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. 2025 Jun 5:14:RP105794.
doi: 10.7554/eLife.105794.

Chalkophore-mediated respiratory oxidase flexibility controls M. tuberculosis virulence

John A Buglino #  1 , Yaprak Ozakman #  1 , Chad E Hatch  2 , Anna Benjamin  1 , Derek S Tan  2   3 , Michael S Glickman  1
Affiliations

Chalkophore-mediated respiratory oxidase flexibility controls M. tuberculosis virulence

John A Buglino et al. Elife. .

Abstract

Oxidative phosphorylation has emerged as a critical therapeutic vulnerability of M. tuberculosis (Mtb). However, it is unknown how intracellular bacterial pathogens such as Mtb maintain respiration during infection despite the chemical effectors of host immunity. Mtb synthesizes diisonitrile lipopeptides that tightly chelate copper, but the role of these chalkophores in host-pathogen interactions is also unknown. We demonstrate that M. tuberculosis chalkophores maintain the function of the heme-copper bcc:aa3 respiratory supercomplex under copper limitation. Chalkophore deficiency impairs Mtb survival, respiration to oxygen, and ATP production under copper deprivation in culture, effects that are exacerbated by loss of the heme-dependent Cytochrome BD respiratory oxidase. Our genetic analyses indicate that the maintenance of respiration is the major cellular target of chalkophore-mediated copper acquisition. M. tuberculosis lacking chalkophore biosynthesis is attenuated in mice, a phenotype that is also severely exacerbated by loss of the CytBD respiratory oxidase. We find that the host immune pressure that attenuates chalkophore-deficient Mtb is independent of adaptive immunity and neutrophils. These data demonstrate that chalkophores counter host-inflicted copper deprivation and highlight a multilayered system by which M. tuberculosis maintains respiration during infection.

Keywords: Copper; M. tuberculosis; infectious disease; microbiology; nutrional immunity; oxidative phosphorylation; tuberculosis.

Plain language summary

When a bacterium known as Mycobacterium tuberculosis infects humans, it can lead to a disease called tuberculosis – one of the leading causes of death from an infectious disease worldwide. The bacteria hide within certain cells in the body so that they are less accessible to the host’s immune system. This approach is so successful that patients typically require long courses of antibiotics lasting many months to eliminate the bacteria. However, the emergence of drug-resistant strains of M. tuberculosis means that new methods to target the bacteria are urgently needed. One possible target for future therapies is a system known as diisonitrile lipopeptide chalkophores, which bind to copper and import the metal into the bacterium from its surroundings. Although certain proteins in bacteria require copper to work properly, the target of the copper acquired by the chalkophores in M. tuberculosis has remained unclear. Here, Buglino, Ozakman et al. used genetic and biochemical approaches to study why diisonitrile lipopeptide chalkophores collect copper in M. tuberculosis. The experiments showed that the chalkophores supply copper to proteins with a key role in respiration, the process by which cells make chemical energy needed for many cell activities. When there was a shortage of copper in their surrounding environment, mutant M. tuberculosis cells lacking the chalkophores were less able to produce chemical energy and more likely to die than healthy M. tuberculosis cells. This effect was more severe if the cells were also missing an enzyme that enables the bacteria to respire without copper. Further experiments in mice found that during a tuberculosis infection, the immune system targeted copper-containing proteins in its attempts to kill the bacteria. The mutant M. tuberculosis cells were less effective at infecting the mice, suggesting that chalkophores help the bacteria defend themselves against the host immune system. Taken together, these findings reveal that copper-containing proteins in M. tuberculosis are a major target of the immune system. In the future, increasing our understanding of these proteins and identifying drugs that interfere with their activities may lead to new, more effective therapies for tuberculosis.

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Conflict of interest statement

JB, YO, CH, AB, DT No competing interests declared, MG Declares equity and consulting fees from Vedanta biosciences and consulting fees from Fimbrion therapeutics

Figures

Figure 1.
Figure 1.. Copper deprivation in chalkophore-deficient M. tuberculosis mimics bcc:aa3 oxidase inhibition.
(A) Heat map of transcripts encoding selected respiratory chain components determined by RNA sequencing of M. tuberculosis wild-type (WT) or Δnrp treated with TTM or Q203. WT_GSE is the published dataset GSE159080 of M. tuberculosis H37Rv treated with Q203. Genes in the chalkophore cluster are boxed in red, genes encoding the cytochrome BD (CytBD) oxidase in blue, and genes encoding components of the bcc:aa3 supercomplex are in green. (B) RT-qPCR of the transcript encoding CydA in M. tuberculosis WT, Δnrp, and complemented strain treated with varying TTM concentrations for 4 hr. Error bars represent the standard error of the mean (SEM). Statistical significance determined via two-way ANOVA with Tukey correction for multiple comparisons. *p<0.05, **p<0.01, ****p<0.0001. (C) RT-qPCR of the transcript encoding CydA in M. tuberculosis WT, Δnrp, and complemented strain treated with 20 μM TTM for 24 hr. Error bars are SEM. Statistical significance determined via two-way ANOVA with Tukey correction for multiple comparisons. **p<0.01, ***p<0.001, ****p<0.0001. (D) Dose-dependent effect of tetrathiomolybdate (TTM) on growth of the indicated M. tuberculosis strains at 7 d post inoculation. The dotted line indicates the starting inoculum. Error bars are SEM. Statistical significance determined via two-way ANOVA with Tukey correction for multiple comparisons. *p<0.05.
Figure 2.
Figure 2.. Chalkophores maintain M. tuberculosis viability through the heme-copper bcc:aa3 oxidase during copper starvation.
(A) Schematic of the terminal respiratory oxidases of M. tuberculosis. The bcc:aa3 oxidase is a heme-copper oxidase and cytochrome BD (CytBD) is a copper-independent heme oxidase. Both transfer electrons to oxygen. Q203 is an inhibitor of bcc:aa3 by targeting the QcrB subunit, whereas ND-011992 targets CytBD. The two oxidases are individually dispensable due to compensation by the other oxidase, but M. tuberculosis lacking both is nonviable. The model to be tested is that copper chelation deprives the bcc:aa3 oxidase of copper and that diisonitrile chalkophores counter this copper deprivation stress. (B) Liquid growth assays of the indicated strains with or without 20 µM TTM treatment. OD600 at day 10 post-inoculation displayed. Dotted line indicates starting inoculum. Error bars are SEM. Statistical significance determined via two-way ANOVA with Tukey correction for multiple comparisons. ****p<0.0001. (C) Bacterial survival of the indicated strains on agar media containing no addition, 1 mM BCS, or 40 μM TTM. Dotted line indicates lower limit of detection (LLOD). Error bars are SEM. Statistical significance determined via two-way ANOVA with Tukey correction for multiple comparisons. **p<0.01, *p<0.05 (D) The copper deprivation sensitivity of M. tuberculosis ΔnrpΔcydAB strain can be rescued with a synthetic diisonitrile chalkophore. Liquid growth assays of ΔnrpΔcydAB with DMSO, 20 μM TTM, or 20 μM TTM with 10 μM of the diisonitrile chalkophore pictured in panel A. Error bars are SEM. (E) The bcc:aa3 oxidase is the only target of copper starvation countered by diisonitrile chalkophores. Liquid growth assays of the indicated strains treated with 10 or 20 µM TTM, or DMSO vehicle control. OD600 at day 10 post-inoculation displayed. Dotted line indicates starting inoculum. Error bars are SEM. Statistical significance determined via two-way ANOVA with Tukey correction for multiple comparisons. ns = not significant, ****p<0.0001. (F) The effect of copper deprivation is masked by inhibition of QcrB subunit of bcc:aa3. Liquid growth assays of the indicated strains treated with Q203 (100 nM) alone or co-treated with 100 nM Q203 and 10 μM TTM. OD600 at day 7 post-inoculation displayed. Dotted line indicates starting inoculum. Error bars are SEM. Statistical significance determined via two-way ANOVA with Tukey correction for multiple comparisons. ***p<0.001, ns = not significant.
Figure 2—figure supplement 1.
Figure 2—figure supplement 1.. Loss of cydAB severely sensitizes chalkophore-deficient M. tuberculosis to copper chelation.
(A, B) Images of triplicate 10-fold serial dilutions of the indicated M. tuberculosis strains on agar media containing no chelator, 1 mM bathocuproinedisulfonic acid (BCS), or 40 µM tetrathiomolybdate (TTM). (C) Quantitation of the agar survival assay in panel B. Error bars are SEM. Statistical significance determined by two-way ANOVA with Tukey correction for multiple comparisons. *p<0.05, **p<0.01, ***p<0.001.
Figure 2—figure supplement 2.
Figure 2—figure supplement 2.. Full growth curves of chalkophore deficient strains with TTM copper chelation.
(A) Quantitation of optical density in liquid culture from Figure 2D at day 10 time point of the ΔnrpΔcydAB strain treated with DMSO, tetrathiomolybdate (TTM), or TTM + synthetic diisonitrile. Error bars are SEM. Statistical significance determined by two-way ANOVA with Tukey correction for multiple comparisons ***p<0.001. Dotted line indicates starting inoculum. (B–F) Full growth curves in liquid media quantitated in Figure 2B for the indicated strains treated with DMSO (gray symbols) or 20 µM TTM (blue symbols). Error bars are SEM and if not visible are within the graphed symbol.
Figure 2—figure supplement 3.
Figure 2—figure supplement 3.. Full growth curves in basal and copper chelation conditions of ctaD mutant strains.
(A-B) Full growth curves in liquid media quantitated in Figure 2E for the indicated strains treated with DMSO (A) or 20 µM tetrathiomolybdate (TTM) (B). Error bars are SEM and are within the symbol if not visible.
Figure 3.
Figure 3.. Chalkophore biosynthesis maintains oxidative phosphorylation through the heme-copper bcc:aa3 oxidase.
(A) Methylene blue decolorization assay of oxygen consumption under copper deprivation (tetrathiomolybdate, TTM) or treatment with Q203 in wild-type (WT) or ΔnrpΔcydAB M. tuberculosis at day 0 (d0) or day 3 (d3) of incubation. Clear vials indicate oxygen consumption by respiration. (B) Quantitative measurement of oxygen consumption using oxygen-sensitive optical sensors. WT M. tuberculosis treated with DMSO, ND-011992, Q203, or both ND-011992 and Q203. Oxygen measurements were taken daily. Each point represents three measurements of two biological replicates. Error bars are SEM. Statistical significance between Q203 and ND-011992 +Q203 determined via two-way ANOVA with Tukey correction for multiple comparisons. ****p<0.0001. (C) Same assay as in panel B with WT and Δnrp M. tuberculosis treated with DMSO or 25 μM TTM. Error bars are SEM. (D) Same assay as in panel B with WT, ΔnrpΔcydAB, or ΔnrpΔcydAB + nrp treated with 25 μM TTM. Error bars are SEM. Statistical significance between WT and ΔnrpΔcydAB treated with 25 μM TTM determined via two-way ANOVA with Tukey correction for multiple comparisons. ****p<0.0001. (E) Cellular ATP levels determined by BacTiter-Glo in the indicated strains treated with DMSO, 20 or 40 μM TTM, or 100 nM Q203. [ATP] determined by standard curve determined in growth media containing the same quantities of DMSO, TTM, or Q203. Error bars are SEM. Statistical significance determined via two-way ANOVA with Tukey correction for multiple comparisons. *p<0.05, ***p<0.001, ****p<0.0001. (F) Relative abundance of a CtaD-ALFA protein in M. tuberculosis of the indicated genotype treated with bathocuproinedisulfonic acid (BCS) or TTM. See Figure 3—figure supplement 1 for primary immunoblot data. Error bars are SEM. Statistical significance determined via two-way ANOVA with Tukey correction for multiple comparisons. ns = not significant.
Figure 3—figure supplement 1.
Figure 3—figure supplement 1.. Chalkophores protect the heme-copper respiratory oxidase in copper-limiting conditions.
(A) Methylene blue decolorization assay in WT M. tuberculosis or M. tuberculosisΔnrp at assay start (d0) or after 3 d of incubation in a sealed tube (d3) treated with DMSO, 50 µM tetrathiomolybdate (TTM), or the combination of Q203 (400 nM) and ND-011992 (50 µM). (B) Copper chelation does not destabilize the CtaD protein M. tuberculosis lacking the CtaD subunit of the bcc:aa3 oxidase and complemented with a fully functional CtaD with a C-terminal ALFA epitope tag (see Figure 2E) either in the wild-type background (WT) or M. tuberculosis Δnrp (KO), which lacks diisonitrile chalkophore biosynthesis, and treated with either bathocuproinedisulfonic acid (BCS) or TTM. Full immunoblots for the ALFA tag or RpoB as a loading control are shown. Images were quantified using ImageJ software. Copper chelation synergizes with CytBD inhibition in the absence of chalkophore biosynthesis. Serial dilutions of M. tuberculosis WT,!1nrp,!1nrp +nrp strains were cultured on agar media containing TTM, ND-011992 (ND), or both. *p=0.0148 by two-way ANOVA.
Figure 4.
Figure 4.. Respiratory chain flexibility is critical for M. tuberculosis virulence.
(A, B) Bacterial titers in the lung (A) or spleen (B) in mice infected with M. tuberculosis wild-type (WT), Δnrp, ΔnrpΔcydAB, or ΔnrpΔcydAB + nrp. Error bars are SEM. Statistical significance determined via two-way ANOVA with Tukey correction for multiple comparisons. Not significant (ns), **p<0.01, and ***p<0.001. (C, D) Copper deprivation by the host is independent of neutrophils. Bacterial titers in the lung (C) or spleen (D) in mice infected with M. tuberculosis WT or ΔnrpΔcydAB, or ΔnrpΔcydAB + nrp treated with isotype control antibodies or anti-Ly6G antibodies to deplete neutrophils. Flow cytometric quantitation of neutrophil depletion is provided in Figure S5. Error bars are SEM. (E, F) Copper deprivation by the host is independent of adaptive immunity. Bacterial titers in the lung (E) or spleen (F) in C57BL/6 J or C57BL/6 SCID mice infected with M. tuberculosis WT or ΔnrpΔcydAB. Error bars are SEM.
Figure 4—figure supplement 1.
Figure 4—figure supplement 1.. Attenuation of chalkophore-deficient M. tuberculosis is independent of neutrophils and adaptive immunity.
(A) Flow cytometric quantitation of neutrophils in the lung at day 7 and 14 post-infection with the indicated M. tuberculosis strains (wild-type, WT or ΔnrpΔcydAB) and treated with isotype control antibody or anti-Ly6G. Neutrophils were defined as CD11B+GR1+ and their percentage among CD45+ cells is graphed. Statistical significance determined by unpaired Welch’s t-test. *p<0.05, **p<0.01. Error bars are SEM (B) Survival curve of C57BL/6 SCID mice infected with WT or ΔnrpΔcydAB M. tuberculosis.
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