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.

Keywords: CtpC; Dictyostelium discoideum; Mycobacterium marinum; ZnTs; infection; zinc poisoning; zinc transporters.

FIG 1

Free Zn²⁺ accumulates inside the MCV. (A) Scheme depicting the different localizations of free Zn²⁺ in D. discoideum: zincosomes (Z), contractile vacuole (CV), and phagosomes (e.g., MCVs [33]). Nu, nucleus. (B) Live cell imaging and illustrations of NBD-TPEA-treated cells expressing AmtA-mCherry (magenta). M. marinum was stained with Vybrant DyeCycle Ruby (cyan) before imaging. Arrows point at Zn²⁺-positive MCVs; arrowheads label bacteria exposed to the D. discoideum cytosol. Scale bars, 5 μm. One example is shown for wt and ΔRD1 M. marinum at 26 and 45 hpi, respectively. The cartoons illustrate the heterogeneity of Zn²⁺ labeling in intact and broken MCVs. (C) Percentage of NBD-TPEA-positive MCVs at different times post-infection with M. marinum wt and ΔRD1. Bars represent the mean and SEM from three independent experiments. About 300 wt and 200 ΔRD1 MCVs were counted in total. Statistical differences were calculated with a Fisher least significant difference (LSD) post hoc test after two-way ANOVA (*, P < 0.05; **, P < 0.01; ****, P < 0.0001).

FIG 2

M. marinum senses and reacts to toxic levels of Zn²⁺ during infection and in vitro. (A) Heat map representing the transcriptional data shown in Table S1A. Cells were infected with GFP-expressing M. marinum wt, and samples were collected at different hpi. The time points with statistically significant differential expression are marked with asterisks (*, P < 0.05; **, P < 0.01). Colors indicate the amplitude of expression of M. marinum ctpC, ctpC-like, ctpV, and ctpV-like (in logarithmic fold change [logFC]) in infected cells compared to M. marinum grown in broth: from dark red (highest expression) to dark blue (lowest expression). (B) Normalized mRNA levels of Mm_ctpC, Mm_ctpC-like, Mm_ctpV, and Mm_CtpV-like in GFP-expressing M. marinum grown in 7H9 with increasing concentrations of ZnSO₄ compared to bacteria grown in 7H9 without extra ZnSO₄ (depicted as dashed gray line). To confirm the successful deletion of ctpC, the mRNA levels in M. marinum ΔctpC were tested as well. Shown are mean and standard deviations from two independent experiments. Statistical differences were calculated with an unpaired t test (*, P < 0.05). (C) Dose-response curves of M. marinum wt, ΔctpC, and ΔctpC::ctpC grown in broth supplemented with increasing concentrations of ZnSO₄ (0, 0.125, 0.25, 0.5, and 1 mM). The fluorescence intensities of GFP and E2-Crimson were used as a proxy for bacterial growth. The areas under the curve were calculated for the three strains, and the values were plotted as a function of ZnSO₄ concentrations. Error bars indicate the SD from eight technical replicates from two independent biological replicates. Statistical differences were calculated with a Fisher LSD test after two-way ANOVA (****, P < 0.0001). (D) D. discoideum was infected with GFP- or E2-Crimson-expressing M. marinum wt, ΔctpC (GFP expressing), or ΔctpC::ctpC (E2-Crimson expressing). The intracellular bacterial growth (in relative fluorescence units [RFUs]) was monitored every hour. Shown is the fold increase in bacterial fluorescence over time of two independent biological replicates. Error bars indicate the SD from eight technical replicates. Statistical differences were calculated with Dunnett’s multiple-comparison test after two-way ANOVA (****, P < 0.0001).

FIG 3

ZntA and ZntB transporters are recruited to the MCV and induced upon infection. (A and B) Immunofluorescence staining at different times postinfection of ZntA- or ZntB-mCherry-expressing cells (shown in magenta) infected with GFP-expressing M. marinum (shown in cyan). MCVs were visualized by staining for the endocytic marker p80 (21) (shown in green). Arrowheads mark Znt-positive MCVs. Scale bars, 5 μm (zoom, 2 μm). (C and D) Quantification of panels A and B, respectively. Two independent experiments were performed. 305 and 246 MCVs were analyzed manually for the presence of ZntA and ZntB at MCVs, respectively. (E) Scheme depicting the localization of the four D. discoideum Znts during infection with M. marinum. CV, contractile vacuole; Nu, nucleus. (F) Heat map representing the transcriptional data shown in Table S1C. Cells were either infected with GFP-expressing M. marinum wt or mock infected, and samples were collected at different hpi. The time points with statistically significant differential expression are marked with asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Colors indicate the strength of expression of zntA to zntD (in logFC) in infected cells compared to mock infected: from dark red (highest expression) to dark blue (lowest expression).

FIG 4

Zn²⁺ accumulates in MCVs of cells lacking ZntA but decreases in zntB KO amoebae. (A and B) Live imaging of NBD-TPEA-treated wt and zntA KO cells (A) or zntB KO cells (B) expressing AmtA-mCherry (magenta). M. marinum was stained with Vybrant DyeCycle Ruby (cyan) before imaging at 3 hpi (B) or 46 hpi (A). Arrows label MCVs. Scale bars, 5 μm. (C and D) Quantification of the normalized integrated intensity of NBD-TPEA inside MCVs per MCV area at 3 hpi in Ax2(Ka) wt and zntA KO cells (C) or Ax4 wt and zntB KO cells (D) infected with mCherry-expressing M. marinum. Images at 3 hpi were taken automatically using an ImageXpress spinning disc confocal microscope (Molecular Devices). To quantify NBD-TPEA inside MCVs, a MetaXpress (Molecular Devices) pipeline that detects NBD-TPEA inside MCVs was set up (see Materials and Methods). Four independent experiments were performed. A total of 7,353, 8,559, 5,713, and 4,679 MCVs were analyzed in infected Ax2(Ka) wt, Ax2(Ka) zntA KO, Ax4 wt, and Ax4 zntB KO cells, respectively. Statistical differences were assessed with an unpaired t test (*, P < 0.05; ***, P < 0.001). (E and F) Summarizing schemes illustrating the mislocalization of Zn²⁺ in infected zntA (E) or zntB (F) KO cells. While depletion of ZntA leads to an accumulation of Zn²⁺ into zincosomes and the MCV, loss of ZntB leads to a reduction of Zn²⁺ inside MCVs. Z, zincosomes; CV, contractile vacuole; MCV, Mycobacterium-containing vacuole; Nu, nucleus.

FIG 5

M. marinum intracellular growth is impaired in cells lacking ZntA or ZntB. Ax2(Ka) wt or zntA KO cells (A) and Ax4 wt or zntB KO cells (B) were infected with luciferase-expressing M. marinum wt or ΔctpC, and the intracellular bacterial growth (in RLUs) was monitored every hour. Shown is the fold increase in bacterial luminescence over time. Error bars indicate the SEM from three independent experiments. Statistical differences of pairwise comparisons were calculated with a Fisher LSD post hoc test after two-way ANOVA (****, P < 0.0001).