntA is killed faster than wild type bacteria. 43

How Phagocytic Cells Kill Different Bacteria: a Quantitative Analysis Using Dictyostelium discoideum

“…We fed bacteria producing fluorescent proteins to D. discoideum cells and measured for each individual bacterium the time between its ingestion and the extinction of its fluorescence. Extinction of bacterial fluorescence has been previously used by us and others as a proxy for bacterial death ( 9 , 21 – 23 ). In this study, we use the term “killing” as synonymous with “extinction of fluorescence.” The representative images shown in Fig.…”