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. 2011 Jan 25;108(4):1621-6.
doi: 10.1073/pnas.1009261108. Epub 2011 Jan 4.

Copper resistance is essential for virulence of Mycobacterium tuberculosis

Frank Wolschendorf  1 , David Ackart, Tej B Shrestha, Laurel Hascall-Dove, Scott Nolan, Gyanu Lamichhane, Ying Wang, Stefan H Bossmann, Randall J Basaraba, Michael Niederweis
Affiliations

Copper resistance is essential for virulence of Mycobacterium tuberculosis

Frank Wolschendorf et al. Proc Natl Acad Sci U S A. .

Abstract

Copper (Cu) is essential for many biological processes, but is toxic when present in excessive amounts. In this study, we provide evidence that Cu plays a crucial role in controlling tuberculosis. A Mycobacterium tuberculosis (Mtb) mutant lacking the outer membrane channel protein Rv1698 accumulated 100-fold more Cu and was more susceptible to Cu toxicity than WT Mtb. Similar phenotypes were observed for a M. smegmatis mutant lacking the homolog Ms3747, demonstrating that these mycobacterial copper transport proteins B (MctB) are essential for Cu resistance and maintenance of low intracellular Cu levels. Guinea pigs responded to infection with Mtb by increasing the Cu concentration in lung lesions. Loss of MctB resulted in a 1,000- and 100-fold reduced bacterial burden in lungs and lymph nodes, respectively, in guinea pigs infected with Mtb. In mice, the persistence defect of the Mtb mctB mutant was exacerbated by the addition of Cu to the diet. These experiments provide evidence that Cu is used by the mammalian host to control Mtb infection and that Cu resistance mechanisms are crucial for Mtb virulence. Importantly, Mtb is much more susceptible to Cu than other bacteria and is killed in vitro by Cu concentrations lower than those found in phagosomes of macrophages. Hence, this study reveals an Achilles heel of Mtb that might be a promising target for tuberculosis chemotherapy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
MctB is required for copper resistance of and maintaining a low intracellular copper concentration in M. smegmatis. (A) Expression of ms3747 in M. smegmatis. Proteins were extracted with 2% SDS from WT M. smegmatis, the ms3747 mutant ML77, and ML77 complemented with the ms3747 expression vector pML451. Proteins were detected in a Western blot by using the monoclonal antibody 5D1.23. (B) Serial dilutions of cultures of M. smegmatis SMR5 (WT), ML77 (ΔmctB), and ML77 complemented with mctB were spotted on 7H10 agar plates without or with CuSO4 at a concentration of 25 μM. (C) M. smegmatis SMR5 (black bars) and the Δms3747 mutant ML77 (gray bars) were grown in self-made Middlebrook 7H9 medium with 0, 6.3, or 25 μM CuSO4. Samples were taken after growth for 36 h. Copper was determined by measuring the absorption of the Cu(II)–dithizone complex at 553 nm.
Fig. 2.
Fig. 2.
MctB is required for copper resistance and maintenance of a low intracellular copper concentration in M. tuberculosis. (A) Schematic representation of the chromosomal rv1698 region of M. tuberculosis H37Rv (WT). A fragment of 39 bp was replaced by the 45-bp loxP site in the 5′ part of the gene introducing stop codons in all three reading frames to construct the rv1698 deletion mutant ML256 by homologous recombination. The genes are drawn to scale. (B) Expression of mctB in Mtb. Proteins were extracted with 2% SDS from WT Mtb and the mctB mutant ML256. ML257 is an ML256 derivative carrying the integrative mctB expression vector pML955. Proteins were detected in a Western blot by using the MctB-specific monoclonal antibody 5D1.23. (C) Copper susceptibility. Serial dilutions of log-phase cultures of Mtb H37Rv (WT), ML256 (ΔmctB), and ML257 (+mctB) were spotted on 7H11/OADC agar without or with CuSO4 at a concentration of 150 μM. (D) Growth defect of Mtb ΔmctB mutant. Mtb WT, the ΔmctB mutant ML256, and the complemented mutant ML257 were grown in 7H10 medium supplemented with peptone (1 g/L) with or without 100 μM CuSO4. The optical density at 600 nm was measured at the indicated time points. (E) Mtb (black bars), the ΔmctB mutant ML256 (light gray bars), and the complemented mutant ML257 (dark gray bars) were grown in minimal medium with 0.8, 6.3, or 25 μM CuSO4. Copper was determined by measuring the absorption of the Cu(II)-dithizone complex at 553 nm.
Fig. 3.
Fig. 3.
MctB-mediated copper resistance is required for virulence of M. tuberculosis in guinea pigs. (A) Cu concentrations in granulomatous lesions from guinea pigs infected with Mtb H37Rv. Tissue Cu was measured by atomic absorption spectroscopy in homogenates of lung parenchyma of five uninfected guinea pigs, and pooled primary granulomas were isolated by microdissection from lungs of five infected guinea pigs. *P ≤ 0.05. (B–D) Guinea pigs were infected with Mtb WT H37Rv (dark gray bars), the ΔmctB mutant ML256 (medium gray bars), and the complemented mutant ML257 (light gray bars). (B) The bacterial burden in lung, lymph node, and spleen was determined from tissue homogenates from guinea pigs 30 d after infection. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; number of infected animals per data point: n = 5. (C) Lung lesions were quantified from the area of total lung parenchyma affected by granulomatous inflammation. Data represents the mean percentage of affected lung for each treatment group or the mean percent area of lesions with necrosis. *P ≤ 0.05, **P ≤ 0.01; n = 5. (D) Lymph node lesions were quantified from the area of total lymph node parenchyma affected by granulomatous inflammation. Data represents the mean percentage of affected lymph node for each treatment group or the mean percent area of lesions with necrosis. *P ≤ 0.05, **P ≤ 0.01; n = 5.
Fig. 4.
Fig. 4.
Role of copper in bactericidal activity of macrophages and model of copper-resistance mechanisms of M. tuberculosis. Cu(II) is taken up across the plasma membrane of macrophages by the high-affinity Cu transporter Ctr1 (32). The Cu-chaperon Atox1 is a cytoplasmic Cu-binding and -transport protein that interacts with the Cu(II)-transporting ATPase Atp7A (33). In macrophages stimulated by IFN-γ or by hypoxia, cytoplasmic vesicles that contain Atp7A fuse with phagosomes (16). As a consequence, Cu is pumped into the phagosome, which contributes to its bactericidal activity (7). MctB is a pore-forming protein located in the outer membrane (OM) of Mtb (13) and prevents the accumulation of Cu within the mycobacterial cell probably by efflux of cuprous ions. Another important component of Cu homeostasis in Mtb is the cytoplasmic Cu-binding metallothionein MymT (12). The inner membrane (IM) transporter CtpV might be a Cu efflux protein (9, 11).
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