Reactive Oxygen Detoxification Contributes to Mycobacterium abscessus Antibiotic Survival

Reviewer #1 (Public review):

Summary:

Persistence is a phenomenon by which genetically susceptible cells are able to survive exposure to high concentrations of antibiotics. This is especially a major problem when treating infections caused by slow growing mycobacteria such as M. tuberculosis and M. abscessus. Studies on the mechanisms adopted by the persisting bacteria to survive and evade antibiotic killing can potentially lead to faster and more effective treatment strategies.

To address this, in this study, the authors have used a transposon mutagenesis based sequencing approach to identify the genetic determinants of antibiotic persistence in M. abscessus. To enrich for persisters they employed conditions, that have been reported previously to increase persister frequency - nutrient starvation, to facilitate genetic screening for this phenotype. M.abs transposon library was grown in nutrient rich or nutrient depleted conditions and exposed to TIG/LZD for 6 days, following which Tn-seq was carried out to identify genes involved in spontaneous (nutrient rich) or starvation-induced conditions. About 60% of the persistence hits were required in both the conditions. Pathway analysis revealed enrichment for genes involved in detoxification of nitrosative, oxidative, DNA damage and proteostasis stress. The authors then decided to validate the findings by constructing deletions of 5 different targets (pafA, katG, recR, blaR, Mab_1456c) and tested the persistence phenotype of these strains. Rather surprisingly only 2 of the 5 hits (katG and pafA) exhibited a significant persistence defect when compared to wild type upon exposure to TIG/LZD and this was complemented using an integrative construct. The authors then investigated the specificity of delta-katG susceptibility against different antibiotic classes and demonstrated increased killing by rifabutin. The katG phenotype was shown to be mediated through the production of oxidative stress which was reverted when the bacterial cells were cultured under hypoxic conditions. Interestingly, when testing the role of katG in other clinical strains of Mab, the phenotype was observed only in one of the clinical strains demonstrating that there might be alternative anti-oxidative stress defense mechanisms operating in some clinical strains.

Strengths:

While the role of ROS in antibiotic mediated killing of mycobacterial cells have been studied to some extent, this paper presents some new findings with regards to genetic analysis of M. abscessus susceptibility, especially against clinically used antibiotics, which makes it useful. Also, the attempts to validate their observations in clinical isolates is appreciated.

Weaknesses:

Amongst the 5 shortlisted candidates from the screen, only 2 showed marginal phenotypes which limits the impact of the screening approach.

While the role of KatG mediated detoxification of ROS and involvement of ROS in antibiotic killing was well demonstrated, the lack of replication of this phenotype in some of the clinical isolates limits the significance of these findings.

Reviewer #2 (Public review):

Summary:

The work set out to better understand the phenomenon of antibiotic persistence in mycobacteria. Three new observations are made using the pathogenic Mycobacterium abscessus as an experimental system: phenotypic tolerance involves suppression of ROS, protein synthesis inhibitors can be lethal for this bacterium, and levofloxacin lethality is unaffected by deletion of catalase, suggesting that this quinolone does not kill via ROS.

Strengths:

The ROS experiments are supported in three ways: measurement of ROS by a fluorescent probe, deletion of catalase increases lethality of selected antibiotics, and a hypoxia model suppresses antibiotic lethality. A variety of antibiotics are examined, and transposon mutagenesis identifies several genes involved in phenotypic tolerance, including one that encodes catalase. The methods are adequate for making these statements.

Weaknesses:

The work can be improved by a more comprehensive treatment of prior work, especially comparison of E. coli work with mycobacterial studies.
Moreover, the work still has some technical issues to fix regarding description of the methods, supplementary material, and reference formating.

Overall impact: Showing that ROS accumulation is suppressed during phenotypic tolerance, while expected, adds to the examples of the protective effects of low ROS levels. Moreover, the work, along with a few others, extends the idea of antibiotic involvement with ROS to mycobacteria. These are field-solidifying observations.

Comments on revisions:

The authors have moved this paper along nicely. I have a few general thoughts.

(1) It would be helpful to have more references to specific figures and panels listed in the text to make reading easier.

(2) I would suggest adding a statement about the importance of the work. From my perspective, the work shows the general nature of many statements derived from work with E. coli. This is important. The abstract says this overall, but a final sentence in the abstract would make it clear to all readers.

(3) The paper describes properties that may be peculiar to mycobacteria. If the authors agree, I would suggest some stress on the differences from E. coli. Also, I would place more stress on novel findings. This might be done in a section called Concluding Remarks. The paper by Shee 2022 AAC could be helpful in phrasing general properties.

(4) Several aspects still need work to be of publication quality. Examples are the materials table and the presentation of supplementary material. Reference formatting also needs attention.

Reviewer #3 (Public review):

Summary:

The manuscript demonstrates that starvation induces persister formation in M. abscesses. They also utilized Tn-Seq for the identification of genes involved in persistence. They identified the role of catalase-peroxidase KatG in preventing death from translation inhibitors Tigecycline and Linezolid. They further demonstrated that a combination of these translation inhibitors leads to the generation of ROS in PBS-starved cells.

Strengths:

The authors used high-throughput genomics-based methods for identification of genes playing a role in persistence.

Weaknesses:

The findings could not be validated in clinical strains.

Comments on revisions: No more comments for the authors.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public review):

Weaknesses:

Only 1 gene (katG) gave a strong and 1 (Mab_1456c) exhibited a minor defect. Two of the clones did not show any persistence phenotype (blaR and recR) and one (pafA) showed a minor phenotype,

We have now carried out more detailed validation studies on the Tn-Seq, with analysis of timedependent killing over 14 d. This more comprehensive analysis shows that 4 of 5 genes analyzed do indeed have antibiotic tolerance defects under the conditions that Tn-Seq predicted a survival defect (Revised Figure 3). In addition, we found that even before actual cell death, several mutants had delayed resumption of growth after antibiotic removal (Figure 3 Supplemental).

Fig 3 - Why is there such a huge difference in the extent of killing of the control strain in media, when exposed to TIG/LZD, when compared to Fig. 1C and Fig. 4. In Fig. 1C, M. abs grown in media decreases by >1 log by Day 3 and >4 log by Day 6, whereas in Fig. 3, the bacterial load decreases by <1 log by Day 3 and <2 log by Day 6. This needs to be clarified, if the experimental conditions were different, because if comparing to Fig. 1C data then the katG mutant strain phenotype is not very different.

We agree with the reviewer that there is variability in the timing and extent of cell death from experiment to experiment. As noted by the reviewer, in Figure 1C the largest decrement in survival is between day 1 - day 3 (also seen in Figure 6A). As they noted in Figure 4 the largest decrement is between day 3 – day 6 (also seen in Figure 3A, Figure 5F). In each experiment with katG mutants we carefully compare the mutant vs. the control strain within that experiment, which is more accurate than comparing the behavior of mutant in one experiment to a control in another experiment.

Reviewer #2 (Public review):

Weaknesses:

.First, word-choice decisions could better conform to the published literature. Alternatively, novel definitions could be included. In particular, the data support the concept of phenotypic tolerance, not persistence.

We appreciate the reviewers comments, text modified.

Second, two of the novel observations could be explored more extensively to provide mechanistic explanations for the phenomena.

We have added several additional experiments, these are detailed below in response to specific comments.

Reviewer #3 (Public review):

Weaknesses:

The findings could not be validated in clinical strains.

We understand the reviewer’s concern that the katG phenotype was only observed in one of the two clinical strains we studied. We feel that our findings are relevant beyond the ATCC 19977 strain for two reasons

(1) We have performed additional analyses of the two clinical isolates and indeed find significant accumulation of ROS following antibiotic exposure in both of these strains (revised Figure 6A).

(2) We do in fact see a role for katG in starvation-induced antibiotic tolerance in Mabs clinical strain-2. It is not surprising that different strains from a particular species may have some different responses to stresses – for example, there is wide strain-specific variability in susceptibility to different phages within a species based on which particular phage defense modules a given strain carries (for example PMID: 37160116). We speculate that different Mabs strains may express varying levels of other antioxidant factors and note that the genes encoding several such factors were identified by our Tn-Seq screen including the peroxidases ahpC, ahpD, and ahpE. Our analysis of the genetic interactions between katG and these other factors is ongoing.

Comments/Suggestions

(1) In Fig1E, the authors show no difference in killing Mtb with or without adaptation in PBS. These data are contrary to the data presented in Figure 1B. These also do not align with the data of M. smegmatis and M. abscesses. Please discuss these observations in light of the Duncan model of persistence (Mol Microbiol. 2002 Feb;43(3):717-31.).’

The above referenced Duncan laboratory study found tolerance after prolonged starvation but did not actually examine tolerance at early time points. While some of the transcriptional and metabolic changes seen by Duncan and others are slow, other groups have described starvation responses in Mtb that are quite rapid. For example, the stringent response mediator ppGpp accumulates within a few hours after onset of starvation in Mtb (PMID: 30906866). We suspect that a rapid signaling response such as this underlies the phenotype we observe. Regarding the difference between Mtb and other mycobacterial species we also find it surprising that Mtb had a much more rapid starvation response. This is a clear species-specific difference that may reflect an adaptation of Mtb to the nutrient-limited physiologic niche within host macrophages.

(2) Line 151, the authors state that they have used an M. abscesses Tn mutant library of ~ 55,000 mutant strains. The manuscript will benefit from the description of the coverage of total TA sites covered by the mutants.

Text modified to add this detail. There are 91,559 TA sites in the abscessus genome. Thus, our Tn density is ~60%.

(3) Line 155: Please explain how long the cells were kept in an Antibiotic medium.

This technical detail was noted above on line 153 in the original text: “…and then exposed them to TIG/LZD for 6 days”. To clarify the overall conditions, we have also revised the text of the manuscript and added the detail of how long cells were passaged after removal of antibiotics.

(4) Line 201: data not shown. Delayed resumption of growth after removal of antibiotic would be helpful in indicating drug resilience. This data could enhance the manuscript.

Data now provided in Figure 3 Supplemental

(5) Figures 4C and 4F represent the kill curve. It will be good to show the date with CFU against the drug concentration in place of OD600. CFU rather than OD600 best reflects growth inhibition.

Figures 4C and 4F are measuring the minimum inhibitory concentration (MIC) to stop the overall growth of the bacterial population. While we agree that CFU could be analyzed, this would be measuring a different outcome – cell death and the minimum bactericidal concentration (MBC). In these experiments we sought to specifically examine the MIC so as to separate growth inhibition from cell death. For this we used the standard method employed by clinical microbiology laboratories for MIC, which is optical density of the culture (PMID: 10325306).

(6) Figure 5C. The authors shall show the effect of TIG/LZD on M. abscesses ROS production without the PBS adaptation. It is important to conclude that TIG/LZD induces ROS in cells. Authors should utilize ROS scavengers such as Thiourea, DFO, etc., to conclude ROS's contribution to bacterial killing following inhibition of transcription and translation.

New data added (revised Figure 5 and Figure 5 Supplemental)

(7) Line 303. Remove "note".

Text revised. We thank the reviewer for identifying this typographical error.

(8) The introduction and Discussion are very similar, and several lines are repeated.

Text revised with overlapping content removed.

Reviewer #1 (Recommendations for the authors):

It appears that the same datasets for PBS adapted cultures were plotted in A-C and D-F. Either this should be specifically mentioned in the legend or it might be better to integrate the non-adapted plots into A-C which would also allow easier comparison.

Appreciate the reviewer’s suggestion; text modified with added clarification to figure legend.

This manuscript is focused on M. abs and the antibiotics TIG/LZD, so the Mtb data or data using the antibiotics INH/RIF/EMB and serves more as a distraction and can be removed

We appreciate the reviewer’s perspective. However, we wish to include these data to show the similarities (and differences) in starvation-induced tolerance between the three organisms.

Fig 3 -As mentioned for Fig. 1, it appears that the same dataset was used for the control in all the figures A-E. This should be explicitly stated in the Figure legend.

Appreciate the reviewer’s suggestion; text modified with added clarification to figure legend.

The divergent results from the clinical strains are extremely interesting. It would be helpful to determine the oxidative stress levels (similar to the cellROX data shown in 5E), to tease out if the difference in katG role is because of lack of ROS induction in these strains or due to expression of alternate anti-oxidative stress defense mechanisms.

We have performed additional cellROX analysis as suggested by the reviewer and found that the ROS induction is indeed present across all three Mabs strains, but that katG is only required in one of the two strains (Strain #2). These data are now included in the revised Figure 6.

Reviewer #2 (Recommendations for the authors):

GENERAL COMMENTS

This is a nice piece of work that uses the pathogen Mabs as a test subject.

The work has findings that likely apply generally to antibiotics and mycobacteria: 1) phenotypic tolerance is associated with suppression of ROS, 2) lethal protein synthesis inhibitors act via accumulation of ROS, and 3) levofloxacin behaves in an unexpected way. Each is a new observation. However, I believe that each topic requires more work to be firmly established to be suitable for eLife.

Phenotypic tolerance: Association with suppression of ROS is important but expected. I would solidify the conclusion by performing several additional experiments. For example, confirm the lethal effect of ROS by reducing it with an iron chelator and a radical scavenger. There is a large literature on effects of iron uptake, levels, etc. on antibiotic lethality that could be applied to this question. In 2013 Imlay argued against the validity of fluorescent probes. Perhaps getting the same results with another probe would strengthen the conclusion.

We have carried out additional experiments with both an iron chelator and small molecule ROS scavengers to further test this idea but note that these experiments have several inherent limitations: 1) These compounds have highly pleiotropic effects. For example while N-acetyl cysteine (NAC) is an antioxidant it also increases mycobacterial respiration and was shown to paradoxically decrease antibiotic tolerance in M. tuberculosis (PMID: 28396391). 2) It has been shown by the Imlay group that small-molecule antioxidants are often ineffective in quenching ROS in bacteria (PMID: 388893820), making negative results difficult to interpret. Nonetheless, we present new experimental data showing that iron chelation does indeed improve the survival of antibiotic-treated Mabs (revised Figure 5). However, small molecule antioxidants such as thiourea do not restore antibiotic tolerance and actually increased bacterial cell death, suggesting that they may be affecting respiration in Mabs in a manner similar to that seen for NAC in Mtb. We also note that our genetic analysis, which identified numerous other genes encoding proteins with antioxidant function (Figure 2) is a strong additional argument in support of the importance of ROS in antibiotic-mediated lethality.

Regarding the concern raised by Imlay about the validity of oxidation-sensitive dyes - this relates to concern bacterial autofluorescence induced by antibiotics that can confound analyses in some species. We have ruled this out in our analyses by using bacteria unstained by cellROX as controls to confirm that there is negligible autofluorescence in Mabs (<0.1%, Figure 5E, Figure 6A).

Protein synthesis inhibitors: At present, this is simply an observation. More work is needed to suggest a mechanism. For example, with E. coli the aminoglycosides are protein synthesis inhibitors that also cause membrane damage. Membrane damage is known to stimulate ROS-mediated killing. Your observation needs to be extended because chloramphenicol, another protein synthesis inhibitor, blocks ROS production. The lethality may be a property of mycobacteria: does it occur with E. coli (note that rifampicin is bacteriostatic with E. coli but lethal to Mtb)?

We agree with the reviewer that the mechanism underlying ROS accumulation following transcription or translational inhibition in Mabs is of significant interest. It is likely to be a mechanism different from E. coli, because in E. coli tetracyclines and rifamycins are both bacteriostatic, whereas in Mabs they are both bactericidal. Determining the mechanism by which translation inhibitors cause ROS accumulation in Mabs is an ongoing effort in our laboratory using proteomics and metabolomics, but is outside the scope of this manuscript.

Levofloxacin: This is also at the observational stage but is unexpected. In other studies, ROS is involved in quinolone-mediated killing of bacteria. Why is this not the case with Mabs? The observation should be solidified by showing the contrast with moxifloxacin, since this compound has been studied with mycobacteria (Shee 2022 AAC). With E. coli, quinolone structure can affect the relative contribution of ROS to killing (Malik 2007 AAC), as is also seen with Mtb (Malik 2006 AAC). What is happening in the present work with levofloxacin, an important anti-tuberculosis drug? Is there a structure explanation (compare with ofloxacin)?

While these are interesting questions, a detailed exploration of the structure-function relationships between different fluoroquinolone antibiotics and their varying activities on Mtb and Mabs is outside the scope of this manuscript.

The writing is generally easy to follow. However, the concept of persistence should be changed to phenotypic tolerance with text changes throughout. I base this suggestion on the definitions of tolerance and persistence as stated in the consensus review (Balaban 2019 Nat Micro Rev). Experimentally, tolerance is seen as a gradual decline in survival following antibiotic addition; the decline is slower than seen with wild-type cells. The data presented in this paper fit that definition. In contrast, persistence refers to a rapid drop in survival followed by a distinct plateau (Balaban 2019 Nat Micro Rev; for example, see Wu Lewis AAC 2012 ). Moreover, to claim persistence, it would be necessary to demonstrate subpopulation status, which is not done. The Balaban review is an attempt to bring order to the field with respect to persistence and tolerance, since the two are commonly used without regard for a consistent definition.

We appreciate the reviewer’s suggestion; text modified in multiple places to clarify.

Another issue requiring clarification is the relationship between resistance and tolerance. Killing by antibiotics is a two-step process, as most clearly seen with quinolones. First a reversible bacteriostatic event occurs. Resistance blocks that bacteriostatic damage. Then a lethal metabolic response to that damage occurs. Tolerance selectively blocks the second, killing event, a distinct process that often involves the accumulation of ROS. Direct antibiotic-mediated damage is an additional mode of killing that also stems from the reversible, bacteriostatic damage created by antibiotics. The authors recognize the distinction but could make it clearer. Take a look at Zheng (JJ Collins) 2020, 2022.

Text modified to clarify this point

Many readers would also like to see a bit more background on Mabs. For example, does it grow rapidly? Are there features that make it a good model for studying mycobacteria or bacteria in general? The more general, the better.

Text modified, background added

Below I have listed specific comments that I hope are useful in bringing the work to publication and making it highly cited.

SPECIFIC COMMENTS

Line 30 unexpectedly. I would delete this word because the result is expected from the ROS work of Shee et al 2022 with mycobacteria. Moreover, Zeng et al 2022 PNAS showed that ROS participates in antimicrobial tolerance, and persistence is a form of tolerance (Balalban et al, 2019, Nat Micro Rev).

Text modified as per review suggestion

Line 39 key goal: this is probably untrue in the general sense stated, since bacteriostatic antibiotics are sufficient to clear infection (Wald-Dickler 2019 Clin Infect Dis). However, it is likely to be the goal for Mtb infections.

We agree with the reviewer that bacteriostatic antibiotics are effective in treating most types of infections and do not claim otherwise in the manuscript. However, from a clinical standpoint, eradication of the pathogen causing the infection is indeed the goal of antibiotic therapy in virtually all circumstances (with the exception of specific scenarios such as cystic fibrosis where it is recognized that the infecting organism cannot be fully eliminated). In most cases, the combination of bacteriostatic antibiotics and the host immune response is sufficient to achieve eradication. We have modified the manuscript text to reflect this nuance noted by the reviewer.

Line 62 several: you list three, but hipAB works via ppGpp, so the sentence needs fixing

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Line 70 uncertain: this uncertainty is unreferenced. Since everything is uncertain, this vague phrase does not add to the story.

The reviewer makes an interesting philosophical argument. However, we would submit that some aspects of biology, for example the regulation of glycolysis, are understood in great detail. However, other mechanisms, such as the precise mechanisms of lethality for diverse antibiotics in different bacterial species, are far more uncertain and remain a subject of debate (for example PMID: 39910302). Text not modified.

Line 72 somewhat controversial: I would delete this, because the points in the Science papers by Lewis and Imlay have been clarified and in some cases refuted by prior and subsequent work.

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Line 72 presumed: this suggests that it is wrong and perhaps a different idea has replaced it. Another, and more likely view is that there is an additional mode of killing. I suggest rephrasing to be more in line with the literature.

Text modified for clarity. In this sentence “presume” refers to the historical concept that direct target inhibition was solely responsible for antibiotic lethality. As the reviewer notes, there is now significant literature that ROS (and perhaps other secondary effects) also contribute to bacterial killing.

Line 73 However and the following might also: this phrasing, plus the presumed, misleads the reader from your intent. I suggest rephrasing.

See above re: line 72

Line 75 citations: these are inappropriate and should be changed to fit the statement. I suggest the initial paper by Collins (Kohanski 2007 Cell) a recent paper by Zhao (Zeng PNAS 2022), and a review Drlica Expert Rev Anti-infect Therapy 2021). The present citations are fine if you want to narrow the statement to mycobacteria, but the history is that the E. coli work came first and was then generalized to mycobacteria. A mycobacterial paper for ROS is Shee 2022 AAC.

We thank the reviewer for noticing that we inadvertently omitted several important E. coli-related references. These have been added.

Line 75 and 76: Conversely ... unresolved. Compelling arguments have been made that show major flaws in the two papers cited, and a large body of evidence has now accumulated showing the validity of the idea promoted by the Collins lab, beginning with Kohanski 2007. In addition to many papers by Collins, see Hong 2019 PNAS and Zeng 2022 PNAS). It is fine if you want to counter the arguments against the Lewis and Imlay papers (summarized in Drlica & Zhao 2021 Expert Rev Anti-infect Therapy), but making a blanket statement suggests that the authors are unfamiliar with the literature.

We agree with the reviewer that the weight of the evidence supports a role for antibiotic-induced ROS as an important mechanism for antibiotic lethality under many (though not all) conditions. We have revised the text to better reflect this nuance.

Line 78. Advantages over what?

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Line 80 exposure: to finish the logic you need to show that E. coli and S. aureus persisters fail to do this.

We thank the reviewer for their suggestion but studying these other organisms is outside the scope of this study.

Line 82 whereas: this misdirects the reader. It would seem that a simple "and" is better

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Line 89 I think this paragraph is about the need to study Mabs, the subject of the present report. This paragraph could use a more appropriate topic sentence to guide the reader so that no guessing is involved. I suggest rephrasing this paragraph to make the case for studying more compelling.

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Line 96. I suggest citing several references after subinhibitory concentration of antibiotic.

The references are in the following sentence alongside the key observations.

Line 99. Genetic analysis: how does this phrase fit with the idea of persister cells arising stochastically?

There are two issues: 1) We would argue that persister formation is not completely stochastic, but rather a probability that can be modified both genetically and by environment (for example hipA PMID: 6348026). 2) Even if persister formation were totally stochastic, the survival of these cells may depend on specific genes – as we indeed find in our Tn-Seq analysis of Mabs.

Line 106. In this paragraph you need to define persister. The consensus definition (Balaban 2019 Nat Micro Rev) is a subpopulation of tolerant cells. Tolerance is defined as the slowing or absence of killing while an antibiotic retains its ability to block growth. See Zeng 2022 PNAS for example with rapidly growing cells. Phenotypic tolerance is the absence of killing due to environmental perturbations, most notably nutrient starvation, dormancy, and growth to stationary phase. By extension, phenotypic persistence would be subpopulation status of a phenotypically tolerant cells. If you have a different definition, it is important to state it and emphasize that you disagree with the consensus statement.

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Line 109 unexpectedly. I would delete this word, because the literature leads the reader to expect this result unless you make a clear case for Mabs being fundamentally different from other bacteria with respect to how antibiotics kill bacteria (this is unlikely, see Shee 2022 AAC). Indeed, lines 111-113 state extensions of E. coli work, although suppression of ROS in phenotypic tolerance and genetic persistence have not been demonstrated.

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Line 124 you might add, in parentheses and with references, that a property of persisters is crosspersistence to multiple antibiotic classes. This is also true for tolerance, both genetic and phenotypic. An addition will support your approach.

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Line 128 minimal

Text not modified. We appreciate the reviewer’s preference but both “minimal” and “minimum” are both widely accepted terms. Indeed, the Balaban et al 2019 consensus statement on definitions cited by the author above also uses “minimum” (PMID: 30980069), as do IDSA clinical guidelines (PMID: 39108079).

Line 130 is MIC somehow connected to killing or did you also measure killing? Note that blocking growth and killing cells are mechanistically distinct phenomena, although they are related. By being upstream from killing, blockage of growth will also interfere with killing.

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Line 133 PBS is undefined

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Line 134 increase in persisters ... you need to establish that these are not phenotypically tolerant cells. Do they constitute the entire population (tolerance)? Your data would be more indicative of persisters if you saw a distinct plateau with the PBS samples, as such data are often used to document persistence (retardation of killing is a property of tolerance, Balaban 2019). Fig. 1B is clearly phenotypic tolerance, as the entire population grows. Your data suggest that you are not measuring persistence as defined in the literature (Balaban 2019). Line 139 persister should be tolerance •

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Lines 142, 143, 144. 159, 163, 171, 181, 211, 226, 238, 246, 277, 279,289 persistent should be tolerant

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Line 146 fig 1E Mtb does not show the adaptation phenomenon and it is clearly tolerant, not persistent. This should be pointed out. As stated, you may be misleading the reader.

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*Line 169. Please make it clear whether these genes are affecting antibiotic susceptibility (MIC will affect killing because blocking growth is upstream) or if you are dealing with tolerance (no change in MIC). These measurements are essential and should included as a table. By antibiotic response, do you mean that antibiotics change expression levels?

Regarding MICs, the data for MICs in control and katG mutant are presented in Figure 4C and 4F. Regarding ‘response’ we have clarified the text of this sentence.

Line 174 Interestingly should be as expected

Text not modified; tetracyclines do not induce ROS in E. coli and oxazolidinones have not been studied in this regard.

Line 183 you need to include citations. You can cite the ability of chloramphenicol to block ROS-mediated killing of E. coli. That allows you to use the word unexpected

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Line 199. All of the data in Fig. 3 shows tolerance, not persistence, requiring word changes in this paragraph.

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Line 226. The MIC experiment is important. You can add that this result solidifies the idea that blocking growth and killing cells are distinct phenomena. You can cite Shee 2022 AAC for a mycobacterial paper

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Line 241. The result with levofloxacin is unexpected, because the fluoroquinolones are widely reported to induce ROS, even with mycobacteria (see Shee 2022 AAC). You need to point this out and perhaps redo the experiment to make sure it is correct.

We appreciate the reviewer’s interest in this question. All experiments in this paper were repeated multiple times. This particular experiment was repeated 3 times and in all replicates the katG mutant was sensitized to translation inhibitors but not levofloxacin. Shee et al examined Mtb treated with moxifloxacin and found ROS generation, but did not assess whether a Mtb katG mutant had impaired survival. Thus, in addition to differences in: i) the species studied and ii) the particular fluoroquinolone used, the two sets of experiments were designed to address different questions (ROS accumulation vs protection by katG) . A cell might accumulate ROS without a katG mutant having impaired survival if genetic redundancy exists – a result we indeed see in our clinical Mabs strains under some conditions (new data included in revised Figure 6A).

Line 269 Additional controls would bolster the conclusion: use of an antioxidant such as thiourea and an iron chelator (dipyridyl) both should reduce ROS effects.

New experiments performed, revised Figure 5.

Line 276 the word no is singular

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Line 284 this suggested ... in fact previous work suggested. This summary paragraph might go better as the first paragraph of the Discussion

Text modified to specify that this is in reference to the work in this manuscript

Lines 294-299 Most of this is redundant and should be deleted.

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Line 299 this species is vague

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Line 310 Do you want to discuss spoT?

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Line 313 paragraph is largely redundant

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Line 314 controversial. As above, I would delete this, especially since it is not referenced and is unlikely to be true. If you believe it, you have the obligation to show why the ROS-lethality idea is untrue. If you are referring to Lewis and Imlay, there were almost a dozen supporting papers before 2013 and many after. This statement does not make the present work more important, so deletion costs you nothing.

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Line 314 direct disruption of targets. This is clearly not a general principle, because the quinolones rapidly kill while inhibition of gyrase by temperature-sensitive mutations does not (Kreuzer 1979 J.Bact; Steck 1985). Indeed, formation of drug-gyrase-DNA complexes is reversible: death is not.

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Line 318 as pointed out above, you have not brought this story up to date. The two papers mainly focused on Kohanski 2007, ignoring other available evidence.’’

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Line 326 you need to cite Shee 2022 AAC

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Line 342 the idea of mutants being protective is not novel, as several have been reported with E. coli studies. Thus, there is a general principle involved.

We agree that this suggests a potential general principle

Line 344. It depends on the inhibitor. For example, aminoglycosides are translation inhibitors and they also cause the accumulation of ROS.

We agree that ROS generation depends on the inhibitor, and indeed upon other variables including drug concentration, growth conditions, and bacterial species as well.

Line 347. You need to point out the considerable data showing that the absence of catalase increases killing

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Line 363 look at Shee 2022 AAC and Jacobs 2021 AAC

Text modified, reference added.

Line 585 I suggest having a colleague provide critical comments on the manuscript and acknowledge that person.

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