Protein kinase PfPK2 mediated signalling is critical for host erythrocyte invasion by malaria parasite

  1. Rahul Singh Rawat
  2. Ankit Gupta
  3. Neelam Antil
  4. Sonika Bhatnagar
  5. Monika Singh
  6. Akanksha Rawat
  7. T. S. Keshava Prasad
  8. Pushkar Sharma

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Abstract

Signalling pathways in malaria parasite remain poorly defined and major reason for this is the lack of understanding of the function of majority of parasite protein kinases and phosphatases in parasite signalling and its biology. In the present study, we have elucidated the function of Protein Kinase 2 (PfPK2), which is known to be indispensable for the survival of human malaria parasite Plasmodium falciparum . We demonstrate that it is involved in the invasion of host erythrocytes, which is critical for establishing infection. In addition, PfPK2 may also be involved in the maturation of the parasite post-invasion. PfPK2 regulates the release of microneme proteins like Apical Membrane Antigen 1 (AMA1), which facilitates the formation of Tight Junction between the merozoite and host erythrocyte- a key step in the process of invasion. Comparative phosphoproteomics studies revealed that PfPK2 may be involved in regulation of several key proteins involved in invasion and signalling. Furthermore, PfPK2 regulates the generation of cGMP and the release of calcium in the parasite, which are key second messengers for the process of invasion. These and other studies have shed light on a novel signalling pathway in which PfPK2 acts as an upstream regulator of important cGMP-calcium signalling, which plays an important role in parasite invasion.

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  1. Version published to 10.1371/journal.ppat.1011770
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    Reply to the reviewers

    A detailed point wise response has been uploaded along with revised manuscript files

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    The authors do not wish to provide a response at this time as only a revision plan is provided at this time.

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    Referee #3

    Evidence, reproducibility and clarity

    Using an elegant set of experiments, the authors probe the function of PfPK2 in asexual blood stage development. Using a rapamycin inducible dimerizable Cre recombinase-based strategy for the conditional knockdown of PK2, coupled with immunofluorescence assay, live cell imaging and phosphoproteomics the authors demonstrate the role of PK2 in invasion of red blood cells and signalling pathways in the parasite. Results show that PK2 is critical for the formation of tight junctions between merozoites and host red blood cells and that PfPK2 regulates cGMP levels and calcium release.

    Major comments:

    1. Is PK2 expressed in other life cycle stages? The authors should discuss what is known about the role of PK2 in other stages of parasite development. Would PK2 inhibitors be expected to have activity against liver or sexual stages of the parasite?
    2. Have any inhibitors of PK2 been reported to date? If so, were these tested against the PK2 cKD parasite line? Do they show similar phenotypic effects to knockdown of PK2? Do these studies give any insight on how vulnerable PK2 is as a target? To what extent does PK2 need to be inhibited/knocked down to lead to parasite death/growth inhibition?
    3. What were the expression yields and purity for the recombinantly expressed wild-type PK2 and mutant/ΔRD and ΔCD deletions? Has the ATP Km for PK2 been reported/determined? How did the deletions affect protein expression and stability? Could these deletions be influencing protein folding/amount of active protein in the preparation rather than regulating catalytic activity? How were protein concentrations normalised for the assay?
    4. Does rapamycin have any effect on wild-type parasites? What controls were included to ensure that observed effects were a result of rapamycin-induced PK2 knockdown, rather than rapamycin acting on other pathways/interfering directly with parasite biology. This should be discussed for readers less familiar with this system. Presumably the cHA-PfPK2-loxP line was used as the control to account for this for at least some of the experiments.

    Minor comments:

    1. "Malaria contributes to almost 400,000 deaths globally (Hay et al, 2004)."It should be made clear that this is the number of deaths reported annually. The references and statistic should be updated based on the most recent WHO Malaria report.
    2. "Several of these kinases are regulated by second messengers like calcium, PIPs, cAMP, cGMP Plasmodium kinases like CDPKs, PKA, PKG etc and are crucial for diverse parasitic functions." This sentence could be reworded for clarity.
    3. "However, the precise function of this protein kinase in parasite life cycle has remained unknown." Add word "the" should be added before "parasite".
    4. "Present studies demonstrate that PfPK2 regulates the invasion of host erythrocyte by P. falciparum." Should be erythrocytes (plural).
    5. "but the sequence similarity is mainly between kinase domains of human CamKIand PfPK2 (~80%)". Change "mainly" to "highest".
    6. "PfPK2 has a long N-terminal extension compared to CamKIand a CaM binding domain was predicted within a postulated regulatory domain present downstream of the kinase domain, which is the case with CamKI (Kato et al, 2008)." Please check this sentence and reword for clarity.
    7. References to "www.plasmodb.org" should be replaced with specific references to the source of the information listed in the database.
    8. Figure 2b. Please explain the relevance of adding/excluding EDTA and the effect of this on PK2 activity.
    9. "Correct integration of the targeting plasmid at the desired locus was confirmed by PCR as amplicons of expected size were obtained parasites from a clone that lacked wild type PfPK2 (Fig. 2B)." Remove the word "parasites".
    10. "Successful expression of GFP-PfPK2 was indicated by Western blotting (Fig. 2D) as well as by IFA (Supp. Fig. S1A), which wasundetectable after rapamycin treatment suggesting efficient depletion of this kinase from the parasite (Fig. 2D and Supp. Fig. S1B)." Write IFA in full the first time it is used in the main text. "was undetectable" should be two words.
    11. "The release of another microneme protein EBA-175, which interacts with glycophorin A on the 11 surface of the erythrocytes (Sim et al, 1994), was also reduced (Fig. 5A, Supp. Fig. S3B). but its localization to the microneme was unaltered (Supp. Fig. S4)." Remove full stop mid-sentence.
    12. "Therefore, we focussed on a possible role of PfPK2 in the release of AMA1, which is involved in late inavsion events like Tight Junction formation and PfPK2 plays a role at this stage of invasion (Fig. 4)." Correct "inavsion" to "invasion".
    13. Figure 5b. What is the difference between upper and lower panels. Please label or include details in figure legend. Figure legends should also include details on the DAPI, DIC and merged images for readers less familiar with microscopy images.
    14. "AMA1 plays a role in the formation of Tight-Junction (TJ) is via its association with rhoptry neck proteins (RONs) that are released by the rhoptries "Just-in-Time" on to erythrocyte surface (Riglar et al, 2011)." Remove "is".
    15. "Interestingly, phosphorylation site S1214 which is regulated by PfPK2 (Fig. 6B, 7B) resides in this insert This phosphositeis conserved only in GC" Add full stop after "insert". Replace "phoshositeis" with "phoshosite is"
    16. Order of Materials and Methods should be consistent with order of the Results section. For example, "Expression of recombinant proteins" and "Kinase assays" sections should come first.
    17. Please ensure all abbreviations, panels and labels in Figures are clearly explained in the relevant figure legends.

    Significance

    This study provides new insight into the role of PK2 in Plasmodium falciparum asexual blood stage development. While this study gives provides information on parasite biology, it does not provide significant advances in terms of understanding the value of PK2 as a drug target. However, this study does lay the groundwork for future studies using PK2 inhibitors as tool compounds for phenotypic validation.

    Audience: Specialised audience interested in parasite biology/signalling pathways.

    Reviewer's expertise: Target-based malaria drug discovery, Plasmodium kinase inhibitors, Target deconvolution for phenotypic hits in Plasmodium, kinase assay development.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate). Please place your comments about significance in section 2.

    The authors present a biochemical and functional characterisation of the Protein Kinase 2 in Plasmodium falciparum (PfPK2). They state that PfPK2 is an active protein kinase, as shown by phosphorylation of a histone protein (Figure 1). The next 6 figures of the paper describe the functional characterisation of PfPK2. It is a protein essential for the propagation of asexual malaria parasites in the clinical blood stage (Figure 2). The protein is expressed in the schizont stage and deletion of the PfPK2 gene by the conditional DiCre-LoxP system prevents parasite invasion, while not affecting egress (Figure 3 and 4), reduces the protein levels of two invasion molecules (Figure 5), modifies phosphorylation status on a variety of schizont stage proteins that play a role in invasion (Figure 6). PfPK2 deletion also leads to the decrease in the molecule cGMP which plays a role in parasite invasion (Figure 7).

    Major comments:

    • Are the key conclusions convincing?

    The conclusions made in Figure 2, 3, 4 and 7 are adequate but I have added questions in my detailed analysis on these figures that should assist in increasing the confidence on the conclusions.
    In my view, conclusions made in Figure 1, 5 and 6 are not convincing as they are missing key quality control data including loading control. Detailed comments on each figure is attached.

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?
      Conclusions drawn from Figures 1,5,6 should be supplemented with the experiments suggested, otherwise the claims should be marked as speculative.
    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.
      The experiments suggested in the detailed figure comments are necessary for supporting the claims of the paper.
    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.
      To improve the phosphoproteomics data, I would strongly suggest the creation or acquisition of a parasite line in which a related kinase has also been conditionally regulated and analysis of the phosphoproteome. I would suggest PfPKA made in the Wilde et al study. These experiments are required in order to qualify the statements made in the discussion section such as
      "A novel pathway was deciphered in which it is an upstream regulator of cGMP-calcium signalling axis, which is critical for invasion"
    • Are the data and the methods presented in such a way that they can be reproduced?

    How many times were the experiments mentioned in the following figures repeated?
    Figure 1C: PfPK2 phosphorylation assay
    Figure 1D: Kinase assay
    Figure 2B,C, D
    How many cells were counted in Figure 2F?
    How many biological replicates were used in Figure 6B?

    • Are the experiments adequately replicated and statistical analysis adequate?
      For figures not stated above, experiments were adequately replicated and analysis is adequate.

    Minor comments:

    • Specific experimental issues that are easily addressable.
    • Are prior studies referenced appropriately?
      Yes.
    • Are the text and figures clear and accurate?
      Axes on Figure 5A require editing as the values are unable to be seen clearly. Other figures are clear and well presented. Specific figure related comments are attached to this document.
    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    This data, in its current state, requires supplementation with necessary controls in order to be convincing and also additional experiments as suggested in the detailed figure related information.

    • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.
      The data included advances the field in characterising the role of PfPK2 and may influence the thinking of researchers studying Apicomplexan invasion biology. The PfPK2 phosphoproteomics and invasion imaging help knowledge gain in this area.
    • Place the work in the context of the existing literature (provide references, where appropriate).
      This work advances the data identified by Kato et al (2008) in the characterisation of PfPK2. Kato et al identified the activity of PfPK2, it's dependence on calmodulin and calcium for activity and the localisation of PfPK2 at the merozoite. Kato et al attempted to delete PK2 in the rodent malaria parasite P. berghei, but were unsuccessful, suggesting that the gene is essential for malaria parasites.
      Rawat et al recapitulate the kinase activity data and advance Kato et al's findings in creating a conditionally deletable PfPK2 parasite strain. Using this strain, they confirm the essentiality of the gene and characterise it's effect on invasion related processes in the parasite.
    • State what audience might be interested in and influenced by the reported findings.
      Researchers in Apicomplexan parasite biology may find this data interesting.
    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.
      My expertise is in malaria parasite biology including parasite invasion, genetic manipulation and protein export.

    Specific Figure related comments:

    Figure 1:

    C: Where is the data on the purification of PfPK2? Looking at Image 1C, I don't know what has been added to the reaction to phosphorylate Histone IIa.
    I would like to see Coomassie or western blots of the His-PfPK2 purification and the presence of Histone IIa so that I can be convinced that only PfPK2 and not a contaminant E-coli protein is responsible for the histone phosphorylation.

    Without knowing this data, I cannot be convinced that PfPK2 is phosphorylating Histone IIa

    Da: Again, without a Coomassie of western blot to show that K140M/WT PfPK2 as well as Histone IIa have been included in the reaction, I can't know that it is the single point mutation that has led to the lack of autophosphorylation of PfPK2 and the lack of Histone IIa phorphorylation.
    Please include this data.

    Db:
    Once again, there is no Coomassie or western to show what proteins are included in each treatment. Please include there.
    Why is there a band of the size of 32P-HIIa in the WT lane lacking HIIa? Is there a breakdown product of PfPK2 that gets autophosphorylated?

    Figure 2:

    A, B: The PCR results are conclusive in showing integration of the plasmid.
    C: The PCR and western blots of excision are conclusive and show efficient excision.
    D: Ideally, I would like to see the growth of the 1G5DC parasite strain on DMSO and rapamycin as well alongside the PK2 parasite line. 200nM of rapamycin is double the concentration used by Collins et al 2013 when creating the 1G5DC strain.

    Supplementary Figure 2A: Figure is missing a loading control here. Rest of the figure is conclusive and the complementation has worked nicely. I expect that the reason the complemented parasite isn't behaving exactly like the DiCre strain is due to the fact the complementation is done via an episomal plasmid which may segregate differentially in daughter merozoites.
    Supp Figure 2C: The Y-axis is hidden behind the legend for the same axis.

    Figure 3

    A + B: In my view the data from Figure 3A and 3B are effectively the one figure and therefore does not require an A/B division. The data shows no change in schizont maturation while invasion rate of rapa treatment is ~50% of DMSO control.
    C: Data shows that parasite invasion is impaired when PK2 is not present and that the cytochalasin D treatment was successful.

    Figure 4:

    The data shows reduction in the invasion rate upon Rapa treatment and an increased rate of prolonged echinocytosis. The video stills demonstrate the defects nicely.

    The data suggest that upon Rapa treatment, 40% of all invasion events are successful (Figure 4B). This result does not seem to match with Figure 2E, where rapa treatment leads to a reduction in parasitemia much less that 40% of control.
    Can the authors comment on this discrepancy?

    Figure 5 and Supp Figure S3

    A loading control is required for culture supernatant and lysate in all conditions (Supp Figure S3A,B,C). Otherwise we cannot interpret the loss of AMA1/EBA175 shedding as well as the non-effect on RhopH3. Without this data, the interpretations made in Figure 5A cannot be assessed.
    B: The figure needs to be better labelled to represent the E64 treatment as well.
    The representative image for PK2-LoxP + Rapa + E64 does not show any level of AMA1 on the surface, but the graph to the right shows 30% of parasites with surface AMA1 levels. Can the authors attach a series of images of this condition, showing the variation of AMA1 on the parasite surface?
    C: This figure shows nicely that AMA1 levels are reduced during invasion in rapa treated parasites.

    Figure 6:

    A. Do the authors identify PfPK2 peptides also being depleted upon PK2 knockdown?
    If so, please update Figure 6B. If not, then it makes the autophosphorylation data from Figure 1D contradictory.

    The data from Figure 6B and 6C identifies many invasion related proteins that are hyper/hypo phosphorylated upon rapamycin addition. It would have been interesting to include another invasion related kinase as a control line in all conditions (For example PfPKAc) that would allow finely dissecting the involvement of PfPK2 vs PfPKA.

    Figure 7A:

    1. What happens to the total protein levels of PDE and GCa upon PfPK2 deletion? Does the level of cGMP reflect the increased stability or loss of these two antagonistic proteins?
      I.E Does PK2 depletion lead to lower GCa activity (=less cGMP production) or increased stability and action of PDE (= more degradation of cGMP)?

    Significance

    This work advances the data identified by Kato et al (2008) in the characterisation of PfPK2. Researchers in Apicomplexan parasite biology may find this data interesting.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary: The manuscript by Rawat et al presents study to define the mechanism of action of an essential Plasmodium protein kinase PfPK2 using a conditional knockout line. The data show that PfPK2 has a role in the invasion of RBC as well as in the intraerythrocytic maturation of the parasite. In addition, quantitative phosphoproteomics was done to identify potential cellular targets and pathway analysis. The manuscript reports several important findings. I have following comments and suggestions:

    Major:

    1. Page 3. The data of malaria mortality is from 2004! Current WHO data needs to be included.
    2. Page 4. As per recent analysis (Adderley and Doerig,2022), number of P. falciparum kinases is higher than 85.
    3. Page 5. Homology analysis references are quite old. Is there any update from recent comparative analysis or alpha fold-based structural similarity.
    4. Page 7, line 4. It should be "Conditional Knockout". Needs to be corrected elsewhere as well.
    5. Page 8, line 18. Based on the methods section, parasites were treated at the ring-stage- needs to stated here as well.
    6. Page 12, line 16. It would be helpful to define "significant" in terms of phosphorylation changes. p<0.5?
    7. Page 12, line 23. It is mentioned that PfPK2-depleted samples have an increase in PPM2 phosphorylation. Is there a reference for these specific phosphosites?
    8. It would be bit of a stretch to say that the differentially phosphorylated proteins are substrate/targets (as in Table 1). PfPK2 depletion occurred in ring stage, and the sample was collected at ~44 HPI, leaving ample time for compensatory changes.
    9. Table 1. Criteria used for selecting proteins for the table needs to be defined in the legend. Example: p<0.5 FC>|1.2|. It would be also useful to see the fold change difference in a column for each protein.
    10. Fig 6B. Looks like there are 8 phosphorites with a greater negative fold change than those annotated. Are these all "unknown proteins"? They may not fall into the invasion or signaling groups in Table 1, but they still need to be included.
    11. Fig 6C. In the STRING analysis, PfPK2 is included as a yellow diamond but not referenced in the legend. Also, it is not clear if PfPK2 was differentially phosphorylated or was placed in the STRING network manually. "CPPUF is a nice abbreviation for "conserved Plasmodium protein, unknown function" but it would be useful to include the PlasmoDb number.
    12. Fig 6C. What is the significance of the line thickness or the circle size? Needs to be specified in the legend.
    13. It is not clear how many replicates of small collection was done for phosphoproteomic analysis.

    Minor:

    1. Page 5, line 3. It would useful to include PlasmoDb ID of PfPK2.
    2. Page 5, line 17: should read "....it has long N and a C-terminal...."
    3. Page 7, line 21: should be " was undetectable"...
    4. Page 9, line 17. It would be better to rephrase as.. "To better understand defects in the invasion following PfPK2 depletion live cell imaging was performed".
    5. Page 11, line 5. should be " late invasion".
    6. Page 30. Expression of recombinant protein and kinases assay should be moved to the beginning of the Methods as it is mentioned first in the results.
    7. Page 31, line 10. Concentration of imidazole needs to be mentioned.

    Significance

    The manuscript defines the role of PFPK2 in parasite invasion as well as in asexual maturation. Although unambiguous PfPK2 targets have not been defined, but the phosphoroteomic analysis alludes to the role of PfPK2 in cellular processes, particularly in invasion.

    The manuscript will be of general interest to readership of the journals listed.

  10. Version published to 10.1101/2023.07.31.551280v1 on bioRxiv