Mycobacterial p(1)-type ATPases mediate resistance to zinc poisoning in human macrophages

Abstract

Mycobacterium tuberculosis thrives within macrophages by residing in phagosomes and preventing them from maturing and fusing with lysosomes. A parallel transcriptional survey of intracellular mycobacteria and their host macrophages revealed signatures of heavy metal poisoning. In particular, mycobacterial genes encoding heavy metal efflux P-type ATPases CtpC, CtpG, and CtpV, and host cell metallothioneins and zinc exporter ZnT1, were induced during infection. Consistent with this pattern of gene modulation, we observed a burst of free zinc inside macrophages, and intraphagosomal zinc accumulation within a few hours postinfection. Zinc exposure led to rapid CtpC induction, and ctpC deficiency caused zinc retention within the mycobacterial cytoplasm, leading to impaired intracellular growth of the bacilli. Thus, the use of P(1)-type ATPases represents a M. tuberculosis strategy to neutralize the toxic effects of zinc in macrophages. We propose that heavy metal toxicity and its counteraction might represent yet another chapter in the host-microbe arms race.

Figure 1

The Microbial and Host Macrophage Transcriptomes Reflect Free Zinc Overload during M. tuberculosis Infection (A) RT-qPCR analysis of ctpC and Rv3269 expression during human macrophage infection. Cells were infected for the indicated time. The data shown are means ±SD of ctpC expression, normalized with respect to sigA, in intracellular bacteria, relative to the inoculum (0). The data shown are from an experiment performed in triplicate and were analyzed with Student's t test. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; the data are representative of four independent biological replicates. (B) Transcriptional profile analysis. Red-blue density display showing the expression levels of the human metallothionein-encoding genes MT1H, MT1M, MT1X, and MT2A at 4, 18, or 48 hr after M. tuberculosis infection in macrophages, as reported in microarray analysis by Tailleux et al. (Tailleux et al., 2008). Genes are ordered in rows, conditions as columns. The colors indicate the strength of expression, running from red (high levels of expression) to blue (low levels of expression; in arbitrary units). (C) RT-qPCR analysis of the expression ratio of the genes encoding the metal transcription factor-1 (MTF1), MT1, MT2, and the zinc exporter ZnT1/SLC30A1 in human macrophages during M. tuberculosis infection, relative to uninfected cells (0). After indicated time of infection, total cellular RNA was extracted and analyzed by RT-qPCR. Data are displayed as fold-change relative to expression before infection, and are normalized relative to hprt. Averaged data (mean ±SD from a triplicate experiment) from one representative donor out of four tested are shown. (D–F) MTF1 localization. (D) MTF1 was immunolocalized (green) in uninfected macrophages (upper panels) and in macrophages infected as in (A) for 24 hr with DsRed-expressing M. tuberculosis (red, lower panels). Cell nuclei are labeled with the fluorescent dye TOPRO-3 (blue). The oblique bars indicate analysis lines as used in (E) to quantify the MTF1 signal. (E) Average MTF1 signal intensity, analyzed as in (D), for 25 cells per condition. (F) MTF1 signal intensity ratio (nucleus/cytoplasm) measured before infection (0) and 2 or 24 hr after infection. Scale white bar, 20 μm. The data shown in (D)–(F) are representative of two independent experiments, and data shown in (E) and (F) represent mean ±SD of values calculated from 40 randomly chosen cells in several fields.

Figure 2

Free Zinc Is Released within Macrophages during M. tuberculosis Infection and Accumulates within the Mycobacterial Phagosome (A) Free zinc labeling in macrophages. Human monocyte-derived macrophages were left uninfected (left panel) or infected for 4 hr as in Figure 1A (middle and right panels), in the absence (left and middle panels) or presence (right panel) of the zinc-chelating agent TPEN. Cells were fixed and stained with the free zinc-specific fluorescent probe FluoZin-3 (FZ3, green). Scale bar, 20 μm. The picture is representative of five independent experiments. (B) FZ3 signal quantification (in arbitrary units) from ∼25 blind-scored cells from three random fields as in (A). The data shown are means ±SD of the signal measured from 20 cells and were analyzed with Student's t test. ^∗∗∗p < 0.001; the data are representative of five independent experiments. (C) Subcellular localization of the FZ3-positive compartments in macrophages. Macrophages were transfected with RFP-reporter plasmids expressing cathepsin D (CathD, top row panels) and LAMP1 (middle row panels), or Rab5 (bottom row panels). After 4 hr of infection with M. tuberculosis, the cells were fixed, stained with FZ3 as in (A), and analyzed by confocal microscopy. Arrows indicate colocalization of the red and green signals. Scale bar is either 20 μm (left column panels) or 5 μm (all panels in the columns to the right). The squared fields depicted in the panels of the left column represent the magnified area shown for all panels in the columns to the right. Representative images from two experiments are shown; images are quantified in (D). (D) Quantification of FZ3 colocalization with RFP-fused CathD, LAMP1, or Rab5, as shown in (C). Percentages of colocalization (means ±SD) were determined by analyzing the proportions of FZ3-positive structures stained for CathD, LAMP1, or Rab5 from high-resolution confocal microscopy pictures. At least 1000 structures from ten cells were counted per condition to calculate mean ±SD. FluoZin-3 colocalization; the data are representative of two independent experiments. (E and F) FZ3 staining of the mycobacterial phagosome. One macrophage infected with DsRed-expressing M. tuberculosis (red), fixed, and stained with FZ3 is shown (E), together with a higher magnification of the mycobacterial vacuole in another macrophage (F). Scale bar is either 10 μm (E) or 1 μm (F). Representative pictures from five independent experiments are shown; images are quantified in (G). (G) Quantification of FZ3 colocalization with DsRed-expressing M. tuberculosis after 2, 4, and 24 hr infection. The mean percentage (±SD) of FZ3-positive phagosomes (as assessed for ∼50 phagosomes in five different fields) is shown; data are representative of three independent experiments.

Figure 3

Expression of the Gene Encoding the M. tuberculosis Metal Cation-Transporting P-Type ATPase CtpC Is Induced by Zinc (A) Transcriptional profile analysis. mRNA levels (in arbitrary units) of the genes most strongly induced by zinc treatment in M. tuberculosis as revealed by microarray analysis. Bacteria were incubated with 0, 50, or 500 μM ZnSO₄ in Sauton medium for 4 hr. Bacterial RNA was prepared for subsequent microarray analysis. The data shown are means ±SD of duplicate experiments and were analyzed with Student's t test; ^∗p < 0.05. (B) RT-qPCR analysis of the expression of the M. tuberculosis ctp genes upon exposure to zinc. Bacteria were incubated with 0 or 500 μM ZnSO₄, and RNA extracted and treated as in (A). The data shown are mean ±SD of a triplicate experiment measuring ctp expression, normalized with respect to sigA, in zinc-treated bacteria, relative to untreated bacteria. Data are representative of at least two independent experiments. (C) RT-qPCR analysis of ctpC expression following the exposure of M. tuberculosis to various divalent metal cations. Bacteria were left untreated or were incubated with 500 μM ZnSO₄, 200 nM CdSO₄, 500 μM CuSO₄, or 200 μM NiSO₄ in Sauton medium for 4 hr. The data are shown as in (B) and are representative of two independent experiments.

Figure 4

CtpC Is Involved in Zinc Efflux in M. tuberculosis (A) Intrabacterial zinc content in M. tuberculosis wild-type and a ctpC null mutant. M. tuberculosis wild-type (GC1237) or a ctpC null mutant (ctpC::Kan^R) was left untreated or incubated with 0.1 mM ZnSO₄ for 1 hr at 37°C, washed twice in PBS, heat inactivated, and the bacterial pellets processed for ICP-MS analysis. The data shown are the means ±SD of intrabacterial zinc concentration expressed as nanograms Zn per grams of bacterial extract in one representative experiment performed in duplicate, out of two independent experiments. (B) Distribution of zinc crystals within macrophages and intraphagosomal bacilli. At selected times postinfection (p.i.) with M. tuberculosis, macrophages were treated for capturing of zinc ions by the AMG technique. Cells were then processed for EM observation. The zinc crystals formed during this procedure appeared as small dense deposits. Panels 1–3, intracellular localization of zinc crystals. The zinc crystals accumulated within mitochondria (M), along the inner face of the nuclear membrane (NMb) over the dense chromatin (panel 1), within lysosomes (L) but not in the Golgi (G) (panel 2), and also along the plasma membrane (PM) and the inner face of the phagosome membrane (PhM) (panel 3). Panels 4–7, localization of zinc crystals in intraphagosomal mycobacteria. Zinc was concentrated in three distinct parts of the Mycobacterium, i.e., on the outer surface (arrows in panel 4), in between the electron translucent layer of the cell wall and the cytoplasmic membrane (arrows in panel 5), and in the cytoplasm (arrows, panels 6 and 7) of live (panel 6) and dead (panel 7) bacilli. In panels 5 and 6, zinc crystals are also observed on the outer surface of the Mycobacterium. Scale bar, 0.5 μm. (C) Zinc distribution in wild-type and ctpC null mutant M. tuberculosis strains harbored in human macrophage phagosomes. Human macrophages were infected with either wild-type M. tuberculosis or its ctpC null mutant counterpart. At 4 and 24 hr p.i., cells were processed as in (B). Bacilli were scored for the presence of zinc at either the outer surface, the cell wall, or the cytoplasm. Quantitations were made on 100–150 bacilli per sample and in two independent experiments. Shown is the mean percent of zinc precipitate localization in one representative out of two experiments.

Figure 5

CtpC Is Involved in Zinc Detoxification and Contributes to the Intracellular Survival of M. tuberculosis (A) Differential sensitivity of M. tuberculosis wild-type and the ctpC null mutant to free zinc. M. tuberculosis wild-type (GC1237), a ctpC null mutant (ctpC::Kan^R), or the cosmid-complemented strain (I437) was allowed to grow in 7H9-ADC medium containing 0.1 mM ZnSO₄ or without zinc supplementation (Control). Bacterial growth was monitored by turbidity measurement (McFarland units). The data are representative of three independent experiments. (B) Differential sensitivity of M. tuberculosis wild-type and the ctpC null mutant to free zinc. M. tuberculosis wild-type (GC1237), a ctpC null mutant (ctpC::Kan^R), or the cosmid-complemented strain (I437) were allowed to grow in 7H9-ADC medium containing 0.5 mM ZnSO₄ or without zinc supplementation (Control). Bacterial growth was monitored by plating on agar and counting CFU. The data are representative of two independent experiments. (C) Differential ability of M. tuberculosis wild-type and the ctpC null mutant to replicate in human macrophages. M. tuberculosis wild-type (GC1237), a ctpC null mutant (ctpC::Kan^R), or a plasmid-complemented strain (CP) was used to infect human macrophages at a multiplicity of infection of one mycobacteria per ten cells. After 4 hr, cells were washed and incubated in fresh medium for 5 days. The data shown are means ±SD of intracellular CFU counts in an experiment carried out in triplicate and analyzed with Student's t test. ^∗p < 0.05; NS, not significant. The data are representative of three independent experiments.

Figure 6

Zinc Accumulates in Classical Phagolysosomes and Contributes to Pathogen Killing (A) Labeling of free zinc in macrophages. Human macrophages were left uninfected (left panel) or infected for 30 min with E. coli (W3110) at a multiplicity of infection of five bacteria per cell (middle and right panels), in the absence (left and middle panels) or presence (right panel) of the zinc-chelating agent TPEN. Cells were fixed and stained with the zinc-specific fluorescent probe FluoZin-3 (FZ3, green), and the plasma membrane was stained with the lectin marker WGA (red). Scale bar, 40 μm. (B) FZ3 signal quantification (in arbitrary units) from ∼25 cells randomly chosen from three fields as in (A). Data are shown as in Figure 2B. (C) FZ3 staining of an E. coli-containing phagolysosome. A macrophage infected with Crimson-expressing E. coli (red), fixed and stained with FZ3, is shown. Scale bar, 10 μm. (D and E) Differential killing of E. coli wild-type and the zntA null mutant (D) or together with the complemented strain (zntA::Cm/zntA, E) by human macrophages. E. coli wild-type (W3110) or a zntA null mutant (zntA::Cm) was used to infect human macrophages at a multiplicity of infection of one bacterium per cell. After 0.5 hr, cells were washed and further incubated in fresh medium. After 1, 2, and 4 hr of infection, cells were lysed and cell lysates were plated onto agar for CFU scoring. The data shown are the means ±SD of intracellular E. coli CFU ratios, relative to CFU contents at 0.5 hr, in a triplicate experiment analyzed with Student's t test. ^∗p < 0.05; ^∗∗p < 0.01. All experiments were performed independently at least two times, and a representative experiment is shown.