C14DM Ablation Leads to Reduced Tolerance to Plasma Membrane Stress and Increased Drug Sensitivity in Leishmania major
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsI have thoroughly reviewed Moitra and co-workers' manuscript, entitled "C14DM ablation leads to reduced tolerance to plasma membrane stress and increased drug sensitivity in Leishmania major". I consider it to be scientifically sound and of interest to the community. Nevertheless, some issues need to be addressed and/or clarified.
1) Please italicize all the scientific names along the manuscript
2) Authors are encouraged to explain more in depth the use of clinical isolate P. aeruginosa strain PA14 in leishmanicidal activity, instead of a bacterium isolated from the vector.
3) Authors don’t describe the cytolytic toxins produced by P. aeruginosa which cause leishmanicidal activity, and the relation with sterol 14α-demethylase
4) Please explain the importance of the deficiency of sterol 14α-demethylase and autophagy
5) Authors should explain why potassium antimony (III) tartrate and ethidium bromide (EtBr) are evaluated at different concentrations (micro and nanomolar). Are they comparable?
6) Please explain why the spent medium from P. aeruginosa was not evaluated against L. mexicana and L. donovani
7) In M&M, authors should include the origin of c14dm‾ (null mutant), and c14dm‾/+C14DM (own laboratory-made or purchased)
8) Authors should analyze the spent medium from P. aeruginosa to confirm that pyocyanin is the primary metabolite (and it is generated by) responsible for the leishmanicidal effect. Since the activity could be attributed to another compound, including a bioactive peptide.
9) Conclusion is not provided in the IJMS journal format
Author Response
Comment 1: Please italicize all the scientific names along the manuscript.
Response 1: Thank you for pointing this out. All the scientific names in the manuscript are now italicized.
Comment 2: Authors are encouraged to explain more in depth the use of clinical isolate P. aeruginosa strain PA14 in leishmanicidal activity, instead of a bacterium isolated from the vector.
Response 2: Thank you for pointing this out. First, we do not have access to those P. aeruginosa strains from the vector. Also, P. aeruginosa has been reported to be associated with Leishmania both in the sandfly and cutaneous lesions. This has been clarified (line 102-107).
Comment 3: Authors don’t describe the cytolytic toxins produced by P. aeruginosa which cause leishmanicidal activity, and the relation with sterol 14α-demethylase.
Response 3: Thank you for the comment. We attribute the leishmanicidal activity to the various apoptosis-inducing toxins and cytolytic toxins (line 244-248).
Comment 4: Please explain the importance of the deficiency of sterol 14α-demethylase and autophagy.
Response 4: Thank you for the comment. Autophagy deficiency could contribute to the observed hypersensitivity of c14dm− to starvation conditions [7] (line 131-137). It may be exploited to develop better anti-Leishmania treatment (e.g., autophagy inducers plus C14DM inhibitors).
Comment 5: Authors should explain why potassium antimony (III) tartrate and ethidium bromide (EtBr) are evaluated at different concentrations (micro and nanomolar). Are they comparable?
Response 5: Thank you for pointing this out. As shown in Figure 5A, L. major WT promastigotes in culture are much more sensitive to EtBr (EC 50: ~250 nM) than to potassium antimony (III) (EC50: ~19 μM). Therefore, we used the appropriate concentrations for these drugs in our evaluation.
Comment 6: Please explain why the spent medium from P. aeruginosa was not evaluated against L. mexicana and L. donovani.
Response 6: Thank you for the comment. Experiments described in Figures 1-5 were done in L. major to extend our previous characterization of the c14dm− mutant which was generated in L. major. Although the assay with P. aeruginosa spent medium could be done with L. mexicana and L. donovani (like other experiments in Figures 1-5), we mainly focused on expanding the drug synergy results to other Leishmania species in Figures 6 and 7 (which has more translational implications).
Comment 7: In M&M, authors should include the origin of c14dm‾ (null mutant), and c14dm‾/+C14DM (own laboratory-made or purchased.
Response 7: Thank you for the comment. These mutants were generated in the PI's lab and the reference [6] is provided (line 304).
Comment 8: Authors should analyze the spent medium from P. aeruginosa to confirm that pyocyanin is the primary metabolite (and it is generated by) responsible for the leishmanicidal effect. Since the activity could be attributed to another compound, including a bioactive peptide.
Response 8: Thank you for this comment. We do not believe that pyocyanin is the primary product responsible for the leishmanicidal effect in the spent medium. As shown in Figure 3A, ~90% of L. major WT promastigotes were lysed after 60 min in spent medium (the Y axis is in log scale); in contrast, it took 24 hours for pyocyanin (250 μM; the typical concentration found in stationary P. aeruginosa culture) to kill 70% of WT parasites (Figure 3B); therefore, other cytotoxic substances such as exotoxins are likely responsible for the rapid killing.
Comment 9: Conclusion is not provided in the IJMS journal format.
Response 9: Thank you for the comment. A conclusion paragraph is added (line 386-395).
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript “C14DM ablation leads to reduced tolerance to plasma membrane stress and increased drug sensitivity in Leishmania major addresses an important question in protozoan biology and chemotherapy: how sterol biosynthesis, particularly C14DM, contributes to Leishmania stress tolerance and drug sensitivity. The work is comprehensive, combining genetic mutants, stress assays, drug response analysis, and synergy testing. It has potential impact for understanding parasite physiology and guiding combination therapies. However, there are several issues that should be addressed to strengthen the manuscript.
- The introduction summarizes known roles of C14DM, but the novelty of the current work could be emphasized more clearly. Many findings (heat sensitivity, mitochondrial dysfunction, pentamidine hypersensitivity) have been reported before. Authors should explicitly delineate what is new in the submitted paper.
- The discussion could benefit from deeper integration with recent literature on sterol-targeting drug combinations in Leishmania and fungi, highlighting translational relevance.
- The link between altered sterol composition and the observed defects (membrane fragility, osmoregulation) remains descriptive. The authors should at least propose mechanistic hypotheses.
- While synergy between ITZ and pentamidine is shown, the range of drug concentrations and the choice of isobologram analysis should be better justified. Authors should clarify whether the observed synergy is robust across multiple dose ratios and whether it could be clinically achievable.
- The paper would benefit from a dedicated paragraph acknowledging key limitations (e.g., in vitro culture systems, use of axenic instead of intracellular amastigotes, lack of in vivo synergy validation). This transparency will improve credibility.
Author Response
Comment 1: The introduction summarizes known roles of C14DM, but the novelty of the current work could be emphasized more clearly. Many findings (heat sensitivity, mitochondrial dysfunction, pentamidine hypersensitivity) have been reported before. Authors should explicitly delineate what is new in the submitted paper.
Response 1: Thank you for the comment. We agree that the novel findings can be more clearly emphasized. This has been addressed (line 49-55).
Comment 2: The discussion could benefit from deeper integration with recent literature on sterol-targeting drug combinations in Leishmania and fungi, highlighting translational relevance.
Response 2: Thank you for pointing this out. A paragraph is added in the discussion (line 282-286).
Comment 3: The link between altered sterol composition and the observed defects (membrane fragility, osmoregulation) remains descriptive. The authors should at least propose mechanistic hypotheses.
Response 3: Thank you for pointing this out. We suspect that the altered sterol composition in c14dm− mutants make their plasma membrane more permeable to certain chemicals and less stable under stress (line 131-233, 244-248).
Comment 4: While synergy between ITZ and pentamidine is shown, the range of drug concentrations and the choice of isobologram analysis should be better justified. Authors should clarify whether the observed synergy is robust across multiple dose ratios and whether it could be clinically achievable.
Response 4: Thank you for the comment. For L. donovani, we tested 7 combinations of ITZ (0-1000 nM) and PENT (0-1000 nM). For L. mexicana, we also tested 7 combinations of ITZ (0-900 nM) and PENT (0-1300 nM). The average FIC values were determined and indicated in Figure 6 (line 195-197). These concentrations (although tested against promastigotes in culture) are clinically achievable.
Comment 5: The paper would benefit from a dedicated paragraph acknowledging key limitations (e.g., in vitro culture systems, use of axenic instead of intracellular amastigotes, lack of in vivo synergy validation). This transparency will improve credibility.
Response 5: Thank you for pointing this out. We have added a conclusion paragraph at the end that addresses the limitations (line 386-395).
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsI have no more comments.
