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Review 1: "Chalkophore Mediated Respiratory Oxidase Flexibility Controls M. Tuberculosis Virulence"

Reviewers found the study highly compelling, providing strong evidence for the crucial role of chalkophores in facilitating copper acquisition by Mycobacterium tuberculosis to maintain the function of the heme-copper bcc:aa3 respiratory oxidase.

Published onMay 16, 2024
Review 1: "Chalkophore Mediated Respiratory Oxidase Flexibility Controls M. Tuberculosis Virulence"

RR:C19 Evidence Scale rating by reviewer:

  • Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.

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Review: On balance, this is a great paper that explores the significance of MTB’s chalkophore system for the electron transport chain. It makes a lot of sense that depriving MTB of copper would impair the effectiveness of the heme-copper oxidase bcc:aa3.  The authors do a very nice job of empirically demonstrating that, in typical laboratory media, the combination of copper chelation and genetic deletion of chalkophore synthesis is necessary to achieve sufficient copper depletion to have a significant phenotype.  The downstream sequelae of disabling bcc:aa3 via copper depletion are elegantly explored in vitro, and are largely as one would expect.  If cytochrome bd is available as an alternate terminal oxidase, MTB is able to survive and even grow, although at a reduced rate; however in the absence of functioning cytochrome bd, MTB suffers catastrophic failure of respiration and ATP production, and consequently cell death.  I especially appreciated the detailed experiments which showed that in the setting of ctaD knockout or Q203 treatment, copper deprivation exerts no additional burden on MTB, thereby clarifying that cytochrome bcc:aa3 is the main pain-point for copper-deprived MTB.  The demonstration that exogenously supplied diisonitrile chalkophore can rescue genetic deletion of nrp is also a very nice additional confirmation of the overall model.

The mouse experiments clearly demonstrate the relevance of these dynamics to at least some in vivo conditions.  A significant fraction of MTB in muro seems to reside in low-copper niche, and depends on its chalkophore system to obtain sufficient copper.  The demonstration that the synergy between genetic ablation of the chalkophore system and cytochrome bd holds up in muro gives more confidence that the dynamics found in vitro may translate to human infections. To solidify this and understand better the role of this respiratory flexibility it will be important to see how the various mutants behave in animal models that develop more human-like pathology.

It is hard to find much to quibble with in this paper. The experiment in which various strains were plated onto copper replete and depleted agar plates (shown in figure 2C) is discussed as demonstrating 6 logs of killing, however I might rather have phrased it as demonstrating that spontaneous resistance of MTB ∆nrp∆cydAB to copper depletion arises at a rate of approximately 1 in 10^6.  It might be interesting to further investigate the colonies that grew on these plates – do they represent a stable genetic change that confers consistent resistance to copper depletion to their progeny, or do they rather represent a phenotypic adaptation that is not stably inherited.  If they represent a genetic mutation, it might be informative to sequence these colonies and determine what mutations they carry.

Overall this is a convincing piece of work that establishes the main role of the chalkophore system in recruiting copper to support one of the main terminal oxidases in Mycobacterium tuberculosis.

PJF and CEB3 are supported by the Intramural Research Program of the NIAID, NIH.

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