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.

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.

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:aa3oxidase 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 TTM or Q203 alone or co-treated with both at the indicated concentrations. 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. ns= not significant.

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. 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.

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.