Coevolution of furA-Regulated Hyper-Inflammation and Mycobacterial Resistance to Oxidative Killing through Adaptation to Hydrogen Peroxide

ABSTRACT Mycobacterium tuberculosis (Mtb) is highly resistant to host oxidative killing. We hypothesized that the evolutionary adaptation of M. smegmatis to hydrogen peroxide (H2O2) would endow the nonpathogenic Mycobacterium persistent in a host. In the study, we screened a highly H2O2-resistant strain (mc2114) via evolutionary H2O2 adaptation in vitro. The MIC of mc2114 to H2O2 is 320 times that of wild-type mc2155. Mouse infection experiments showed that mc2114, similar to Mtb, was persistent in the lungs and caused high lethality in mice with restricted responses of NOX2, ROS, IFN-γ, decreased macrophage apoptosis, and overexpressed inflammatory cytokines in the lungs. Whole-genome sequencing analysis revealed that mc2114 harbored 29 single nucleotide polymorphisms in multiple genes; one of them was on the furA gene that caused FurA deficiency-mediated overexpression of KatG, a catalase-peroxidase to detoxify ROS. Complementation of mc2114 with a wild-type furA gene reversed lethality and hyper-inflammatory response in mice with rescued overexpression of KatG and inflammatory cytokines, whereas NOX2, ROS, IFN-γ, and macrophage apoptosis remained reduced. The results indicate that although FurA regulates KatG expression, it does not contribute significantly to the restriction of ROS response. Instead, FurA deficiency is responsible for the detrimental pulmonary inflammation that contributes to the severity of the infection, a previously nonrecognized function of FurA in mycobacterial pathogenesis. The study also indicates that mycobacterial resistance to oxidative burst results from complex mechanisms involving adaptive genetic changes in multiple genes. IMPORTANCE Mycobacterium tuberculosis (Mtb) causes human tuberculosis (TB), which has killed more people in human history than any other microorganism. However, the mechanisms underlying Mtb pathogenesis and related genes have not yet been fully elucidated, which impedes the development of effective strategies for containing and eradicating TB. In the study, we generated a mutant of M. smegmatis (mc2114) with multiple mutations by an adaptive evolutionary screen with H2O2. One of the mutations in furA caused a deficiency of FurA, which mediated severe inflammatory lung injury and higher lethality in mice by overexpression of inflammatory cytokines. Our results indicate that FurA-regulated pulmonary inflammation plays a critical role in mycobacterial pathogenesis in addition to the known downregulation of NOX2, ROS, IFN-γ responses, and macrophage apoptosis. Further analysis of the mutations in mc2114 would identify more genes related to the increased pathogenicity and help in devising new strategies for containing and eradicating TB.

2. Fig 2A, was the initial bacteria load for Day 0 same for the 2 strains? Looks like 1-day post-infection itself the numbers are different for the 2 strains.
3. Fig 2B, I am not convinced why the difference at 6 and 24 hrs is significant but not at 120 hrs pi? Please explain. 4. Fig 2C, authors need to perform viability assay for THP1 cells (eg MTT) to make sure that the survival difference is not because of differential viability of THP1 cells between the 2 bacterial strains. Reviewer #2 (Comments for the Author): The manuscript titled "Co-evolution of furA-regulated hyper-inflammation and mycobacterial resistance to oxidative killing through adaptation to hydrogen peroxide" by Fan et al., describes the generation of an H2O2-resistant mutant of M. smegmatis (named as mc2114) with multiple mutations through an adaptive evolutionary screen. The Msemg mc2114 mediated severe inflammatory lung injury and higher lethality in mice associated with overexpression of inflammatory cytokines. Further data suggest that FurA-regulates pulmonary inflammation in mice along with down-regulation of NOX2, ROS, IFN-γ responses, and macrophage apoptosis. This is a fascinating and well-written manuscript with carefully designed experiments. I have the following comments/suggestions for the authors. 1. Figure 1D and 6B. Please clarify if the data are from one experiment only, as there are no error bars/statistical analysis. 2. Figure 2C, data shall be presented as CFU/million THP-1 or CFU/104 THP-1 cells. CFU/well of THP-1 does not represent the correct way of representation as it could lead to misinterpretation of data. 3. Mycobacterium is a genus and hence shall be italicized in lines 84 and 28. 4. The sentence in lines 138-141 lacks clarity. Please modify. 5. Line 149-151, please mention the infection dosage to clarify the text. Although the dosage is mentioned in the figure legend, bringing it here as well will make it aptly clear. 6. Lines 166-168, is it possible to analyze the levels of NADPH or its activity? 7. Line 220-222, the authors have made a clean mutant, as claimed in the supplementary data. But mentioning ectopic expression confuses the readers. Please clarify. 8. Discussion section. Data presented in the manuscript suggest a correlation between the capability of mycobacterial strains to cause apoptosis and the protection of the host. The discussion section may be improved further but discussing these findings.

Preparing Revision Guidelines
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 raised by the reviewers in a file named "Response to Reviewers," NOT 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.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website. We add lines as "Although Mtb has a diminished capacity to restore endogenous 2 redox balance in comparison with Msmeg (1) it tolerates exogenous ROS and can persist in phagocytes. Whereas Msmeg is cleared from the lungs promptly, suggesting that Msmeg is less resistant to exogenous ROS (2), probably activities of these genes in Msmeg differ markedly (page 4, lines 86-90). The CFUs of mc 2 114 were higher than that of mc 2 155 but not statistically significant because of variation in CFU numbers (We add P-value in the revised Fig.   2B).

Fig 2C, authors need to perform viability assay for THP1 cells (eg MTT) to
make sure that the survival difference is not because of differential viability of

THP1 cells between the 2 bacterial strains.
Viability assay was performed with a CCK-8 Assay Kit (KGA317, KeyGEN BioTECH, China) according the manufacturer's instructions and showed that following PMA stimulation, proliferation of THP-1 cells in non-infected group was lower than that in infected group, indicating that infection with Msmeg promotes growth of THP-1. When the two infected groups were compared, we found that proliferation of THP-1 was significantly higher in mc 2 155 infected than in mc 2 114 infected group at all examined time points (Fig. 1), ruling out that higher numbers of intracellular mc 2 114 is due to increased THP-1 cells and suggesting that mc 2 114 inhibits phagocyte proliferation and phagocytic killing, thus makes mc 2 114 survived better in macrophages (page 7, lines 153-156). We thank the reviewer for the comment. All the comparisons in figures are changed using lines for clarity and consistency.

Response to reviewer #2:
The manuscript titled "Co-evolution of furA-regulated hyper-inflammation and mycobacterial resistance to oxidative killing through adaptation to hydrogen   Figure 2C, data shall be presented as CFU/million THP-1 or CFU/10 4 THP-1

cells. CFU/well of THP-1 does not represent the correct way of representation as it could lead to misinterpretation of data.
Thank you for the correction. The Y-axis labeling is changed as suggested.

Mycobacterium is a genus and hence shall be italicized in lines 84 and 28.
Thanks for the correction. They are italicized in the revised manuscript (page 2, lines 28 and page 4, lines 84).

The sentence in lines 138-141 lacks clarity. Please modify.
We agree with the reviewer and modified the statement for clarity in the revised manuscript (page 6, lines 140-145).

Line 149-151, please mention the infection dosage to clarify the text. Although
the dosage is mentioned in the figure legend, bringing it here as well will make it aptly clear.
Thanks for the suggestion. The infection dosage is included in the text (page 6, lines 138-140).

Lines 166-168, is it possible to analyze the levels of NADPH or its activity?
We thank the reviewer for this suggestion and measured NADPH activity. The results showed that NADP + /NADPH ratios were similar in mc 2 114 and mc 2 155 5 infected mice though higher levels of NADPH and NADP + were in mc 2 114 infected than in mc 2 155 infected group. More statement and a new figure (Fig. S1)  Thanks. We changed the sentence to avoid confusion(page 10, lines 232-234).
8. Discussion section. Data presented in the manuscript suggest a correlation between the capability of mycobacterial strains to cause apoptosis and the protection of the host. The discussion section may be improved further but discussing these findings.
We appreciate the reviewer's suggestion very much. A paragraph discussing mc 2 114 induced restriction of apoptosis is added in the revised manuscript (page 13, lines 309-317).
The manuscript titled "Co-evolution of furA-regulated hyper-inflammation and mycobacterial resistance to oxidative killing through adaptation to hydrogen peroxide" by Fan et al., has improved through the revision. However, I still have the following concerns. 1. In figure 1D and 6B, scanning the same image 3 times to get the statistics is not appropriate. Data from different biological replicates shall be quantified and image shown could be a representative image.
Staff Comments:

Preparing Revision Guidelines
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: 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.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.

Response to Reviewer #2
Reviewer #2 (Comments for the Author): The manuscript titled "Co-evolution of furA-regulated hyper-inflammation and mycobacterial resistance to oxidative killing through adaptation to hydrogen peroxide" by Fan et al., has improved through the revision. However, I still have the following concerns. figure 1D and 6B, scanning the same image 3 times to get the statistics is not appropriate. Data from different biological replicates shall be quantified and image shown could be a representative image.

In
We understand and accept the reviewer's comments. The Msmeg strains were cultured, then the expression and enzymatic activity of KatG of the strains were measured ( Fig. 1 and 2). The data in figures 1D and 6B were from three independent experiments (one done previously and two newly repeated). The figure legends of