Fig 1.
Enzyme activity and drug inhibition assays.
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.
Fig 2.
Subcellular localization of endogenous PbPP6 and PbPP6-GFP fusion protein.
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.
Fig 3.
The sexual stage phenotype of pbpp6 gene knockout clones.
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).
Table 1.
Reduced oocyst intensity and infection prevalence in mosquito fed on Δpbpp6-infected mice and sporogonic development.
Fig 4.
PbPP6 disruption impairs the cGMP-PKG-Ca2+ signaling pathway.
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.
Fig 5.
Quantification of phosphosites of Δpbpp6 parasites at gametocyte stages.
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.