Oxidative stress drives potent bactericidal activity of pyrazinamide against Mycobacterium tuberculosis

Reviewer #1 (Public review):

Summary

Pyrazinamide (PZA) is a key drug in the anti-TB arsenal, yet despite over 50 years of clinical use, its precise mechanism of action remains unclear. This study offers valuable insights into the in vitro potentiating effect of PZA when used with exogenous oxidative agents. The authors suggest that oxidative stress, specifically thiol oxidation, may be a primary driver of PZA/POA's bactericidal activity. Although the work is substantial, conceptually innovative, and timely, the evidence supporting the authors' conclusions requires further investigation with additional controls and experiments to fully validate the proposed mechanism of action. Once revised, this work will undoubtedly be of significant interest to the TB drug discovery community and researchers focusing on mycobacterial diseases.

Strengths

The authors have long-standing experience in the field of PZA mode of action, with several publications that have been highly relevant to the field. They are particularly well aware of the literature, and this is clearly visible in the introduction of the manuscript which is beautifully articulated. The biological question(s) and their hypotheses are also well-formulated in the introduction section.

The understanding of PZA mode of action is a long-lasting question in the TB community, therefore studies reporting well-conducted research that aims at deciphering the underlying mechanism responsible for PZA peculiar activity is always appreciated. Since PZA/POA are poorly active in conventional 7H9 media, but very potent in cellulo or in vivo; looking at host-mediated stress that can eventually lead to an increased vulnerability is extremely relevant. In that context, most of the work has been focused on host-cell endolysosomal pH but very little information is available on other stress. Thus, investigating the contribution of oxidative stress and ROS as specific host environments that might contribute to PZA/POA activity is overall novel and conceptually very interesting.

To address this question, the authors combine multiple approaches including conventional antimicrobial susceptibility profiling, CFU-based counting, and checkerboard assays to report the potentiating effect of PZA pre-treatment on hydrogen peroxide- and diamide-mediated antibacterial action. The use of multiple reference strains including Mtb H37Ra, Mtb H37Rv, M.bovis BCG, and M.bovis BCG::pncA is a great asset of the manuscript, even though they might have been more appropriately used to get further mechanistical insights on the proposed model of action. The findings are reported in 4 major figures that are clear and in an order that appears logical for the understanding of the story.

Weaknesses

Although the manuscript is conceptually very interesting and contains intriguing results, it sometimes fails to fully convince and some additional controls/experiments might help to better back up the authors' claims and really strengthen the study. Indeed, some conclusions seem premature therefore leading to some molecular assumptions regarding a potential mode of action that is not fully supported by the presented data.
The rationale behind some of the experiments is not always clearly explained which makes difficult to follow the authors ideas, the biological hypothesis/model that they test, and therefore the overall scientific story.

The authors conclude their study by proposing a mechanism by which the active form of the drug POA acts in concert with exogenous ROS to promote cellular oxidative damage. This is tested within two models of macrophage infection where they propose that IFN-γ mediated ROS production is essential for PZA activity. Unfortunately, the in cellulo part presents some weaknesses and inconsistencies that the authors need to carefully address.

Finally, the in vitro experiments performed in this manuscript mainly report that PZA pre-treatment increases H2O2-mediated killing or inhibition. There is no direct evidence that clearly shows that oxidative stress drives the potent bactericidal activity of PZA. In these settings, the oxidative stress is always applied after PZA pre-treatment and is therefore likely displaying the major lethal effect.

Reviewer #2 (Public review):

Summary:

The authors tested how ROS and PZA affected Mycobacterium survival to determine if ROS could have a role in the remarkable in vivo efficacy of PZA.

Strengths:

This is a well-written and clear manuscript convincingly demonstrating the synergy between PZA and reactive oxygen species in the inhibition of growth and survival of Mycobacterium tuberculosis.

Weaknesses:

The manuscript would benefit from a clear statement of the rationale for the protocols used to examine the synergy of PZA with ROS, the possible models their protocols could be testing, and then how their data supports or disproves the models being tested. The manuscript appears to propose, as stated in the title, that "Oxidative stress drives potent bactericidal activity of pyrazinamide...". However their experimental design more likely tests the effect of PZA on ROS sensitivity. Indeed, by the last figure, the authors begin the present their data as PZA sensitizing the bacteria to ROS. More clarity on these possible models and the different interpretations of the data should be considered.

Impact:

The data provide important insight to expand our understanding of the in vivo efficacy of PZA in the treatment of tuberculosis.

Author response:

We thank the reviewers for their thoughtful and constructive assessment of our manuscript. We agree that additional clarity on some key points in the manuscript will be valuable additions to this work. Both reviewers expressed a related concern regarding the basis for design and interpretation of our pyrazinamide ROS synergy experiments.

Reviewer 1:

The in vitro experiments performed in this manuscript mainly report that PZA pre-treatment increases H2O2-mediated killing or inhibition. There is no direct evidence that clearly shows that oxidative stress drives the potent bactericidal activity of PZA. In these settings the oxidative stress is always applied after PZA pre-treatment and is therefore likely displaying the major lethal effect.

Reviewer 2:

The manuscript would benefit from a clear statement of the rationale for the protocols used to examine the synergy of PZA with ROS, the possible models their protocols could be testing, and then how their data supports or disproves the models being tested. The manuscript appears to propose, as stated in the title, that "Oxidative stress drives potent bactericidal activity of pyrazinamide...". However their experimental design more likely tests the effect of PZA on ROS sensitivity. Indeed, by the last figure, the authors begin the present their data as PZA sensitizing the bacteria to ROS. More clarity on these possible models and the different interpretations of the data should be considered.

We agree that the data presented in the current version of the manuscript is incomplete in supporting our assertion that oxidative stress drives bactericidal activity of pyrazinamide. As both reviewers note, pretreatment of bacilli with pyrazinamide followed by challenge with ROS indicates that pyrazinamide enhances susceptibility to oxidative stress but does not address whether oxidative stress enhances susceptibility to pyrazinamide. Further, we neglected to provide information regarding why we chose to pretreat bacilli with pyrazinamide before ROS exposure. Over the course of our work, we had found that pyrazinoic acid, the active form of pyrazinamide, showed potent synergy with hydrogen peroxide. In contrast to the time-dependent synergy that we observed between pyrazinamide and peroxide, synergy between pyrazinoic acid and peroxide did not require pretreatment. We will revise our manuscript to include results that address these key issues and we will carefully consider revising our interpretations accordingly.