Review 3: "Chalkophore Mediated Respiratory Oxidase Flexibility Controls M. Tuberculosis Virulence"

RR:C19 Evidence Scale rating by reviewer:

• Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.

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Review: Buglino and Ozakman present a very interesting study that expands our understanding on the Mycobacterium tuberculosis respiratory chain network. Combining bacterial genetics, treatments with small molecules, measurement of oxidative phosphorylation related parameters and animal models of infection, the authors were able to produce a convincing argument that copper starvation negatively impacts the function of the major terminal oxidase cytochrome bc:aa3 oxidase. This work is of interest for the tuberculosis drug development field as it shines light to a previously unsuspected aspect of M. tuberculosis respiratory chain – a drug target space that has received considerable attention due to the approval of bedaquiline (ATP synthase inhibitor) for clinical use. It will also be of broad interest to the bacterial physiology field as it may be a mechanism used by other bacteria to protect respiration.

Nevertheless, there are a few issues that, in my opinion, should be addressed by the authors:

1. In the results section it is stated that “Assays on copper chelated agar media revealed a dramatic sensitization of chalkophore deficient M. tuberculosis by loss of the secondary oxidase with 6 logs of killing”. In the correspondent methods section, it is explained: “Washed cells were (...) spotted on to 7H10-OADC plates containing the indicated concentrations of TTM, BCS, or 314 DMSO vehicle control in triplicate.” This assay, as it is, cannot differentiate growth inhibition from killing. To make a claim on killing, the authors could, for example, spot the bacteria on filters placed on top of solid chelated medium and after treatment transfer the filter to rich media for outgrowth.

2. In Figure 2F, why is there different TTM concentration when in combination with Q203? The lack of synergy between Q203 and TTM does indicate that cytochrome bcc:aa3 is the major target of copper deficiency, but the data would be stronger if TTM was in the same concentration on both situations.

3. If a) cytochrome bcc:aa3 is the only target of copper starvation, and b) cytochrome bd oxidase can compensate for a compromised cytochrome bcc:aa3 during mouse infection; why does nrp knockout have an attenuated phenotype in vivo (with more prominence in the spleen)? This may indicate that copper deficiency have other (minor) targets beyond cytochrome bcc:aa3 contributing to the attenuated phenotype in vivo.

4. With the level of respiration inhibition observed in Fig. 3D for ΔnrpΔcydABtreated with TTM (similar to WT treated with ND-011992 + Q203 - a combination that is lethal to M. tuberculosis), I think it would be expectable to see killing. Yet, in Fig. 2B there is only growth inhibition. Do the authors have an explanation for this?

5. In the same line of thought, why are nrp and cydAB not synthetically lethal during mouse infection? Is copper depletion level not enough to trigger the phenotype? Are there possible compensatory effects to nrp?