Dear Mr Lee,

Thank you very much for submitting your manuscript "Predicting nitroimidazole antibiotic resistance mutations in Mycobacterium tuberculosis with protein engineering" (PPATHOGENS-D-19-01241) for review by PLOS Pathogens. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the manuscript as it currently stands. These issues must be addressed before we would be willing to consider a revised version of your study. We cannot, of course, promise publication at that time.

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The question whether this enzyme is a specific menaquinone reductase is relevant. Thus, it is warranted to probe a few substrates as suggested. In addition, it is important to note that fitness was never demonstrated. Measurement of fitness by a competitive assay under the relevant in vitro conditions could substantially strengthen this work. This can be done in vitro for the hypoxia work with the appending conclusions then limited to exit from hypoxia by performing fitness assessment (competition assay of fit versus impaired fitness mutants) with analysis done by allele-specific PCR. An ideal experiment would of course be competitive experiments in mice with for example 1:5 and 5:1 ratios to see which strain gets out-competed since this would really substantiate the claims about in vivo fitness.

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: This manuscript by Lee and colleagues presents a careful and fairly thorough molecular and biochemical analysis of the role of Ddn variants in resistance of Mycobacterium tuberculosis to pretomanid and delamanid. The authors make the interesting observation that reduction of menaquinone (menadione), pretomanid and delamanid have distinct structural requirements. The implication of this finding is that resistance to pretomanid and delamanid can occur without ablation of native function of Ddn. Therefore, resistance need not impair fitness. Moreover, while there is overlap in some requirements for pretomanid and delamanid reduction, some variant retain activity for one but not the other. Thus, resistance to one does not necessarily confer resistance to both. Importantly, the authors interrogate numerous genome sequences from clinical isolates, many of which are not expected to have been exposed to pretomanid and delamanid. The authors are able to confirm resistance based on their variant analysis. This study is important and interesting, and the study was well designed and well presented. I have some minor suggestions to improve clarity for readers.

Reviewer #2: This manuscript aims at predicting and understanding the role of resistance to nitroimidazole compounds by mutation in Mycobacteirum tuberculosis. One of the great strengths of this work is the use of natural polymorphisms in a large number of M. tuberculosis strains as a source of natural variability, which is of course highly relevant, given that nitroimidazole drugs will be used against such strains in practice. Most of the experiments appear to have been carefully designed, executed, analysed and interpreted.

Reviewer #3: Background: The rapid emergence of drug resistant variants of the causative agent of tuberculosis (TB), Mycobacterium tuberculosis, has created an urgent need for new therapeutic options for this disease. Among the new and repurposed drugs that have been introduced for TB are the nitroimidazoles, delamanid and pretomanid, which are prodrugs that require activation by the deazaflavin-dependent nitroreductase (Ddn). Resistance to delamanid is acquired through mutations in the biosynthesis of F420, which serves as a cofactor for Ddn, or by direct abrogation/reduction in Ddn activity. With nitroimidazoles and another new TB drug, bedaquiline, resistance has been reported clinically in individuals who have not been exposed to the drug. Considering this, the authors set out to determine the effect of possible resistance conferring mutations on Ddn activity and the resulting resistance to nitroimidazoles.

Summary of findings: The authors first demonstrate that Ddn displays F420-dependent menaquinone-1 reductase activity that appears to be somewhat weak in vitro and they speculate the activity within the native membrane environment in M. tuberculosis is most likely enhanced. In addition to M. tuberculosis, the authors demonstrate that Ddn orthologs from a variety of mycobacterial species display menaquinone-1 reductase activity but only Ddn from M. tuberculosis and M marinum can activate nitroimidazoles. The fact that Ddn reductase activity can be found without nitroimidazole activation activity suggests that the former function is not coupled to the latter. Consistent with this, those organisms from which Ddn was unable to activate nitroimidazoles were naturally resistant to these compounds. The authors then interrogated genomes of M. tuberculosis isolates and report 46 non-synonymous substitutions and 2 deletions in ddn. They engineer these mutations and create a panel of recombinant proteins. Using this, the authors demonstrate that these mutants retained menadione reductase activity and the majority of these were also able to activate pretomanid. Ddn mutations L49P, S78Y, K79Q, W88R, and Y133C were unable to activate pretomanid, two of these - S78Y and Y113C - were able to activate delaminid. The authors conduct a phylogenetic analysis of these mutations in clinical isolates and suggest that as S78Y mutation is present in clinically successful strains, it does not have a large fitness cost. In a Beijing isolate with the S78Y mutation, the authors demonstrate robust delamanid resistance. Next, the authors create a panel of recombinant Ddn mutants with point mutations in the substrate binding site and test these for native activity and nitroimidazole activation. From the 26 mutants, all retain varying levels of menadione activity and 16 did not display the ability to activate pretomanid. Using molecular docking, the authors demonstrate that binding of pretomanid to Ddn is different to delamanid. Using mutant strains of M. tuberculosis that were selected from mice on pretomanid monotherapy and from a previous study, the authors demonstrate that some of the point mutations identified earlier (M1T, L49P, L64P, R112W, C149Y, S22L, W88R) in their analysis are sufficient to confer resistance and were associated with reduced pretomanid activation. Some of these mutants were defective in their ability to recover from hypoxia with no associated survival defects under these conditions and in the murine model of TB infection.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: None

Reviewer #2: The authors need to improve their description of the enzymology results and its interpretations.

For example, on the first paragraph of results, the authors "Ddn catalyzed menaquinone reduction

in vitro with moderate efficiency (kcat/KM = 8.6 x 102 M-1 s-1)...". 10^2 M-1s-1 is not moderate efficiency, it is low efficiency. One would expect a value at around 10^6 M-1s-1 for a metabolic enzyme. Also, " and physiologically relevant affinity (KM = 22.4 ± 3.8 μM) (Table 1);", what is the concentration of menaquinone in the cells? Only by knowning that, the authors can judge if this is or isn't physiologic. Finally, Km does not equal to Kd, in most cases I have seen. Therefore, the authors must swap "affinity" by "Km".

On the second paragraph of the results, the authors leap to a conclusion without any supporting data, by stating "Having established that Ddn is a menaquinone reductase,...". To this point, the authors demonstrated that Ddn can reduce menaquinone, but that does not prove that Ddn is a menaquinone reductase. To prove that, the authors must test a number of other substrates for enzymes that reduce quinone and show that kcat/Km values are smaller than what they get for menaquinone. Showing that an enzyme act (very poorly) on a substrate does not prove anything. The authors must use a variety of other quinone reductase substrates to probe the substrate specificity and therefore assign the likely bona fide substrate specificity of Ddn, if that is what they are claiming they are doing.

All other parts of the manuscript appear to be well interpreted.

Reviewer #3: This is an exceptional biochemical analysis of amino acid sequence - structure - function relationships in Ddn with respect to its native reductase activity and the ability to activate nitroimidazoles. The study is well-executed from a biochemical perspective and has important implications for surveillance of the reported mutations in the clinical setting which would ultimately assist in better stewardship of nitroimidazoles as therapeutic agents for TB. That said, there are some shortfalls, detailed below

1. The authors use the term fitness several times but make no substantive measure of fitness in laboratory experiments. Most conclusions are derived from clinical and epidemiological information. Several aspects may be at play in the clinical settings and these remarks should be reconsidered. The claims of fitness costs or lack thereof are not support by the data presented in this manuscript.

2. Similarly, hypoxia seems to used interchangeably with dormancy. Dormancy is a complex phenomenon associated with metabolic quiescence. It is unclear if hypoxia in a test tube faithfully reflects this state. Moreover, recovery from such conditions in the lab is not a measure of fitness or transmissibility. This is all speculative and should be revised.

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: 1. One of the interesting and important points of the manuscript is that resistance to these new bicyclic nitroimidazoles potentially occurs through genetic drift rather than by selection. The authors might consider bringing this point into the title and abstract.

2. In author summary, change "Strikingly, when analysed >15,000 M. tuberculosis..." to "Strikingly, when we analysed >15,000 M. tuberculosis..."

3. "In order to spread effectively and endanger health, these resistant strains would need to retain sufficient fitness to survive all stages of the lifecycle of M. tuberculosis, including recovery from dormancy." Formal proof of the importance of dormancy for fitness of M. tuberculosis in the host is non-existent. We know that such populations exist, but, whether this mode is essential for transmission and pathogenesis is unknown. The authors should modify this sentence.

4. Change "M. tuberculosis H37Rv and M. marinum, which were the only two strains that encoded Ddn orthologs with in vitro pretomanid activation activity," to "M. tuberculosis (H37Rv) and M. marinum, which were the only two species that encoded Ddn orthologs with in vitro pretomanid activation activity,"

5. "Every mutant tested was able to reduce menadione, indicating that there was selective pressure to maintain the physiological function of Ddn." Probably true, but, without the isoprenyl tail, menadione may be a more promiscuous substrate, it is overreaching to state that this observation indicates maintenance of function, would need to test MQ (but not necessary to do this). May be better to state that it is consistent with selective pressure for maintenance of the physiological function of Ddn...

6. "could readily occur and spread with sufficient selection pressure." to "could readily occur and spread with sufficient selective pressure."

7. "...will result in substantial loss of fitness given that the ability to recover from dormancy is such an important aspect of M. tuberculosis pathogenesis." While this might be the case, to my knowledge, there is no direct evidence, and the references provided do not provide such evidence. Maintenance of this function supports its role in fitness, but, that role might be distinct from that which has been modeled in the laboratory.

8. "The activation of the prodrugs pretomanid and delamanid by Ddn is a promiscuous activity that is not coupled to its native activity." Statement needs modification, hydride transfer is part of the native activity of Ddn

9. "It has long been known that promiscuous activities are more susceptible to mutation than native functions..." Modify, activities don't mutate.

10. Figure 3 title, "sequence" and "sequences" is redundant, for figure 3 legend, should describe what color represents.

11. Figure 4, increase font size of SNPs

12. Table 2 seems redundant with figures that show these data. However, I do favor inclusion -perhaps as supplemental. Also, the meaning of no activity (below limit of detection?) and no expression is unclear.

13. Table 3, rate of 0 should be expressed as less than the limit of detection. Need to define N/A, not sure why it says N/A for M. marinum when Table 2 shows activity.

14. Table 4 seems redundant with figure 4.

15. Same data is shown in multiple figures and tables, most journals require footnotes for repeated data.

16. For modeling, please show top docking scores.

17. Figure 7, replicates and statistics?

18. Need to provide additional information for readers to understand what is presented in Table S2.

19. Check th

Reviewer #2: There are a few typos in the manuscript. For example, on Figure 1A (M. ulcerans, not M. ulceran) and the title of the figure (binding, not bindng).

Please, change "Wt" by the strain name used, as for this case, clearly H37Rv or analogous strain is not a wild-type.

Reviewer #3: (No Response)

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Reviewer #1: Yes: Anthony Baughn

Reviewer #2: Yes: Luiz Pedro Carvalho

Reviewer #3: No

Dear Mr Lee,

We are pleased to inform that your manuscript, "Predicting nitroimidazole antibiotic resistance mutations in Mycobacterium tuberculosis with protein engineering", has been editorially accepted for publication at PLOS Pathogens. 

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***********************************************************

The authors have addressed the reviewers' concerns. This is a manuscript that describes the importance of Ddn to pathogenesis based on its role in respiration. The results demonstrate which mutations confer resistance to Pretomanid or Delamanid without impairing the respiratory function of the enzyme.

Reviewer Comments (if any, and for reference):