PI4‐kinase and PfCDPK7 signaling regulate phospholipid biosynthesis in Plasmodium falciparum
2021; Springer Nature; Volume: 23; Issue: 2 Linguagem: Inglês
10.15252/embr.202154022
ISSN1469-3178
AutoresRanjana Maurya, Anuj Tripathi, Manish Kumar, Neelam Antil, Yoshiki Yamaryo‐Botté, Praveen Kumar, Priyanka Bansal, Christian Doerig, Cyrille Y. Botté, Thottethodi Subrahmanya Keshava Prasad, Pushkar Sharma,
Tópico(s)Mosquito-borne diseases and control
ResumoArticle6 December 2021free access Source DataTransparent process PI4-kinase and PfCDPK7 signaling regulate phospholipid biosynthesis in Plasmodium falciparum Ranjana Maurya Ranjana Maurya orcid.org/0000-0002-3339-1429 Eukaryotic Gene Expression laboratory, National Institute of Immunology, New Delhi, India Search for more papers by this author Anuj Tripathi Anuj Tripathi orcid.org/0000-0002-3697-0207 Eukaryotic Gene Expression laboratory, National Institute of Immunology, New Delhi, India Search for more papers by this author Manish Kumar Manish Kumar Eukaryotic Gene Expression laboratory, National Institute of Immunology, New Delhi, India Institute of Bioinformatics, International Tech Park, Bangalore, India Manipal Academy of Higher Education, Manipal, India Search for more papers by this author Neelam Antil Neelam Antil orcid.org/0000-0002-5154-2825 Institute of Bioinformatics, International Tech Park, Bangalore, India ApicoLipid Team, Institute of Advanced Biosciences, CNRS UMR5309, Université Grenoble Alpes, INSERM U1209, Grenoble, France Amrita School of Biotechnology, Amrita Vishwa Vidyapeetham, Kollam, India Search for more papers by this author Yoshiki Yamaryo-Botté Yoshiki Yamaryo-Botté Center for Systems Biology and Molecular Medicine, Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, India Search for more papers by this author Praveen Kumar Praveen Kumar Eukaryotic Gene Expression laboratory, National Institute of Immunology, New Delhi, India Search for more papers by this author Priyanka Bansal Priyanka Bansal Eukaryotic Gene Expression laboratory, National Institute of Immunology, New Delhi, India Search for more papers by this author Christian Doerig Christian Doerig orcid.org/0000-0002-3188-094X NIMHANS IOB Proteomics and Bioinformatics Laboratory, Neurobiology Research Centre, National Institute of Mental Health and Neuro Sciences, Bangalore, India Search for more papers by this author Cyrille Y Botté Cyrille Y Botté Center for Systems Biology and Molecular Medicine, Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, India Search for more papers by this author T S Keshava Prasad T S Keshava Prasad orcid.org/0000-0002-6206-2384 Institute of Bioinformatics, International Tech Park, Bangalore, India Amrita School of Biotechnology, Amrita Vishwa Vidyapeetham, Kollam, India School of Health and Biomedical Sciences, RMIT University, Bundoora, Vic., Australia Search for more papers by this author Pushkar Sharma Corresponding Author Pushkar Sharma [email protected] orcid.org/0000-0001-6861-0508 Eukaryotic Gene Expression laboratory, National Institute of Immunology, New Delhi, India Search for more papers by this author Ranjana Maurya Ranjana Maurya orcid.org/0000-0002-3339-1429 Eukaryotic Gene Expression laboratory, National Institute of Immunology, New Delhi, India Search for more papers by this author Anuj Tripathi Anuj Tripathi orcid.org/0000-0002-3697-0207 Eukaryotic Gene Expression laboratory, National Institute of Immunology, New Delhi, India Search for more papers by this author Manish Kumar Manish Kumar Eukaryotic Gene Expression laboratory, National Institute of Immunology, New Delhi, India Institute of Bioinformatics, International Tech Park, Bangalore, India Manipal Academy of Higher Education, Manipal, India Search for more papers by this author Neelam Antil Neelam Antil orcid.org/0000-0002-5154-2825 Institute of Bioinformatics, International Tech Park, Bangalore, India ApicoLipid Team, Institute of Advanced Biosciences, CNRS UMR5309, Université Grenoble Alpes, INSERM U1209, Grenoble, France Amrita School of Biotechnology, Amrita Vishwa Vidyapeetham, Kollam, India Search for more papers by this author Yoshiki Yamaryo-Botté Yoshiki Yamaryo-Botté Center for Systems Biology and Molecular Medicine, Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, India Search for more papers by this author Praveen Kumar Praveen Kumar Eukaryotic Gene Expression laboratory, National Institute of Immunology, New Delhi, India Search for more papers by this author Priyanka Bansal Priyanka Bansal Eukaryotic Gene Expression laboratory, National Institute of Immunology, New Delhi, India Search for more papers by this author Christian Doerig Christian Doerig orcid.org/0000-0002-3188-094X NIMHANS IOB Proteomics and Bioinformatics Laboratory, Neurobiology Research Centre, National Institute of Mental Health and Neuro Sciences, Bangalore, India Search for more papers by this author Cyrille Y Botté Cyrille Y Botté Center for Systems Biology and Molecular Medicine, Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, India Search for more papers by this author T S Keshava Prasad T S Keshava Prasad orcid.org/0000-0002-6206-2384 Institute of Bioinformatics, International Tech Park, Bangalore, India Amrita School of Biotechnology, Amrita Vishwa Vidyapeetham, Kollam, India School of Health and Biomedical Sciences, RMIT University, Bundoora, Vic., Australia Search for more papers by this author Pushkar Sharma Corresponding Author Pushkar Sharma [email protected] orcid.org/0000-0001-6861-0508 Eukaryotic Gene Expression laboratory, National Institute of Immunology, New Delhi, India Search for more papers by this author Author Information Ranjana Maurya1,†, Anuj Tripathi1,†, Manish Kumar1,2,3, Neelam Antil2,4,5, Yoshiki Yamaryo-Botté6, Praveen Kumar1, Priyanka Bansal1, Christian Doerig7, Cyrille Y Botté6,‡, T S Keshava Prasad2,5,8,‡ and Pushkar Sharma *,1,‡ 1Eukaryotic Gene Expression laboratory, National Institute of Immunology, New Delhi, India 2Institute of Bioinformatics, International Tech Park, Bangalore, India 3Manipal Academy of Higher Education, Manipal, India 4ApicoLipid Team, Institute of Advanced Biosciences, CNRS UMR5309, Université Grenoble Alpes, INSERM U1209, Grenoble, France 5Amrita School of Biotechnology, Amrita Vishwa Vidyapeetham, Kollam, India 6Center for Systems Biology and Molecular Medicine, Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, India 7NIMHANS IOB Proteomics and Bioinformatics Laboratory, Neurobiology Research Centre, National Institute of Mental Health and Neuro Sciences, Bangalore, India 8School of Health and Biomedical Sciences, RMIT University, Bundoora, Vic., Australia † These authors contributed equally to this work ‡ These authors contributed equally to this work *Corresponding author. Tel: 91-11-26703791; Fax: 91-11-26742125; E-mail: [email protected] EMBO Reports (2022)23:e54022https://doi.org/10.15252/embr.202154022 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract PfCDPK7 is an atypical member of the calcium-dependent protein kinase (CDPK) family and is crucial for the development of Plasmodium falciparum. However, the mechanisms whereby PfCDPK7 regulates parasite development remain unknown. Here, we perform quantitative phosphoproteomics and phospholipid analysis and find that PfCDPK7 promotes phosphatidylcholine (PC) synthesis by regulating two key enzymes involved in PC synthesis, phosphoethanolamine-N-methyltransferase (PMT) and ethanolamine kinase (EK). In the absence of PfCDPK7, both enzymes are hypophosphorylated and PMT is degraded. We further find that PfCDPK7 interacts with 4'-phosphorylated phosphoinositides (PIPs) generated by PI4-kinase. Inhibition of PI4K activity disrupts the vesicular localization PfCDPK7. P. falciparum PI4-kinase, PfPI4K is a prominent drug target and one of its inhibitors, MMV39048, has reached Phase I clinical trials. Using this inhibitor, we demonstrate that PfPI4K controls phospholipid biosynthesis and may act in part by regulating PfCDPK7 localization and activity. These studies not only unravel a signaling pathway involving PfPI4K/4'-PIPs and PfCDPK7 but also provide novel insights into the mechanism of action of a promising series of candidate anti-malarial drugs. Synopsis PfPI4K and PfCDPK7 regulate the development of the malaria parasite Plasmodium falciparum by promoting phospholipid biosynthesis. PI4-Kinase regulates the localization and activity of protein kinase PfCDPK7 via 4’-PIPs. PfCDPK7 promotes phosphatidylcholine (PC) synthesis by targeting key enzymes involved in the synthesis of this phospholipid. PI4-K promotes PL biosynthesis in P. falciparum and its regulation of PfCDPK7 activity contributes to this process. Introduction The malaria parasite Plasmodium spp is transmitted to the human host through the bite by an infected female Anopheles mosquito, which allows the entry of sporozoites into the bloodstream of the human host. The sporozoites then infect hepatocytes in the host liver to expand their population during an asymptomatic period of asexual schizogonic division, during which thousands of merozoites are formed. Merozoites enter the bloodstream and invade erythrocytes to initiate the intra-erythrocytic stages of parasite development, which gives rise to malaria pathogenesis. After the invasion of red blood cells (RBCs), the intracellular parasites mature from rings to trophozoites followed by asexual schizogonic division that results in the generation of ~30 merozoites per schizont within 48 h in the case of P. falciparum. Freshly egressed merozoites can infect fresh RBCs to start a new round of asexual division. Some parasites withdraw from proliferation and undergo sexual differentiation to male or female gametocytes, which can be ingested by the mosquito, in which sporogony occurs, resulting in the generation of infective sporozoites (Cowman et al, 2016). CDPKs, a family of kinases that are restricted to plants and Alveolates (the taxon that includes Ciliates and Apicomplexa), are major effectors of calcium signaling in Plasmodium as well as the related apicomplexan parasite Toxoplasma gondii, and collectively regulate crucial processes, including host cell invasion, egress, growth, and sexual differentiation (Billker et al, 2004, 2009; Dvorin et al, 2010; Lourido et al, 2010; Sebastian et al, 2012; Gaji et al, 2014; Kumar et al, 2014, 2017; Morlon-Guyot et al, 2014; Treeck et al, 2014). Classical CDPKs contain a protein kinase domain and C-terminal calmodulin (CaM)-like domain, which are connected by a regulatory Junction domain (Billker et al, 2009; Ahmed et al, 2012). The domain architecture of PfCDPK7 diverges from that of other members of the CDPK family. It has two N-terminal EF-hand calcium-binding motifs connected by a long linker to a PH-domain, with the kinase domain located at the C terminus (Kumar et al, 2014; Morlon-Guyot et al, 2014; Bansal et al, 2021). Despite the presence of the EF-hands, there is no experimental evidence of its regulation by calcium. This atypical kinase is conserved in Toxoplasma and Plasmodium and is able to interact with phosphoinositides PI4P and PI(4,5)P2 via its PH domain (Kumar et al, 2014; Morlon-Guyot et al, 2014). Previously, we have demonstrated that the PH domain via which PfCDPK7 interacts with PI(4,5)P2 is important for its localization to vesicular structures (Kumar et al, 2014). CDPK7 is critical for the development of both the rodent malaria parasite P. berghei and P. falciparum; it is indispensable for P. berghei (Tewari et al, 2010) and its knockout in P. falciparum is achievable (Solyakov et al, 2011) but severely impairs asexual parasite development (Kumar et al, 2014). Additional studies suggested that CDPK7 regulates the division of P. falciparum and T. gondii (Kumar et al, 2014; Morlon-Guyot et al, 2014) as well as the transition from ring to trophozoite during the P. falciparum erythrocytic cycle (Kumar et al, 2014). The underlying mechanisms through which PfCDPK7 regulates parasite development remain unknown. In the present study, we have identified PfCDPK7-dependent molecular processes in P. falciparum blood stages. Quantitative phosphoproteomics of wild-type and PfCDPK7 knockout parasites revealed that the phosphorylation levels of proteins predicted to be involved in lipid metabolism and protein/lipid trafficking were significantly altered. Furthermore, we provide evidence that the biosynthesis of a major phospholipid, phosphatidylcholine (PC), is regulated by PfCDPK7. Finally, we show that the parasite’s PI4-kinase PfPI4K regulates PfCDPK7 and is therefore also involved in PL biosynthesis. Results Identification of PfCDPK7-regulated pathways in P. falciparum asexual parasites by comparative phosphoproteomics In order to elucidate the mechanism through which PfCDPK7 regulates the development of P. falciparum, we used a previously reported parasite line in which the PfCDPK7 gene is disrupted by homologous recombination-dependent insertion. This line (PfCDPK7-KO) exhibits significant defects in parasite growth relative to the parental 3D7 clone, which is due to impaired ring to trophozoite maturation and division (Kumar et al, 2014). We performed quantitative phosphoproteomics on PfCDPK7-KO and wild-type 3D7 parasites to identify potential targets of PfCDPK7 signaling. Since PfCDPK7-KO parasites were defective in ring to trophozoite maturation, late ring–early trophozoites were used for this experiment (see Methods). iTRAQ-labeled peptides from PfCDPK7-KO and wild-type parasites were analyzed on high-resolution mass spectrometer. In all, 82 phosphosites from 68 proteins exhibited hypophosphorylation and 15 phosphosites from 13 proteins showed hyperphosphorylation in the PfCDPK7-KO parasites as compared to the wild-type (Dataset EV1). Fig 1A illustrates the number of phosphosites identified in the analysis and their respective fold-change in the mutant parasites. A subset of proteins that were found to be differentially phosphorylated across biological replicates with significant P-value (< 0.05) are annotated (Dataset EV1). Gene Ontology (GO) analysis suggested the major classes of proteins that exhibited altered phosphorylation were involved in the synthesis of lipids and metabolites such as sugars (Fig 1B). Interestingly, several trafficking-related proteins were also differentially phosphorylated in PfCDPK7-KO, including Rab11b, beta-coatamer, Sec61, and VPS18 (Fig 1A and C, and Dataset EV1). Figure 1. Comparative phosphoproteomics to identify processes regulated by PfCDPK7 Comparative phosphoproteomic and proteomic analyses were performed on late ring/early trophozoite stage wild-type (3D7) or PfCDPK7-KO parasites. The phosphorylation fold-change ratio of identified phosphopeptides was normalized to total protein abundance fold-change. The fold-change ratios for all phosphopeptides from various replicates are provided in Dataset EV1. The S-curve for the normalized data is provided for some of the significantly altered hyper-(blue) and hypo-(red) phosphorylated sites belonging to key proteins (Dataset EV1) are indicated. Phosphorylation sites of key enzymes involved in PL biosynthesis that exhibited reduced phosphorylation in PfCDPK7-KO parasites are indicated in green. Pathway analysis of proteins that exhibited reduced phosphorylation upon PfCDPK7 disruption and the possible metabolic pathways these proteins may regulate (Dataset EV1). The pathways are represented based on their P-values (log 2), as estimated by the gene ontology tool available at PlasmoDB. Protein–protein interactions were predicted between differentially phosphorylated proteins using the STRING resource. The analysis exhibited high confidence interactions between the candidate proteins (Appendix Fig S1 and Dataset EV1). A major signaling module that involved enzymes including EK and PMT implicated in PC/PE metabolism was identified, in addition to a network involving trafficking protein PfRab11b. Hyper- and hypophosphorylated proteins are indicated in blue and red respectively. Proteins that are relevant to the topic of this paper are encircled. Proteins involved in PL metabolism and trafficking are indicated in red and blue, respectively. Download figure Download PowerPoint Strikingly, the phosphorylation of ethanolamine kinase (EK) and phosphoethanolamine-N-methyltransferase (PMT) was altered significantly (Fig 1A and Dataset EV1, and Appendix Figs S1 and S3). EK phosphorylates ethanolamine to generate the phosphoethanolamine polar head group used for PE synthesis via the Kennedy pathway (Dechamps et al, 2010b). PfPMT is most closely related to plant orthologues (Dechamps et al, 2010a, 2010b) and is absent in T. gondii and in the rodent malaria parasite P. berghei (Ramakrishnan et al, 2013); this enzyme methylates phosphoethanolamine to generate phosphocholine, which can be used as a precursor for PC synthesis via the Kennedy pathway (Reynolds et al, 2008; Witola et al, 2008). These enzymes are thus implicated in PC and PE biosynthesis (Fig 2C) (Ramakrishnan et al, 2013), and the aforementioned observations were first indicators of a potential role of PfCDPK7 in the metabolism of these two PL classes. In addition, a phosphatidate phosphatase (PAP) homologue exhibited reduced phosphorylation in PfCDPK7-KO. PAP is putatively capable of generating diacylglycerol (DAG) from phosphatidic acid (PA), which is then used as the central precursor of PC and PE in the Kennedy pathway. Figure 2. PfCDPK7 is involved in PC biosynthesis in Plasmodium falciparum Total lipids were extracted from PfCDPK7-KO or 3D7 parasites and separated by HPTLC. After quantification by GC-MS content relative to total fatty acids was determined. Relative abundance of individual phospholipids was quantified. Disruption of PfCDPK7 caused a significant decrease in PC (mean ± SEM, n = 3; biological replicates, ***P < 0.001, t-test). Total fatty acids were extracted and separated before quantification by GC-MS analysis. The analysis shows an increase of C18:1 as well as the general decrease of PUFAs (inset) and more particularly C20:4 (mean ± SEM, n = 3; biological replicates, ***P < 0.001, t-test). Metabolic pathways depicting the synthesis of PC and PE in P. falciparum. PMT and EK, which are putative targets of PfCDPK7, are indicated in red (Ramakrishnan et al, 2013). Metabolic labeling to monitor PC and PE synthesis. Equal number of WT (3D7) and PfCDPK7-KO (KO) parasites were incubated with 14C-Eth or 14C-Cho (in RPMI) for 12 h. Subsequently, parasites were harvested and lipids were extracted and separated by thin-layer chromatography (TLC). The radiolabeled lipids were detected by phosphorimaging (upper panel). Bottom panel, radiolabeled PC and PE were quantitated by performing densitometry on phosphorimage of TLC plates in above panel and % change in PC or PE formation in PfCDPK7-KO parasites with respect to 3D7 (100%) was calculated (mean ± SEM, n = 4; biological replicates, **P < 0.01, ANOVA; ns, nonsignificant). 3D7 or PfCDPK7-KO parasites were cultured in the presence or absence of 200 μM choline in culture medium. After 3 days, parasitemia was determined by counting parasites on thin blood smears (mean ± SEM, n = 6; biological replicates, *P < 0.05, ***P < 0.001, ANOVA). Download figure Download PowerPoint Protein–protein interaction (PPI) networks among the differentially phosphorylated proteins were predicted using the STRING database (Szklarczyk et al, 2015). The analysis indicated that the potential substrates or downstream effectors of PfCDPK7 might be involved in direct or indirect interactions with each other (Fig 1C and Appendix Fig S1). Some of these proteins were further shown to interact with PfCDPK7 using co-immunoprecipitation, which is discussed in the later sections. PfCDPK7 disruption impairs phosphatidylcholine synthesis in P. falciparum asexual parasites Since phosphorylation of proteins/enzymes involved in PL biosynthesis was significantly altered in PfCDPK7-KO line (Fig 1), we investigated if PfCDPK7 regulates phospholipid composition in P. falciparum. To this end, mass spectrometry-based lipidomics analyses were conducted on lipids extracted from wild-type 3D7 or PfCDPK7-KO. The analysis of the parasite phospholipid composition revealed a significant drop in total phosphatidylcholine (PC) content, whereas no other phospholipid class was significantly altered in the PfCDPK7-KO parasites (Fig 2A). As mentioned above, multiple pathways are involved in the synthesis of PC in P. falciparum (Fig 2C). Ethanolamine as well as choline can be utilized for the formation of PC. PfPMT enzyme is capable of generating phosphocholine from phosphoethanolamine, which is then used to generate PC via the regular Kennedy pathway (Reynolds et al, 2008; Witola et al, 2008). To further validate the lipidomics data, the de novo synthesis of PC and PE via the parasite Kennedy pathway (Fig 2C) was monitored by metabolic labeling and thin-layer chromatography (TLC). For this purpose, 14C-Ethanolamine (Eth) and 14C-Choline (Cho) were used as precursors of both PE and PC in intracellular parasites, respectively. Total lipids were extracted and labeled lipids were detected and quantified by high precision–thin-layer chromatography (HPTLC) (Fig 2D). Consistent with our data and previous analyses (Witola et al, 2008), 14C-Eth labeling indicated the de novo synthesis of both PE and PC by using Eth (Fig 2D). While the change in PE levels was marginal and not significant, PC synthesis was significantly impaired in PfCDPK7-KO parasites (Fig 2D). Interestingly, the PC content was also reduced in PfCDPK7-KO when 14C-Cho was used as the precursor (Fig 2D). These results are in agreement with our lipidomics analyses. Collectively, these data suggested that PfCDPK7 regulates PC biosynthesis in P. falciparum. We also examined the fatty acid (FA) profile of PfCDPK7-KO parasites and compared it to that from the parental 3D7 line. Long polyunsaturated fatty acid (PUFA) chains were significantly reduced in PfCDPK7-KO parasites, and more specifically levels of arachidonic acid, C20:4 (Fig 2B), while there was a slight significant increase in C18:1 cis species, likely reflecting overall changes in FA homeostasis and compensatory mechanisms. Long-chain PUFAs are mainly acquired from the host by blood-stage P. falciparum (Botte et al, 2013; Gulati et al, 2015), and our data suggested that FA uptake might thus be slightly impaired in PfCDPK7-KO parasites. We determined the FA content of PC and PE; analysis of FA from PC suggested a decrease in C16:0 and a slight increase in C18:1 upon PfCDPK7 disruption (Appendix Fig S2A). However, PE from PfCDPK7-KO contained slightly higher C16:0 FA (Appendix Fig S2B). In contrast, C18:1 and C18:2 FA were significantly reduced. The PE levels do not change significantly in PfCDPK7-KO; therefore, the changes in FA do not seem to alter PE levels. It is possible that the altered phosphorylation of enzymes like EK and PMT (Fig 1A and C and Appendix Fig S3) that are directly involved in PC biosynthesis (Fig 2C) may contribute to the impairment of lipid metabolism in PfCDPK7-KO parasites. Supplementation of P. falciparum culture medium with exogenous choline can overcome growth defects caused by perturbation in PC biosynthesis (Reynolds et al, 2008; Brancucci et al, 2017). We complemented both parental and PfCDPK7-KO parasite lines with excess choline in culture medium and monitored the parasite growth. A moderate but significant restoration of PfCDPK7-KO growth was observed suggesting that the loss of PC may impair parasite development in the absence of PfCDPK7 (Fig 2E). Taken together, our results suggest that PfCDPK7 is involved in regulating PC biosynthesis, which contributes to parasite development. PfCDPK7 regulates PfPMT and PfEK We next addressed the mechanisms through which PfCDPK7 regulates PC biosynthesis. Phosphoproteomic analysis revealed that PfCDPK7 knockout leads to reduced phosphorylation of PMT and EK (Fig 1A and Appendix Fig S3). First, we tested if PfCDPK7 interacts with these enzymes, by performing co-immunoprecipitation assays using parasite lysates, using a specific antisera raised against recombinant 6xHis-PMT. For studies related to EK, a parasite line overexpressing EK as a GFP fusion protein was generated and anti-GFP antibody was used to detect the EK-GFP fusion protein. PfCDPK7 co-immunoprecipitated with both PMT (Fig 3A) and EK (Fig 3B), suggesting that PfCDPK7 may interact with EK and PMT in the parasite. This corroborated well with the data showing that they are hypophosphorylated at specific sites in PfCDPK7-KO parasites (Appendix Fig S3A and B). Figure 3. PfCDPK7 interacts with PMT and EK in the parasite and regulates PMT protein expression A, B. 3D7 (A) or parasites overexpressing EK-GFP (B) were used to prepare protein lysates followed by immunoprecipitation using anti-PfCDPK7 or anti-GFP antibody, respectively. Subsequently, protein lysate or indicated IP were subjected to Western blotting using anti-PMT (A), anti-GFP (B). PMT was found to co-IP with PfCDPK7 (A, lane 2) and EK-GFP with PfCDPK7 (B, lane 3). C. 3D7 and PfCDPK7-KO parasites at schizont stage (~40 h) were used for Western blotting using anti-PMT antisera. Actin served as a loading control. Right panel: Densitometry was performed for bands corresponding to PMT in Western blots and the measurements were normalized to Actin. Fold change in PMT was determined with respect to 3D7 (mean ± SEM, n = 4; biological replicates, **P < 0.01, t-test). Source data are available online for this figure. Source Data for Figure 3 [embr202154022-sup-0003-SDataFig3.pdf] Download figure Download PowerPoint Interestingly, immunoblotting revealed that PMT protein levels (Fig 3C) were significantly lower in PfCDPK7-KO parasites. However, the levels of PMT transcript were almost unchanged (Appendix Fig S5), suggesting that PfCDPK7 may regulate PMT post-translationally, possibly by preventing its degradation. The aforementioned decrease in PMT phosphorylation in PfCDPK7-KO (Fig 1A) was particularly apparent on residue S49 (Appendix Fig S3A). IFA studies revealed that PfCDPK7 resides mainly in punctate structures (Appendix Fig S4A and B) as described previously (Kumar et al, 2014). Since PMT staining is spread throughout the parasite cytoplasm, it is difficult to determine if a pool of that protein co-localizes with PfCDPK7, although overlap can be observed (Appendix Fig S4B). EK-GFP was seen in the cytoplasm as well as in some punctate structures, which were often co-incidental with PfCDPK7 puncta (Appendix Fig S4A). Collectively, these data suggested that PfCDPK7 interacts with PMT and may participate in its regulation. Phosphoproteomics indicated that the phosphorylation of EK S37 is decreased in PfCDPK7-KO parasites (Appendix Fig S3B). To further analyze the role of S37 phosphorylation, we generated parasite lines overexpressing a phospho-deficient (S37A) mutant of EK. The comparison of growth rates of parasites episomally overexpressing WT or EK-S37A mutant revealed that there was a significant decrease in the growth of parasites expressing the phosphomutant (Fig 4A). While analyzing the Giemsa smears, we noted that several S37A mutant-expressing parasites exhibited abnormal morphology and most of these abnormal parasites were at the ring to trophozoite transition or early trophozoite stage (Fig 4B). To investigate this further, IFAs were performed using antibodies against MSP1 and RAP1 as these proteins reside on the parasitophorous vacuole membrane (PVM) and in parasitophorous vacuole (PV) post-invasion, respectively (Riglar et al, 2011). Typically, WT EK-GFP parasites attained “round” or “amoeboid” shape 3–12 h post-invasion with MSP1 and RAP1 in close proximity as described previously (Kumar et al, 2014). In contrast, a significant number of S37A mutant expressing parasites possessed “abnormal” shapes in which fragmented or discontinuous MSP1 staining was observed (Fig 4C and D). In addition, blobs and extensions containing RAP1 were found in the host RBC outside the PV in infected erythrocytes (Fig 4C). These observations corroborated well with stunted mutant parasites observed on Giemsa-stained smears (Fig 4B). In addition to these defects, there was a significant reduction in the number of merozoites per schizont, which suggested defects in parasite replication (Fig 4E). Figure 4. Phosphorylation of EK S37 is important for parasite development Parasites overexpressing EK-GFP or its S37A mutant were synchronized and parasitemia was determined at the end of first (48 h.p.i) and second cycle (96 h.p.i). Fold change in parasitemia of EK_S37A parasites with respect to WT EK overexpressing parasites was compared, which was significantly reduced (mean ± SEM, n = 3; biological replicates, *P < 0.05, ANOVA). Giemsa stained thin blood smears prepared at indicated hours post-infection (h.p.i) from assay. Arrows indicate parasites exhibiting abnormal morphology, which were observed at the beginning of each replication cycle. IFA was performed on parasites expressing WT EK-GFP or its S37A mutant 6–12 h post-invasion to detect (i) RAP1, which is transferred to PV post-invasion, and (ii) MSP1 which is located at the PVM. Parasite expressing WT EK-GFP exhibited typical round ring like shape. In contrast, several parasites expressing the S37A mutant exhibited irregular morphology with much smaller vacuolar space and in some parasites RAP1 was found outside the PV (Scale bar, 1 μm). Quantitation of abnormal parasites in IFA described in panel C (mean ± SEM, n = 3; biological replicates, ***P < 0.001, t-test). Parasites EK-GFP or its S37A mutant were synchronized and parasites were allowed to mature to schizonts/segmentors. The graph shows average number of merozoites per schizont from three biological
Referência(s)