In Vitro Resistance against DNA Gyrase Inhibitor SPR719 in Mycobacterium avium and Mycobacterium abscessus

ABSTRACT The aminobenzimidazole SPR719 targets DNA gyrase in Mycobacterium tuberculosis. The molecule acts as inhibitor of the enzyme’s ATPase located on the Gyrase B subunit of the tetrameric Gyrase A2B2 protein. SPR719 is also active against non-tuberculous mycobacteria (NTM) and recently entered clinical development for lung disease caused by these bacteria. Resistance against SPR719 in NTM has not been characterized. Here, we determined spontaneous in vitro resistance frequencies in single step resistance development studies, MICs of resistant strains, and resistance associated DNA sequence polymorphisms in two major NTM pathogens Mycobacterium avium and Mycobacterium abscessus. A low-frequency resistance (10−8/CFU) was associated with missense mutations in the ATPase domain of the Gyrase B subunit in both bacteria, consistent with inhibition of DNA gyrase as the mechanism of action of SPR719 against NTM. For M. abscessus, but not for M. avium, a second, high-frequency (10−6/CFU) resistance mechanism was observed. High-frequency SPR719 resistance was associated with frameshift mutations in the transcriptional repressor MAB_4384 previously shown to regulate expression of the drug efflux pump system MmpS5/MmpL5. Our results confirm DNA gyrase as target of SPR719 in NTM and reveal differential resistance development in the two NTM species, with M. abscessus displaying high-frequency indirect resistance possibly involving drug efflux. IMPORTANCE Clinical emergence of resistance to new antibiotics affects their utility. Characterization of in vitro resistance is a first step in the profiling of resistance properties of novel drug candidates. Here, we characterized in vitro resistance against SPR719, a drug candidate for the treatment of lung disease caused by non-tuberculous mycobacteria (NTM). The identified resistance associated mutations and the observed differential resistance behavior of the two characterized NTM species provide a basis for follow-up studies of resistance in vivo to further inform clinical development of SPR719.

Aragaw et al identify frequency and likely cause of resistance against the DNA gyrase inhibitor SPR719 in two NTM species. This work is highly valuable for our understanding of emergence of drug resistance in pathogenic mycobacteria. The experimental approach is technically sound and the methods are described adequately. The authors find that in M. avium, a very low frequency resistant mutant emerges against SPR719, with moderate resistance while in M. abscessus, a higher frequency resistant mutant with stronger resistance emerges. While the authors describe mutations identified by sanger sequencing of gyrB and results of the WGS, their study does not adequately answer the findings of differences in degree of resistance. Specific concerns: 1. The authors do not discuss the GyrB T518C mutation in the resistance clones of M. avium nor of GyrB C506A in resistant clones M. abscessus. 2. In the absence of functional data for any of these mutations on the resistance mechanism, it would be difficult to explain the differences in degree of resistance across the two species, or across the small and big colonies in case of M. abscessus. 3. What explains the relatively weaker resistance of the smaller colonies that harbour MAB-4384 mutations?
Reviewer #3 (Comments for the Author): The present study by Aragaw et al titled "In vitro resistance against DNA gyrase inhibitor SPR719 in Mycobacterium avium and Mycobacterium abscessus" investigates in vitro resistance against a novel drug SPR719 in non-tuberculous mycobacteria.
The authors have identified and characterized spontaneous mutations in M. avium and M. abscessus that they concluded to be associated with SPR719 resistance. A missense mutation mapped to the ATPase domain of the Gyrase B subunit in both bacteria. It is interesting to note the second high frequency resistance mechanism involving regulation of drug efflux system in M. abscessus. To sum up, the study throws light into the possible mechanisms of resistance against SPR719 in two NTM strains that could be useful in treatment of NTMs.
General Comments: Overall the manuscript reads well. However, at some points the text appears repetitive and thus writing could be improved in some sections.
Some supplementary figures could be moved to the main body to support the Table data. It will be easier for the reader to follow the data if some of the growth curves were included as main figures Specific Comments: 1. There is insufficient proof that the spontaneous mutations identified in the study are directly responsible for the observed resistance against SPR719.
2. No data is provided regarding the effect of these mutations and SPR719 resistance in vivo. To better understand the resistance mechanisms under clinical settings, the authors should consider experimental approaches in vivo or using ex vivo cell culture models to help support the conclusions.
3. Statistical analysis is missing.

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The present study by Aragaw et al titled "In vitro resistance against DNA gyrase inhibitor SPR719 in Mycobacterium avium and Mycobacterium abscessus" investigates in vitro resistance against a novel drug SPR719 in non-tuberculous mycobacteria.
The authors have identified and characterized spontaneous mutations in M. avium and M. abscessus that they concluded to be associated with SPR719 resistance. A missense mutation mapped to the ATPase domain of the Gyrase B subunit in both bacteria. It is interesting to note the second high frequency resistance mechanism involving regulation of drug efflux system in M. abscessus. To sum up, the study throws light into the possible mechanisms of resistance against SPR719 in two NTM strains, that could be useful in treatment of NTMs.

General Comments:
Overall the manuscript reads well. However, at some points the text appears repetitive and thus writing could be improved in some sections.
Some supplementary figures could be moved to the main body to support the Table data. It will be easier for the reader to follow the data if some of the growth curves were included as main figures.

Specific Comments:
1. There is insufficient proof that the spontaneous mutations identified in the study are directly responsible for the observed resistance against SPR719.
2. No data is provided regarding the effect of these mutations and SPR719 resistance in vivo. To better understand the resistance mechanisms under clinical settings, the authors should consider experimental approaches in vivo or using ex vivo cell culture models to help support the conclusions. 1

Response to editor and reviewers: Spectrum01321-21 (In vitro resistance against DNA gyrase inhibitor SPR719 in Mycobacterium avium and Mycobacterium abscessus)
Editor's comment (16 Sept 21) The work is interesting and our reviewers are generally favorable. However, all of them pointed out that further consideration of this work requires genetic complementation of mutations. I agree with our reviewers and request you and coauthors to experimentally revise the manuscript in line with the comments.

Authors clarification to the editor (17 Sept 21)
Regarding the point that the reviewers raised: complementation of the resistant strains to confirm that the observed polymorphisms indeed cause the observed phenotype. This is a good point. In fact, we discuss this point rather extensively in our discussion as a limitation. We also discuss why we think that this limitation is acceptable. Regarding the mutations in the gyrase genes of M. abscessus and M. avium: the mutant strains were isolated in independent selection experiments; and the same (identical) amino acid substitutions have been described to cause resistance against ATPase inhibitors previously. Thus, it is highly plausible that these mutations are causal for resistance. A similar rationale can be applied to the indirect resistance mechanism observed for M. abscessus. FYI: We just published earlier this year (as an example) a manuscript using the same rationaleand that was acceptable by AAC standards ( Editor's comment on authors' clarification (20 Sept 21) Thanks for your email and your response clarifying reviewers' comments. I broadly agree with your clarifications. Our reviewers are also convinced about the gyrase B subunit mutations as these have been previously reported in other bacteria. However, polymorphism in the transcriptional repressor is unique and requires further characterization. Our decision is largely based on the reviewers' comments and in this case all three reviewers unanimously raised similar concerns. My suggestion is to address the reviewers' concerns as much as possible and/or provide an in-depth discussion or any new analysis on why these mutations are likely responsible for resistance in the response letter and in the revised manuscript.
Authors: Thank you for your understanding and thank you for agreeing that confirmation of the resistance mechanism due to mutations in gyrase B is not essential. We carried now out complementation for the loss of function mutations in the transcriptional repressor as requested and added the data to the manuscript. We also added the corresponding materials and methods. - Reviewer comments: Reviewer #1 (Comments for the Author): Authors Aragaw et al. characterized the in vitro resistance of a known GyrB inhibitor SPR719 against the two non-tuberculous mycobacterial (NTM) pathogens Mycobacterium avium and Mycobacterium abscessus by raising the spontaneous resistant mutants which allowed them to determine the frequencies of resistance, MICs of resistant mutants, and mapping of mutations.
While the MS reads very well it lacks in novelty (and in-depth investigation of the mechanism of resistance/action -such as validation of resistant mechanism by complementation of resistant mutants with wt copy of the gyrB, analysis of mutated GyrB vs wt GyrB for enzymatic activity and inhibition kinetics with SPR719, on-target activity using conditional knockdown etc. -most of these experiments are done by this lab in their recent publication) and for this reason, I find it very preliminary.
Authors: thank you for your comments. Regarding 'the work lacks novelty and in-depth investigation'. We don't agree with the statement that the work 'lacks novelty' but we do agree that this work does not present an 'in-depth investigation' (of mechanisms). Characterization of in vitro resistance in NTM against the clinical candidate SPR719/720 has not been reported. Thus, this work contains novel data. These novel data are highly relevant to the further clinical development of this candidate (for instance one may favor to develop the drug for M. avium first). Indeed, this work does not describe an in-depth investigation of mechanisms. But this was not the objective of the current study. For further clinical development it is important to determine frequencies of resistance, classes of resistance mutants, possible in vitro fitness effects of resistance mutations, and the resistance associated DNA polymorphisms. That was the objective of the current study (as stated in the abstract and in the introduction; see label in marked-up manuscript) and these data were delivered.
The gyrase B mutations we find associated with SPR719 resistance have been identified in other bacteria to cause resistance against ATPase inhibitors. Thus, it is highly likely, that these mutations are also causal for resistance against NTM. We discuss this point extensively in the discussion and also clearly state that the lack of complementation presents as a limitation of the study (see label in the discussion of the marked-up manuscript).
Taken together, we would like to emphasize that this study is not about in-depth analyses of mechanisms but a standard (and rather comprehensive) in vitro resistance analysis supporting and guiding clinical development of a drug candidate.
Aragaw et al identify frequency and likely cause of resistance against the DNA gyrase inhibitor SPR719 in two NTM species. This work is highly valuable for our understanding of emergence of drug resistance in pathogenic mycobacteria. The experimental approach is technically sound and the methods are described adequately. The authors find that in M. avium, a very low frequency resistant mutant emerges against SPR719, with moderate resistance while in M. abscessus, a higher frequency resistant mutant with stronger resistance emerges. While the authors describe mutations identified by sanger sequencing of gyrB and results of the WGS, their study does not adequately answer the findings of differences in degree of resistance.
Thank you for your comments and positive evaluation.
Specific concerns: 1. The authors do not discuss the GyrB T518C mutation in the resistance clones of M. avium nor of GyrB C506A in resistant clones M. abscessus.
Authors: That is correct. We do not discuss (address the possible reasons for) why we are getting two distinct missense mutations and why the respective resistance levels differ. The differences in the resistance levels associated with the (different) GyraseB missense mutations in the different mycobacterial species is indeed intriguing. We state the difference in our manuscript, and also say that the reason for this difference remains to be determined (see label in results of the marked-up manuscript). The reason we don't discuss this difference in more detail is because we don't have anything reasonable to say or to speculate. The finding is 'puzzling' and requires detailed structural (Xray structures of wild types and mutants, with and without bound inhibitor) and biochemical analyses of the two DNA gyrases. We think that this detailed analysis is beyond the scope of the current work. We would like to note that our finding that M. avium appears to develop only low level resistance due to mutations in the DNA gyrase is of high relevance for the further clinical development of the drug candidate. If confirmed in vivo (in patients) this property would make the drug very useful for the treatment of M. avium infections (high level, clinically significant resistance may only emerge at ultra low frequencies).
2. In the absence of functional data for any of these mutations on the resistance mechanism, it would be difficult to explain the differences in degree of resistance across the two species, or across the small and big colonies in case of M. abscessus.

Authors: Agree. Detailed structural, biochemical (and genetic) analyses would be required to address these questions. Again, as this study is focused on in vitro NTM
resistance characterization to support further clinical development, we feel that these additional works are beyond the scope of the current manuscript.
3. What explains the relatively weaker resistance of the smaller colonies that harbour MAB-4384 mutations?

Authors: Good questionand we don't know. Detailed mechanistic studies are required to answer this question. In our experience in the lab, we typically observe higher resistance levels in on-target missense mutants (reducing/preventing binding) when compared to indirect resistance mechanisms (such as pumps). This may be similar here.
Without any supporting data we prefer not to speculate.
Reviewer #3 (Comments for the Author): The present study by Aragaw et al titled "In vitro resistance against DNA gyrase inhibitor SPR719 in Mycobacterium avium and Mycobacterium abscessus" investigates in vitro resistance against a novel drug SPR719 in non-tuberculous mycobacteria.
The authors have identified and characterized spontaneous mutations in M. avium and M. abscessus that they concluded to be associated with SPR719 resistance. A missense mutation mapped to the ATPase domain of the Gyrase B subunit in both bacteria. It is interesting to note the second high frequency resistance mechanism involving regulation of drug efflux system in M. abscessus. To sum up, the study throws light into the possible mechanisms of resistance against SPR719 in two NTM strains that could be useful in treatment of NTMs.
General Comments: Overall the manuscript reads well. However, at some points the text appears repetitive and thus writing could be improved in some sections.
Authors: Thank you for your comment. We carried out two projectsresistance characterization in M. avium and in M. abscessus. We followed the same general procedure twice. This may explain the impression that the text feels somewhat repetitive. However, the results we obtained for the two mycobacterial species when we executed the two resistance characterization analyses were very different. This, we feel, required a detailed description of the process and the results. We also wanted to ensure that our work can be reproduced, again requiring detailed description of process and results. This again, may contribute to the impression of being somewhat repetitive. We would prefer not to shorten the write up as we think this would affect the ability of other groups to repeat our work. Of note: many published resistance mutant selection experiments are described very briefly which makes interpretation and reproducibility of the data difficult. We wanted to avoid this.  Taken together, and considering the redundancy of information, we find it more appropriate to show these data for reference in the supplemental materials. We hope this makes sense and is acceptable. We did put the new data figure (Fig. 3) showing the results of the complementation experiment of the MAB_4384 mutant in the main text.

Some supplementary figures could be moved to the main body to support the
Specific Comments: 1. There is insufficient proof that the spontaneous mutations identified in the study are directly responsible for the observed resistance against SPR719.
Authors: Please see discussion with the editor above; here re-inserted.
Regarding the point that the reviewers raised: complementation of the resistant strains to confirm that the observed polymorphisms indeed cause the observed phenotype. This is a good point. In fact, we discuss this point rather extensively in our discussion as a limitation. We also discuss why we think that this limitation is acceptable. Regarding the mutations in the gyrase genes of M. abscessus and M. avium: the mutant strains were isolated in independent selection experiments; and the same (identical) amino acid substitutions have been described to cause resistance against ATPase inhibitors previously. Thus, it is highly plausible that these mutations are causal for resistance. A similar rationale can be applied to the indirect resistance mechanism observed for M. abscessus. FYI: We just published earlier this year (as an example) a manuscript using the same rationaleand that was acceptable by AAC standards ( Editor's comment on authors' clarification (20 Sept 21) Thanks for your email and your response clarifying reviewers' comments. I broadly agree with your clarifications. Our reviewers are also convinced about the gyrase B subunit mutations as these have been previously reported in other bacteria. However, polymorphism in the transcriptional repressor is unique and requires further characterization. Our decision is largely based on the reviewers' comments and in this case all three reviewers unanimously raised similar concerns. My suggestion is to address the reviewers' concerns as much as possible and/or provide an in-depth discussion or any new analysis on why these mutations are likely responsible for resistance in the response letter and in the revised manuscript.
Authors: Thank you for agreeing that confirmation of the resistance mechanism due to mutations in gyrase B is not essential.
We carried now out complementation for the loss of function mutations in the transcriptional repressor and added the data to the manuscript (please see labelled part in results section and new Figure 3). We also added the additional materials and methods. The results show that adding a wild type copy of MAB_4384 into MAB_4384mutant background reverses the resistance phenotype. This suggests that the polymorphisms observed in MAB_4384 are indeed causing the SPR719 resistance phenotype.
2. No data is provided regarding the effect of these mutations and SPR719 resistance in vivo.
To better understand the resistance mechanisms under clinical settings, the authors should consider experimental approaches in vivo or using ex vivo cell culture models to help support the conclusions.
Authors: That is a very good point. The characterization of in vitro resistance is only a first step (as we state in the manuscript). We feel that isolation and characterization of in vivo mutants would be beyond the scope of the current work. Importantly, resistance will be monitored in the clinical trials in patients. Our in vitro resistance data provide a valuable baseline for the study of clinical (in vivo) resistance. Please note that we mention the limitation of the in vitro work in the manuscript. We state that the relevance of the polymorphisms identified in vitro for the clinical use remains to be determined (see label in discussion of the marked-up manuscript).

Statistical analysis is missing.
Authors: We carried out a number of dose response determinations and (drug free) growth curve measurements. All these experiments were carried out three times independently and mean values with standard deviations are shown. This is stated in the legends. (see labels in Table legends

Data in
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Editor, Microbiology Spectrum Reviewer comments: Reviewer #2 (Comments for the Author): My questions are answered by the authors adequately in the response letter. Some of the points made by the authors in the response letter pertaining to reasons for differences in resistance mechanisms between the two NTM species studied should find a place in the discussion section, indeed being pointed out as speculative. Data in Figure 3 should also include comparison with the wild type strain.

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To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary.
Here are a few examples of required updates that authors must address: • point-by-point responses to the issues I raised in your cover letter • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file.
• Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
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