https://orcid.org/0000-0002-9064-586X
Figures
Abstract
Protein phosphorylation plays a critical role during the development of malaria parasites. Here, we performed a functional analysis of the Plasmodium berghei Ser/Thr protein phosphatase 6 (PbPP6), which is associated with the plasma membrane of macrogametes and ookinetes. Compared to wild-type P. berghei, the genetic disruption of pbpp6 (∆pbpp6) resulted in reduced asexual growth of the parasites and prolonged survival of infected mice. The ∆pbpp6 parasites showed impaired gametogenesis, particularly affecting male gametogenesis, which substantially decreased both ookinete formation and mosquito transmission. Transcriptomic analysis revealed an over 11-fold downregulation of nek3, a regulator of MAPK2 within the PKG-Ca2⁺ signaling cascade, foreshadowing pathway dysregulation that was further evidenced by significantly diminished intracellular cGMP levels, decreased cytosolic Ca2⁺ mobilization, and reduced DNA replication in activated Δpbpp6 gametocytes. Phosphoproteomic analysis detected increased phosphorylation at the Ser508 site of guanylyl cyclase alpha (GCα), indicating that PbPP6 regulates cGMP-PKG-Ca2+ signaling through modulation of GCα activity during gametogenesis. Additionally, we observed altered expression of messenger ribonucleoproteins in the Δpbpp6 parasites, which may affect the translational repression of stored mRNAs in female gametocytes and impact post-fertilization development in mosquitoes. Collectively, this study highlights the potential of targeting PP6 to disrupt malaria transmission.
Author summary
Malaria transmission begins when Plasmodium gametocytes, ingested by a female Anopheles mosquito, activate and differentiate into gametes in the midgut. Through genetic and multi-omics analyses, we identified the protein phosphatase PbPP6 as a key regulator of the cGMP-PKG-Ca2⁺ signaling cascade, specifically via the dephosphorylation of GCα. Disruption of the pbpp6 gene impaired male gametogenesis and affected post-fertilization development, likely due to dysregulated mRNP-mediated translational repression. Our findings demonstrate that genetic disruption of PbPP6 drastically reduced parasite transmission, highlighting protein phosphatases as potential targets for blocking malaria transmission.
Citation: Feng Y, Gao W, Wang C, Shi S, Zhou D, Sun L, et al. (2025) Loss of serine/threonine protein phosphatase 6 severely impairs sexual stage development in malaria parasite Plasmodium berghei. PLoS Pathog 21(7): e1013318. https://doi.org/10.1371/journal.ppat.1013318
Editor: Tobias Spielmann, Bernhard-Nocht-Institute for Tropical Medicine, GERMANY
Received: April 23, 2025; Accepted: June 19, 2025; Published: July 7, 2025
Copyright: © 2025 Feng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The raw RNA-seq data has been deposited in the GEO database (accession number: GSE271105). The MS/MS raw data and peptide details (sequence, unique peptides, spectral counts, sequence coverage, and Mascot scores) were deposited in the ProteomeXchange via the PRIDE database (proteomic accession number: PXD053412; phosphoproteomic asscession number: PXD063164).
Funding: This work was supported by the National Natural Science Foundation of China (Grants 82372280 and 81971961 to X.Z.) and the Department of Science & Technology of Liaoning Province (Grant 2022-MS-239 to X.Z.). L.C. was supported by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA (Grant U19AI089672). Y.C. was supported by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA (Grant R01 AI150533-01). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The World Health Organization has launched the “Global Technical Strategy for Malaria 2016 – 2030” to accelerate malaria elimination [1]. However, malaria chemotherapy, one of the technical pillars of malaria control and elimination, is challenged by the emergence of multidrug-resistant parasites. Of particular concern is the emergence and spread of Plasmodium falciparum parasites resistant to artemisinins and their partner drugs in Southeast Asia [2]. Thus, developing new antimalarial drugs is a high priority. In recognition of the significance of interrupting malaria transmission, it is desired that antimalarial candidates not only clear asexual parasitemia but also block parasite transmission. For drug discovery, a better understanding of parasite biology is critical for identifying novel drug targets [3].
Nearly half of the total proteins from humans, mouse and yeast are subject to phosphorylation, the most frequent post-translational modification [4]. Reversible phosphorylation, mediated by the opposing actions of kinases and phosphatases, also plays essential roles in malaria parasites, regulating cell division, propagation, stage conversion, and pathogenesis [5,6]. The P. falciparum genome encodes approximately 85 putative protein kinases and 30 protein phosphatases [7–9]. Compared with protein kinases, protein phosphatases in malaria parasites have received much less attention and deserve more detailed investigations, given their recognized potentials as therapeutic targets. The Plasmodium phosphatome is highly conserved among species [9]; thus, studies using the genetically more amenable rodent malaria parasite Plasmodium berghei may yield information that is directly relevant to the human malaria parasites. The Plasmodium protein phosphatases are divided into five families: 11 phosphoprotein phosphatases (PPPs), 10 metallo-protein phosphatases (PPMs), 4 protein tyrosine phosphatases (PTPs) and a PTP-like phosphatase, and 5 NLI-interacting factor-like phosphatases (NIFs) [8,9]. A systematic functional analysis of the P. berghei phosphatome revealed that 16 out of the 30 predicted protein phosphatases are essential for asexual stage development, while six are required for transmission to mosquitoes [9]. Although the study indicated that all major members of the PPP subfamily (PP1–PP8) are essential for asexual erythrocytic development [9], we found that PbPP5 is not required for this process but is crucial for male gamete formation and fertility [10]. Within the PPP subfamily, P. berghei protein phosphatase 6 (pbpp6, PbANKA_0412100) is also expressed in gametocytes and ookinetes [11], suggesting its potential involvement in sexual development. PP6 members are conserved among eukaryotes, and they are involved in intracellular signaling [12–14]. In Toxoplasma gondii (T. gondii), TgPP6C is essential for the parasite replication and its deletion could significantly attenuate the parasite virulence in mice [15]. In yeast, PP6 plays a non-redundant function in regulating cell cycle progression from G1 to S phase, which cannot be complemented by other closely-related protein phosphatases such as PP2A and PP4 [16,17].
In this study, we performed functional characterization of PbPP6 during the asexual erythrocytic cycle and sexual development of P. berghei. We found that PbPP6 was up-regulated during sexual development and located underneath the parasite plasma membrane at the gamete and ookinete stages. Functional analyses showed that pbpp6 deletion significantly impaired sexual development and infection of mosquito midguts. To understand the underlying mechanisms, we explored the effect of pbpp6 deletion on transcriptome, proteome and phosphoproteome in gametocytes.
Results
Validation of protein phosphatase activity and expression of the PbPP6 protein
PbPP6, encoded by the PbANKA_041210 gene, is a protein of 308 amino acids (aa) with a predicted molecular mass of 35.8 kDa. The phosphatase domain (PP2Ac, 21–292 aa) contains several identifiable motifs, such as the protein kinase C phosphorylation sites (TKK and SVK) and tyrosine kinase phosphorylation site (KYCTDIFDY) (S1A Fig). The PP2Ac domain shares significant homology to PP6-like proteins from different organisms, with six highly conserved core motifs ‘GDIHG’, ‘GDYVDRG’, ‘GNHE’, ‘HGG’, ‘RG’ and ‘H’ at the proposed catalytic site (S1B Fig). Although the sequence identify between PbPP6 and TgPP6C is only 24% when comparing the full legth protein sequence, the sequence identity of PP2Ac domain between these two protein is of 74% (S1B Fig). Furthermore, PP6 is highly conserved in Plasmodium spp., PbPP6 is 95.1% identical to its ortholog PfPP6 in P. falciparum. Phylogenetic analysis based on the full-length proteins of the PP6 orthologs revealed that the apicomplexan PP6 proteins are monophyletic (S1C Fig).
To confirm the prediction of PbPP6 as a PP2Ac phosphatase, we expressed the full-length PbPP6 in the Pichia expression system. With a His tag fused to the C-terminus of the protein, the recombinant PbPP6 protein (rPbPP6) was purified by using Ni-NTA chromatography. Analysis of rPbPP6 on an SDS-PAGE gel revealed a protein band of ~38 kDa, consistent with its predicted molecular weight (Fig 1A). Using the phosphorylated Ser/Thr PPase R110 as a substrate, the rPbPP6 exhibited obvious phosphatase activity, in stark contrast to the control recombinant His-tag protein (P < 0.001, Fig 1B). rPbPP6 exhibited high sensitivity to okadaic acid, with an IC50 (the concentration that inhibits 50% of enzymatic activity) as low as 0.86 nM (Fig 1C), which is lower than that of rPbPP5 [10].
A. Recombinant PbPP6 (rPbPP6) was expressed and purified, followed by SDS-PAGE analysis. The arrow indicates the expected ~38 kDa band. B. The phosphatase activity of rPbPP6 and a His-tagged Trx-A protein (control) was measured using a phosphorylated S/T PPase R110 substrate (-FLU, fluorescence light units). C. The dose-dependent inhibition of rPbPP6 by okadaic acid (OKA) was assessed. The error bars represent ±SD (biological triplicates). D. Western blot analysis of PbPP6 expression profile. The endogenous PbPP6 was detected using anti-PbPP6 sera (predicted band: 35.8 kDa). R, Ring; T, Trophozoite; S, Schizont; G, Gametocyte; O, Ookinete. Equal loading of samples was verified using mouse anti-Hsp70 sera. Arrows denote the expected bands. Representative data from three biological replicates are shown. E. The relative protein expression levels of PbPP6 (normalized to Hsp70) were quantified from (D) using ImageJ software.
To determine the expression and subcellular localization of PbPP6, we generated polyclonal antisera against PbPP6 by immunizing mice with rPbPP6. Since transcriptomic analysis showed pbpp6 expression during the life cycle of malaria parasites [18], we purified different parasite stages for analysis (S2 Fig). Western blots with protein extracts from purified rings, trophozoites, schizonts, gametocytes, and ookinetes using the anti-rPbPP6 antisera detected PbPP6 expression as a 36 kDa protein band in all stages analyzed (Fig 1D). During asexual intraerythrocytic development, the PbPP6 expression level was relatively low in the ring and trophozoite stages, but it increased by approximately 2-fold during the schizont stage. In contrast, PbPP6 was predominantly expressed in the gametocyte and ookinete stages (Fig 1D and 1E).
Localization and association of PbPP6 with the peripheral membrane in sexual stages
To determine the subcellular locations of the PbPP6 protein, we performed an indirect immunofluorescence assay (IFA) in wild-type (WT) P. berghei ANKA strain using antisera against rPbPP6 and antibodies against proteins associated with known cellular compartments. In the ring and trophozoite stages, PbPP6 fluorescence showed a diffused pattern with one or a few brighter puncta, largely overlapping with the cytoplasmic marker P. berghei glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Fig 2A). In schizonts, whereas the PbPP6 showed substantial co-localization with the merozoite surface protein 1 (MSP1), much of the fluorescence also emanated from the cell periphery suggesting a plasmalemma location pattern (Fig 2A). In both male and female gametocytes, the fluorescence was distributed throughout the cytoplasm with many prominent puncta. In microgametes, the fluorescence signal was associated with both the flagella and the residual body, co-localized with the α-tubulin II protein (Fig 2A). In macrogametes, zygotes and ookinetes, however, the PbPP6 signal pattern resembled both the plasma membrane (CDPK1) and the IMC marker (GAP40).
A-B. IFA analysis for location of endogenous PbPP6 (A) and PbPP6-GFP fusion protein (B) at various stages of Plasmodium berghei. Troph, Trophozoite; Sch, Schizont; MGc, Male gametocyte; FGc, Female gametocyte; MGa, Male gamete; FGa, Female gamete; Ook, Ookinete. (A) Endogenous PbPP6 was detected using Alexa Fluor 488 (Green). Phase contrast (DIC), DAPI-stained nuclei (blue), Alexa Fluor 594 channel (magenta; stage-specific markers), and merged images are presented. (B) PbPP6-GFP fluorescence (green) is shown alongside DIC and DAPI (blue). Scale bar = 5 μm. Note that different images were taken by non-consistence exposure time, which were not meant for quantitative comparison. C. IFA analysis assessing PbPP6 localization in gametes and ookinetes following fixation with (+) or without (-) treatment using 0.1% Triton X-100 (TX-100). Fixed Parasites were treated with (+)/(-) TX-100 before being stained with anti-GFP (detected via Alexa Fluor 488; green). After staining, all samples were universally permeabilized with TX-100 and stained with co-localization markers (CDPK1 or Pbs21; Alexa Fluor 594; magenta). The DAPI channel (blue) was used to visualize nucleus. Alexa488 and Alexa594 images were merged to show concordance. Scale bar = 5 μm. D. Subcellular fractionation of PbPP6-GFP. Parasite lysates were separated into soluble cytosolic (obtained through hypotonic lysis; Hypo) and peripheral membrane (sodium carbonate-extracted; Na₂CO₃) fractions. PbPP6-GFP (black arrow) was detected by immunoblotting using an anti-GFP antibody. Anti-GAPDH and anti-CDPK1 antibodies were used as controls for cytosolic and membrane-associated proteins, respectively.
To perform cross-validation, we engineered a transgenic line of P. berghei (designated PbPP6-GFP) in which the endogenous PbPP6 protein was tagged at the C-terminally with 2 × FKBP, followed by a green fluorescent protein (GFP) (S3A Fig). The correct integration of the tags at the pbpp6 locus was confirmed by diagnostic PCR (S3B Fig). Western blot analyses of protein extracts from mixed asexual stage parasites, using an anti-GFP monoclonal antibody (mAb), revealed a protein band of approximately 90 kDa, which aligns with the expected size of the PbPP6-GFP fusion protein (S3C Fig). The PbPP6-GFP parasites did not show any noticeable growth defects during the blood stage (S3D Fig) and showed no significant difference in virulence (S3E Fig), nor did they interfere with transmission through Anopheles stephensi mosquitoes (S3F and S3G Fig). IFA of the PbPP6-GFP line, using anti-GFP antibodies, revealed similar location patterns of PbPP6-GFP in various developmental stages as PbPP6 in WT parasites detected with the anti-rPbPP6 antisera (Fig 2B). Additionally, sporozoites dissected from the salivary glands of infected mosquitoes at 21 days post infection (dpi) displayed cytosolic GFP fluorescence (Fig 2B).
To further examine the association of PbPP6 with the plasma membrane in macrogametes and ookinetes, we conducted co-localization studies with and without treatment using Triton X-100 for membrane permeabilization. PbPP6-GFP was only detected following Triton X-100 treatment, suggesting that PbPP6 was located beneath the parasite plasma membrane of the parasite (Fig 2C). To determine whether PbPP6-GFP was directly associated with the cytoplasmic membrane in ookinetes, purified ookinetes were fractioned into the cytosolic and membrane-associated fractions using a hypotonic reagent and sodium carbonate, respectively. The proteins in both fractions were then analyzed using SDS-PAGE and western blotting. The results showed the presence of PbPP6-GFP only in the carbonate-soluble fraction (which contains peripheral membrane proteins), suggesting that PbPP6 is a membrane-associated protein in ookinetes (Fig 2D).
Perturbation of sexual development upon pbpp6 knockout
Previous functional studies of human and rodent malaria parasites suggested that pbpp6 gene is possibly essential [9]. To investigate the function of PbPP6, we attempted to knock out the pbpp6 gene by using both double and single crossover homologous recombination strategies. In both experiments, we were able to obtain parasite clones that had pbpp6 either completely deleted or partially disrupted (deleting aa 45–308), as verified by genotyping PCR, western blots, and IFA analysis (S4A–C Fig). Two pbpp6 knockout clones, Δpbpp6 K1 (K1) and Δpbpp6 K2 (K2), from two independent transfection attempts, were selected for further analysis. To determine whether pbpp6 deletion affected asexual parasite growth, mice were infected by injection of 106 parasitized red blood cells (RBCs) of the WT P. berghei, K1 and K2, respectively. The two Δpbpp6 clones showed indistinguishable growth curves, and the initial daily parasitemias were also similar between the Δpbpp6 and WT parasites (S4D Fig). However, from 6 dpi onwards, the daily parasitemias were significantly lower in mice infected with the Δpbpp6 parasites than in mice infected with the WT parasites (S4D Fig, P < 0.01, Student’s t-test). Specifically, the mean parasitemias at 6 and 14 dpi in the two Δpbpp6 groups reached 7.1% and 41.3%, respectively, compared to 8.1% and 45.8% in the WT group (S4D Fig). Correspondingly, mortality of Δpbpp6-infected mice was also delayed for about two days (S4E Fig, P < 0.01, Kaplan–Meier’s survival analysis).
Given the predominant expression of PbPP6 in gametocytes and ookinetes, we wanted to determine the impact of pbpp6 deletion on sexual development. We observed similar levels of gametocytemia and sex ratio at 3 dpi in WT and Δpbpp6 parasites, when gametocytemia normally peaks, indicating that pbpp6 knockout did not affect gametocytogenesis (Fig 3A and 3B, Student’s t test). However, the gametogenesis efficiencies of both male and female gametocytes were significantly affected. Compared to the WT, the proportion of macrogametocytes forming macrogametes was reduced by 8.3% and 17.4% in the K1 and K2 lines, respectively, whereas the proportion of microgametocytes forming exflagellation centers was reduced by 48.5% and 49.5% in the K1 and K2 lines, respectively (Fig 3C). We also scored the proportion of positive male-female gamete interactions, defined as male-female attachment lasting for more than 3 sec, during a period of 20 min from 10 min after induction for gametogenesis. The results revealed that the proportion of positive male-female interactions was reduced by 44.3% and 44.1% in Δpbpp6 K1 and K2, respectively (Fig 3D). We further determined the in vitro ookinete conversion rates of the WT and mutant lines. At 24 h of the ookinete culture, 20.3% and 19.5% of the Δpbpp6 macrogametocytes were converted into morphologically mature ookinetes in K1 and K2, respectively, significantly lower than the rate (92.1%) in the WT (Fig 3E, P < 0.001, Student’s t test). We then performed a time-course analysis to determine the step(s) in the zygote-ookinete development affected by the pbpp6 deletion. Compared with WT, the Δpbpp6 K1 parasite began to show a delay in the morphological transformation from 4 h post-fertilization (Fig 3F). At 16 h post-fertilization, 4.0% of WT parasites appeared as mature ookinetes compared to none in the Δpbpp6 K1 parasites. At 24 h, the majority (92.2%) of the WT parasites developed into mature ookinetes, which was in sharp contrast to 72.8% of the Δpbpp6 K1 parasites remaining in the “retort” stage (Fig 3F). These results indicated that PbPP6 had essential functions in gametogenesis, fertilization, and post-zygotic development of P. berghei.
A. Gametocytemia in WT and ∆pbpp6 parasites at day 3 post-infection (p.i.). B. Ratios of male to female gametocytes at day 3 p.i. C. Efficiency of macro- and microgamete formation. D. Interaction between male and female gametes. E. Ookinete conversion rates, measured as the percentage of Pbs21-positive parasites that developed into mature ookinetes, identified through anti-Pbs21 mAb staining. F. Developmental progression of Δpbpp6 and WT parasites during ookinete differentiation, with zygotes (cyan), retorts (dark grey), and mature ookinetes (light grey) quantified using fluorescence microscopy (anti-Pbs21 mAb). G. Ookinete conversion rates in genetic crosses between Δpbpp6 and female-defective (Δp47) or male-defective (Δp48/45) mutants. H. Oocyst counts per mosquito midgut at day 10 post-infection. For (A-H), data represent mean ± SD from three biological replicates unless otherwise noted. In (F), means were derived from two independent experiments (≥300 parasites scored per time point). Statistical significance versus WT is indicated as follows: * P < 0.05, ** P < 0.01, *** P < 0.001; for intergroup comparisons: n.s. (not significant), ## P < 0.01, ### P < 0.001. The samples sizes for mosquitoes were as follows: Experiment 1 (n = 85), Experiment 2 (n = 95), Experiment 3 (n = 120).
To investigate whether the defects in sexual development upon pbpp6 deletion were sex-specific, we conducted in vitro genetic crossover experiments by mixing equal numbers of mature gametocytes of Δpbpp6 K1 with either the male-defective (Δp48/45) or female-defective (Δp47) gametocytes and culturing them for 24 h. Whereas the cross between the Δp48/45 or Δp47 parasites restored ookinete conversion rate to the level of WT parasites, the cross between Δpbpp6 and Δp48/45 parasites did not rescue the ookinete conversion defects of the Δpbpp6 parasite line (Fig 3G). Although the cross between Δpbpp6 and Δp47 parasites could partially rescue the phenotype, the ookinete conversion defects is still 40.2% lower than the cross between Δp48/45 and Δp47 parasites, suggesting that both male and female gametes were impaired in the Δpbpp6 lines (Fig 3G).
In mosquito feeding assays, An. stephensi mosquitoes feeding on Δpbpp6-infected mice showed a 76.9% reduction in midgut oocyst intensity compared to those feeding on WT P. berghei-infected mice (Mann–Whitney U test, P < 0.001, Fig 3H and Table 1). In addition, the infection prevalence of mosquitoes feeding on Δpbpp6-infected mice (82.8%) was also significantly reduced as compared to mosquitoes feeding on WT-infected mice (97.4%) (Fisher’s exact test, P < 0.001, Table 1). Likewise, the sporozoite load in the infected salivary glands were also drastically reduced in mosquitoes feeding on Δpbpp6-infected mice (Table 1). To determine sporozoite viability, we purified sporozoites from the mosquito salivary glands at 20 dpi and injected them intravenously into BALB/c mice at 3000 sporozoites/mouse. Under these conditions, the Δpbpp6 and WT sporozoites displayed similar efficiencies in infecting mice; all injected mice developed blood-stage infections with similar pre-patent periods and parasitemia levels (Table 1). Together, these results showed that pbpp6 is essential for sexual stage development in mosquitoes.
With such a transmission reducing effect of pbpp6 deletion, we wondered whether inhibition of the PbPP6 enzyme activity would have a similar effect. We assessed the exflagellation and in vitro ookinete conversion of WT P. berghei in the presence of okadaic acid. At the final concentration of 1 × IC50 (0.86 nM) and 1.5 × IC50 (1.29 nM), okadaic acid dramatically inhibited exflagellation and ookinete conversion in a concentration-dependent manner (S5 Fig).
Integrated omics analysis of Δpbpp6 gametocytes
To elucidate the molecular mechanisms underlying the gametogenesis defect in the Δpbpp6 line, transcriptomic and proteomic analyses were performed on activated gametocytes (15 min post-activation). High reproducibility among biological replicates was confirmed by hierarchical clustering of RNA-seq read counts (S1A Table) and Pearson correlation coefficients of proteomic replicates (WT: r = 0.81–0.85; Δpbpp6: r = 0.85; S7A Fig and S2A Table).
Transcriptomic analysis identified 493 significantly altered transcripts (151 up-regulated, 342 down-regulated; adjusted P-value < 0.1, |log₂FC| > 1) out of 5,159 detected genes (S6A Fig and S1B–C Table). Down-regulated genes included those involved in gametogenesis and transmission, such as NEK3 [19–21], MDV1 [22], and LCCL proteins (LAP5, CCp1) [23], as well as AP2-Z, IMP4, and P25/28, all exhibiting fold changes greater than 4.8 (S6B Fig and S1C Table). GO enrichment analysis revealed that up-regulated transcripts were associated with protein homeostasis and ribosomal functions, whereas down-regulated transcripts were linked to cellular signaling processes (specifically cyclic nucleotide metabolic process and cyclic-nucleotide phosphodiesterase activity), motility, and cytoskeletal organization (S6C Fig and S1D Table). Reduced expression of phosphodiesterases (PDEα, γ, δ) (S1C Table) indicated disruption of the cGMP-PKG-Ca2⁺ signaling cascade in Δpbpp6 gametocytes, potentially explaining the observed developmental deficiency.
Proteomic analysis identified 1,896 P. berghei proteins, with 327 differentially expressed (162 up-regulated, 165 down-regulated; |log₂FC| > 0, P-adj < 0.1; S7B–C Fig and S2C Table). Up-regulated proteins included gamete egress regulators (MDV1, GEST, MiGS, MAPK2, G377, GEP) [24–26], while down-regulated proteins were implicated in endocytosis, DNA metabolism, phosphorylation, and inner membrane complex formation (KIC transporters, histones, CDPK6, IMC components) [7,27–29] (S7D Fig and S2C Table). Sex-specific analysis revealed 51.1% shared dysregulation, 8.3% (27/327) male-specific and 10.1% (33/327) female-specific dysregulation (S2C Table). Critically, significant alterations were observed for the female-specific transcription factors AP2-FG (0.8-fold downregulation) and PFG/FD2 (1.2-fold upregulation). GO analysis highlighted enrichment of up-regulated proteins in ribosome-associated processes and protein folding, while down-regulated proteins were predominantly membrane-associated (S7E Fig and S2D Table).
Comparative analysis of transcriptomic and proteomic revealed a moderate correlation between transcript and protein levels (Spearman’s r = 0.28, P = 2.2e-16; S8A Fig), with only 41.4% (1,300 genes) of transcripts (FPKM >20 in either the WT or Δpbpp6 across triplicates) corresponding to detectable proteins (S8B Fig). The remaining 58.6% (1,840 genes) included female-enriched, translationally repressed transcripts, such as pbs28, plasmepsin (pm)8/9, cpw-wpc family members, palmitoyl-S-acyltransferases proteins dhh3/10, rom3, and trap, representing 45.6% of the known Plasmodium TR transcripts [30,31] (S3A–B Table). Stabilization of these TR genes requires the mRNP complex in gametocytes [32]. Notably, 13 out of the 16 major subunits of the mRNP particle were identified in our proteomic analysis (the transcript-protein correlation: r = 0.05, P = 2.29e-11). With the exception of HoMu protein, all detected mRNP subunits were up-regulated upon pbpp6 deletion (S2B Table and S8C Fig), providing insight into the mechanisms of translational dysregulation.
Pbpp6 disruption impairs the cGMP-PKG-Ca2+ signaling pathway during gametogenesis
The cGMP-PKG-Ca2+ signaling cascade is vital for gametogenesis [33]. The deletion of the pbpp6 gene disrupts several components of this cascade, prompting us to investigate the role of PbPP6 in this signaling pathway. We compared intracellular cGMP concentrations in activated gametocytes from WT and Δpbpp6 lines. Treatment with XA, the phosphodiesterase (PDE) inhibitor zaprinast, or a pH of 8.0 significantly increased cGMP levels in WT gametocytes but not in the Δpbpp6 line, suggesting that PbPP6 is involved in cGMP synthesis (Fig 4A). Additionally, we observed a roughly 1.2-fold increase in the cytosolic Ca2+ signal in WT gametocytes approximately 5 seconds after XA stimulation. However, this response was absent in the Δpbpp6 line. This indicates that the deletion of pbpp6 impairs downstream Ca2+ mobilization during gametogenesis (Figs 4B and S9A). To further investigate whether the deletion of pbpp6 affects genome replication during male gametogenesis, we analyzed the DNA content in gametocytes after XA induction using flow cytometry. In XA-treated WT parasites, we noted an increase in fluorescence signal from 7.6% to 57.3% (Student’s t-test, P < 0.001; Figs 4C and S9B). In contrast, the Δpbpp6 line showed only a minor increase, from 6.6% to 12.4% (Student’s t-test, P < 0.01; Figs 4C and S9B). Collectively, our results demonstrated that parasites with disrupted PbPP6 cannot efficiently generate cGMP, leading to severe impairment in downstream Ca2+ signaling during gametogenesis (Fig 4D).
A. The cGMP levels in WT and Δpbpp6 gametocytes after stimulation with DMSO (control), 100 μM xanthurenic acid (XA), 100 μM zaprinast (Zap), or pH 8.0, measured 2 min post-treatment (n = 3). Data represent mean ± SD (three biological replicates). Statistical significance: ns (not significant), ** P < 0.01, *** P < 0.001 vs. respective controls; ### P < 0.001 for WT vs. Δpbpp6 (Student’s t-test). B. Cytosolic Ca2⁺ kinetics in Fluo-8-loaded gametocytes monitored by flow cytometry for 30 s before and 90 s after XA/DMSO treatment. Signals were normalized to baseline Ca2⁺ levels in DMSO controls. Black arrows indicate treatment timepoints. Representative data from three biological replicates are shown. C. Genomic DNA replication in XA-activated gametocytes (8 min post-treatment) assessed by Hoechst 33342 fluorescence. The data illustrate the proportion (left) and percentage of gametocytes with replicated DNA (right). Representative data from three biological replicates are shown. Statistical comparisons within the WT and Δpbpp6 groups indicated significance, with ** P < 0.01 and *** P < 0.001 for XA- vs. DMSO-treated groups (Student’s t-test). D. A hypothetical location of PbPP6 in the cGMP-PKG-Ca2+ signaling cascade during gametogenesis is illustrated.
Phosphoproteomic analysis of PbPP6-regulated signaling during gametogenesis
To identify the potential perturbation of protein phosphorylation upon pbpp6 deletion during gametogenesis, we conducted a quantitative phosphoproteome analysis of activated WT and Δpbpp6 gametocytes, focusing on samples taken 2 min post-activation. Equal amounts of protein extracts from both WT and Δpbpp6 gametocytes were subjected to trypsin digestion and phosphopeptide enrichment, and TMT-labeled peptides were quantified by LC-MS/MS (S4 Table). After normalization of the TMT-labeled proteomic data, 9935 unique peptides were identified, of which 6955 were phosphopeptides. These phosphopeptides contained 9490 phosphosites, of which 6374 were quantifiable, and mapped to 1717 proteins at site level with confidence > 0.75 [34] (S4A Table). By selecting log2FC higher or lower than 0 consistently in all biological replicates and average adjusted P-value < 0.1, we identified 50 up-regulated phosphosites on 46 proteins and 74 down-regulated phosphosites on 62 proteins, referred to herein as regulated sites (Fig 5A–B and S4A Table). GO analysis of the proteins with significantly up-regulated phosphosites revealed several biological process terms, including microtubule-based movement, cGMP metabolic process, and signal transduction. Additionally, we identified molecular function terms such as microtubule motor activity, cyclic nucleotide binding, and purine nucleotide binding (Fig 5C and S4B Table). Notably, the enriched GO terms for cyclic nucleotide metabolic process and cyclic-nucleotide phosphodiesterase activity included up-regulated phosphosites in proteins such as guanylyl cyclase alpha (with a ~ 39-fold increase at Ser508) and PDEα (with a ~ 16-fold increase at Ser494), which are involved in the synthesis and hydrolysis of cGMP during gametogenesis (Fig 5 and S4 Table). Overall, these findings indicate that the deletion of pbpp6 disrupts the phosphorylation status of proteins involved in cGMP synthesis and hydrolysis within the cGMP-PKG-Ca2+ signaling pathways during gametogenesis.
A. The volcano plot illustrates the significantly regulated phosphorylation sites (red, up-regulated phosphorylation sites; blue, down-regulated phosphorylation sites) upon PbPP6 deletion. Refer to S4A Table. B. Quantification of the numbers of regulated phosphosites and proteins in the Δpbpp6 strain compared to the WT strain. Refer to S4A Table. C. GO analysis of significantly up-regulated phosphorylation proteins in the Δpbpp6 strain compared to the WT strain. Related to S4B Table.
Discussion
We identified a critical role of a novel protein phosphatase, PbPP6, in sexual development of the malaria parasite P. berghei. It has been reported that the PbPP6 homologue in Toxoplasma gondii, TgPP6C regulates parasite replication and virulence [15]. Although the sequence identity of the PP2Ac domain between these two proteins is high, the low sequence identity of full-length protein between PbPP6 and TgPP6C implies that PbPP6 may function distinctly from TgPP6C, probably by interacting with different substrates. Our previous study in assessing the transmission blocking activity of 25 phosphatase inhibitors showed that two phosphatase inhibitors, BVT-948 and alexidine dihydrochloride, significantly inhibited sexual stage development from gametogenesis to ookinete maturation, indicating that phosphatases can be considered as promising targets for drug development [35]. A PP2A family inhibitor okadaic acid showed potent inhibition of the rPbPP6 enzyme and significant transmission-blocking activity, suggesting that Plasmodium PP6 is a promising target for disrupting malaria transmission. It is encouraging that two natural products, fostriecin and LB100, acting as highly selective inhibitors for PP2Ac, have entered phase I human clinical trials for anti-tumor treatment [36,37]. Further studies may alleviate the general toxicity of PPP family inhibitors to all eukaryotic cells and lead to the development of highly selective and specific inhibitors of PPPs targeting the parasite development.
Deletion of pbpp6 had a profound effect on gametogenesis, a key step within mosquitos that is essential for malaria parasite transmission. After a blood meal by mosquito, gametogenesis is triggered by temperature drop and the presence of xanthurenic acid (XA) in mosquito [38,39]. It has been well-established that cGMP-PKG-Ca2+ signaling cascade controls XA-triggered gametogenesis event in Plasmodium species [33,40–42]. In the present work we obtained clear evidence that PbPP6 disruption impairs cGMP-PKG-Ca2+ signaling pathway in male gametogenesis. Our global transcriptomic analysis also revealed that the down-regulated transcripts of Δpbpp6 mutant were enriched in GO terms related to signaling transduction (cGMP metabolic process and cyclic nucleotide metabolic process), and within these GO terms, we observed components of cGMP-PKG-Ca2+ signaling cascade, including PDEα, PDEγ, and PDEδ. Of these, PDEδ is predominantly expressed in gametocyte stages and has a specific cGMP hydrolytic activity [43]. Consistent with the gametogenesis deficiency observed in Δpbpp6 mutants, previous reports showed that the activation of male gametocytes is dramatically reduced in PDEδ– mutant [43]. Once the cGMP-PKG-Ca2+ signaling cascade is activated, the differentiation of male gametocytes into gametes results in DNA replication, followed by parasitophorous vacuole membrane (PVM) rupture and gametes egress from the host cell [44]. Our proteomic analysis revealed a compensatory upregulation of egress-related effectors in Δpbpp6 parasites, including MDV1 and GEST, which are responsible for mediating PVM disruption [22,45]; G377 and MiGS, which are essential for biogenesis of osmiophilic bodies and gamete egress [45–47]; and GEP, which plays a role in executing PKG-dependent egress steps [25,48]. This coordinated proteomic shift suggests a compensatory mechanism due to impaired PKG-Ca2⁺ signaling activity. A critical step in the release of male gametes is the activation of a male-specific protein kinase known as MAPK2, which acts subsequent to CDPK4 during cytokinesis, chromatin condensation, and axoneme motility in male gametogenesis [20,49]. MAPK2 activation is known to be modulated by Nek3 kinase [19–21], whose transcript levels were significantly reduced in Δpbpp6 parasites according to our RNA-seq analysis. While the direct transcriptional regulation of PKG-Ca2⁺ signaling components by PbPP6 remains unverified, both Nek3 and MAPK2 have been established as downstream effectors of PKG-mediated Ca2⁺ mobilization during gametogenesis and ookinete gliding [40,50]. Therefore, the dyregulation of these proteins in Δpbpp6 parasites likely reflects secondary effects of compromised cGMP-PKG-Ca2⁺ signaling.
Interestingly, we found that the phosphorylation levels of both the Ser508 site in GCα and the Ser494 site in PDEα were significantly increased in Δpbpp6 gametocytes just 2 minutes after activation. The levels of cGMP, which are crucial for initiating gametogenesis, are tightly controlled by GCs and PDEs. A previous study found that PDEα and PDEδ do not directly raise cGMP levels in response to XA or pH changes. Instead, they play a role in preventing the premature activation of gametocytes in the vertebrate host at 37°C [43]. In contrast, GCα is the primary enzyme that synthesizes cGMP in response to XA or elevated extracellular pH [43]. Previous research has shown that phosphorylation plays a key role in regulating the activity of soluble GCs [51,52]. Meanwhile, a recent research on P. falciparum shows that PP1 regulates the activity of GCα during merozoite egress [53], indicating that protein phosphatases play a role in the regulation of cGMP-PKG-Ca2+ signaling pathways in malaria parasites. Therefore, we hypothesized that the knockout of PbPP6 leads to hyperphosphorylation and functional deregulation of GCα, ultimately resulting in decreased cGMP production. The observed decrease in calcium mobilization in the Δpbpp6 line is likely a secondary effect rather than a direct consequence of PbPP6 disruption. We propose that the use of a cGMP analog could help bypass this blockage and allow us to further test our hypothesis in future studies. As GCα functions as a membrane-anchoring protein, PbPP6 may associate with the plasma membrane through a transient or indirect interaction with GCα during gametogenesis. However, biochemical validation through co-immunoprecipitation and membrane fractionation remains essential to identify PbPP6 interactors. Collectively, these findings highlight the role of the serine/threonine phosphatase PbPP6 in regulating cGMP-PKG-Ca2+ signaling pathways during gametogenesis.
The ApiAp2 family contains the APETALA-2 (AP2) DNA binding domain that recognizes multiple and distinct palindromic DNA sequences, which accounts for tighter regulation of many stage-specific gene expressions [54]. It has been reported that two female-specific transcription factors (TFs), AP2-FG and PFG (FD2) form a complex and regulate a broad repertoire of target genes, which are involved in fertilization, meiosis, and ookinete development, such as AP2-Z that regulates zygote development [54–57]. Disruption of either of these two TFs could impair female gametocyte development, and produce abnormal morphology and reduced number of ookinetes [55,56]. In our proteomic analysis, we observed dysregulated expression of both two TFs. As genes under the control of these two TFs partially overlapped, the down-regulated expression of AP2-FG may be responsible for the female gametogenesis deficiency observed in the pbpp6 deletion parasites, while the upregulated expression of PFG maybe a compensatory mechanism for parasite to complete its sexual stage development. However, the relatively modest (~20% variation) expression shifts may also reflect biological variation or secondary effects of impaired gametogenesis. To determine whether TF dysregulation directly drives the gametogenesis deficiency in PP6-deficient parasites, further studies, including complementation assays and mechanistic investigations, will be necessary in future analysis.
In addition to the functions of PbPP6 during gametogenesis, pbpp6 deletion also affected post-fertilization ookinete development. Consistently, several genes including kinesin-13, -20 and pbs25/28 that are involved in zygote/ookinete development, midgut epithelium traversal and oocyst development [58,59], were also down-regulated upon pbpp6 deletion. In gametocytes, we observed highly abundant transcripts without proteome evidence, which is consistent with the presence of a large set of TR transcripts in female gametocytes of both P. falciparum and P. berghei [30,31]. Some of the down-regulated putative TR transcripts (e.g., pbs28, dhhc and cpw-wpc family members) have been shown to be essential for zygote/ookinete development [59–61]. In addition, TR transcripts include genes (e.g., pm8/9, rom3 and trap), whose disruption results in a phenotypic effect on oocyst and/or sporozoites development [62–64]. CDPK1 was shown to be important for translation derepression during ookinete development [65]. Although not significant, we found down-regulated expression of CDPK1 in activated gametocyte of pbpp6 deletion parasites, suggesting the translational regulation of sexual stage-specific mRNA expression may also be affected. Translational repression is a major and conserved mechanism in post-fertilization development, and the storage of these TR transcripts in female gametocytes is known to require mRNP complex, whose two core components DOZI and CITH play an essential roles in ookinete motility [32]. We have observed disturbance of the mRNP complex upon pbpp6 deletion, which includes upregulation of 12 out of 13 detected mRNP subunits and down-regulation of HoMu [31]. In neural stem cells, HoMu inhibits translation initiation by competing with eIF4G for poly(A) binding protein (PABP)-binding [66]. Therefore, there is a possibility that the overall disturbance of the mRNP complex and reduced expression of HoMu could affect mRNP function in female gametocytes, leading to post-fertilization development deficiency in the mosquito. Alternatively, the apparent upregulation of mRNP proteins in the Δpbpp6 strain may simply be a downstream effect of incomplete gametogenesis. In WT strain, these proteins are likely rapidly degraded upon activation, which releases translational repression. However, in the Δpbpp6 strain, this degradation might not occur due to the incomplete gametogenesis, leading to an apparent increase in protein levels when comparing the knockout (KO) and WT strains, even if the actual levels haven’t changed. Additionally, deletion of pbpp6 resulted in reduced mosquito infection rates and oocyst densities. The reduced oocyst numbers are consistent with the observed defect in ookinete conversion and likely represent a follow-up phenotype to this earlier impairment.
Conclusion
Our results identified PbPP6 as a crucial factor in controlling several essential biological steps in the life cycle of malaria parasites. Although the precise function of PbPP6 in sexual stage development remains to be solved and the molecular targets of PbPP6 need to be verified, it is tempting to speculate that PbPP6 plays a critical function through regulating the function of GCα in male gametogenesis and possibly midgut invasion of ookinetes. Meanwhile, by altering the translation profile of mRNP, PbPP6 may play a role in regulating female gametogenesis and the following post-fertilization development in the mosquito. It is now widely accepted that drugs targeting both asexual and sexual stages are highly desirable for malaria control [67]. Although deletion of PbPP6 has only a mild effect on asexual blood-stage growth, it severely impairs parasite transmission in the mosquito. Therefore, targeting PbPP6 activity holds promise primarily as a strategy to block parasite transmission. Further elucidation of PbPP6’s involvement in signaling pathways could lead to novel transmission-blocking strategies, and the determination of substrates of PbPP6 is therefore a logical next step.
Materials and methods
Ethics statement
All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of China Medical University (Approval No. CMU2022189) and performed in compliance with institutional ethical guidelines for animal research.
Parasite and mosquito maintenance
The Plasmodium berghei ANKA parasites were propagated in female Kunming or BALB/c mice (BEIJING HFK BIOSCIENCE Co., Ltd., China), aged 6–8 weeks [68]. Colonies of Anopheles stephensi were maintained under controlled conditions: 25°C, 50–80% relative humidity, and a 12-hour photoperiod 12-hour light/dark cycle. Adult female mosquitoes were provided with 10% (w/v) glucose solution [68].
Sequence analysis
The PP6 sequences in P. falciparum (PfPP6, PF3D7_0314400) and P. berghei (PbPP6, PbANKA_041210) were retrieved from PlasmoDB (plasmodb.org); PP6 sequence in Toxoplasma gondii (TgPP6, TGARI_301010) was retrieved from ToxoDB (toxodb.org); PP6 sequence in Cryptosporidium parvum (CpPP6, GY17_00001243-t36_1) was retrieved from CryptoDB (cryptodb.org); and PP6 homologs in Homo sapiens PP6C (HsPP6C, NP_002712.1), Drosophila melanogaster PPV (DmPPV, NP_511061.1), Saccharomyces cerevisiae SIT4 (ScSIT4, NP_010236.1) and Schizosaccharomyces pombe Ppe1 (SpPpe1, NP_588420.1) were retrieved from GenBank. The conserved domains and motifs of PbPP6 were analyzed using the SMART software (http://smart.embl-heidelberg.de/) and the Motif Scan software (https://myhits.isb-sib.ch/cgi-bin/motif_scan) [69]. Sequence alignment was performed with MUSCLE software (https://www.ebi.ac.uk/Tools/msa/muscle/). The BioEdit Sequence Alignment Editor was used for visualization of the alignment. For the phylogenetic analysis of PP6 sequences, the Neighbor-Joining method in MEGA 7.0 was employed and modified using Eolview v3 [70,71].
Recombinant protein expression and antisera production
The complete open reading frame (ORF) of PbPP6, optimized for expression in the Pichia expression system, was expressed in the GS115 his4 strain (Genecreate, Wuhan, China). The recombinant PbPP6 protein (rPbPP6) was purified using HisPur Ni-NTA (ThermoFisher, MA, USA). SDS-PAGE was performed on the protein samples to assess their purity, and these samples were subsequently used for immunization in mice according to a standard immunization protocol [68]. A control recombinant Trx-A protein with 6 × His tag that was present on the pET-32a (+) plasmid was expressed and purified as previously described [10]. Rabbit anti-PbCDPK1 sera were custom-made against recombinant PbCDPK1 protein (amino acid position: 260 – 523) by Genecreat (Wuhan, China).
PbPP6 phosphatase assay
The recombinant PbPP6 (rPbPP6) phosphatase activity was measured using the ProFluor Ser/Thr PPase Assay kit (Promega, WI, USA). rPbPP6 and a His-tagged control protein were diluted to a concentration of 30 nM and mixed with the Peptide Solution (containing 5 × Reaction Buffer B [200 mM Tris-HCl, pH 7.5, 0.5 mg/ml BSA] and S/T PPase R110 Substrate [10 mM]) for 15 seconds, followed by incubation at room temperature (RT) for 10 minutes. Protease Solution was subsequently added, and the mixture was incubated at RT for an additional 90 minutes. Following the addition of Stabilizer Solution, fluorescence was measured using a Biomek 2000 workstation (Beckman Coulter, CA, USA) with an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Inhibition assays using okadaic acid were conducted as described [10].
Western blot and immunofluorescence assays
Parasites at various developmental stages (ring, trophozoite, schizont, gametocyte, and ookinete) were purified as described [72]. Briefly, mice pretreated with phenylhydrazine (6 mg/ml) were intravenously injected with 1 × 108 parasites. At 3 days post-infection (d.p.i.), sulfadiazine (20 mg/ml, Sigma, MO, USA) was administered in drinking water for 48 hr to eliminate asexual-stage parasites. Gametocytes were then isolated through cardiac puncture and separated from uninfected erythrocytes using a 48% NycoDenz gradient. Schizonts and ookinetes were obtained by culturing the infected blood at 37°C overnight and at 20°C for 24 hr, respectively, followed by purification on 55% or 62% NycoDenz gradients. For the ring and trophozoite stages, purified schizonts were injected intravenously into naïve mice, and the parasites were collected at 4 hr (for rings) or 12 hr (for trophozoites) post-invasion after two replication cycles.
For protein extraction, purified parasites were lysed using 0.15% saponin (Sigma) in PBS. After centrifugation at 13,000 × g for 5 min, the samples were washed with PBS. Proteins were extracted using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher) and quantified with the Pierce BCA Protein Assay Kit (Thermo Fisher), followed by resolution via 10% SDS-PAGE. Western blotting was performed as described [10] using mouse anti-PbPP6 sera, anti-GFP mAb (Abcam, Cambridge, UK), or anti-Hsp70 mAb (Abcam), followed by HRP-conjugated goat anti-mouse IgG (H + L) antibodies (Thermo Fisher). Signal detection was accomplished using SuperSignal West Pico PLUS (Thermo Fisher) on a Tanon 4200 imaging system (Tanon, Shanghai, China).
For the solubility analysis, ookinetes from PbPP6-GFP parasites were lysed in 5 mM Tris-HCl (pH 8.0) and then frozen at −80°C [73]. After thawing, soluble proteins were collected through centrifugation at 16,000 × g for 10 min. The resulting pellet was resuspended in 0.1 M Na2CO3 and incubated on ice for 30 min to extract peripheral membrane proteins. The proteins were then resolved by SDS-PAGE and probed with anti-GFP mAb, anti-GAPDH (Abcam), or anti-CDPK1 serum.
For immunofluorescence assays (IFA), both WT and PbPP6-GFP parasites were subjected to fixation utilizing a solution composed of 4% paraformaldehyde (PFA) and 0.0075% glutaraldehyde in PBS. Following fixation, the samples were permeabilized with 0.1% Triton X-100 for 5 min on ice, and subsequently neutralized with NaBH4 at a concentration of 0.1 mg/ml. After blocking with 5% non-fat milk, the samples were incubated with a series of primary antibodies: mouse anti-PbPP6 (1:500), rabbit anti-GAPDH, rabbit anti-MSP1 (1:500), rabbit anti-CDPK1 (1:500), rabbit anti-Pbg377 (female marker, 1:500), rabbit anti-α-tubulin II (male marker, 1:500), mouse anti-GAP40 (glideosome marker, 1:500), rabbit anti-GFP mAb (Abcam), and mouse anti-Pbs21 (ookinete marker, 1:500). Secondary antibodies conjugated to Alexa Fluor 488 and 594 (1:500, Thermo Fisher) were subsequently employed. The samples were then mounted with ProLong Diamond Antifade Mountant containing DAPI for nuclear counterstaining. Imaging was conducted using a Nikon C2 confocal microscope, and data analysis was performed using ImageJ
Generation of transgenic parasites
The pbpp6 gene in P. berghei parasites was disrupted in two independent experiments using either a double- or single-crossover homologous recombination strategy [74,75]. For the double-crossover approach, a 1460 bp fragment of the 5′ untranslated homologue recombinant region (5R) (-1502 – -71 bp) and an 1157 bp fragment from the 3R (33 bp – 1162 bp) of the pbpp6 gene were amplified using the parasite genomic DNA as a template (primers are listed in S5 Table), and cloned into the HindIII/PstI and XhoI/NotI sites of the pL0034 vector, which contains the hdhfr selection cassette, resulting in the creation of the pL0034-PbPP6KO plasmid. For the single-crossover approach, amined at disrupting and C-terminally tagging PbPP6 with a 2 × FKBP followed by GFP (referred to as dPbPP6), a 408 bp fragment (25–432 bp) from the pbpp6 open reading frame (ORF) and an 898 bp fragment (26–924 bp) were amplified and cloned into the NotI/AvrII sites of pSLI-2 × FKBP-GFP vector [75] to generate the pSLI-dPbPP6–2 × FKBP-GFP and pSLI-PbPP6tag-2 × FKBP-GFP plasmids, respectively.
Transfection, selection and cloning of transgenic parasites were performed using genetic modification technology developed for P. berghei [74,76]. Resistant parasites were selected by 70 mg/L pyrimethamine (Sigma) supplied in the drinking water for 7 days. For pbpp6 gene disruption and C-terminal tagging with GFP, resistant parasites selected by pyrimethamine were adjusted to 1% parasitemia and further selected by intraperitoneal injection of G418 (Sigma) at 40 mg/kg. Successful integration of the plasmids was confirmed by genotyping PCR using primers in S5 Table.
Phenotype analysis
The phenotypic characterization of transgenic parasite lines (Δpbpp6 K1, Δpbpp6 K2, PbPP6-GFP C1) and WT-infected RBCs (iRBCs) was injected into 6–8-week-old BALB/c mice. Daily parasitemia was monitored by Giemsa-stained tail blood smears, with concurrent documentation of mouse survival rates. At 3 dpi, gametocytemia was quantified by enumerating mature gametocytes per 1,000 RBCs, and the male-to-female gametocyte ratio was determined using Giemsa-stained smears.
To evaluate gamete formation rates, blood samples were activated with a gametocyte activation buffer (GAB, RPMI 1640, penicillin, streptomycin, 20% fetal calf serum, heparin, and 100 μM XA, pH 7.4) for 30 min at 25°C, followed by sex-specific staining (anti-α-tubulin II/Ter-119 for male gametes and anti-Pbg377/Ter-119 for female gametes). Gametes were considered to have egressed/not-egressed if they were negative/positive for Ter119 staining. Exflagellation centers and gamete interactions were quantified as described [10].
The ookinete conversion was assessed in vitro using Pbs21 surface marker staining [72,77]. The mosquito transmission experiments were performed by feeding 30–60 An. stephensi per replicate on infected mice, with oocyst counts at 10 days post-feeding [78]. Gamete fertility was evaluated through crosses with Δp47 (female-defective) or Δp48/45 (male-defective) parasite lines [10]. For the sporogonic development analysis, 3,000 salivary gland sporozoites (from either WT or Δpbpp6 strains) were injected intravenously into BALB/c mice at a dosage of 100 μL, with parasitemia monitored via Giemsa-stained blood smears from day 4 post-injection. The exflagellation and ookinete conversion inhibition assays were performed as described [10]. Each assay included three biological replicates to ensure valid and reliable results.
Flow cytometry assay
Cytosolic Ca2⁺ mobilization was assessed as described [25,79]. Purified WT and Δpbpp6 gametocytes were incubated with 5 μM Fluo-8 (ATT Bioquest, CA, USA) in calcium-free buffer (CFB: 137 mM NaCl, 4 mM KCl, 20 mM glucose, 20 mM HEPES, 4 mM NaHCO₃ [pH 7.2-7.3], 0.1% BSA) for 20 min at 37°C. Following three washes with CFB, baseline fluorescence was recorded for 30 sec on a FACS Celesta flow cytometer. Subsequently, the samples were stimulated with 100 μM xanthurenic acid (XA, Sigma) and fluorescence measurements were taken for an additional 90 sec. The data were analyzed using FlowJo 10 software (BD Pharmingen, NJ, USA) to ensure precise quantification of cytosolic Ca2⁺ dynamics.
For DNA content analysis, gametocytes were either subjected to immediate fixation with 4% PFA or were activated in a GAB buffer for 8 min at 25°C prior to fixation. The fixed samples were stained with Hoechst 33342 for 30 min at 37°C and were subsequently analyzed using a FACS Celesta flow cytometer with FlowJo v10.8.1 software (BD Pharmingen).
Measurement of cellular cGMP levels
The intracellular concentrations of cGMP in WT and Δpbpp6 gametocytes were measured following stimulation with XA as described [79]. Briefly, 2 × 107 WT and Δpbpp6 gametocytes were purified and incubated for 2 min in gametocyte maintenance buffer (GMB: 137 mM NaCl, 4 mM KCl, 1 mM CaCl2, 20 mM glucose, 20 mM HEPES, 4 mM NaHCO3, 0.1% BSA, pH 7.24–7.29) supplemented with 100 μM XA, 100 μM Zap, pH 8.0, or DMSO as a vehicle control. Subsequent lysis was performed using ice-cold 0.2 M HCl for 10 min, followed by mechanical disruption through sequential passages in a 27-gauge needle. The cGMP levels were determined using a cGMP ELISA kit (Cayman Chemical, MI, USA), following the acetylated protocol.
Transcriptome analysis of gametocytes
Total RNA was extracted from activated gametocytes (25°C for 15 min) of both Δpbpp6 and WT parasites using the Qiagen RNeasy kit (Qiagen, Dusseldorf, Germany). The quantity, purity and integrity of the RNA were assessed through 1.5% agarose gel electrophoresis and analyzed with a Nanodrop OneC spectrophotometer (ThermoFisher). Stranded mRNA-seq libraries were prepared from 2 μg of total RNA per sample using the KC-Digital Stranded mRNA Library Prep Kit (Wuhan Seqhealth Co., Hubei, China) [80]. RNA-seq was conducted using three biological replicates of both the Δpbpp6 and WT parasites. The library products were sequenced on a Hiseq X 10 sequencer (Illumina, San Diego, USA).
Raw sequencing data were filtered using Trimmomatic (v0.36) and processed with in-house scripts to eliminate duplication bias introduced during library preparation and sequencing. The RNA-seq reads from each sample were aligned to the PlasmoDB-67 reference genome of the P. berghei ANKA using the STAR RNA-seq alignment tool [81]. The HTSeq tool (v.0.6.1) was employed to count the transcripts for each gene [82]. Normalization was performed using Cuffdiff (v2.1) [83], and differential expression analysis was conducted with DESeq2 (v.1.28.1) in R (v.4.04) [84]. Genes with an false discovery rate (FDR) below 0.1 and a minimal log2Fold change (log2FC) of 1 were considered significantly differentially expressed. Clustering and PCA were carried out in R to estimate sampling distribution for each experiment. GO analysis for differentially expressed genes was performed in PlasmoDB [85], with an adjusted P-value threshold of less than 0.1 to determine statistically significant enrichment. The raw RNA-seq data has been deposited in the GEO database (accession number: GSE271105).
Quantitative proteomic and phosphoproteomic analyses
Proteins from purified activated gametocytes (15 minutes post-activation) were extracted by sonicating in 8 M urea, which was followed by reduction with 5 mM DTT at 56 °C and alkylation with 11 mM iodoacetamide at room temperature. The sample then underwent overnight digestion with trypsin, followed by desalting and drying. The reconstituted samples in 0.5 M TEAB were incubated with TMT reagent for 2 hours, then desalting and drying were repeated. The samples were separated into 60 fractions using an Agilent 300 Extend C18 column with an acetonitrile gradient. Finally, the fractions were combined into 18 and dried using a SpeedVac concentrator.
For phosphoproteomic analysis, we collected activated gametocyte samples (2 min post-activation). The peptide mixtures were incubated at 37°C for 1 h with Ti-IMAC microsphere (ReSyn Biosciences, Beijing, China) to enrich for phosphopeptides. After centrifuging at 12,000 g for 5 min, we washed the microspheres sequentially with 50% acetonitrile/6% TFA and 30% acetonitrile/0.1% TFA to remove non-specific binding. The enriched phosphopeptides were eluated with an elution buffer containing 10% NH4OH and then lyophilized.
For LC-MS/MS analysis, peptide fractions were dissolved in solvent A (0.1% formic acid in 2% acetonitrile) and directly loaded onto a reversed-phase analytical column (15 cm in length and 75 μm in diameter). LC was performed using the EASY-nLC 1000 Ultra-Performance Liquid Chromatography (UPLC) system (ThermoFisher). The peptides were subjected to nanoelectrospray ionization, followed by MS/MS in a Q Exactive Plus mass spectrometer (ThermoFisher), which was coupled online with the UPLC. The resulting MS/MS data were processed using Maxquant search engine (v.1.5.2.8) and searched against the PlasmoDB-67 reference database for P. berghei, using Trypsin/P cleavage with up to 2 missed cleavages. The mass tolerances were set to 20 ppm for initial and 5 ppm for main searches, with a 0.02 Da tolerance for fragment ions. The FDR was adjusted to less than 1%, and peptide scores were > 40. The MS/MS raw data and peptide details (sequence, unique peptides, spectral counts, sequence coverage, and Mascot scores) were deposited in the ProteomeXchange via the PRIDE database (proteomic accession number: PXD053412; phosphoproteomic asscession number: PXD063164).
Statistical analyses
For the phenotype analysis of the Δpbpp6 parasite line, we conducted statistical analyses using GraphPad Prism. We performed a two-tailed, unpaired Student’s t-test to compare various metrics, including parasitemia, exflagellation, gametocyte activation, male-female gamete interactions, and ookinete conversion. To assess survival differences in infected mice, we applied Kaplan–Meier’s survival analysis. Oocyst intensity was analyzed using the Mann–Whitney U test, while infection prevalence was evaluated with Fisher’s exact test.
Supporting information
S1 Fig. Sequence analysis of PbPP6 protein.
A. Schematic representation highlighting the phosphatase domain (PP2Ac) within the PbPP6 protein. B. Multiple sequence alignment of conserved PP2Ac domains across PP6 orthologs, performed using CLUSTALW with manual refinement. Included species: Plasmodium berghei PP6 (PbPP6 [Pb], PbANKA_0412100), Plasmodium falciparum PP6 (PfPP6 [Pf], PF3D7_0314400), Toxoplasma gondii PP6 (TgPP6 [Tg], TGARI_301010), Cryptosporidium parvum PP6 (CpPP6 [Cp], GY17_00001243), Homo sapiens PP6C (HsPP6C [Hs], NP_002712.1), Caenorhabditis elegans PPh6 (CePPh6 [Ce], NP_497714.2), Drosophila melanoqaster PPV (DmPPV [Dm], NP_511061.1), Saccharomyces cerevisiae SIT4 (ScSIT4 [Sc], NP_010236.1), Schizosaccharomyces pombe Ppe1 (SpPpe1 [Sp], NP_588420.1). The conserved catalytic motifs and active-site residues are marked with boxed. C. Phylogenetic reconstruction of PP6 orthologs was performed using the neighbor-joining method with 1,000 bootstrap replicates.
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S2 Fig. Purified rings, trophozoites, schizonts, gametocytes, and ookinetes.
P. berghei schizonts were cultured and enriched using a 55% Nycodenz cushion (v/v). The purified schizonts were injected intravenously into naïve mice. After 48 h (equivalent to two cell cycles), the infected mice were bled by cardiac puncture at 4 h and 12 h (the third cycle) to collect the ring and trophozoite stages, respectively. Gametocytes were harvested from sulfadiazine-treated mice and enriched on a 48% Nycodenz cushion (v/v) at 37°C. Ookinetes were obtained by culturing gametocyte-infected blood and enriched on a 62% Nycodenz cushion (v/v). The resulting smears were stained with Giemsa. Scale bar = 5 µm.
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S3 Fig. Phenotype analysis of PbPP6-GFP parasites.
A. Schematic of C-terminal endogenous pbpp6 tagging with 2 × FKBP-GFP. Primer positions (p1, p2, p3) are indicated by arrows. B. Diagnostic PCR of the PbPP6-GFP C1 clone. Lanes 1–2: wild-type (WT); lanes 3–4: C1 clone. PCR products: p1 + p2 (1092 bp, lanes 1/3); p1 + p3 (1776 bp, lanes 2/4). C. Western blot of mixed blood-stage parasites using anti-GFP mAb. PbPP6-GFP is marked (arrow); β-actin served as the loading control. (D–E) No significant differences were observed between PbPP6-GFP and WT parasites in (D) daily parasitemia or (E) mouse survival (n = 9 per group). (F–G) PbPP6-GFP parasites exhibited (F) comparable ookinete conversion rates in vitro and (G) similar oocyst numbers per midgut in Anopheles stephensi mosquitoes (Mann-Whitney U test).
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S4 Fig. Pbpp6 gene deletion/disruption and analysis.
A. Targeted deletion of pbpp6. Upper panel: Double-crossover replacement strategy. Lower left panel: Diagnostic PCR results of pbpp6 deletion. Lane 1/4: cF1 + cR2 (WT, 4031 bp; Δpbpp6 parasite, 5787 bp); Lane 2/5: cF2 + cR2 (1919 bp); Lane 3/6: cF1 + cR1 (2293 bp). Lower right panel: Immunoblot of parasite lysates probed with anti-PbPP6 sera (arrow indicates PbPP6); Hsp70 served as loading control. B. Single-crossover homologous recombination strategy for pbpp6 disruption. Upper panel: Targeting strategy. Lower left panel: Diagnostic PCR results: p1 + p3 (1284 bp, red arrow); p4 + p2 (724 bp, black arrow). Lower right panel: Western blot detecting truncated PbPP6 (dPbPP6, ~ 71.5kDa) in Δpbpp6 parasites using anti-GFP antibody; Hsp70 confirmed equal loading. C. IFA analysis of WT and Δpbpp6 parasite of schizont and ookinete stages using anti-PbPP6 sera. D. Asexual stage growth curves. Growth of asexual blood stages of the WT and two Δpbpp6 clones was monitored daily for 15 days (mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001). E. Survival analysis of infected mice (n = 9/group) by Kaplan-Meier method (WT vs Δpbpp6 K1: χ² = 7.569, P = 0.006; WT vs Δpbpp6 K2: χ² = 8.676, P = 0.003). Data represent three independent experiments.
https://doi.org/10.1371/journal.ppat.1013318.s004
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S5 Fig. Effect of okadaic acid (OKA) on exflagellation and ookinete formation.
A. Effect of OKA on gametocyte exflagellation. B. Effect of OKA on ookinete conversion. 1 × IC50 = 0.86 nM; 1.5 × IC50 = 1.29 nM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
https://doi.org/10.1371/journal.ppat.1013318.s005
(TIF)
S6 Fig. Global transcriptional analysis for activated gametocytes of Δpbpp6 parasites by RNA-seq.
A. Volcano plot displaying the estimated log2 fold changes for Δpbpp6 versus WT versus the –log10 adjusted P-values. Each point represents a P. berghei gene from our analysis. The significantly differentially transcribed genes (i.e., genes with a P-adj < 0.1 and greater than 2-folds difference) are highlighted in orange (up-regulated genes) and green (down-regulated) colored points, respectively. B. Heatmap illustrating the significantly down-regulated male gametogenesis-related genes identified in the current study. Related to S1C Table. C. Gene Ontology (GO) analysis was performed on both the up-regulated (upper panel) and down-regulated (lower panel) genes following the deletion of PbPP6. Bar graphs present a generic description of the gene sets associated with these functions. Refer to S1D Table. The expected number of genes in a specific GO term was calculated as the fraction of genes categorized as this GO term in P. berghei multiple the number of genes up- or down- regulated.
https://doi.org/10.1371/journal.ppat.1013318.s006
(TIF)
S7 Fig. Distribution of predicted proteins from the P. berghei proteomes of activated gametocytes.
A. Hierarchical clustering of Pearson correlation coefficients shows the relationship between all quantified genes for each pair of samples, representing three biological replicates of WT and Δpbpp6 parasite proteomes. B. Heatmap displaying of all proteins that exhibit significant changes in Δpbpp6 gametocytes compared to WT gametocytes. Refer to S2 Table. C. The volcano plot demonstrates significantly regulated proteins, with orange indicating upregulated proteins and green indicating downregulated proteins following PbPP6 deletion. Refer to S2C Table. D. Heatmap illustrating the significantly up- and down-regulated proteins involved in egress, endocytosis, DNA metabolic process, phosphorylation and inner membrane complex location identified in the current study. Related to S2C Table. E. GO analysis of significant differentially-regulated proteins in Δpbpp6 strain when comparing WT strain. The up-regulated and down-regulated GO terms are displayed by orange and green color, respectively. Refer to S2D Table.
https://doi.org/10.1371/journal.ppat.1013318.s007
(TIF)
S8 Fig. Comparison of the transcriptome and proteome identified in this study.
A. Scatter plot showing transcript abundance (log2 fold change) versus protein abundance (log2 fold change) for genes identified in the transcriptome and proteome. The spearman’s correlation value is displayed in the corner of the plot. B. Venn diagrams showing the overlap of genes identified in both the transcriptome and proteome. C. Heatmap of gene expression, featuring the identified components of the CITH/DOZI complex on the vertical axis and the strains (WT and ∆pbpp6) on the horizontal axis.
https://doi.org/10.1371/journal.ppat.1013318.s008
(TIF)
S9 Fig. Flow cytometry gating strategies for calcium mobilization and genomic DNA replication analysis.
A. Gating approach for intracellular Ca2⁺ flux quantification in activated gametocytes pre-loaded with Fluo-8 AM. The black arrow denotes XA or DMSO addition time point. Data were acquired continuously from 30 s pre-stimulation to 90 s post-stimulation (corresponding to Fig 4B). B. Gating methodology for genomic DNA replication analysis in Hoechst 33342-stained gametocytes (corresponding to Fig 4C).
https://doi.org/10.1371/journal.ppat.1013318.s009
(TIF)
S1 Table. Transcriptome analysis of Δpbpp6 and WT P. berghei gametocytes.
A. Hierarchical clustering of Pearson correlation coefficients between each pair of samples from three biological replicates. B. Raw sequencing counts per gene in ∆pbpp6 transcriptome analysis. C. Transcriptome analysis of significantly 2-fold regulated genes in ∆pbpp6 parasites. D. GO analysis of significantly altered transcripts in ∆pbpp6 parasites compared to WT. Related to S6C Fig.
https://doi.org/10.1371/journal.ppat.1013318.s010
(XLSX)
S2 Table. Quantitative proteomic analysis of Δpbpp6 and WT P. berghei gametocytes.
A. Pearson’s correlation of quantification from all quantified proteins between each pair of samples. B. Total list of identified proteins in LC-MS/MS analysis. C. List of significant differentially expressed proteins in ∆pbpp6 parasites as measured by LC-MS/MS analysis. Related to S7 Fig. D. GO analysis of significantly dysregulated proteins in ∆pbpp6 parasites compared to WT. Related to S7E Fig.
https://doi.org/10.1371/journal.ppat.1013318.s011
(XLSX)
S3 Table. The putative translationally repressed (TR) analysis of Δpbpp6 and WT P. berghei gametocytes.
A. The putative TR genes identified in our study compared with sex-specific transcriptional regulation in P. falciparum [31]. B. The overlapped putative TR genes of female gametocytes identified in this study and previous reports in P. falciparum [31] and P. berghei [30].
https://doi.org/10.1371/journal.ppat.1013318.s012
(XLSX)
S4 Table. Phosphoproteomic analysis of Δpbpp6 and WT P. berghei gametocytes.
A. Quantification of phosphosites of Δpbpp6 parasites in activated gametocytes (2 min post-activation). B. GO analysis of significantly dysregulated phosphoproteins in Δpbpp6 strain when comparing WT strain.
https://doi.org/10.1371/journal.ppat.1013318.s013
(XLSX)
S5 Table. Oligonucleotides used for plasmid construction and genotype checking.
https://doi.org/10.1371/journal.ppat.1013318.s014
(XLSX)
Acknowledgments
We would like to thank Ms Jun Liu at the China Medical University for technical support and assistance in this work.
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