Chalkophore mediated respiratory oxidase flexibility controls M. tuberculosis virulence

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:aa ₃ 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 maintenance of respiration is the only 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.

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 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 CytBD oxidase in 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 hours. Error bars represent 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 hours. 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 TTM on growth of the indicated M. tuberculosis strains at 7 days 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. 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 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 DMSO, 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 (100nM) alone or co-treated with 100nM Q203 and 10μM 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 3. Chalkophore biosynthesis maintains oxidative phosphorylation through the heme-copper bcc:aa₃ oxidase

A. Methylene blue decolorization assay of oxygen consumption under copper deprivation (TTM) or treatment with Q203 in 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 biologic 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 BCS or TTM. See Figure S4 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 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 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/6J or C57BL/6 SCID mice infected with M. tuberculosis WT or ΔnrpΔcydAB. Error bars are SEM.