
==== Front
Int J Parasitol Drugs Drug Resist
Int J Parasitol Drugs Drug Resist
International Journal for Parasitology: Drugs and Drug Resistance
2211-3207
Elsevier

S2211-3207(24)00044-7
10.1016/j.ijpddr.2024.100563
100563
Article
The phosphatase inhibitor BVT-948 can be used to efficiently screen functional sexual development proteins in the malaria parasite Plasmodium berghei
Jia Xitong ab1
Wang Yong cd1
Wang Meilian b
Min Hui c
Fang Zehou e
Lu Haifeng b
Li Jiao b
Cao Yaming ymcao@cmu.edu.cn
c⁎⁎⁎
Bai Lunhao bailh@sj-hospital.org
a⁎⁎
Lu Jinghan lujh1@sj-hospital.org
a⁎
a Department of Orthopedic Surgery, Shengjing Hospital of China Medical University, Shenyang, 110000, China
b Department of Pathogen Biology, College of Basic Medical Sciences, China Medical University, Shenyang, Liaoning, 110122, China
c Department of Immunology, College of Basic Medical Sciences, China Medical University, Shenyang, Liaoning, 110122, China
d Department of Family Medicine, Shengjing Hospital of China Medical University, Shenyang, 110000, China
e The Second Clinical College of China Medical University, Shenyang, Liaoning, 110122, China
⁎ Corresponding author. lujh1@sj-hospital.org
⁎⁎ Corresponding author. bailh@sj-hospital.org
⁎⁎⁎ Corresponding author. ymcao@cmu.edu.cn
1 These authors contributed equally to the work.

14 8 2024
12 2024
14 8 2024
26 1005638 5 2024
12 8 2024
14 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Background

Studying and discovering the molecular mechanism of Plasmodium sexual development is crucial for the development of transmission blocking drugs and malaria eradication. The aim of this study was to investigate the feasibility of using phosphatase inhibitors as a tool for screening proteins essential for Plasmodium sexual development and to discover proteins affecting the sexual development of malaria parasites.

Methods

Differences in protein phosphorylation among Plasmodium gametocytes incubated with BVT-948 under in vitro ookinete culture conditions were evaluated using phosphoproteomic methods. Gene Ontology (GO) analysis was performed to predict the mechanism by which BVT-948 affected gametocyte–ookinete conversion. The functions of 8 putative proteins involved in Plasmodium berghei sexual development were evaluated. Bioinformatic analysis was used to evaluate the possible mechanism of PBANKA_0100800 in gametogenesis and subsequent sexual development.

Results

The phosphorylation levels of 265 proteins decreased while those of 67 increased after treatment with BVT-948. Seven of the 8 genes selected for phenotype screening play roles in P. berghei sexual development, and 4 of these were associated with gametocytogenesis. PBANKA_0100800 plays essential roles in gametocyte–ookinete conversion and transmission to mosquitoes.

Conclusions

Seven proteins identified by screening affect P. berghei sexual development, suggesting that phosphatase inhibitors can be used for functional protein screening.

Graphical abstract

Image 1

Highlights

• BVT-948 influences microtubule associated activity and transcription process.

• PBANKA_0100800 plays essential roles in transmission of Plasmodium berghei.

• Phosphatase inhibitors and phosphoproteomics are valid tools for screening for functional proteins.

Keywords

Malaria
Plasmodium berghei
Phosphatase inhibitor
Transmission
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pmc1 Introduction

Despite continuous efforts to eliminate malaria, malaria remains associated with high levels of morbidity and mortality worldwide, with 249 million cases and 608,000 deaths in 2022 alone (WHO, 2023). The emergence of Plasmodium spp. resistant to front-line antimalarials and anopheline mosquitoes resistant to insecticides makes the development of new antimalarial medicines urgent (Mok et al., 2023). The lack of understanding of the molecular mechanisms of Plasmodium growth has greatly limited the development of antimalarials; therefore, it is very important to elucidate the molecular mechanism of Plasmodium development (Su et al., 2019).

Malaria parasites have a complex lifecycle that involves both mammalian and anopheline mosquito vector hosts. In vertebrates, such as humans, most parasites undergo asexual development, which is responsible for the clinical symptoms of malaria and subsequent death. During sexual development, a small fraction of the parasites convert into gametocytes during the asexual replication cycle. When mosquitoes feed on blood, female/male gametocytes enter the mosquito midguts. In the midgut, both sexes of gametocytes undergo gametogenesis to form female/male gametes, which can fertilize to form zygotes. Zygotes develop into ookinetes, which can penetrate the midgut epithelium and finally undergo sporogonic development to form oocysts in the basal lamina (Meibalan and Marti, 2017).

Drugs that target the sexual stage of the parasite and interrupt transmission between humans and mosquito vectors are called transmission blocking drugs. Thus far, most antimalarials that are both in clinical use and in the research stage target the asexual blood stage. The application of and research on transmission blocking drugs are lacking and insufficient (Birkholtz et al., 2022; Yu et al., 2022). To achieve the goal of eliminating malaria, it is very important to develop transmission blocking drugs. Resistance to transmission blocking drugs may occur more easily if the compound is dually active and blocks the same target in both the asexual and sexual stages, such as the case with primaquine (Birkholtz et al., 2022; Camarda et al., 2019). However, transmission blocking drugs with unique drug mechanisms that target only the sexual stages of malaria parasites can circumvent this problem (van der Watt et al., 2022).

Drugs that target molecular mechanisms specific to microbial or parasitic development are good targets for new drug development (Zhang and Lin, 2021). The sporogonic stages (gametes, zygotes, ookinetes, oocysts and sporozoites) themselves remain insufficiently studied as drug targets (Siqueira-Neto et al., 2023; Yu et al., 2022). The transcriptional master switch AP2-G and the epigenetic modulator GDV1, as well as some metabolic signals that modulate their activities, determine the differentiation of asexual parasite differentiation into nonreplicative male and female gametocytes; this progress is called sexual commitment (Bechtsi and Waters, 2017; Guttery et al., 2022; Neveu et al., 2020). The sex determination of Plasmodium gametocytes occurs at the same time as or shortly after sexual commitment and is regulated by AP2-G (Bancells et al., 2019; Silvestrini et al., 2000; Smith et al., 2000). Both male and female gametogenesis depend strongly on reversible phosphorylation, which is regulated by numerous stage-specific kinases and phosphatases (Guttery et al., 2014; Invergo et al., 2017). Recent studies also revealed essential proteins for ookinete development, such as P230, chitinase 1 (CHT1), ApiAp2-O (Santos et al., 2015; Viswanath et al., 2021; Yuda et al., 2009). Although an increasing number of functional proteins have been found to be involved in sexual development, the molecular mechanism underlying the sexual development of the malaria parasite has not been fully elucidated.

Previous studies have shown that the phosphatase inhibitor BVT-948 has good transmission blocking activity, but the mechanisms by which it affects sexual parasites are unknown (Jia et al., 2022). In this study, we used phosphoproteomic analysis to investigate the molecular mechanisms by which BVT-948 inhibits the transformation of gametocytes into ookinetes. We evaluated the functions of 8 proteins in sexual development by deleting these genes in the rodent malaria parasite Plasmodium berghei and performing a phenotype screen of the mutants. The phenotype screen showed that 7 of the 8 proteins were involved in Plasmodium berghei sexual development. This study validated that phosphatase inhibitors with transmission blocking activity can act as a medium to discover Plasmodium parasite functional proteins involved in sexual development and identified essential components of Plasmodium sexual development.

2 Materials and methods

2.1 Chemicals and compounds

The phosphatase inhibitor BVT-948, sourced from MedChemExpress (MCE, NJ, USA) was solubilized in dimethyl sulfoxide (DMSO) to generate a potent 10 mM stock solution and cryopreserved at −80 °C (Jia et al., 2022).

2.2 Bioinformatics

Genomic sequences were sourced from PlasmoDB (https://www.plasmodb.org). Putative domains were unveiled through SMART (http://smart.embl-heidelberg.de/). A diagram was generated by TBtools-II (Chen et al., 2023). The alignment of multiple sequences was orchestrated with the RPS-BLAST program of the NCBI conserved domains search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The Clustal Omega program (http://www.uniprot.org/align/) was used to compute sequence identity. The interaction network was generated by STRING version 12.0 (https://cn.string-db.org/).

2.3 Mice, mosquitoes, and parasites

Female BALB/c mice from Beijing Animal Institute (Beijing, China) were chosen based on their age (six to eight weeks) and weight (18–25 g). Adult female Anopheles stephensi mosquitoes were reared within an insectary environment, maintaining an equilibrium of temperature (20 ± 2 °C), light exposure (12-h light/dark cycle), and humidity (50–80%). The mosquitoes were provided sustenance through a 10% (w/v) glucose solution absorbed in cotton balls. The murine malaria parasite P. berghei ANKA strain 2.34 was maintained by serial passages. The authorized ethics protocol for animal experiments was approved by China Medical University on November 6, 2023 under the number CMU20231421.

2.4 Phosphoproteomic analysis

2.4.1 Sample preparation

BALB/c mice were pretreated with 6 mg/mL phenylhydrazine in saline to cause an increase in reticulocytosis. After three days, mice were intravenously (i.v.) inoculated with 5 × 106 parasite-infected erythrocytes. Three days postinfection (dpi), the mice were treated with sulfadiazine (Sigma‒Aldrich) at 20 mg/L in potable water over a duration of 48 h to eliminate parasites other than gametocytes (Beetsma et al., 1998). Heparinized blood was mixed at the volume ratio of 1:9 with the complete ookinete culture medium (RPMI 1640, 50 mg/L penicillin, 50 mg/L streptomycin, 100 mg/L neomycin, 25% [v/v] heat-inactivated fetal calf serum, 6 U/mL heparin, 100 μM xanthurenic acid, pH 8.0) supplemented with either 0.1% DMSO or 10 μM BVT-948, and cultured in plastic Petri dishes at 19 °C for 1 h. The gametocytes were harvested underwent a centrifugation process at 400×g for 10 min. 0.15% saponin (Sigma‒Aldrich) was utilized to lyse erythrocytes for a duration of 10 min on ice. The parasites were collected and rinsed in ice-cold phosphate-buffered saline (PBS) supplemented with a protease inhibitor cocktail (Thermo Fisher), followed by centrifugation at 1700×g for 10 min at 4 °C to eliminate haemoglobin. The samples were subjected to three rounds of sonication in lysis buffer (comprising 8 M urea containing 1% protease inhibitor cocktail) on ice utilizing a high-intensity sonicator (Scientz), and then spun at 14,000×g for 10 min at 4 °C to eliminate debris. The protein solution was treated with 5 mM dithiothreitol (DTT) (Sigma‒Aldrich), 11 mM iodoacetamide and trypsin to reduce, alkylate and digest overnight at 37 °C. Peptides from each sample were solubilized in 0.5 M tetraethylammonium bromide (TEAB). Each peptide channel was then labelled with its corresponding TMT reagent (Thermo Fisher) and incubated at room temperature for 2 h consistent with previously established protocols (Paulo and Gygi, 2015). Upon confirming the labelling efficiency, the TMT-labelled samples were desalted using a Strata X C18 solid-phase extraction column (Phenomenex, Beijing, China) and dried via vacuum centrifugation. Each protein extract was derived by three independent gametocyte preparations (biological replicates) and the phosphoproteomics analysis was conducted on three replicates of the protein extracts in total (technique replicates).

2.4.2 Phosphopeptide enrichment

Phosphopeptide enrichment was executed utilizing Ti-immobilized metal ion affinity chromatography (IMAC) microsphere (ReSyn Biosciences, Beijing, China). The Ti-IMAC microspheres underwent thorough washing with solutions of increasing acetonitrile concentration and decreasing trifluoroacetic acid (TFA) content. The phosphopeptides bound to Ti-IMAC microspheres were separated by centrifugation at 12,000×g for 5 min. Then the Ti-IMAC microspheres underwent sequential washing with 50% acetonitrile/6% trifluoroacetic acid (TFA) and 30% acetonitrile/0.1% TFA. Following isolation, phosphopeptides were eluted with 1 mL of 10% NH4OH and lyophilized.

2.4.3 Liquid chromatography–tandem mass spectrometry (LC‒MS/MS) analysis

The LC‒MS/MS analysis was performed following established protocols (Gaji et al., 2015). In summary, the tryptic peptides were solubilized in solvent A, containing 0.1% formic acid and introduced onto a self-made reversed-phase analytical column (15 cm in length and 75 μm in diameter). Employing an EASY-nLC 1200 UPLC system, the peptides underwent meticulous separation at a consistent flow rate of 500 nL/min. The peptides were then directed into a Q Exactive HF-X mass spectrometer manufactured by Thermo Fisher for comprehensive mass scanning. A comprehensive mass scan was performed, covering the mass spectrum from 400 to 1500 m/z in profile mode, utilizing a resolution of 120,000. MS/MS analysis was performed on selected peptides utilizing an NCE setting of 28, with detected fragments analysed in the Orbitrap at a resolution of 15,000. The resulting MS/MS data underwent thorough processing using MaxQuant version 1.6.15.0 (Cox and Mann, 2008). The tandem mass spectra underwent interrogation against a composite database comprising the UniProt P. berghei proteome alongside a reverse decoy database. Trypsin/P was specified as the cleavage enzyme with an allowance of up to two missed cleavages. At the analysis of proteomic data, the precursor ion mass tolerance was initially set at 20 ppm. A refinement to 4.5 ppm was implemented for the primary search stage. The fragment ion mass tolerance was established at 0.02 Da. Fixed modifications included cysteine carbamidomethylation, whereas variable modifications encompassed methionine acetylation, oxidation, and phosphorylation of serine, threonine, and tyrosine residues. This calibration of the false discovery rate (FDR) was to a precise threshold of 1% at both the peptide and protein levels.

Utilizing the improved Gaussian model, comprehensive protein alterations induced by phosphatase inhibitor treatment were elucidated (Hawkins et al., 2021; Li et al., 2019; Zhang et al., 2010). The log2 (fold change) of normalized protein/peptide intensities was subjected to Gaussian distribution modeling. To mitigate variance fluctuations stemming from differences in protein/peptide intensities, a low-to-hight-intensity sliding window approach was employed. Subsequently, parameters within the Gaussian distribution were optimized after excluding the top and bottom 5% of entities within each sliding window iteration. Significant alterations in global protein phosphorylation were delineated by fold changes ≥1.5 or ≤1/1.5, plus a reliability score of ≥0.7 in all three biological replicates.

2.4.4 Bioinformatic analysis

For the assessment of relative abundances of phosphorylated peptides, phosphorylation intensity for each phosphorylated peptide was standardized at the same protein expression level within the global proteome of gametocyte samples. An improved Gaussian model was utilized to dissect changes in phosphopeptide expression post-drug administration. Proteins exhibiting significant fluctuations in phosphorylation levels were identified according to the criteria including a log2 (fold change) exceeding or falling below 0, alongside an adjusted P value < 0.1, consistently observed across all three biological replicates. The rectification of the P value involved the utilization of the FDR. To depict alterations in phosphorylation patterns consequent to BVT-948 treatment, a heatmap encompassing all identified peptides and their phosphorylation sites was generated. Gene Ontology (GO) enrichment analyses were performed by the clusterProfiler R package with an FDR-adjusted P value lower than 0.05.

2.5 Generation of transgenic parasites

The approach utilized for generating transgenic parasite lines were generated via double-crossover homologous recombination by electroporation described previously (Janse et al., 2006). To knock out the genes (KO), the 5′ and 3′ fragments (named the 5′UTR and 3′UTR, respectively) were amplified from P. berghei genomic DNA (gDNA) and incorporated into the designated restriction enzyme cleavage sites within the pL0034 plasmid. The linearized plasmid (10 μg) was introduced into purified P. berghei schizonts utilizing the precise methodology of a Nucleofector II instrument (Lonza, Basel, Switzerland) in combination with Basic Parasite Nucleofector® kit (Lonza). Subsequently, the transfected parasites were i.v. administered into BALB/c mice. To restore the genes (RE) in the KO lines, we used the “GIMO” method (Lin et al., 2011). Briefly, the primers KO-5UTR-F and KO-3UTR-R were used to amplify a reconstructed fragment. The purified PCR fragments were digested with their specific restriction enzymes and then transfected into the KO lines schizonts as described above. For each gene, KO C1 line was used in transfections to obtain its RE line. To facilitate the selection of parasites harboring the desired genetic modifications, KO and RE parasites were selected with pyrimethamine (70 μg/mL, Sigma‒Aldrich, St. Louis, MO, USA) via drinking water and 5-fluorocytosine (0.4 mg/kg/day, Sigma‒Aldrich, St. Louis, MO, USA) by intraperitoneal injection for mice respectively 24 h after transfection. To isolate parasite clones harboring the targeted modifications, limiting dilution was employed. Verification of successful gene knockout and restoration was performed by extracting parasite gDNAs from blood-stage parasites using a DNeasy Blood Kit (Qiagen), followed by diagnostic PCR analysis. The primers and restriction enzymes utilized in this analysis are listed in Table S1. For each gene, two independent transfections of knockout were performed and both clones were analysed in phenotypic analysis.

2.6 Phenotypic analysis and functional complementation assay

Mice underwent pretreatment with a 6 mg/mL phenylhydrazine solution, followed by infection with 5 × 106 wild-type (WT) or KO (two clones: C1, C2) infected red blood cells (iRBCs) three days later.

At the 3-day post infection, gametocytaemia, characterized by the presence of mature gametocytes per 1000 RBCs. The female-to-male gametocyte ratio underwent determination through Giemsa-stained smears by at least 100 mature gametocyte counting per sample.

The quantification of exflagellation centers of male gametocytes adhered to a protocol delineated in previous literature (Guttery et al., 2014). In a succinct procedural sequence, 10 μL of tail blood from infected mice was combined with 40 μL of standard ookinete medium. Following a 15-min incubation period at 25 °C, 1.5 μL of the cultured media was transferred onto a coverslip for subsequent analysis. Under the observation of a light microscope, magnified to 100 times, the presence of an exflagellation center was characterized by the engagement of an exflagellating male gametocyte with a minimum of four RBCs. The number of exflagellation centers in continuous observation of 10 fields was counted. And 6 independent observations were obtained for each sample.

The quantification of in vitro ookinete formation was conducted following established procedures (Tewari et al., 2010). In brief, 10 μL of tail blood from infected mice was mixed with 90 μL of standard ookinete medium and subjected to a 24-h incubation period at 19 °C. Upon completion of the incubation, a minute volume of 1 μL from the cultured mixture was deposited onto a slide, where the cells were fixed using 4% paraformaldehyde. The samples underwent a washing procedure and were subjected to a blocking process with 3% bovine serum albumin in PBS (pH 7.4) for 1 h at ambient temperature. The cells were probed with mouse anti-Pbs21 (1:1000) for an hour. After PBS washes, the cells were incubated with Alexa Fluor® 594 goat anti-mouse IgG (Molecular Probes, diluted at 1:1000) for another hour. Following three additional PBS washes, the enumeration of ookinetes within 1 μL of culture media was conducted using a Nikon Upright E800 fluorescence microscope, magnified at 100 times.

During the direct mosquito feeding assay, mosquitoes were allowed to feed on mice infected with either WT or KO parasites at 3 days post-infection as described in previous studies (Jia et al., 2022). Across three independent experimental iterations, infection prevalence and the quantity of oocysts per midgut were assessed using a sample size of 30 mosquitoes on day 10 post-feeding.

Functional complementation assays were carried for genes showing significant differences after knockout in phenotypic analysis. Mice were infected with WT, KO (C1) or RE iRBCs as described above. Gametocytemia, exflagellation centers, ookinete and oocyst formation were observed to certify the functions of these genes.

2.7 Statistical methods

Statistical comparisons across groups, encompassing parameters such as gametocytaemia, gametocyte ratio, exflagellation center, and ookinete count, were conducted employing Student's t-tests through GraphPad Prism software. The analysis of mosquito infection intensity, measured in terms of oocysts per midgut, underwent examination through the Mann‒Whitney U test. The infection prevalence was analysed by Fisher's exact test. These statistical analyses were facilitated by the utilization of SPSS version 21.0. All data were derived from three distinct and independent experimental trials.

3 Results

3.1 BVT-948 dysregulates the phosphorylation of proteins involved in signalling pathways during sexual development

Our data showed that BVT-948 significantly inhibited P. berghei gametocyte-to-ookinete conversion and oocyst formation in the mosquito midgut (Jia et al., 2022). To determine whether these effects are related to the phosphatase-inhibiting activity of BVT-948, we performed phosphoproteomic analysis of blood obtained from infected mice 3 dpi after treatment with 10 μM BVT-948 or 0.1% DMSO for 1 h at 19 °C (Table S2). Principal component analysis (PCA) of the phosphoproteomic data showed distinct clustering of the groups treated with DMSO and BVT-948 (Fig. S1). Three-way comparative analysis revealed differences in protein phosphorylation patterns. A total of 265 proteins were downregulated, and 67 proteins were upregulated at their phosphorylation sites after treatment with BVT-948 (Fig. 1A and B). We performed a thorough GO pathway analysis of the phosphoproteome and found that the biological processes that were differentially affected between the BVT-948 and DMSO groups were mainly related to microtubule-based movement, protein‒DNA complex assembly and transcription regulation. In terms of molecular function, processes related to microtubule motor activity and RNA binding were those predominantly different between the BVT-948 and DMSO groups. In terms of cellular components, components associated with the microtubule-associated complex, dynein complex and plasma membrane exhibited major differences (Fig. 2).Fig. 1 Eight proteins with increased phosphorylation levels after the administration of BVT-948 were chosen for phenotype screening. (A) Hierarchical clustering of all genes with significant changes in phosphorylation levels. Clustering was based on Spearman correlation coefficients, and the data were plotted using the R program. Duplicates from each experimental group clustered independently (upper dendrogram). The gene IDs of the proteins chosen for phenotype screening were listed. (B) Volcano plot showing the extent and significance of the upregulated (yellow) and downregulated (purple) genes (absolute log2-fold change >1). The gene IDs of the proteins chosen for phenotype screening were listed. (C) Domain organizations of proteins encoded by the eight selected genes from P. berghei. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 1

Fig. 2 Mechanism prediction of BVT-948 in inhibiting Plasmodium sexual development. Proteins whose phosphorylation levels at certain phosphorylation sites significantly changed after incubation with BVT-948 at 19 °C for 1 h compared to those after incubation with DMSO were selected for enrichment tests to reveal the enriched Gene Ontology terms. The significant molecular functions, biological processes and cellular components of the top 25 enriched terms are shown. The enrichment level was measured by rich factor. Rich factor = Number of differentially expressed genes located under the pathway term/number of all annotated genes located under the pathway term. A higher value of rich factor indicates a higher degree of enrichment. Different colours represent different GO categories. (A) Circle chart. (B) GO terms. (C) Bubble chart. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

To evaluate the effectiveness of using the phosphatase inhibitor BVT-948 to screen for functional proteins involved in P. berghei sexual development, we selected proteins whose phosphorylation levels increased after the administration of BVT-948 and whose functions were unknown. The gene IDs corresponding to these 8 proteins were PBANKA_0100800, PBANKA_0314900, PBANKA_0408400, PBANKA_0704700, PBANKA_0836800, PBANKA_0927700, PBANKA_1000600 and PBANKA_1320100 (Fig. 1A and B). Domain prediction of these proteins indicated that they had a variety of structures and suggested their diverse functions (Fig. 1C–Table S3).

3.2 Gene deletions impair P. berghei sexual development

To explore the function of each selected protein in the life cycle of P. berghei, we generated two gene knockout lines (KO) for each gene using a double-crossover homologous recombination strategy from two independent transfection experiments and restoration lines (RE) for genes showing significant differences in phenotypic analysis (Fig. S2A). Successful modifications of the genes were confirmed by integration-specific PCR (Fig. S2B). We next examined whether these proteins play roles in sexual development using these KO and RE lines.

We first evaluated gametocyte development in these 16 KO lines. The PBANKA_0100800 KO, PBANKA_0704700 KO, and PBANKA_0836800 KO clones showed no significant differences from WT parasites. However, PBANKA_0314900, PBANKA_0408400, PBANKA_0927700 and PBANKA_1320100 KO significantly affected gametocytogenesis, and the PBANKA_1000600 KO lines showed significantly higher gametocyte density than the WT parasites (Fig. 3A). Notably, PBANKA_0100800, PBANKA_0704700, PBANKA_0836800 and PBANKA_1000600 did not have essential roles in gametocyte sex differentiation (Fig. 3B).Fig. 3 Phenotypic analyses of KO and RE lines. (A) Gametocytogenesis of the wild-type (WT), knockout (KO) and restoration (RE) parasites at 3 dpi. (B) Female/male gametocyte ratio of the WT and KO lines at 3 dpi. (C) The number of exflagellation centers per 10 fields in the WT, KO and RE parasites. Each independent experiment includes 6 technical repeats. (D) Ookinete numbers in the WT, KO and RE parasites. (E) Oocyst number per mosquito midgut in the WT, KO and RE parasites. WT C1 and C2 represent two independent data from two WT-infected mice. KO C1 and C2 represent two clones. * and ** indicate P < 0.05 and P < 0.01, respectively, compared with the WT or RE. The data are presented as the means ± SDs, and t tests were performed for three experiments for A, B, C and D; the data are presented as the means ± SEMs for E. The intensity of mosquito infection (oocysts/midgut) was analysed using the Mann‒Whitney U test.

Fig. 3

To determine whether knocking out PBANKA_0100800, PBANKA_0704700, PBANKA_0836800 or PBANKA_1000600 affected subsequent sexual development, we performed in vitro ookinete culture using the WT and KO clones. In vitro analysis revealed an over 90% reduction in the number of exflagellation centers formed in male gametocytes of both PBANKA_0100800 KO lines compared with those in WT control gametocytes. There were no defects in the egress of male gametes in the PBANKA_0704700, PBANKA_0836800 or PBANKA_1000600 KO lines (Fig. 3C). An in vitro ookinete culture assay showed that PBANKA_0100800 was essential for ookinete formation. Deletion of PBANKA_0100800 completely reduced the ookinete number compared to that in the WT line. The PBANKA_0704700 and PBANKA_1000600 deletion lines also showed significant reductions in ookinete formation, with ∼10% and ∼40% reductions in ookinete numbers, respectively (Fig. 3D).

In direct mosquito feeding assays, complete reductions in the percentage of infected mosquitoes and oocyst density per mosquito midgut were observed in those feeding on PBANKA_0100800 KO-infected mice compared to the WT control. Mosquitoes fed on mice infected with the PBANKA_0704700 KO lines showed modest reductions in the infection rate compared to those fed on mice infected with the WT parasites. There were slight reductions in the mean number of oocysts per midgut in mosquitoes fed on mice infected with the PBANKA_0704700 and PBANKA_1000600 KO lines compared to those infected with the WT strain. However, the differences in these two indices were not significant between these two KO lines and the WT control (Fig. 3E–Table 1).Table 1 Ookinete numbers, oocyst densities and prevalence in mosquitoes fed on WT, PBANKA_0100800 KO, PBANKA_0704700 KO, and PBANKA_1000600 KO parasites.

Table 1		Mosquitoes Infected/Dissected	Prevalence of infection (%)a	Reduction in prevalence (%)b	Oocysts intensity (mean ± SEM)c	Reduction in oocyst intensity (%)d	
WT	C1	28/30	93.3		145.9 ± 16.8		
C2	28/30	93.3		147.8 ± 18.0		
0100800 KO	C1	0/30	0.0	100.0	0.0	100.0	
C2	0/30	0.0	100.0	0.0	100.0	
0704700 KO	C1	26/30	86.7	7.1	118.3 ± 17.0	19.5	
C2	27/30	90.0	3.3	129.9 ± 17.7	11.6	
1000600 KO	C1	27/30	90.0	3.3	134.5 ± 18.0	8.4	
C2	29/30	96.7	0.0	138.3 ± 18.4	5.8	
(a) Prevalence of mosquito infection = number of infections/number of dissected samples × 100%.

(b) Reduction in prevalence = % prevalenceWT - % prevalenceKO.

(c) Number of oocysts per midgut.

(d) Reduction in oocyst density = (mean oocyst densityWT – mean oocyst densityKO)/mean oocyst densityWT × 100%.

All phenotypic differences were restored with gene restoration in RE lines.

3.3 Functional prediction of the PBANAK_0100800 gene

As the deletion of PBANKA_0100800 significantly interrupted P. berghei sexual development and blocked transmission, we performed a bioinformatics analysis of PBANKA_0100800 to explore its possible role in P. berghei sexual development. To predict the underlying mechanism, we first analysed the interaction protein network of the protein encoded by PBANKA_0100800. The interaction network showed that PBANKA_0100800 might interact with PBANKA_0713800, PBANKA_0922200 and PBANKA_1141700 and their associated genes (Fig. 4B). GO analysis revealed that the biological processes related to PBANKA_0100800 were mainly peptide biosynthetic processes. In terms of molecular function, the processes predominantly related to PBANKA_0100800 were structural constituents of ribosomes and structural molecule activity. In terms of cellular components, components associated with ribosomes, especially the intracellular ribonucleoprotein complex, were the most enriched (Fig. 4C and D).Fig. 4 Prediction of the function of PBANKA_0100800 in Plasmodium parasites. (A) Alignment of the C3H1 domain (66–92 aa) of protein encoded by PBANKA_0100800 in P. berghei to those in P. falciparum and P. vivax. Amino acids conserved across three species are marked as follows: identical (red background), similar (red letters), and different (black letters). (B) STRING association network of PBANKA_0100800 with a significance analysis of the interactome (SAINT) score probability of >0.4. Table S4 shows a complete list of putative interactors with the gene IDs. (C, D) Putative proteins in the STRING association network of PBANKA_0100800 were selected for enrichment analysis to reveal the enriched gene ontology terms. The significantly different molecular functions, biological processes and cellular components of the top 25 terms are shown. Different colours represent different GO categories. C and D are the circle chart and GO term list, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

To evaluate the reference value of this functional analysis of PBANKA_0100800 in P. berghei for other Plasmodium species that infect humans, we analysed the conservation of the C3H1 domain (66–92 aa) of the protein encoded by PBANKA_0100800 in P. berghei with P. falciparum and P. vivax. Multiple sequence alignment revealed that this protein was highly conserved among these Plasmodium species and that the C3H1 domain was completely the same in all three (Fig. 4A and Fig. S3).

4 Discussion

In this work, we investigated the molecular mechanism by which BVT-948 inhibits the sexual development of P. berghei and explored the potential of using phosphatase inhibitors with transmission blocking activity to discover functional Plasmodium parasite proteins involved in sexual development. We found that 7 proteins whose phosphorylation levels increased after the administration of BVT-948 affected the sexual development of P. berghei at different stages. The proteins encoded by PBANKA_0314900, PBANKA_0408400, PBANKA_0927700 and PBANKA_1320100 play essential roles in gametocytogenesis, the proteins encoded by PBANKA_0704700 and PBANKA_1000600 play roles in ookinete formation, and the protein encoded by PBANKA_0100800 had an important effect on P. berghei male gamete formation, ookinete development and subsequent transmission to mosquitoes.

Gametocytogenesis is initiated at committed merozoites and results in the development of gametocytes. Both host and parasite factors can influence gametocytogenesis (Bousema et al., 2007, 2011). Host immunity affects gametocytogenesis both through acquired immunity and specific immunity. (Alemayehu, 2023; Nyarko and Claessens, 2021). Gametocytogenesis starts with sexual commitment regulated by the transcriptional master switch AP2-G and its modulators, such as GDV1, PfHda2, and HP1 (Bechtsi and Waters, 2017; Bui et al., 2021; Coleman et al., 2014; Neveu et al., 2020). Reverse phosphorylation plays important roles in sexual development including gametocytogenesis (Guttery et al., 2015). Kinases and phosphatases were found to be essential for gametocytogenesis. Casein kinase 2, glycogen synthase kinase (GSK3) and Nima-related kinase (NEK4) were required for gametocytogenesis or maturation in Plasmodium falciparum (Alder et al., 2022; Hitz et al., 2021; Reininger et al., 2012). Protein phosphatase NIF4 and PPM2 affected the gametocyte development. Phosphorylation of PfGAP40 was required for PfGAP40 function for gametocyte formation (He et al., 2023). In this study, we found that the deletion of PBANKA_0314900, PBANKA_0408400, PBANKA_0927700 and PBANKA_1320100 led to the complete loss of gametocytes in P. berghei. These findings suggest that these four genes play essential roles in gametocytogenesis. To elucidate the molecular mechanism of gametocytogenesis, further studies may explore the correlations between these four genes and AP2-G and its modulators or with host immune responses. We would investigate the relevance of phosphorylation levels to their functions of these four proteins in future studies.

Plasmodium parasites have a complex life cycle and stage-to-stage conversions occur rapidly. Reverse phosphorylation and enzymes that regulate the phosphorylation level participate in gametocyte-to-ookinete formation and the following oocyst development. Protein phosphatase PPM1 was essential for microgamete formation (Guttery et al., 2014). Our work demonstrated that the deletion of PBANKA_0100800 blocks gametocyte–ookinete conversion and transmission which showed the same phenotype with Δppm1. In-depth studies whether phosphorylation state influencing the function of PBANKA_0100800 and whether the protein is a target of PPM1 or a functional protein downstream in PPM1 pathway may help better understand the mechanism of action of PBANKA_0100800. Informatics analysis of PBANKA_0100800 speculated that this gene may have a function related to ribosome-mediated peptide biosynthesis. Further studies targeting PBANKA_0100800 to demonstrate the prediction are needed and may help to elucidate the effect of ribosome protein synthesis on the sexual development of Plasmodium parasites and the role of phosphorylation in sexual development of Plasmodium parasites.

Here, we used the phosphatase inhibitor BVT-948 to block the initial period of gametocyte–ookinete conversion and screened for proteins whose phosphorylation levels significantly changed. We speculated that the inhibition of P. berghei gametocyte-to-ookinete development by BVT-948 during the initial period was related to microtubule-associated activity and transcriptional progress through phosphoproteomic analysis. A phenotype screen revealed that 7 of the 8 proteins whose phosphorylation levels increased played roles in P. berghei sexual development. These findings indicate that phosphatase inhibitors are good tools for screening Plasmodium parasite proteins involved in sexual development. Future studies targeting other phosphatase inhibitors could verify the high efficiency of this screening method, and more functional proteins may be discovered to further understand the molecular mechanism of Plasmodium sexual development and provide new targets for the development of novel drugs.

CRediT authorship contribution statement

Xitong Jia: Writing – review & editing, Writing – original draft, Resources, Methodology, Data curation. Yong Wang: Writing – review & editing, Project administration, Methodology. Meilian Wang: Writing – review & editing, Methodology. Hui Min: Writing – review & editing, Methodology. Zehou Fang: Writing – review & editing, Methodology. Haifeng Lu: Writing – review & editing, Software, Methodology. Jiao Li: Writing – review & editing, Software, Methodology. Yaming Cao: Writing – review & editing, Conceptualization. Lunhao Bai: Writing – review & editing, Conceptualization. Jinghan Lu: Writing – review & editing, Conceptualization.

Declaration of competing interest

Declarations of interest: none.

Appendix A Supplementary data

The following are the Supplementary data to this article.Multimedia component 1

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Fig. S1 Principal components analysis for phosphoproteomic samples.

Fig. S1

Fig. S2 Generation of gene knockout lines. (A) Scheme depicting the generation of the gene knockout and restoration parasites. The yFCU:hDHFR box represents the yeast cytosine deaminase and uridyl-phosphoribosyltransferase: human dihydrofolate reductase expression cassette in the pL0034 plasmid, which was positively selected by pyrimethamine. (B) Genotyping PCR confirming gene knockout in KO parasites and gene restoration in RE parasites. The band sizes of the diagnostic PCR products are listed in Table S1. Lane 1 indicates amplification with primers P1 and P2, lane 2 indicates amplification with primers P1 and P3, and lane 3 indicates amplification with primers P5 and P6.

Fig. S2

Fig. S3 Alignment of protein encoded by PBANKA_0100800 in P. berghei to those in P. falciparum and P. vivax. Amino acids conserved across three species are marked as follows: identical (red background), similar (red letters), and different (black letters).

Fig. S3

Acknowledgments

This study was supported by the 10.13039/100000060 National Institute of Allergy and Infectious Diseases (grants R01AI150533 ).

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijpddr.2024.100563.
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References

Alder A. Wilcke L. Pietsch E. von Thien H. Pazicky S. Low C. Mesen-Ramirez P. Bachmann A. Burda P.C. Kunick C. Sondermann H. Wilson D. Gilberger T.W. Functional inactivation of Plasmodium falciparum glycogen synthase kinase GSK3 modulates erythrocyte invasion and blocks gametocyte maturation J. Biol. Chem. 298 2022 102360
Alemayehu A. Biology and epidemiology of Plasmodium falciparum and Plasmodium vivax gametocyte carriage: implication for malaria control and elimination Parasite Epidemiol Control 21 2023 e00295
Bancells C. Llora-Batlle O. Poran A. Notzel C. Rovira-Graells N. Elemento O. Kafsack B.F.C. Cortes A. Revisiting the initial steps of sexual development in the malaria parasite Plasmodium falciparum Nat Microbiol 4 2019 144 154 30478286
Bechtsi D.P. Waters A.P. Genomics and epigenetics of sexual commitment in Plasmodium Int. J. Parasitol. 47 2017 425 434 28455236
Beetsma A.L. van de Wiel T.J. Sauerwein R.W. Eling W.M. Plasmodium berghei ANKA: purification of large numbers of infectious gametocytes Exp. Parasitol. 88 1998 69 72 9501851
Birkholtz L.M. Alano P. Leroy D. Transmission-blocking drugs for malaria elimination Trends Parasitol. 38 2022 390 403 35190283
Bousema J.T. Drakeley C.J. Kihonda J. Hendriks J.C. Akim N.I. Roeffen W. Sauerwein R.W. A longitudinal study of immune responses to Plasmodium falciparum sexual stage antigens in Tanzanian adults Parasite Immunol. 29 2007 309 317 17518949
Bousema T. Sutherland C.J. Churcher T.S. Mulder B. Gouagna L.C. Riley E.M. Targett G.A. Drakeley C.J. Human immune responses that reduce the transmission of Plasmodium falciparum in African populations Int. J. Parasitol. 41 2011 293 300 20974145
Bui H.T.N. Passecker A. Brancucci N.M.B. Voss T.S. Investigation of heterochromatin protein 1 function in the malaria parasite Plasmodium falciparum using a conditional domain deletion and swapping approach mSphere 6 2021
Camarda G. Jirawatcharadech P. Priestley R.S. Saif A. March S. Wong M.H.L. Leung S. Miller A.B. Baker D.A. Alano P. Paine M.J.I. Bhatia S.N. O'Neill P.M. Ward S.A. Biagini G.A. Antimalarial activity of primaquine operates via a two-step biochemical relay Nat. Commun. 10 2019 3226 31324806
Chen C. Wu Y. Li J. Wang X. Zeng Z. Xu J. Liu Y. Feng J. Chen H. He Y. Xia R. TBtools-II: a "one for all, all for one" bioinformatics platform for biological big-data mining Mol. Plant 16 2023 1733 1742 37740491
Coleman B.I. Skillman K.M. Jiang R.H.Y. Childs L.M. Altenhofen L.M. Ganter M. Leung Y. Goldowitz I. Kafsack B.F.C. Marti M. Llinas M. Buckee C.O. Duraisingh M.T. A Plasmodium falciparum histone deacetylase regulates antigenic variation and gametocyte conversion Cell Host Microbe 16 2014 177 186 25121747
Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification Nat. Biotechnol. 26 2008 1367 1372 19029910
Gaji R.Y. Johnson D.E. Treeck M. Wang M. Hudmon A. Arrizabalaga G. Phosphorylation of a myosin motor by TgCDPK3 facilitates rapid initiation of motility during toxoplasma gondii egress PLoS Pathog. 11 2015 e1005268
Guttery D.S. Poulin B. Ramaprasad A. Wall R.J. Ferguson D.J. Brady D. Patzewitz E.M. Whipple S. Straschil U. Wright M.H. Mohamed A.M. Radhakrishnan A. Arold S.T. Tate E.W. Holder A.A. Wickstead B. Pain A. Tewari R. Genome-wide functional analysis of Plasmodium protein phosphatases reveals key regulators of parasite development and differentiation Cell Host Microbe 16 2014 128 140 25011111
Guttery D.S. Roques M. Holder A.A. Tewari R. Commit and transmit: molecular players in Plasmodium sexual development and zygote differentiation Trends Parasitol. 31 2015 676 685 26440790
Guttery D.S. Zeeshan M. Ferguson D.J.P. Holder A.A. Tewari R. Division and transmission: malaria parasite development in the mosquito Annu. Rev. Microbiol. 76 2022 113 134 35609946
Hawkins L.M. Naumov A.V. Batra M. Wang C. Chaput D. Suvorova E.S. Novel CRK-cyclin complex controls spindle assembly checkpoint in toxoplasma endodyogeny mBio 13 2021 e0356121
He L. Qiu Y. Pang G. Li S. Wang J. Feng Y. Chen L. Zhu L. Liu Y. Cui L. Cao Y. Zhu X. Plasmodium falciparum GAP40 plays an essential role in merozoite invasion and gametocytogenesis Microbiol. Spectr. 11 2023 e0143423
Hitz E. Gruninger O. Passecker A. Wyss M. Scheurer C. Wittlin S. Beck H.P. Brancucci N.M.B. Voss T.S. The catalytic subunit of Plasmodium falciparum casein kinase 2 is essential for gametocytogenesis Commun. Biol. 4 2021 336 33712726
Invergo B.M. Brochet M. Yu L. Choudhary J. Beltrao P. Billker O. Sub-minute phosphoregulation of cell cycle systems during Plasmodium gamete formation Cell Rep. 21 2017 2017 2029 29141230
Janse C.J. Ramesar J. Waters A.P. High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei Nat. Protoc. 1 2006 346 356 17406255
Jia X. Liu F. Bai J. Zhang Y. Cui L. Cao Y. Luo E. Phosphatase inhibitors BVT-948 and alexidine dihydrochloride inhibit sexual development of the malaria parasite Plasmodium berghei Int J Parasitol Drugs Drug Resist 19 2022 81 88 35792443
Li M. Tu S. Li Z. Tan F. Liu J. Wang Q. Zhang Y. Xu J. Zhang Y. Zhou F. Shao Z. MAP: model-based analysis of proteomic data to detect proteins with significant abundance changes Cell Discov 5 2019 40 31636953
Lin J.W. Annoura T. Sajid M. Chevalley-Maurel S. Ramesar J. Klop O. Franke-Fayard B.M. Janse C.J. Khan S.M. A novel 'gene insertion/marker out' (GIMO) method for transgene expression and gene complementation in rodent malaria parasites PLoS One 6 2011 e29289
Meibalan E. Marti M. Biology of malaria transmission Cold Spring Harb Perspect Med 7 2017
Mok S. Yeo T. Hong D. Shears M.J. Ross L.S. Ward K.E. Dhingra S.K. Kanai M. Bridgford J.L. Tripathi A.K. Mlambo G. Burkhard A.Y. Ansbro M.R. Fairhurst K.J. Gil-Iturbe E. Park H. Rozenberg F.D. Kim J. Mancia F. Fairhurst R.M. Quick M. Uhlemann A.C. Sinnis P. Fidock D.A. Mapping the genomic landscape of multidrug resistance in Plasmodium falciparum and its impact on parasite fitness Sci. Adv. 9 2023 eadi2364
Neveu G. Beri D. Kafsack B.F. Metabolic regulation of sexual commitment in Plasmodium falciparum Curr. Opin. Microbiol. 58 2020 93 98 33053503
Nyarko P.B. Claessens A. Understanding host-pathogen-vector interactions with chronic asymptomatic malaria infections Trends Parasitol. 37 2021 195 204 33127332
Paulo J.A. Gygi S.P. A comprehensive proteomic and phosphoproteomic analysis of yeast deletion mutants of 14-3-3 orthologs and associated effects of rapamycin Proteomics 15 2015 474 486 25315811
Reininger L. Garcia M. Tomlins A. Muller S. Doerig C. The Plasmodium falciparum, Nima-related kinase Pfnek-4: a marker for asexual parasites committed to sexual differentiation Malar. J. 11 2012 250 22849771
Santos J.M. Kehrer J. Franke-Fayard B. Frischknecht F. Janse C.J. Mair G.R. The Plasmodium palmitoyl-S-acyl-transferase DHHC2 is essential for ookinete morphogenesis and malaria transmission Sci. Rep. 5 2015 16034
Silvestrini F. Alano P. Williams J.L. Commitment to the production of male and female gametocytes in the human malaria parasite Plasmodium falciparum Parasitology 121 Pt 5 2000 465 471 11128797
Siqueira-Neto J.L. Wicht K.J. Chibale K. Burrows J.N. Fidock D.A. Winzeler E.A. Antimalarial drug discovery: progress and approaches Nat. Rev. Drug Discov. 22 2023 807 826 37652975
Smith T.G. Lourenco P. Carter R. Walliker D. Ranford-Cartwright L.C. Commitment to sexual differentiation in the human malaria parasite, Plasmodium falciparum Parasitology 121 Pt 2 2000 127 133 11085232
Su X.Z. Lane K.D. Xia L. Sa J.M. Wellems T.E. Plasmodium genomics and genetics: new insights into malaria pathogenesis, drug resistance, epidemiology, and evolution Clin. Microbiol. Rev. 32 2019
Tewari R. Straschil U. Bateman A. Bohme U. Cherevach I. Gong P. Pain A. Billker O. The systematic functional analysis of Plasmodium protein kinases identifies essential regulators of mosquito transmission Cell Host Microbe 8 2010 377 387 20951971
van der Watt M.E. Reader J. Birkholtz L.M. Adapt or die: targeting unique transmission-stage biology for malaria elimination Front. Cell. Infect. Microbiol. 12 2022 901971
Viswanath V.K. Gore S.T. Valiyaparambil A. Mukherjee S. Lakshminarasimhan A. Plasmodium chitinases: revisiting a target of transmission-blockade against malaria Protein Sci. 30 2021 1493 1501 33934433
WHO World Malaria Report 2023 2023
Yu S. Wang J. Luo X. Zheng H. Wang L. Yang X. Wang Y. Transmission-blocking strategies against malaria parasites during their mosquito stages Front. Cell. Infect. Microbiol. 12 2022 820650
Yuda M. Iwanaga S. Shigenobu S. Mair G.R. Janse C.J. Waters A.P. Kato T. Kaneko I. Identification of a transcription factor in the mosquito-invasive stage of malaria parasites Mol. Microbiol. 71 2009 1402 1414 19220746
Zhang H. Lin G. Microbial proteasomes as drug targets PLoS Pathog. 17 2021 e1010058
Zhang Y. Askenazi M. Jiang J. Luckey C.J. Griffin J.D. Marto J.A. A robust error model for iTRAQ quantification reveals divergent signaling between oncogenic FLT3 mutants in acute myeloid leukemia Mol. Cell. Proteomics 9 2010 780 790 20019052
