Review 5: "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: It is essential for Mycobacterium tuberculosis (Mtb) to scavenge trace metals from its host to survive. Mtb synthesizes small molecular lipopeptides termed chalkophores, that chelate host copper for import, whereby the copper is incorporated into Mtb metalloproteins. However, the role of chalkophores in Mtb biology and their targeted metalloproteins are unknown. This study investigates Mtb proteins that require chalkophores for copper incorporation and their effect on Mtb virulence. By using a chalkophore deficient mutant and RNA sequencing under copper starvation conditions, the paper shows that Mtb has a gene expression profile that mimics inhibition of the membrane enzyme complex, the bcc:aa3 supercomplex, the heme-copper respiratory oxidase. This suggests that chalkophores are required for copper insertion into the Mtb respiratory protein to ensure a functional heme-copper bcc:aa3 respiratory oxidase under copper limiting conditions. Using a series of Mtb mutants and inhibitors of Mtb respiratory oxidases, experiments demonstrate that the alternate copper-independent respiratory oxidase CytBD supercomplex can compensate for bcc:aa3 oxidase, and these oxidases were individually dispensable; however Mtb lacking both oxidases was non-viable. The study went on to demonstrate that oxygen consumption was abolished when bcc:aa3 oxidase was the only functional respiratory oxidase in the absence of the chalkophore. Furthermore, when bcc:aa3 oxidase is the sole respiratory oxidase in the absence of copper, Mtb cannot produce ATP, suggesting that the chalkophore maintains oxidative phosphorylation under copper starvation. Finally, mice infected with the Mtb chalkophore deficient mutant results in a mild Mtb attenuation phenotype. However, when mice were infected with chalkophore/CytBD deficient Mtb mutant, this resulted in a severe Mtb attenuation in the spleen, and complementation with the chalkophore biosynthetic pathway restored Mtb virulence. These results suggest that chalkophore mediated protection of the respiratory chain is critical to Mtb virulence. The redundant respiratory oxidases within Mtb provides respiratory chain flexibility may promote host adaptation. In short, this manuscript shows that Mtb chalkophores are required to scavenge for host copper to maintain a fully functional membrane bound supercomplex, the heme-copper bcc:aa3 respiratory oxidase. This new information about Mtb biology may be leveraged for drug discovery, highlighting that the Mtb respiratory pathway is a promising drug target, where one could target the Mtb chalkophore biosynthetic pathway in concert with CytBD, to obliterate Mtb.   

The experiments are extremely well thought-out and executed, with extensive literature comparisons. The manuscript is well-written. The conclusions of this body of research are exciting and well justified and will lead to further studies within this field. Enthusiasm for this body of work and the manuscript is high.