Dear Dr. Prigge:

Thank you very much for submitting your manuscript "A mevalonate bypass system facilitates elucidation of plastid biology in malaria parasites" (PPATHOGENS-D-19-01463) for review by PLOS Pathogens. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. All three reviewers found your topic compelling and approach innovative, but identified some aspects of the manuscript that should be improved.

We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer. In particular, please note the need to include three or more replicates for all experimental findings.

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Sincerely,

Audrey Ragan Odom John, MD, PhD

Pearls Editor and guest Associate Editor

PLOS Pathogens

Kami Kim

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Grant McFadden

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-2556-3526

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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: Swift et al have generated an alternative tool for IPP rescue by genetic engineering parasites to utilize the lower mevalonate pathway. The major strengths of this study are the successful metabolic engineering of Plasmodium parasites and thorough characterization of the engineered strain. This is a tricky bit of genetic engineering in a difficult organism, including optimization and fusing of pathway genes. An easily-addressable weakness is that the authors do not clearly state the advantages and disadvantages of this alternative compared to the current tool, IPP rescue, making it difficult to assess significance. The main advantage of PfMev the authors discuss is the lower cost of the rescue compound, but this comes at the disadvantage of additional genetic manipulation of parasites which itself is costly in time and resources. Several of the applications (i.e. genetic transfection, compound screening, and forward genetic screens) mentioned by the authors have been performed using IPP rescue, suggesting that IPP rescue is sufficient to “elucidate plastid biology” for these types of experiments and not “prohibitive.” Unfortunately, the previous literature is not sufficiently discussed or cited in the manuscript and therefore do not allow for a fair evaluation of how PfMev improves upon the existing tool. I agree with authors that the application where IPP rescue is indeed prohibitive is metabolic labeling because of lack of availability of isotope-labeled reagents. Although not necessary to validate this tool, it would increase the significance of PfMev if more proof-of-concept for identification of isoprenoid products could be included. In addition, this engineering is halfway to a bypass in P. berghei for which chemical supplementation cannot easily be performed in mice; this would indeed open up many studies of plastid biology in this in vivo model. In terms of significance, metabolic labeling of isoprenoid products and facilitating study of apicoplast biology in in vivo malaria models are what I find most exciting for this new tool. This work should certainly be shared with the community, but I would recommend a more balanced presentation about its utility compared to the existing tool of IPP rescue.

Reviewer #2: The manuscript by Swift et al. describes an elegantly conceptualized and executed body of work that is likely to provide an excellent tool to understand plastid biology in Plasmodium falciparum. They have generated transgenic parasites, called PfMev line, expressing four enzymes able to utilize mavelonate to synthesize isopentenyl pyrophosphate (IPP). These enzymes were judiciously chosen from three different organisms and were physically fused to generate an artificial multifunctional enzyme complex, which then was expressed through a p15a origin of replication containing vector integrated at a nonessential chromosomal locus. These transgenic parasites were shown to become independent of apicoplast localized IPP synthesis as long as mavelonate was provided in the medium. These parasites could be made to lose their apicoplast when treated with azithromycin. The transcriptomic and metabolomic analysis of these apicoplast-minus parasites revealed little alterations: transcription of genes encoding apicoplast-targeted proteins was unaltered as were most of the metabolites. They were also able to ablate an essential gene involved in apicoplast-localized IPP synthesis in PfMev parasites. The manuscript is well written, experiments provide clear results, and the findings have far-reaching implications. There are a few minor points that I would suggest to improve the manuscript:

1. Authors do not provide rationale as to of how they came up to choose the four enzymes from 3 species to generate their bypass system. It would be important to describe this in the Results section. One has to go to the Methods section to even find out that the genes were different species. The rationale for their choices would be instructive to readers even outside the parasitology field.

2. They should provide a fully annotated DNA sequence of the plasmid used to generate PfMev parasites. There are many interesting and important aspects of this novel vector that are not apparent from the Supplementary Fig. 1, and a deposit in GenBank is not the same as a color-coded DNA sequence indicating various features of the plasmid included in the Supplementary information.

3. They do see some of the metabolites that seem to be altered in PfMev line . It would help to provide some information about them in the Results text in addition to being listed in Fig. 8.

4. It would help to provide a structure of mavelonolectone in Fig. 2 and state that this is what was used to supplement the culture medium in the Results section.

5. It would be worthwhile to point out that one possible reason for the lack of apicoplast biosynthetic activity in apicoplast-minus parasites, in spite of having most of the apicoplast-directed proteins in fragmented vesicles, could be the absence of a chaperone encoded by the apicoplast genome. This could render the targeted proteins unable to form functional enzymes.

Reviewer #3: This is a very well-written manuscript describing the generation, and characterisation, of a P. falciparum parasite line that can replicate normally after the apicoplast has been disrupted provided that mevalonate is present in the culture medium. This parasite line can synthesise isoprenoid precursors via an exogenous, mevalonate-dependent pathway, bypassing the parasite’s endogenous methylerythritol phosphate pathway. In characterising the parasite line, the authors were able to delete a normally-essential enzyme (the target of fosmidomycin). The authors also carried out metabolomic and transcriptomic studies to investigate the consequences for the parasite of apicoplast disruption, generating valuable new information. I believe the manuscript will be of high interest to the readership of PLOS Pathogens and the parasite line the authors have generated will be in demand by a number of malaria research labs around the world. I do not have any major criticism of the work but have identified a number of minor issues which I hope the authors will address. These are listed in order of appearance below.

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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: No additional experiments are required to validate the study conclusions that the lower MVA pathway has been engineered into a P. falciparum strain generating PfMev.

Reviewer #2: (No Response)

Reviewer #3: No new experiments are required, but I recommend that some of the experiments are repeated once more to generate averages from three independent experiments (see details below).

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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: In Introduction, establishing what is possible with IPP rescue prior to this manuscript, key citations are missing:

Use of IPP for drug screens: numerous, including Bowman et al. Anti-apicoplast and gametocytocidal screening to identify the mechanisms of action of compounds within the Malaria Box, Uddin et al. Validation of Putative Apicoplast-Targeting Drugs Using a Chemical Supplementation Assay in Cultured Human Malaria Parasites, Gisselberg et al. Specific Inhibition of the Bifunctional Farnesyl/Geranylgeranyl Diphosphate Synthase in Malaria Parasites via a New Small-Molecule Binding Site

Use of IPP for making gene knockouts and grow otherwise non-viable transfectants: Florentin et al. The Clp proteolytic system acts as an essential nexus for Plasmodium plastid biogenesis (https://www.biorxiv.org/content/10.1101/718452v1.full), Gisselberg et al. The suf iron-sulfur cluster synthesis pathway is required for apicoplast maintenance in malaria parasites

Use of IPP for forward genetic screens: Tang et al. A mutagenesis screen for essential plastid biogenesis genes in human malaria parasites (https://doi.org/10.1371/journal.pbio.3000136)

In Results, Design and validation of a mevalonate dependent isoprenoid synthesis pathway, the authors mention the need for IPP isomerase (idi) for proper balancing of IPP/DMAPP levels in E. coli. However, there is a parasite-encoded IPP isomerase annotated and no additional isomerase is required for IPP rescue. It seems the idi may be superfluous in the parasite. Was this tested?

In Results, Transcriptomic analysis of apicoplast disrupted parasites, the continued expression and detection of nuclear-encoded apicoplast proteins upon loss of the apicoplast under IPP rescue has already been demonstrated in previous papers. These should be cited, since the literature already indicated that expression of nuclear-encoded apicoplast proteins is not affected by apicoplast loss. The advance of this study is to show evidence for expression of many nuclear-encoded apicoplast genes using transcriptomic analysis.

Was there any attempt to reconstitute the upper MVA pathway (so no chemical supplementation is required)? Please discuss in context of potential for use in P berghei where chemical supplementation is difficult or not possible. This would increase the significance of this work.

To show utility of strain for identification of isoprenoid products, show evidence of labeled final products, for example the volatile terpenoid compounds described in Kelly et al. This would be very exciting and increase the significance of this work.

Fig 5. Please show the mass spectrum of the products compared to chemical standards to validate the peaks are correctly identified.

Reviewer #2: (No Response)

Reviewer #3: Minor issues.

1) In the “author summary”, IPP is referred to as “relatively unstable”. This term is too vague. Relative to what?

2) In several places the authors refer to “as little as 10 µM”. I would suggest that the authors refrain from using terms such as this, or explain why they consider 10 µM to be “little”. I assume they are comparing it to how much IPP has been used in the past to allow disruption of the apicoplast, but this is not clear (and the comparison unnecessary).

3) The last paragraph of the Discussion is a bit repetitive of other parts of the manuscript and can be shortened.

4) In the legend of Figure 2, the authors should clarify that the following sentence refers to parasite treated with fosmidomycin: “PfMev parasites not supplemented with mevalonate failed to grow”.

5) The data shown in Figures 2 and 4 are from two independent experiments and the error bars shown are SEM. SEM should not be used if the experiment has only been carried out twice. I would encourage the authors to repeat these experiments a third time.

6) Legends of Figures 3, 4 and 6. The following sentence should be modified: “Images are 10 microns long by 10 microns wide”. The actual images are much bigger than that (what the authors mean is that the size of the image can be used to represent 10 microns).

7) Legend of Figure 5. To be consistent, the authors should avoid using [12C]-mevalonate and refer to it as unlabeled mevalonate.

8) Figure 6. There is no indication of the number of independent experiments that were carried out to generate the data shown in part D. The legend also does not indicate what the error bars represent.

9) Figure 8 legend. The word “the” needs to be inserted in the following sentence: “The hierarchal clustering method is THE same as described…”

10) The references needs to be carefully checked to italicise species names. Reference 32 has errors within the title. Reference 47 has a minor error next to the citation details.

11) Figures 4, 6 and S4. Include units for 1000 and 500.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Prof Kevin J Saliba

Dear Dr. Prigge,

We are pleased to inform that your manuscript, "A mevalonate bypass system facilitates elucidation of plastid biology in malaria parasites", has been editorially accepted for publication at PLOS Pathogens. 

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Best regards,

Audrey Ragan Odom John, MD, PhD

Pearls Editor

PLOS Pathogens

Kami Kim

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewer Comments (if any, and for reference):