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::KanR) 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.