Pf SRPK1 Regulates Asexual Blood Stage Schizogony and Is Essential for Male Gamete Formation

Abstract

Serine/arginine-rich protein kinases (SRPKs) are cell cycle-regulated serine/threonine protein kinases and are important regulators of splicing factors. In this study, we functionally characterize SRPK1 of the human malaria parasite Plasmodium falciparum. P. falciparum SRPK1 (PfSRPK1) was expressed in asexual blood-stage and sexual-stage gametocytes. Pfsrpk1^- parasites formed asexual schizonts that generated far fewer merozoites than wild-type parasites, causing reduced replication rates. Pfsrpk1^- parasites also showed a severe defect in the differentiation of male gametes, causing a complete block in parasite transmission to mosquitoes. RNA sequencing (RNA-seq) analysis of wild-type PfNF54 and Pfsrpk1^- stage V gametocytes suggested a role for PfSRPK1 in regulating transcript splicing and transcript abundance of genes coding for (i) microtubule/cilium morphogenesis-related proteins, (ii) proteins involved in cyclic nucleotide metabolic processes, (iii) proteins involved in signaling such as PfMAP2, (iv) lipid metabolism enzymes, (v) proteins of osmophilic bodies, and (vi) crystalloid components. Our study reveals an essential role for PfSRPK1 in parasite cell morphogenesis and suggests this kinase as a target to prevent malaria transmission from humans to mosquitoes. IMPORTANCE Plasmodium sexual stages represent a critical bottleneck in the parasite life cycle. Gametocytes taken up in an infectious blood meal by female anopheline mosquito get activated to form gametes and fuse to form short-lived zygotes, which transform into ookinetes to infect mosquitoes. In the present study, we demonstrate that PfSRPK1 is important for merozoite formation and critical for male gametogenesis and is involved in transcript homeostasis for numerous parasite genes. Targeting PfSRPK1 and its downstream pathways may reduce parasite replication and help achieve effective malaria transmission-blocking strategies.

Keywords: RNA-seq; SRPK1; exflagellation; gametocyte; mosquito; transmission.

FIG 1

Expression and localization of PfSRPK1 in asexual and sexual parasite stages. (a) Schematic for PfSRPK1 protein shows that its single kinase domain has a spacer region dividing it into kinase domains I and II. The peptide region used for antisera generation is indicated in green. (b) IFAs were performed on asexual stages (ring, trophozoite, schizont) using thin culture smears and anti-PfSRPK1 antisera (green) in combination with anti-PfCDPK4 antibodies (red). The parasite DNA was visualized with DAPI (blue). Scale bar, 5 μm. Images are shown from representative experiments. Merge I, merged image for red and green channels; merge II, merged image for red, green, and DAPI (blue) channels. (c) IFAs were performed on sexual stages (stage II to V gametocytes) and 10 min postactivation using smears and anti-PfSRPK1 antisera (green) in combination with anti-PfCDPK4 (red). The parasite DNA was visualized with DAPI (blue). (d) IFAs were performed on stage V gametocytes using anti-PfSRPK1 antisera (green) in combination with anti-Pfg377 antibodies (red; for female gametocytes). PfSRPK1 staining was negative for female gametocytes. The parasite DNA was visualized with DAPI (blue). Scale bar, 5 μm. Representative images are shown. Merge I, merged image for red and green panels; merge II, merged image for red, green, and DAPI (blue) channels. DIC, differential interference contrast; DAPI, 4′,6-diamidino-2-phenylindole.

FIG 2

Asexual blood-stage and sexual-stage phenotypes of Pfsrpk1⁻ parasites. (a) Percentage of parasitemia showing the increase in parasitemia in two subsequent generations of WT PfNF54 and Pfsrpk1⁻ (clones 1B4 and 2C8). Pfsrpk1⁻ parasites showed reduced growth compared to WT PfNF54. The means ± standard deviations (SDs) (error bars) of three biological replicates are shown. (b) Number of nuclei per schizont in WT PfNF54 and Pfsrpk1⁻ parasites (clones 1B4 and 2C8). (Duplicate experiments; n = 50 cells for each condition; bars are SDs). (c) IFAs were performed on mature schizont stages for WT PfNF54 and Pfsrpk1⁻ using thin culture smears with anti-PfMSP1 antisera, which labels the merozoite surface (red) in combination with anti-tubulin X antibodies, which would mark the subpellicular microtubules (green). Representative images are shown. The parasite DNA was visualized with DAPI (blue). Scale bar, 5 μm. Merge I, merged image for red and green panels; merge II, merged image for red, green, and DAPI (blue) channel. DIC, differential interference contrast; DAPI, 4′,6-diamidino-2-phenylindole. (d) Gametocytemia for WT PfNF54 and Pfsrpk1⁻ parasites (clones 1B4 and 2C8) was measured on day 15 using Giemsa-stained thin culture smears. Pfsrpk1⁻ gametocytes showed reduced gametocytemia compared to WT PfNF54. The means ± SDs (error bars) of three biological replicates are shown. (e) IFAs were performed on WT PfNF54 and Pfsrpk1⁻ mature stage V gametocytes culture thin smears using anti-PfP230p antisera, which labels the stage V male gametocytes (green) in combination with anti-Pfg377 antisera, which labels female gametocytes (red). Representative images are shown. The parasite DNA was visualized with DAPI (blue). Scale bar, 5 μm. Merge I, merged image for red and green panels; merge II, merged image for red, green, and DAPI (blue) channel. Symbols for male and female gametocytes are shown on the left sides of the image panels.

FIG 3

Pfsrpk1⁻ parasites do not form male gametes and cannot infect the mosquito vector. (a) Number of exflagellation centers (vigorous flagellar beating of emerging microgametes in clusters of RBCs) per field in 15 random fields of view at 15 min postactivation. Pfsrpk1⁻ (clones 1B4 and 2C8) gametocytes did not show any formation of exflagellation centers. Data were averaged from three biological replicates and are presented as the mean ± standard deviation (SD). (b) The cultures of mature stage V gametocytes were activated for 20 min in vitro by addition of human serum and RBCs for WT PfNF54 or Pfsrpk1⁻ parasites. The activated and nonactivated parasites were stained for α-tubulin (green), a male-specific marker. Note the male gamete emerging from an exflagellating male gametocyte in the WT PfNF54. No emerging microgametes were observed in activated Pfsrpk1⁻ gametocytes. DIC, differential interference contrast; DAPI, 4′,6-diamidino-2-phenylindole. Symbols for male gametocytes are shown on the left sides of the image panels. (c) The activated and nonactivated parasites were stained for PfUIS4 (red), a marker for parasitophorous vacuole, and anti-Pfs25 (green), a marker for female gametes in an IFA. α-Pfs25 staining (green) showed female gamete formation for WT PfNF54 and Pfsrpk1⁻. (d) WT PfNF54 and Pfsrpk1⁻ gametocytes were fed to A. stephensi mosquitoes, and the numbers of oocysts per mosquito midgut were enumerated on day 7 postfeed. The plot depicts the number of oocysts per mosquito fed from 3 independent experiments (n = 3). Data were averaged from three biological replicates with a minimum of 50 mosquito guts and presented as the mean ± standard deviation (SD). (e) Oocyst formation of WT PfNF54, Pfsrpk1⁻, Pfcdpk4⁻, Pfmacfet⁻, Pfsrpk1⁻ × Pfcdpk4⁻, Pf srpk1⁻ × Pfmacfet⁻, and Pfcdpk4⁻ × Pfmacfet⁻. In vitro genetic crosses demonstrated that the Pfsrpk1⁻ showed productive cross-fertilization with the Pfmacfet⁻ parasites (which produces functional males only) and not with Pfcdpk4⁻ (which produces functional females only) (error bar indicates mean ± SD; n = 2).

FIG 4

Disruption of PfSRPK1 results in extensive perturbation of transcript abundance. (a to c) Gene ontology terms for biological processes (a), cellular components (b), and molecular functions (c) of transcripts with reduced abundance are provided and highlight transcript perturbations for proteins involved in key biological processes that are impacted by PfSRPK1 deletion. Log₂ (P values) are indicated on the x axes for all the categories. (d to f) Biological processes (d), molecular functions (e), and cellular components (f) of transcripts with increased transcript abundance are provided and highlight transcript perturbations for proteins involved in key biological processes that are impacted by PfSRPK1 deletion. Log₂(P values) are indicated on the x axes for all the categories.

FIG 5

Deletion of PfSRPK1 results in perturbation of transcript abundance for genes encoding proteins involved in microtubule/cilium formation and signaling. (a) Heatmaps showing differentially expressed genes (DEGs) encoding microtubule/cilium formation proteins that are downregulated in Pfsrpk1⁻ gametocytes. (b) Heatmaps showing DEGs encoding gametocytogenesis-related genes that are downregulated in Pfsrpk1⁻ gametocytes. Scale bar indicates log₂ fold change of transcript-per-million (TPM) values of the samples in expression. (c) Heatmaps showing that DEGs encoding proteins involved in cell signaling and cyclic nucleotide metabolism are downregulated in Pfsrpk1⁻ gametocytes. Scale bar indicates log₂ fold change of TPM values of the samples in expression. (d) Western blot analysis of PfMAP2 in WT PfNF54 and Pfsrpk1⁻ gametocytes, showing reduced protein abundance for PfMAP2 in Pfsrpk1⁻ gametocytes. PfCDPK4 abundance is shown as the loading control. 1, 2, 3, and 4 represent lysates prepared from two independent experiments. 1 and 3, WT PfNF54; 2 and 4, Pfsrpk1⁻ gametocytes.

FIG 6

Disruption of PfSRPK1 results in perturbation of transcript abundance for genes encoding constituent proteins of the inner membrane complex (IMC), osmophilic bodies, and crystalloid. (a) Heatmaps showing DEG transcripts for IMC components that are downregulated in Pfsrpk1⁻ gametocytes. (b) Heatmaps showing DEG transcripts encoding osmophilic body components that are downregulated in Pfsrpk1⁻ gametocytes. Scale bar indicates log₂ fold change in expression. (c) Heatmaps showing that DEG transcripts encoding ookinete/crystalloid components are downregulated in Pfsrpk1⁻ gametocytes. Scale bar indicates log₂ fold change of TPM values of the samples in expression.

FIG 7

Model of the links between transcriptome perturbations in PfSRPK1-deficient parasites, (a)sexual-stage phenotype, and PfSRPK1 function. Possible upstream kinases PfCRK4-mediated phosphorylation likely regulate PfSRPK1 activity. PfSRPK1 phosphorylates SR proteins, which regulate mRNA splicing and thereby control expression of proteins involved in microgametogenesis and also splicing of mRNAs destined for storage and translation postfertilization. PfSRPK1 might also be involved in controlling gene expression by regulating the chromatin state and cell cycle by phosphorylating chromatin regulators. Annotated and predicted protein function was based on gene ontology terms and manual curation. An example of a protein in each category is given (red). Letter P inside circle denotes phosphorylation.