PPATHOGENS-D-26-00737

FIKK1, a member of the FIKK kinase family, phosphorylates VAR2CSA and regulates adhesion of Plasmodium falciparum-infected erythrocytes to the placental receptor CSA

PLOS Pathogens

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The authors should pay particular attention to the Reviewer's suggestions for additional controls.

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1) Please ensure that the CRediT author contributions listed for every co-author are completed accurately and in full.

At this stage, the following Authors/Authors require contributions: Dominique Dorin-Semblat, Jean-Philippe Semblat, Heledd Eavis, Hugo Belda, Romain Hamelin, Clara-Eva Paquereau, Christian Doerig, Moritz Treeck, and Benoît Gamain. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

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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: In this manuscript, Dorin Semblat and colleagues show that the well-known P. falciparum adhesin VAR2CSA can be phosphorylated by the plasmodial kinase FIKK1. Using a FIKK1-HA conditional knockout transgenic line, recombinant proteins and phosphorylation assays, they show that phosphorylation by FIKK1 increases the binding of VAR2CSA to the placental receptor CSA. The authors propose that FIKK1 contributes to placental malaria virulence through RBC adherence in the placenta mediated by VAR2CSA phosphorylation.

These findings are interesting and relevant for the malaria community. The experiments performed and reported were thorough and convincing. In most cases, data and methods were clearly explained. The data provide the evidence needed for the authors to make their conclusions. I only have some minor points or questions that should be addressed.

Reviewer #2: The manuscript by Dorin-Semblat et al provides in vitro evidence to delineate the function of FIKK1 in phosphorylating VAR2CSA which impacts cytoadhesion property to CSA. The work described here follows a similar line of work recently published by the authors on VAR2CSA phosphorylation by PfCK2. This is a well-planned study that supports the conclusions drawn. The authors are aware of the limitations of the conclusions drawn from in vitro studies and refrained from over-interpreting their results. I recommend publication of this work with some modifications.

Specific comments:

Conclusions of this study are derived from orthogonal experiments that include, co-localization, co-IP, in vitro binding experiments, and kinase assays. Importantly, the authors demonstrate that the knockout of FIKK1 causes reduction in CSA adhesion while VAR2CSA expression remains unchanged. Authors recognize the possibility of another protein kinase (PfCK2?) co-immunoprecipitating that may contribute to VAR2CSA phosphorylation. Mass spectrometric analysis of the IP could have ruled out that possibility. Additionally, phosphoproteomic analysis of VAR2CSA in wild type vs FIKK1 cKO would have strengthened the conclusion.

It is not clear if there are any phenotypic change during the induction by rapamycin.

In Fig 6B, there are residual phosphorylation in DBL1-6 mutants, suggesting additional phosphosites may be involved.

There is a possibility that FIKK1 can mediate changes in adhesion through its functions at the erythrocyte membranes rather than exclusively through VAR2CSA phoshphorylation. Although the data presented in the manuscript supports the conclusions drawn, a robust discussion on other possibilities, such as changes erythrocyte cytoskeleton causing remodeling RBC membrane architecture, would improve the manuscript.

Reviewer #3: The current manuscript by Dorin-Semblat et al shows that the P. falciparum kinase phosphorylates the parasite ligand var2csa increasing the parasite binding affinity to csa. The study is elegantly done using a combination of molecular tools, including knockout parasites, pull down and phosphorylation assays on parasite lysates and recombinant proteins, proteomics approaches and parasite binding studies. The authors show that the phosphorylation residues on the N-terminal multidomain region of the var2csa, and that preventing phosphorylation significantly reduces the parasite binding to csa. The study is sound, logical and experiments were well controlled. Although I consider that no additional experiments are needed for publication, there were some points that would strengthen the manuscript. Although they could be considered minor, these suggestions would significantly improve the clarity of the manuscript.

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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: NA

Reviewer #2: None in particular.

Reviewer #3: (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: My main comment is that the authors identified several phosphosites (such as S429, S433) as phosphorylated by FIKK1, however they were also shown to be phosphorylated by PfCK2 in their previous paper (Dorin Semblat, Plos pathogens 2025). How do the authors explain these results? Do they think that these residues are phosphorylated by both kinases? They should discuss this point.

The selection for a clone positive for VAR2CSA expression at the IE surface is not clearly explained. It would help the reader to better explain their protocol.

Interestingly, Figure 5B suggests that FIKK1 phosphorylates the endogenous VAR2CSA (band indicated with a *). However, it would be more convincing to load immuno-conjugated material (DMSO and RAPA-treated) incubated with ATP32 alone to confirm this observation.

There is a typo line 242

Reviewer #2: It is suggested to include alternative possibilities of FIKK1-mediated changes in adhesion.

Reviewer #3: - Quantification of immunofluorescences: I would suggest for good scientific practice and for better representation of experimental variability that immunofluorescences in the manuscript are quantified. This is especially important for the images shown in Figure 1C in which co-localization between var2csa and SBP1 seem to be higher than with FIKK1HA. How many iRBC were quantified in this experiment? Similarly, quantification of Figure 3A is missing, and in Figure 3E I suggest to move the FACS quantification to the main figure, as this is the most representative experiment of the phenotype. Likewise, I would clearly show that Figure 4A and B correspond to the same experiment.

- I would suggest to indicate the wall shear stress instead of flow rate, as the later is dependent on the dimensions of the channel/flow chamber. If the authors use an IBIDI set up, a conversion should be available in the vendors website.

- Clarity of figures: The figures in the manuscript are not self-explanatory and this impacts the reading of the manuscript. Simple experimental schematics or labeling the protein gels and western blot lines, instead of just numbering them will be helpful for the reader.

- PfEMP1 terminology. The var2csa structure is not shown, which might make difficult the understanding certain sections of the manuscript for non var/PfEMP1 aficionados. This is especially important for Figures 5 and 6. Also, the authors use interchangeably the term extracellular domain of VAR2CSA and rDBL1.6, this could be confusing when different terms are used in the result section and in the figure.

- Implications for future development of drugs. The unphosphorylated form of var2csa still presents cytoadhesion. Although the authors do not claim that a pan-FIKK inhibitor would prevent cytoadhesion, the reference in the text (line 407-409) appears a bit out of the blue. It could be good to add a couple of sentences to contextualize it with the results of the manuscript.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Maria Bernabeu

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Dear Dr. Gamain,

We are pleased to inform you that your manuscript 'FIKK1, a member of the FIKK kinase family, phosphorylates VAR2CSA and regulates adhesion of Plasmodium falciparum-infected erythrocytes to the placental receptor CSA' has been provisionally accepted for publication in PLOS Pathogens.

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Leann Tilley

Academic Editor

PLOS Pathogens

Tracey Lamb

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

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Reviewer Comments (if any, and for reference):