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

Abstract

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 P 1B-type ATPase ZntA are killed faster than wild-type bacteria.

Time-resolved RNA-seq profiling of the infection of Dictyostelium discoideum by Mycobacterium marinum reveals an integrated host response to damage and stress

“…Additionally, the late time points showed a clear downregulation of lytic enzymes and ATP hydrolysis-coupled proton transport and an upregulation of genes belonging to the TOR pathway (regulation of the intracellular signal transduction). Lipid metabolism (ceramide synthases) and transport (ABC transporters) seem to play a central role during the infection time-course as genes belonging to these groups were upregulated at various time points, which is in perfect agreement with the documented interface between the host and the pathogen lipid metabolisms (figure 7 E-G) (Barisch et al, 2015;Barisch and Soldati, 2017).…”

Dictyostelium discoideumflotillin homologues are essential for phagocytosis and participate in plasma membrane recycling and lysosome biogenesis

“…Despite this faster maturation, phagosomes of vacuolin KO mutants are Role of vacuolins in phagocytosis and phagosome biogenesis 9 still bactericidal and as proteolytic as in wt cells. This confirms once again, that acidification is not the only factor required for killing bacteria, which also requires proteolysis, oxidation and/or toxic metals Barisch et al, 2018). Surprisingly, the phenotypes described here do not reproduce what had been found with the previously described vacAand vacBmutants .…”

“…To this effect, during maturation, the phagosome, by fusing with lysosomes, acquires the proton pump vacuolar-ATPase (v-ATPase), which acidifies the compartment, followed by delivery of lysosomal enzymes, activated at low pH (Souza et al, 1997;Clarke et al, 2002). In addition, phagocytic cells produce reactive oxygen species (ROS) via the NADPH-oxidase complex and pump toxic levels of metals inside the phagosome (Soldati and Neyrolles, 2012;Dunn et al, 2018;Barisch et al, 2018), rendering the newly-formed phago-lysosome a highly bactericidal environment. In D. discoideum, lysosomes eventually mature into neutral and nondegradative compartments, called postlysosomes (PLs), by retrieval of the v-ATPase and, later, of the Role of vacuolins in phagocytosis and phagosome biogenesis 2 lysosomal hydrolases.…”

Uropathogenic Escherichia coli employs both evasion and resistance to subvert innate immune-mediated zinc toxicity for dissemination

“…tuberculosis (16). Recently, the zinc toxicity antibacterial pathway was shown to be operational in the soil amoeba Dictyostelium discoideum (40), thus indicating that this is an evolutionarily conserved innate immune response. Here we show, through the use of newly developed zinc stress reporter systems as well as zinc-sensitive mutants, that UPEC strain EC958 both evades and resists innate immune-mediated zinc toxicity in vitro and in vivo.…”