PPATHOGENS-D-24-02287

Gene deletion as a possible strategy adopted by New World Leishmania infantum to maximize geographic dispersion

PLOS Pathogens

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Dawn M Wetzel, MD, PhD

Academic Editor

PLOS Pathogens

Margaret Phillips

Section Editor

PLOS Pathogens

 Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Additional Editor Comments:

The authors have effectively revised their manuscript from an original submission to Review Commons to address prior reviewer comments. Two of the reviewers have commented on the revised manuscript below. The 3rd was not available.

Several deficiencies remain from the prior reviews, including:

1) One ongoing concern that remains from the unavailable reviewer is that there could be other unknown factors that could explain the differences seen in Del vs non-DEL strains. This concern would be mitigated through several approaches. Please improve the discussion of any statistics or analysis used to ensure that it was the lack of 3NU/NT activity in the clinical isolates that was responsible for reduced infectivity and increased transmissibility and not another factor related to the other deleted genes. Were any other changes among the DEL vs non-DEL strain groups noted in prior manuscripts or analysis that might affect conclusions here? In addition - if parasites with higher and lower levels of SHERP, META1 or META2 are grouped together instead of high vs low 3NU/NT activity, are there any differences seen in metacyclogenesis? Finally, the authors should make clear in the discussion that another, more conclusive means to show that it is specifically the lack of 3NU/NT activity that results in reduced infectivity and increased transmissibility would be through use of genetics in the same Leishmania background strain - eg, by introducing the locus/gene of interest in their DEL strain OR removing that locus from a non DEL strain - and explain why this was not done and how it will be done in the future.

2) Please otherwise ensure that conclusions match the data provided and do not overstate them, as discussed by the reviewers.

3) Please address the other concerns below from the 2 reviewers.

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Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: 1) The authors have clarified all the points concerning sand fly infections and improved the data visualizations.

2) The use of PNA to purify L. infantum metacyclics remains a topic of debate. It would be useful to include images of both PNA- and PNA+ parasites to support findings. Alternatively, direct readers to relevant literature that demonstrates enrichment of L. infantum metacyclics in the PNA- population (e.g., Soares et al 2002; Alcolea et al 2016).

3) It remains to be confirmed if the DEL genotype is linked to parasite attenuation, as there were no attempts to engineer transfectants to reconstitute the NonDEL phenotype. However, I appreciate that such genetics studies are not trivial and may be beyond the scope of the current paper.

4) I recommend that the following limitations in the current work should also be made clear in the Discussion.

(a) The BALB/c iv infections did not show significant decrease in parasite loads in the DEL strain vs the NonDEL strain in the bone marrow, spleen or liver. This suggests a similar pattern of dissemination between DEL and NonDEL strains. Of note, no pathology measure was provided in those infections (e.g., splenomegaly), which would have been valuable to assess experimental disease severity.

(b) Although other findings seem to indicate changes in the infection competence of the purified metacyclics, the unchanged expression of all but one of the metacyclic markers between DEL and NonDEL suggests that there is little apparent intrinsic differences between the metacyclic populations.

(c) At this stage, it is uncertain whether the DEL strains truly ‘possess a remarkable ability to colonize the vector and undergo metacyclogenesis’, in the context of more mature infections in prior work using Lu. longipalpis-Linf combinations (e.g., Rogers et al 2010; Giraud et al 2019). Additionally, an association with ‘higher transmissibility by Lu. longipalpis’ has yet to be confirmed.

Lastly, although I acknowledge that some sections of the manuscript do have a more speculative tone, I must clarify that the ‘Significance’ paragraph included in the #3 section of ‘Response to Reviewers’ was not submitted by me.

Reviewer #2: The authors have responded and made changes in the conclusions. However, some of the experiments which were suggested by the reviewers they were not able to do, which they claim are part of the ongoing studies.

Given the fact that it is novel finding that L. infantum species lacking the 3NUT gene has resulted in the reduction in infectivity and increase transmissibility. The authors have speculated that such parasites may cause asymptomatic infections and could provide immunity without causing the disease. That needs to be seen in future studies.

With this in mind I would suggest that the paper may be accepted.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: None

Reviewer #2: (No Response)

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: *Typos*

Supp. Fig. 5 (bone marrow panel): ‘semanas’ -> weeks

Fig.4 not referenced in the text (Error)

Reviewer #2: None

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Reviewer #1: No

Reviewer #2: No

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Dear Mrs Boité,

We are pleased to inform you that your manuscript 'Gene deletion as a possible strategy adopted by New World Leishmania infantum to maximize geographic dispersion' has been provisionally accepted for publication in PLOS Pathogens.

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Best regards,

Dawn M Wetzel, MD, PhD

Academic Editor

PLOS Pathogens

Margaret Phillips

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):