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

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

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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 …
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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 …

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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 …