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. 2020 Apr 9;11(1):1764.
doi: 10.1038/s41467-020-15479-3.

An intracellular membrane protein GEP1 regulates xanthurenic acid induced gametogenesis of malaria parasites

Yuanyuan Jiang #  1 , Jun Wei #  1 , Huiting Cui #  1 , Chuanyuan Liu  1 , Yuan Zhi  1 , ZhengZheng Jiang  1 , Zhenkui Li  1 , Shaoneng Li  1 , Zhenke Yang  1 , Xu Wang  1 , Pengge Qian  1 , Cui Zhang  1 , Chuanqi Zhong  1 , Xin-Zhuan Su  2 , Jing Yuan  3   4
Affiliations

An intracellular membrane protein GEP1 regulates xanthurenic acid induced gametogenesis of malaria parasites

Yuanyuan Jiang et al. Nat Commun. .

Abstract

Gametocytes differentiation to gametes (gametogenesis) within mosquitos is essential for malaria parasite transmission. Both reduction in temperature and mosquito-derived XA or elevated pH are required for triggering cGMP/PKG dependent gametogenesis. However, the parasite molecule for sensing or transducing these environmental signals to initiate gametogenesis remains unknown. Here we perform a CRISPR/Cas9-based functional screening of 59 membrane proteins expressed in the gametocytes of Plasmodium yoelii and identify that GEP1 is required for XA-stimulated gametogenesis. GEP1 disruption abolishes XA-stimulated cGMP synthesis and the subsequent signaling and cellular events, such as Ca2+ mobilization, gamete formation, and gametes egress out of erythrocytes. GEP1 interacts with GCα, a cGMP synthesizing enzyme in gametocytes. Both GEP1 and GCα are expressed in cytoplasmic puncta of both male and female gametocytes. Depletion of GCα impairs XA-stimulated gametogenesis, mimicking the defect of GEP1 disruption. The identification of GEP1 being essential for gametogenesis provides a potential new target for intervention of parasite transmission.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Membrane proteins screening identified gep1 essential for gametogenesis.
a In vitro XA stimulated exflagellation rates for P. yoelii 17XNL wild type (WT) and 45 mutant strains each with a specific gene disruption. The exflagellation rate of each mutant was normalized with that of WT parallelly tested each time. The numbers for the gene name are the gene IDs derived in PlasmoDB. Data are shown as mean ± SD from n = 3 independent experiments for strains except n = 5 for ∆1315200, ∆1339400, ∆1342800, ∆1366100 and ∆1463300, and n = 6 for ∆1240600. b Representative images of XA stimulated exflagellation centers (ECs, white arrows) under light microscope (10×). Scale bar = 20 μm. c Images of the exflagellated male gametes (Black arrow) after Giemsa staining under light microscope (100×). Scale bar = 5 μm. d Diagrams of WT gep1 gene structure and various mutants: S1 (∆gep1), deletion in C-terminus; S2 (∆gep1/gep1::6HA), reconstructed gep1 with a 6HA tag; S3 (∆gep1n), deletion in N-terminus; S4 (∆gep1fl), deletion of the full coding region; S5 (∆gep1mScarlet), coding region replaced with mScarlet gene. e XA-stimulated EC counts from WT and the gep1 mutants. c1 and c2 are two clones of S2 parasite. n is the numbers of microscopic fields counted (40×). f Oocyst counts from WT and the gep1 mutants. Oocysts are counted from the mosquito midguts 7 days post blood feeding. x/y on the top is the number of mosquito containing oocyst/the number of mosquito dissected; the percentage number is the mosquito infection prevalence. Experiments were independently repeated six times in b, and three times in c, e, and f. Two-tailed unpaired Student’s t test was applied in a, e, and f. Source data of a, e, and f are provided as a Source Data file.
Fig. 2
Fig. 2. GEP1 is essential for gametogenesis of both sexes.
a Predicted GEP1 protein structure with 14 TM domains (green bar) and cytoplasmic N-termini and C-termini. b IFA analysis of GEP1 expression in asexual blood stages (ABS), gametocytes, ookinetes, oocysts, and sporozoites of the 6HA::gep1 parasite using anti-HA antibody. Hoechst 33342 (Blue) is used for nuclear acid stain for all images in this figure. c Western blot analysis of GEP1 in ABS and gametocytes of the 6HA::gep1 parasite. BiP as loading control. d IFA analysis of GEP1 in the 4Myc::gep1 parasite using anti-Myc antibody. e mScarlet fluorescence protein expression driven by the endogenous gep1 promoter in ABS and gametocytes of the ∆gep1mScarlet parasite. f Co-staining of GEP1 and α-Tubulin (male gametocyte specific) in the non-activated (NAG) 6HA::gep1 gametocytes. x/y in the figure is the number of cell displaying signal/the number of cell tested. g and h, P28 expression during in vitro gametocyte to ookinete differentiation. P28 expression is detected in female gametes, fertilized zygotes, and ookinetes in IFA (g) and western blot (h). mpa: minute post activation; hpa, hour post activation. i Day 7 midgut oocyst counts from mosquitoes infected with parasites, including 17XNL, ∆gep1, ∆nek4, or ∆map2 parasite alone, as well as mixtures of ∆gep1/∆nek4, ∆gep1/∆map2, or ∆map2/∆nek4 parasites. ∆nek4 and ∆map2 are female and male gamete-defect parasites, respectively. x/y on the top is the number of mosquito containing oocyst/the number of mosquito dissected; Mosquito infection prevalence is shown above. Scale bar = 5 μm for all images in this figure. Experiments were independently repeated three times in b, c, d, e, f, g, and two times in i. Two-tailed unpaired Student’s t test in i.
Fig. 3
Fig. 3. GEP1 acts upstream of PKG in the cGMP-PKG-Ca2+ signaling cascade.
a α-Tubulin expression and distribution in differentiating male gametocytes from 17XNL, ∆gep1 and ∆map2 parasites after XA stimulation. mpa: minute post XA activation. b Flow cytometry analysis of genomic DNA content in XA-stimulated male gametocytes of 17XNL, ∆gep1 and ∆cdpk4 parasites. The parasites were fixed with 4% paraformaldehyde at indicated time and stained with Hoechst. c Representative images of gametocytes stained by anti-mouse TER119 antibody 0 and 30 min post XA stimulation (mpa). d Flow cytometry detection of cytosolic Ca2+ in gametocytes using Fluo-8 probe. Purified gametocytes were preloaded with Fluo-8, and signals were collected 30 s before addition of XA or DSMO. Black arrows indicate the time for DMSO or XA addition. e Representative IFA images of the sep1::4Myc and sep1::4Myc/∆gep1 gametocytes stained by anti-Myc antibody. f Proposed location of GEP1 in the XA-PKG-Ca2+ signal cascade of gametogenesis. GEP1 depletion causes defect in both Ca2+-dependent and Ca2+-independent cellular events of gametogenesis. EM: erythrocyte membrane, PVM: parasitophorus vacuole membrane, PPM: parasite plasma membrane. x/y in a, c, and e are the number of cell displaying representative signal/the number of cell analyzed. Scale bar = 5 μm for all images in this figure. All experiments in this figure were repeated three times independently with similar results.
Fig. 4
Fig. 4. Impaired activity of cGMP synthesis in GEP1 deficient gametocytes.
a Enzyme immunoassay detecting intracellular cGMP level in XA-stimulated gametocytes of the 17XNL, ∆gep1, and ∆map2 parasites. Cells were incubated with 100 μM XA at 22 °C for 2 min before assay. Ctl are control groups without XA stimulation. b Exflagellation center counts of 17XNL, ∆gep1, and ∆map2 parasites after treatment with XA (100 μM), Zaprinast (Zap, 100 μM), or pH 8.0 alone at 22 °C, or at the presence of compound 2 (C2, 5 μM). n is the numbers of microscopic fields counted (40×). c Enzyme immunoassay detecting intracellular cGMP level in Zap-treated gametocytes of the 17XNL, ∆gep1, and ∆map2 parasites. Cells were incubated with 100 μM Zap at 22 °C for 2 min before assay. Ctl are control groups without Zap stimulation. d Proposed role of GEP1 in regulating cGMP synthesis activity of guanylyl cyclase in gametogenesis. All source data are provided as a Source Data file. Experiments in a, b, and c were repeated three times independently. Data are shown as mean ± SD; two-tailed unpaired Student’s t test.
Fig. 5
Fig. 5. GEP1 interacts with GCα in gametocytes.
a Top 10 GEP1 interacting proteins in the gametocytes of the 6HA::gep1 parasite detected by immunoprecipitation and mass spectrometry (MS), including guanylyl cyclase α (GCα) with 15 peptides detected. b MS2 spectrum of a representative peptide of the GCα protein. c Co-immunoprecipitation of Myc::GEP1 and GCα::HA proteins in gametocytes of the double tagged parasite 4Myc::gep1/gcα::6HA (DTS1). IP-Myc, anti-Myc antibody was used. d Co-immunoprecipitation of HA::GEP1 and GCα::Myc proteins in gametocytes of the double tagged parasite 6HA::gep1/gcα:: 4Myc (DTS2). IP-Myc, anti-Myc antibody was used. e Two-colored IFA of GEP1 and GCα proteins in the DTS1 gametocytes using anti-HA (GCα) and anti-Myc (GEP1) antibodies (left panel). Cross sections (white dash line) of the cells show the co-localization of GEP1 and GCα (right panel). Scale bar = 5 μm. f Pearson coefficient analysis for GEP1 and GCα co-localization shown in e, data are shown as mean ± SD from n = 10 cells measured. Experiments in c, d, and e were repeated three times independently with similar results.
Fig. 6
Fig. 6. GCα knockdown in gametocytes results in gametogenesis defect.
a Diagram showing a promoter swap strategy to knockdown gcα expression in gametocytes, generating HA-tagged gcαkd mutant with endogenous gcα promoter replaced with the sera1 promoter. b Western blotting of GCα expression in asexual blood stages and gametocytes of the gcαkd parasite. The 6HA::gcα as a control. c Quantitative analysis of GCα protein expression in b. d Intracellular cGMP level in XA-stimulated gametocytes of the 17XNL and gcαkd parasites. Cells were incubated with 100 μM XA at 22 °C for 2 min before assay. Ctl are control groups without XA stimulation. e In vitro exflagellation rates for 17XNL, 6HA::gcα, and two clones of the gcαkd parasite after XA stimulation. f Day 7 midgut oocyst counts in mosquitos infected with 17XNL, 6HA::gcα, and two clones of the gcαkd parasites. Mosquito infection prevalence is shown above. g A proposed model of GEP1/GCα interaction essential for XA-stimulated cGMP synthesis and gametogenesis. Experiments were independently repeated three times in b, d, e, and f. Data are shown as mean ± SD in c, d, and e. Two-tailed unpaired Student’s t test in c, d, e, and f. Source data of c, d, e, and f are provided as a Source Data file.
Fig. 7
Fig. 7. GCα expression and localization in the GEP1-depleted gametocytes.
a RT-PCR analysis of gcα transcript in gametocytes of the 17XNL, ∆gep1, and ∆cdpk4 parasites. b Western blotting detecting GCα protein in gametocytes of the 17XNL, gcα::6HA, gcα::6HA/∆gep1, and gcα::6HA/∆cdpk4 parasites. c Western blotting detecting GEP1 (Myc) and GCα (HA) proteins expression in gametocytes of DTS1 parasite 2 min post XA stimulation. Ctl are control groups without XA stimulation. d Co-staining of GEP1 and α-Tubulin expressions in gametocytes of the 6HA::gep1 parasite 2 min post XA stimulation. NAG: non-activated, AG: XA stimulation. e Co-staining of GCα and α-Tubulin expressions in the gcα::6HA and 6HA::gcα/∆gep1 gametocytes 2 min post XA stimulation. NAG: non-activated, AG: XA stimulation. f Co-staining of α-Tubulin and HA-tagged GEP1 or GCα expressions in the 6HA::gep1 (upper panel) and gcα::6HA (lower panel) gametocytes 2 min post XA stimulation plus C2 treatment. x/y in d, e, and f are the number of cell displaying representative signal/the number of cell analyzed. Scale bar = 5 μm for all images in this figure. All experiments in this figure were repeated three times independently.
Fig. 8
Fig. 8. XA stimulation likely enhances the interaction between GEP1 and GCα.
a Proximity Ligation Assay (PLA) detecting protein interaction between GEP1 and GCα in DTS1 gametocytes. NAG: non-activated, AG: 2 min after XA stimulation. Activated male gametocytes were observed with enlarged nucleus containing replicated genome. Scale bar = 5 μm. b Number of PLA signal dot in each cell shown in a, n is the number of cells counted. c Fluorescence intensity value for each PLA signal dot shown in a. n is the number of PLA signal dot measured. Source data are provided as a Source Data file. Experiment was repeated three times independently. Data are shown as mean ± SD; two-tailed unpaired Student’s t test.
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