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Sex-Specific Biology of the Human Malaria Parasite Revealed from the Proteomes of Mature Male and Female Gametocytes*

  • Jun Miao
    Correspondence
    To whom correspondence should be addressed: Jun Miao,Department of Entomology, The Pennsylvania State University, 539 ASI Building, University Park, PA 16802. Tel.:1-814-863-7660; E-mail: [email protected]; and Liwang Cui,Department of Entomology, The Pennsylvania State University, 537 ASI Building, University Park, PA 16802, USA. Tel.:1-814-863-7663,.
    Affiliations
    From the Department of Entomology, The Pennsylvania State University, 501 ASI Building, University Park, Pennsylvania 16802;
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  • Zhao Chen
    Affiliations
    Department of Statistics, The Pennsylvania State University, 413 Thomas Building, University Park, Pennsylvania 16802
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  • Zenglei Wang
    Affiliations
    From the Department of Entomology, The Pennsylvania State University, 501 ASI Building, University Park, Pennsylvania 16802;
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  • Sony Shrestha
    Affiliations
    From the Department of Entomology, The Pennsylvania State University, 501 ASI Building, University Park, Pennsylvania 16802;
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  • Xiaolian Li
    Affiliations
    From the Department of Entomology, The Pennsylvania State University, 501 ASI Building, University Park, Pennsylvania 16802;
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  • Runze Li
    Affiliations
    Department of Statistics, The Pennsylvania State University, 413 Thomas Building, University Park, Pennsylvania 16802
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  • Liwang Cui
    Correspondence
    To whom correspondence should be addressed: Jun Miao,Department of Entomology, The Pennsylvania State University, 539 ASI Building, University Park, PA 16802. Tel.:1-814-863-7660; E-mail: [email protected]; and Liwang Cui,Department of Entomology, The Pennsylvania State University, 537 ASI Building, University Park, PA 16802, USA. Tel.:1-814-863-7663,.
    Affiliations
    From the Department of Entomology, The Pennsylvania State University, 501 ASI Building, University Park, Pennsylvania 16802;
    Search for articles by this author
  • Author Footnotes
    * This study was supported by funding from the National Institute of Allergy and Infectious Diseases, NIH (R21AI098058, R01AI104946 and U19AI089672). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
    This article contains supplemental material.
      The gametocytes of the malaria parasites are obligate for perpetuating the parasite's life cycle through mosquitoes, but the sex-specific biology of gametocytes is poorly understood. We generated a transgenic line in the human malaria parasite Plasmodium falciparum, which allowed us to accurately separate male and female gametocytes by flow cytometry. In-depth analysis of the proteomes by liquid chromatography-tandem mass spectrometry identified 1244 and 1387 proteins in mature male and female gametocytes, respectively. GFP-tagging of nine selected proteins confirmed their sex-partitions to be agreeable with the results from the proteomic analysis. The sex-specific proteomes showed significant differences that are consistent with the divergent functions of the two sexes. Although the male-specific proteome (119 proteins) is enriched in proteins associated with the flagella and genome replication, the female-specific proteome (262 proteins) is more abundant in proteins involved in metabolism, translation and organellar functions. Compared with the Plasmodium berghei sex-specific proteomes, this study revealed both extensive conservation and considerable divergence between these two species, which reflect the disparities between the two species in proteins involved in cytoskeleton, lipid metabolism and protein degradation. Comparison with three sex-specific proteomes allowed us to obtain high-confidence lists of 73 and 89 core male- and female-specific/biased proteins conserved in Plasmodium. The identification of sex-specific/biased proteomes in Plasmodium lays a solid foundation for understanding the molecular mechanisms underlying the unique sex-specific biology in this early-branching eukaryote.
      The life cycle of the protozoan malaria parasites encompasses multiple developmental stages and alternates between a human host and an Anopheles mosquito. In human blood, the majority of parasites assumes the asexual replication cycle, starting from merozoite invasion of a red blood cell (RBC)
      The abbreviations used are: RBC, red blood cell; MG, Male gametocytes; FG, Female gametocytes; GFP, Green fluorescent protein; FDR, False discovery rate; HPLC, High-performance liquid chromatography; LC-MS/MS, Liquid chromatography coupled with tandem mass spectrometry; SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel electrophoresis; PCR, Polymerase chain reaction.
      1The abbreviations used are: RBC, red blood cell; MG, Male gametocytes; FG, Female gametocytes; GFP, Green fluorescent protein; FDR, False discovery rate; HPLC, High-performance liquid chromatography; LC-MS/MS, Liquid chromatography coupled with tandem mass spectrometry; SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel electrophoresis; PCR, Polymerase chain reaction.
      to the maturation of a schizont. Asexual multiplication of the parasites in erythrocytes is responsible for the clinical symptoms of malaria, and thus most antimalarial treatments are directed toward the asexual stages. Meanwhile, a small proportion of the parasites, in response to poorly defined triggers, enters the sexual pathway and develops into male and female gametocytes. Subsequently, circulating gametocytes in the human blood, after ingestion by a vector, develop into gametes in the mosquito midgut. As an essential link to the life stages in mosquitoes and for continued transmission of the parasites, gametocytes have increasingly been recognized as a prime target for interruption of transmission, especially during the malaria elimination phase (
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      ), but the underpinning molecular mechanism is unknown. Plasmodium gametocyte population is generally female-biased, but the sex ratio can be modulated by host and environmental factors (
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      • Thompson J.
      • Gardiner D.L.
      • Trenholme K.R.
      Sex in Plasmodium: a sign of commitment.
      ,
      • Alano P.
      Plasmodium falciparum gametocytes: still many secrets of a hidden life.
      ). Once inside the mosquito midgut, male gametocytes (MG) are prepared for rapid DNA replication and mitosis to produce eight motile microgametes, whereas the female gametocytes (FG) are ready for mitosis and subsequent zygote-ookinete development (
      • Alano P.
      Plasmodium falciparum gametocytes: still many secrets of a hidden life.
      ,
      • Janse C.
      • Waters A.P.
      Sexual development of malaria Parasites.
      ). Consistent with this functional division, the MG- and FG-specific proteomes of the rodent parasite P. berghei revealed distinctive sex-specific biology (
      • Khan S.M.
      • Franke-Fayard B.
      • Mair G.R.
      • Lasonder E.
      • Janse C.J.
      • Mann M.
      • Waters A.P.
      Proteome analysis of separated male and female gametocytes reveals novel sex-specific Plasmodium biology.
      ). Sex specificity in this parasite is underlined by the identification of 36% of the 650 MG proteome and 19% of the 541 FG proteome as sex-specific. Remarkably, the two sexes only share 69 common proteins. Despite that conservation in gametocyte-specific gene sets across Plasmodium species foretells similarity in sex-specific biology in all malaria parasites, the distinctive features of P. falciparum gametocytes in morphology, development, and sequestration suggest significant differences in the induction, differentiation and development of the two sexes in this parasite (
      • Sinden R.E.
      The cell biology of malaria infection of mosquito: advances and opportunities.
      ,
      • Sinden R.E.
      • Carter R.
      • Drakeley C.
      • Leroy D.
      The biology of sexual development of Plasmodium: the design and implementation of transmission-blocking strategies.
      ). Efforts toward dissecting the differences in MG and FG at the molecular level are thwarted by the difficulties in clearly separating the two sexes. An attempt to identify sex-partitioned proteomes of the P. falciparum gametocytes relied heavily on species-species conservation and bioinformatic predictions (
      • Tao D.
      • Ubaida-Mohien C.
      • Mathias D.K.
      • King J.G.
      • Pastrana-Mena R.
      • Tripathi A.
      • Goldowitz I.
      • Graham D.R.
      • Moss E.
      • Marti M.
      • Dinglasan R.R.
      Sex-partitioning of the Plasmodium falciparum stage V gametocyte proteome provides insight into falciparum-specific cell biology.
      ), and thus might have missed important information underlying the true biological differences between these species. Most recently, a comprehensive proteome analysis of separated P. falciparum MG and FG coupled with transcriptome analysis was reported (
      • Lasonder E.
      • Rijpma S.R.
      • van Schaijk B.C.
      • Hoeijmakers W.A.
      • Kensche P.R.
      • Gresnigt M.S.
      • Italiaander A.
      • Vos M.W.
      • Woestenenk R.
      • Bousema T.
      • Mair G.R.
      • Khan S.M.
      • Janse C.J.
      • Bartfai R.
      • Sauerwein R.W.
      Integrated transcriptomic and proteomic analyses of P. falciparum gametocytes: molecular insight into sex-specific processes and translational repression.
      ). This study identified divergent MG and FG proteomes similar to the sex-specific proteomes in the gametocytes of P. berghei. We report here accurate separation of mature MG from FG in P. falciparum based on differential expression of the green fluorescent protein (GFP) in MG and FG using flow cytometry and in-depth analysis of the proteomes. The sex-specific proteomes of MG and FG exhibited significant difference that is consistent with the functional division of MG and FG. Compared with the P. berghei sex-specific proteomes, the P. falciparum sex-specific proteomes had an overall conservation of features as in P. berghei, while they also exhibited considerable inter-species differences which may underpin the different biology of gametocytogenesis in these species.

      EXPERIMENTAL PROCEDURES

       Parasite Culture and Gametocyte Induction

      Cultures of P. falciparum parasite lines were maintained in complete RPMI 1640 medium supplemented with 10% human serum and hypoxanthine as described previously (
      • Trager W.
      • Jensen J.B.
      Human malaria parasites in continuous culture.
      ,
      • Ponnudurai T.
      • Meuwissen J.H.
      • Leeuwenberg A.D.
      • Verhave J.P.
      • Lensen A.H.
      The production of mature gametocytes of Plasmodium falciparum in continuous cultures of different isolates infective to mosquitoes.
      ). A modified induction scheme for gametocytogenesis was used to obtain highly synchronous gametocyte cultures (
      • Wang Z.
      • Liu M.
      • Liang X.
      • Siriwat S.
      • Li X.
      • Chen X.
      • Parker D.M.
      • Miao J.
      • Cui L.
      A flow cytometry-based quantitative drug sensitivity assay for all Plasmodium falciparum gametocyte stages.
      ,
      • Fivelman Q.L.
      • McRobert L.
      • Sharp S.
      • Taylor C.J.
      • Saeed M.
      • Swales C.A.
      • Sutherland C.J.
      • Baker D.A.
      Improved synchronous production of Plasmodium falciparum gametocytes in vitro.
      ). Briefly, asexual parasites were synchronized twice in two successive asexual erythrocytic cycles by 5% d-sorbitol treatment of ring-stage parasites. Synchronous cultures at the trophozoite stage were set up at a parasitemia of 2.5–3.2% and a hematocrit of 3%. On the second day (day -2), ring-stage parasitemia typically reaches 8–12%. A half of spent medium was replaced by fresh medium. On the third day (day -1), stressed schizont cultures including spent medium were adjusted with fresh RBCs and medium to a parasitemia of ∼2% and a hematocrit of 3%. From day 1 to 3, 10 units/ml of heparin were added to the culture to block invasions of RBCs from residual contaminating asexual stages (
      • Miao J.
      • Wang Z.
      • Liu M.
      • Parker D.
      • Li X.
      • Chen X.
      • Cui L.
      Plasmodium falciparum: generation of pure gametocyte culture by heparin treatment.
      ).

       Flow Cytometry and Fluorescence-activated Cell Sorting

      A transgenic parasite line, 3D7α−tubII/GFP, where GFP expression is under the control of α-tubulin II promoter (
      • Miao J.
      • Fan Q.
      • Parker D.
      • Li X.
      • Li J.
      • Cui L.
      Puf mediates translation repression of transmission-blocking vaccine candidates in malaria parasites.
      ,
      • Wang Z.
      • Liu M.
      • Liang X.
      • Siriwat S.
      • Li X.
      • Chen X.
      • Parker D.M.
      • Miao J.
      • Cui L.
      A flow cytometry-based quantitative drug sensitivity assay for all Plasmodium falciparum gametocyte stages.
      ), was cultured and induced for synchronous gametocytes. On day 12 of induction, stage V gametocytes were purified on a 35%/50% Percoll gradient and immediately sorted on a Beckman Coulter MoFlo Astrios system at 488 nm excitation. Parasites were suspended in a warm “suspended animation buffer”, which inhibits the activation of gametocytes to gametes (
      • Ogwan'g R.
      • Mwangi J.
      • Gachihi G.
      • Nwachukwu A.
      • Roberts C.R.
      • Martin S.K.
      Use of pharmacological agents to implicate a role for phosphoinositide hydrolysis products in malaria gamete formation.
      ), and injected into the cell sorter under sterile conditions. Forward angle light scatter height and side angle light scatter height and area (used to discriminate doublets) were set at 280 V 1.0 gain and 240 V 1.0 gain photomultiplier tube (PMT) power, respectively. By gating the parasites which were negative for an irrelevant detector (664/22 at 596 V PMT) but positive on the GFP detector (513/26 at 458 V PMT), it was possible to accurately sort the high- and low-GFP parasites into individual tubes.

       Examination of Sexual Features of Sorted Gametocytes

      Sorted gametocytes were used to make thin smears, stained with Giemsa, and examined by microscopy to confirm the sexual characters of sorted gametocytes with high- and low-GFP signals (
      • Miao J.
      • Li J.
      • Fan Q.
      • Li X.
      • Cui L.
      The Puf-family RNA-binding protein PfPuf2 regulates sexual development and sex differentiation in the malaria parasite Plasmodium falciparum.
      ,
      • Talman A.M.
      • Paul R.E.
      • Sokhna C.S.
      • Domarle O.
      • Ariey F.
      • Trape J.F.
      • Robert V.
      Influence of chemotherapy on the Plasmodium gametocyte sex ratio of mice and humans.
      ). The five classic criteria are (1) males are larger than females, (2) the ends of the cells are angular in females and round in males, (3) the nucleus is smaller in females than in males, (4) the granules of malaria pigment are centrally located in females and more widely scattered in males, and (5) the cytoplasm stains deep blue in females and pale purple in males (
      • Talman A.M.
      • Paul R.E.
      • Sokhna C.S.
      • Domarle O.
      • Ariey F.
      • Trape J.F.
      • Robert V.
      Influence of chemotherapy on the Plasmodium gametocyte sex ratio of mice and humans.
      ). Sorted parasites were also used for inducing exflagellation as described previously (
      • Miao J.
      • Li J.
      • Fan Q.
      • Li X.
      • Cui L.
      The Puf-family RNA-binding protein PfPuf2 regulates sexual development and sex differentiation in the malaria parasite Plasmodium falciparum.
      ) to further confirm the high-GFP parasites as mature MG.

       Protein Extraction and Digestion

      The sorted parasites (3.5 × 106) were lysed in SDT-lysis buffer (4% SDS, 100 mm Tris/HCl pH 7.6, 0.1 m dithiothreitol - DTT) for complete dissolution (
      • Liebler D.C.
      • Ham A.J.
      Spin filter-based sample preparation for shotgun proteomics.
      ). The lysate was separated in a 10% Bis-Tris SDS-PAGE gel. Ten slices were excised and in-gel digestion was performed using a robot (ProGest, DigiLab, Marlborough, MA). Briefly, gel bands were washed using 25 mm ammonium bicarbonate followed by acetonitrile, and then were reduced using 10 mm DTT at 60 °C followed by alkylation with 50 mm iodoacetamide at room temperature. Finally, the gel bands were digested with trypsin (Promega, Madison, WI) at 37 °C for 4 h followed by peptide extraction with formic acid in acetonitrile.

       LC-MS/MS Analysis

      The extracted peptides were analyzed by nano LC/MS/MS with a Waters NanoAcquity HPLC system interfaced to a Q Exactive™ Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific). Peptides were loaded on a trapping column and eluted over a 75 μm analytical column at 350 nL/min. MS and MS/MS were performed at 70,000 FWHM and 17,500 FWHM resolution, respectively. The 15 most abundant ions were selected for MS/MS and Mascot Generic Files (MGF) from RAW files were extracted by Proteome Discoverer v1.4 (Thermo Scientific). Parasite proteins were identified by searching the Uniprot P. falciparum protein database (v01/2014, 5369 entries) concatenated with the SWISS-PROT Human database (20,160 entries). The combined database (25,529 entries) was reversed and appended back to the forward database (51,058 entries total). All peak list files (MGFs) were analyzed using the Mascot (Matrix Science; London, UK; version 2.5.1). Search parameters included trypsin digestion (C-terminal K and R cleavage) full cleavage with 2 missed sites, fixed modification: +57 on C (carbamidomethyl), variable modifications: −17 on n (Gln->pyro-Glu), +1 on NQ (deamidated), +16 on M (oxidation), +42 on n (acetyl), fragment ion mass tolerance of 0.02 Da, peptide mass tolerance of 10 ppm. Data were filtered at 1% spectra, 1% protein, 0.2% peptide false discovery rate (FDR), and at least two unique peptides per protein. FDR was calculated by counting the number of reverse entries and dividing that by the total number of forward and reverse entries. Peptides of interest were independently manually verified. Mascot DAT files were parsed into the Scaffold software for validation and filtering to create a nonredundant list per sample. Normalization of protein abundance using normalized spectral abundance factors (NSAFs) was performed based on protein sizes (
      • Pavelka N.
      • Fournier M.L.
      • Swanson S.K.
      • Pelizzola M.
      • Ricciardi-Castagnoli P.
      • Florens L.
      • Washburn M.P.
      Statistical similarities between transcriptomics and quantitative shotgun proteomics data.
      ,
      • Lundgren D.H.
      • Hwang S.I.
      • Wu L.
      • Han D.K.
      Role of spectral counting in quantitative proteomics.
      ,
      • Zybailov B.L.
      • Florens L.
      • Washburn M.P.
      Quantitative shotgun proteomics using a protease with broad specificity and normalized spectral abundance factors.
      ). The relative abundance of identified proteins was also calculated by spectral counts (
      • Lundgren D.H.
      • Hwang S.I.
      • Wu L.
      • Han D.K.
      Role of spectral counting in quantitative proteomics.
      ). The raw data and peptide information of proteomes were deposited in the ProteomeXchange via the PRIDE database (data set identifier PXD003556).

       Validation of Sex-specific Protein Expression

      To validate the sex-specific expression of nine proteins identified from the proteomic analysis, GFP was tagged to these endogenous proteins. For each gene, ∼ 1 kb fragment from the C terminus end (without the stop codon) was amplified and subcloned to fuse with the GFP and 3′ UTR region of P. berghei dhfr-ts gene, and cloned into the transfection vector pHD22Y with the human DHFR cassette (supplemental Fig. S1A, supplemental Table S1A) (
      • Fidock D.A.
      • Wellems T.E.
      Transformation with human dihydrofolate reductase renders malaria parasites insensitive to WR99210 but does not affect the intrinsic activity of proguanil.
      ,
      • Miao J.
      • Fan Q.
      • Cui L.
      • Li X.
      • Wang H.
      • Ning G.
      • Reese J.C.
      • Cui L.
      The MYST family histone acetyltransferase regulates gene expression and cell cycle in malaria parasite Plasmodium falciparum.
      ). Parasite transfection, drug selection and cloning were performed as described (
      • Miao J.
      • Li J.
      • Fan Q.
      • Li X.
      • Cui L.
      The Puf-family RNA-binding protein PfPuf2 regulates sexual development and sex differentiation in the malaria parasite Plasmodium falciparum.
      ,
      • Miao J.
      • Fan Q.
      • Cui L.
      • Li X.
      • Wang H.
      • Ning G.
      • Reese J.C.
      • Cui L.
      The MYST family histone acetyltransferase regulates gene expression and cell cycle in malaria parasite Plasmodium falciparum.
      ). Positive clones were verified by integration-specific PCR and detection of GFP expression by fluorescence microscopy (supplemental Fig. S1B, supplemental Table S1B). Gametocytes from these tagged parasite lines were examined by microscopy (
      • Miao J.
      • Li J.
      • Fan Q.
      • Li X.
      • Cui L.
      The Puf-family RNA-binding protein PfPuf2 regulates sexual development and sex differentiation in the malaria parasite Plasmodium falciparum.
      ). Because bright-field microscopy does not always clearly separate MG from FG by morphology, sex-specific expression of the GFP-fusion proteins was analyzed by sorting the GFP-positive and -negative gametocytes, or high- and low-GFP gametocytes, which were stained with Giemsa and subjected to sex identification using the sex-specific morphological characters described above.

       Experimental Design and Statistical Rationale

      Three sorting experiments were performed for the separation of MG and FG from three different gametocyte cultures, which represent three biological replicates for this study. The separated gametocytes (sample number n = 3 for low-GFP gametocytes and n = 3 for high-GFP gametocytes) were used for LC-MS/MS analysis. Spearman correlation was used to assess the correlation between different proteome profiles. The numbers of proteins and their abundance in the MG and FG proteomes were compared by Pearson's chi-square test. To compare the MG and FG proteomes in different functional groups, one-sided two-sample t test was applied. To compare the differences between shared MG and FG proteins, we analyzed the protein abundance using DESeq implemented in the R/Bioconductor package (
      • Anders S.
      • Huber W.
      Differential expression analysis for sequence count data.
      ), which test the difference between spectral counts of shared MG and FG proteins based on the negative binomial distribution, with mean and variance linked by local regression, resulting in the corresponding marginal p value.

      RESULTS

       Separation of P. falciparum Male and Female Gametocytes

      To separate P. falciparum MG from FG, we generated a transgenic 3D7 parasite line (3D7α−tubII/GFP), where GFP expression is under the control of the α-tubulin II promoter (
      • Miao J.
      • Fan Q.
      • Parker D.
      • Li X.
      • Li J.
      • Cui L.
      Puf mediates translation repression of transmission-blocking vaccine candidates in malaria parasites.
      ,
      • Wang Z.
      • Liu M.
      • Liang X.
      • Siriwat S.
      • Li X.
      • Chen X.
      • Parker D.M.
      • Miao J.
      • Cui L.
      A flow cytometry-based quantitative drug sensitivity assay for all Plasmodium falciparum gametocyte stages.
      ). The GFP expression cassette was integrated into the parasite genome and a single clone was selected to ensure homogeneous expression of GFP (
      • Wang Z.
      • Liu M.
      • Liang X.
      • Siriwat S.
      • Li X.
      • Chen X.
      • Parker D.M.
      • Miao J.
      • Cui L.
      A flow cytometry-based quantitative drug sensitivity assay for all Plasmodium falciparum gametocyte stages.
      ). No noticeable GFP expression was found in asexual stages of the 3D7α−tubII/GFP, whereas all mature gametocytes showed GFP expression with two distinct populations that differed in GFP fluorescence intensity by >10 folds (Fig. 1A). This expression pattern is consistent with a significantly higher level of expression of α-tubulin II in mature MG than in FG (
      • Schwank S.
      • Sutherland C.J.
      • Drakeley C.J.
      Promiscuous expression of alpha-tubulin II in maturing male and female Plasmodium falciparum gametocytes.
      ). Giemsa staining of the gametocytes separated by fluorescence-activated cell sorting revealed that the low- and high-GFP populations were consistent with the classic morphological characteristics of FG and MG, respectively (Fig. 1B) (
      • Miao J.
      • Li J.
      • Fan Q.
      • Li X.
      • Cui L.
      The Puf-family RNA-binding protein PfPuf2 regulates sexual development and sex differentiation in the malaria parasite Plasmodium falciparum.
      ,
      • Talman A.M.
      • Paul R.E.
      • Sokhna C.S.
      • Domarle O.
      • Ariey F.
      • Trape J.F.
      • Robert V.
      Influence of chemotherapy on the Plasmodium gametocyte sex ratio of mice and humans.
      ). In agreement with a female-biased sex ratio, stage V 3D7α−tubII/GFP gametocytes had a MG:FG ratio of ∼1:5. Activation of sorted parasites resulted in exflagellation of the gametocytes in the high-GFP population (Fig. 1C,D), further corroborating this gametocyte population as MG. Based on the difference in GFP fluorescence, we sorted equal numbers of stage-V MG and FG and used them for in-depth proteomic studies. In total, three independent experiments with a fixed number (3.5 × 106) of sorted MG and FG each were performed for proteome analysis. Because there was no GFP signal in asexual stage parasites and no or a very low number of asexual parasites existed in long-term gametocyte cultures, contamination with asexual parasites in the sorted gametocytes was rare.
      Figure thumbnail gr1
      Fig. 1Separation of P. falciparum stage V MG and FG by flow cytometry based on differential expression of the GFP reporter under the control of α-tubulin II promoter. A, Representative plot of GFP fluorescence intensities from 3D7α−tubII/GFP stage V gametocytes shows the separation of low- and high-GFP populations (circled). Stage V gametocytes were first isolated by Percoll and then analyzed by flow cytometry. FCS: Forward scattered light. B, Sorted gametocytes from low- and high-GFP populations shown in (A) stained with Giemsa show the typical characteristics of mature FG (left) and MG (right). MG appear pink with rounded cell ends and more widely scattered pigments, whereas FG appear blue with angular cell ends and more centralized pigment. C, Pictures of exflagellating gametocytes from the high-GFP population. D, Percentage of exflagellated gametocytes from the high- and low-GFP populations. Data are shown as mean ± s.d. from three replicates.

       Proteomes of Mature Male and Female Gametocytes

      We determined the proteomes of MG and FG by high-accuracy LC-MS/MS (Table I, supplemental Table S2). The proteome data from three biological replicates showed high reproducibility, with strong correlations among the MG (r = 0.68–0.86) and FG (r = 0.81–0.91) proteome profiles (supplemental Table S2A). By using a threshold of 1% protein, 0.2% peptide FDR and at least two unique peptides per protein, we identified a total of 25524 unique peptides, corresponding to 1506 proteins from the sorted stage-V gametocytes. Among them, 1244 and 1387 proteins were from MG and FG, respectively (Table I, supplemental Table S2H and S2I). The MS/MS raw date and peptides information, including peptide sequence, number of unique peptide and spectrum count, percentage sequence coverage and Mascot scores, were deposited in the ProteomeXchange via the PRIDE database (data set identifier PXD003556) for open access.
      Table ISummary of mass spectrometry data of mature male and female gametocytes in P. falciparum
      MS analysisReplicate 1Replicate 2Replicate 3Total
      MGFGMGFGMGFGMGFG
      # of total spectra count34,05843,63614,96027,69127,18534,46173,397105,415
      # of exclusive spectra count33,4744303914,74827,45326,83433,99470,33498,819
      # of unique spectra count17,51922,9497,89716,13512,89118,15922,57630,290
      # of unique peptide13,95917,3646,72612,16610,03313,34317,24021,805
      Unique proteins identified1,1171,2698071,0209081,0581,2441,387
      Compared with earlier proteomic studies in P. falciparum, our combined FG and MG proteome data set showed the strongest correlation (r = 0.71–0.80) with two recently published mature gametocyte proteomes (
      • Silvestrini F.
      • Lasonder E.
      • Olivieri A.
      • Camarda G.
      • van Schaijk B.
      • Sanchez M.
      • Younis Younis S.
      • Sauerwein R.
      • Alano P.
      Protein export marks the early phase of gametocytogenesis of the human malaria parasite Plasmodium falciparum.
      ,
      • Tao D.
      • Ubaida-Mohien C.
      • Mathias D.K.
      • King J.G.
      • Pastrana-Mena R.
      • Tripathi A.
      • Goldowitz I.
      • Graham D.R.
      • Moss E.
      • Marti M.
      • Dinglasan R.R.
      Sex-partitioning of the Plasmodium falciparum stage V gametocyte proteome provides insight into falciparum-specific cell biology.
      ) (supplemental Table S3A). The present gametocyte proteome data set showed 1099 proteins shared with earlier data sets and 97 new gametocyte proteins (supplemental Fig. S2A, supplemental Table S3B). Further comparison of the mature gametocyte proteomes with a combination of published asexual-stage proteomes of 2380 proteins led to the classification of 348 proteins as gametocyte specific (supplemental Table S3C), among which 44 were newly found in this study (supplemental Table S3D). These gametocyte-specific proteins contain many known gametocyte proteins such as Pfs25, Pfs230p, Pfs47, ABCG2, dyneins, PfNEK4, and LCCL domain-containing proteins (supplemental Table S3C) (
      • van Schaijk B.C.
      • van Dijk M.R.
      • van de Vegte-Bolmer M.
      • van Gemert G.J.
      • van Dooren M.W.
      • Eksi S.
      • Roeffen W.F.
      • Janse C.J.
      • Waters A.P.
      • Sauerwein R.W.
      Pfs47, paralog of the male fertility factor Pfs48/45, is a female specific surface protein in Plasmodium falciparum.
      ,
      • Eksi S.
      • Williamson K.C.
      Male-specific expression of the paralog of malaria transmission-blocking target antigen Pfs230, PfB0400w.
      ,
      • Tran P.N.
      • Brown S.H.
      • Mitchell T.W.
      • Matuschewski K.
      • McMillan P.J.
      • Kirk K.
      • Dixon M.W.
      • Maier A.G.
      A female gametocyte-specific ABC transporter plays a role in lipid metabolism in the malaria parasite.
      ,
      • 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.
      ).

       Sex-specific Features of Mature Gametocyte Proteomes

      Despite that abundance of each protein in MG and FG showed a high level of correlation, the total spectral counts obtained from FG were 1.4 times higher than those from MG (p > 0.05) (Table I, Fig. 2A). This is consistent with the ratio between the amounts of total proteins extracted from the same number of sorted FG and MG (supplemental Table S3E). The MG and FG protein profiles discovered in this study clearly illustrate the sex-specific biology in P. falciparum. Cluster analysis by the GO terms showed that the number of proteins in each of the 13 clusters was not significantly different between FG and MG with the exception of proteins associated with the axonemes and flagella (p < 0.01) (supplemental Fig. S2B, supplemental Table S4). However, the relative abundance of certain clusters clearly differed between the FG and MG proteomes (Fig. 2B, supplemental Table S4). The FG proteome was more abundant in apicoplast (FG 4111: MG 2781), mitochondrial (FG 5921: MG 4111) (p < 0.05), and ribosomal proteins (FG 5170: MG 2405) (p < 0.001) (supplemental Table S4). FG biases were also identified in functional groups of metabolic enzymes, proteases, chaperones, proteins associated energy generation and utility, and proteasome- and redox-associated proteins. Noteworthy, the proteases identified in both sexes include several falcipains and plasmepsins, some of which might be important for the egress of the parasite from the erythrocyte during gametogenesis (
      • Sologub L.
      • Kuehn A.
      • Kern S.
      • Przyborski J.
      • Schillig R.
      • Pradel G.
      Malaria proteases mediate inside-out egress of gametocytes from red blood cells following parasite transmission to the mosquito.
      ,
      • Wirth C.C.
      • Pradel G.
      Molecular mechanisms of host cell egress by malaria parasites.
      ). In contrast, all proteins associated with DNA replication were enriched in the MG proteome (p < 0.001) (supplemental Table S4). Although the majority of proteins associated with axonemes were found in MG only or enriched in MG, the overall protein abundance was even slightly higher in FG because of a particular dynein (PF3D7_0729900), which was highly abundant in the FG proteome (supplemental Table S4).
      Figure thumbnail gr2
      Fig. 2Comparison between the P. falciparum MG and FG proteomes. A, Correlation of protein abundance (log2 transformed spectral counts) between MG and FG. The regression line (dashed line) indicates the degree of similarity in the abundance of individual proteins between MG and FG. Two paralleling lines above and below the diagonal line mark the boundaries of 2 fold difference in protein abundance in MG and FG, respectively. The color scale indicates enrichment in MG (positive values) and FG (negative values), respectively. B, Relative abundance of proteins in each GO category in the MG and FG proteomes. Protein abundance is shown in spectral count (SC). *, **, and *** indicate significant differences between MG and FG at p = 0.05, 0.01, and 0.001, respectively.
      To further dissect the sex dichotomy in protein expression, detailed comparison was performed on MG- and FG-specific proteins (proteins identified only in one sex). Comparison of the MG and FG proteomes identified 119 (10%) and 262 (19%) proteins as MG- and FG-specific, respectively, and 1125 as shared proteins between the two sexes (Fig. 3A, supplemental Table S5A–S5C). After filtering out proteins also expressed in asexual stages by comparing the gametocyte proteomes with the proteome from synchronous trophozoites of the gametocyte-less parasite clone F12 (
      • Silvestrini F.
      • Lasonder E.
      • Olivieri A.
      • Camarda G.
      • van Schaijk B.
      • Sanchez M.
      • Younis Younis S.
      • Sauerwein R.
      • Alano P.
      Protein export marks the early phase of gametocytogenesis of the human malaria parasite Plasmodium falciparum.
      ), 91 (7.3%) and 171 (12%) proteins were left as gametocyte-specific that are expressed in MG and FG only, and 349 as shared gametocyte-specific proteins between the two sexes (Fig. 3B, supplemental Table S5D–S5F).
      Figure thumbnail gr3
      Fig. 3P. falciparum sex-specific proteomes of mature gametocytes. A, Comparison of the P. falciparum sex-specific proteomes from mature MG (GFPtub MG) and FG (GFPtub FG). B, Comparison of the P. falciparum sex-specific proteomes from mature MG and FG and asexual proteome from synchronous trophozoites of the gametocyte-less clone F12 (FS 3D7 Trophozoite) (Silvestrini et al. 2010, ref.
      • Silvestrini F.
      • Alano P.
      • Williams J.L.
      Commitment to the production of male and female gametocytes in the human malaria parasite Plasmodium falciparum.
      ). C, Functional differences between MG- and FG-specific proteins in protein abundance shown as percentiles in spectral count (SC). *** indicates significant differences between MG and FG at p = 0.001.
      In agreement with the biology of MG in the preparation for rapid DNA replication and formation of motile gametes, the MG-specific proteome (119 proteins) contains significantly more proteins associated with DNA replication and the axonemes (p < 0.001). Specifically, nine proteins are potentially involved in DNA replication, whereas ten proteins are associated with motility. In comparison, the FG-specific proteome (262 proteins) contains significantly more proteases and proteins involved in translation in both rankings of protein numbers and their spectral counts (p < 0.05, Fig. 3C and supplemental Fig. S2C), which are in accordance with higher abundance of ribosomes in FG. Although the numbers of protein kinases and phosphatases were similar between the two sex-specific proteomes, their relative abundance in spectral count was significantly higher in FG (p < 0.001). In addition, significantly more mitochondrial and apicoplast proteins were identified in the FG-specific proteome (p < 0.05) (Fig. 3C, supplemental Table S5A and S5B). Gametocyte-specific, MG- and FG-specific proteins (Fig. 3C) exhibited similar sex-specific features as shown above (supplemental Table S5D and S5E).
      We also compared the MG and FG proteomes to obtain a more conserved data set of proteins that showed biased expression in one sex. The relative abundance of individual protein levels was determined by fold change in spectral counts between the two sexes (supplemental Table S5C). We used 2-fold cutoff as it corresponded to ∼96.5 percentile of the MG/FG ratio. Fifteen proteins associated with DNA replication were among the top 40 proteins with > 2-fold higher abundance in MG than in FG. In comparison, 392 proteins showed > 2-fold higher abundance in FG than in MG, which include proteins associated with translation, transcription, proteasome, redox system, and enzymes in the metabolic pathways (supplemental Table S5C). Interestingly, the RNA-binding protein PfPuf1, all components of DOZI complex found in P. berghei, two epigenetic regulators Sir2A and protein arginine methyltransferase 1 (PRMT1) were also enriched in FG (supplemental Table S5C and S5G). Altogether, 159 MG-specific/biased proteins and 654 FG-specific/biased proteins were identified in this study.

       Confirmation of Expression of Selected Proteins by GFP Tagging

      Based on the results of this study and comparison with the P. berghei gametocyte proteomes, we selected three MG-specific/biased and six FG-biased genes based on their spectral counts for experimental validation. The criteria used for the selection include (1) male-specific proteins identified here not yet validated by GFP tagging before, (2) female-biased proteins shown as female-specific proteins in the P. berghei proteome data set, (3) female-biased proteins shown as male-specific proteins in the P. berghei proteome data set, and (4) proteins newly identified only in our data set as compared with the P. berghei data set. Using a single-crossover strategy, we tagged each of the nine selected proteins with GFP at the C terminus (Fig. 4). Stable integration of the plasmids at endogenous loci of the respective genes was confirmed by integration-specific PCR (supplemental Fig. S1, supplemental Table S1). GFP-positive and -negative mature gametocytes or gametocytes with high and low GFP intensities from these tagged parasite lines were sorted in order to identify their sex by Giemsa staining (supplemental Fig. S3). Consistent with the proteomic data, PF3D7_1122900 (dynein heavy chain) and PF3D7_0208900 (P230p) were only detected in MG, whereas PF3D7_0219600 (replication factor C subunit 1) was predominantly expressed in MG (Fig. 4). The expression patterns of these three MG-specific or -biased proteins were consistent with those of their orthologs in P. berghei (
      • Khan S.M.
      • Franke-Fayard B.
      • Mair G.R.
      • Lasonder E.
      • Janse C.J.
      • Mann M.
      • Waters A.P.
      Proteome analysis of separated male and female gametocytes reveals novel sex-specific Plasmodium biology.
      ,
      • Tao D.
      • Ubaida-Mohien C.
      • Mathias D.K.
      • King J.G.
      • Pastrana-Mena R.
      • Tripathi A.
      • Goldowitz I.
      • Graham D.R.
      • Moss E.
      • Marti M.
      • Dinglasan R.R.
      Sex-partitioning of the Plasmodium falciparum stage V gametocyte proteome provides insight into falciparum-specific cell biology.
      ). Among the six FG-biased proteins selected, only DOZI was consistently enriched in the FG proteomes of both parasite species, whereas others showed incompatible expression patterns between the two species (Fig. 4). GFP-tagged PfCCP1 (PF3D7_1475500), a LCCL domain-containing protein, showed 1.5-fold enrichment in FG in P. falciparum (Fig. 4), whereas its ortholog was only detected in the P. berghei FG (
      • Khan S.M.
      • Franke-Fayard B.
      • Mair G.R.
      • Lasonder E.
      • Janse C.J.
      • Mann M.
      • Waters A.P.
      Proteome analysis of separated male and female gametocytes reveals novel sex-specific Plasmodium biology.
      ). Recent re-analysis of the P. berghei sex-specific proteomes revealed over 100-fold enrichment of this protein in FG (
      • Tao D.
      • Ubaida-Mohien C.
      • Mathias D.K.
      • King J.G.
      • Pastrana-Mena R.
      • Tripathi A.
      • Goldowitz I.
      • Graham D.R.
      • Moss E.
      • Marti M.
      • Dinglasan R.R.
      Sex-partitioning of the Plasmodium falciparum stage V gametocyte proteome provides insight into falciparum-specific cell biology.
      ). The punctuate GFP foci of PfCCP1-GFP associated with the surface of mature gametocytes were concordant with the pattern previously described by using the anti-PfCCP1 antibodies (
      • Pradel G.
      • Hayton K.
      • Aravind L.
      • Iyer L.M.
      • Abrahamsen M.S.
      • Bonawitz A.
      • Mejia C.
      • Templeton T.J.
      A multidomain adhesion protein family expressed in Plasmodium falciparum is essential for transmission to the mosquito.
      ,
      • Scholz S.M.
      • Simon N.
      • Lavazec C.
      • Dude M.A.
      • Templeton T.J.
      • Pradel G.
      PfCCp proteins of Plasmodium falciparum: gametocyte-specific expression and role in complement-mediated inhibition of exflagellation.
      ). Similarly, PF3D7_0217500 (calcium-dependent protein kinase 1, CDPK1) and PF3D7_0503400 (actin-depolymerizing factor 1) displayed 2.9- and 3.4-fold enrichment in FG in P. falciparum, respectively (Fig. 4), whereas their P. berghei orthologs were identified as weakly MG-biased in the P. berghei proteomes (
      • Khan S.M.
      • Franke-Fayard B.
      • Mair G.R.
      • Lasonder E.
      • Janse C.J.
      • Mann M.
      • Waters A.P.
      Proteome analysis of separated male and female gametocytes reveals novel sex-specific Plasmodium biology.
      ,
      • Tao D.
      • Ubaida-Mohien C.
      • Mathias D.K.
      • King J.G.
      • Pastrana-Mena R.
      • Tripathi A.
      • Goldowitz I.
      • Graham D.R.
      • Moss E.
      • Marti M.
      • Dinglasan R.R.
      Sex-partitioning of the Plasmodium falciparum stage V gametocyte proteome provides insight into falciparum-specific cell biology.
      ). Myosin C (PF3D7_1329100) showed 4.1-fold enrichment in FG (Fig. 4), but its ortholog was not identified in the P. berghei proteome. Given that the present and earlier proteomic studies detected the major ookinete surface protein Pfs25 in gametocytes (
      • Silvestrini F.
      • Bozdech Z.
      • Lanfrancotti A.
      • Di Giulio E.
      • Bultrini E.
      • Picci L.
      • Derisi J.L.
      • Pizzi E.
      • Alano P.
      Genome-wide identification of genes upregulated at the onset of gametocytogenesis in Plasmodium falciparum.
      ,
      • Tao D.
      • Ubaida-Mohien C.
      • Mathias D.K.
      • King J.G.
      • Pastrana-Mena R.
      • Tripathi A.
      • Goldowitz I.
      • Graham D.R.
      • Moss E.
      • Marti M.
      • Dinglasan R.R.
      Sex-partitioning of the Plasmodium falciparum stage V gametocyte proteome provides insight into falciparum-specific cell biology.
      ), we evaluated its expression by GFP-tagging, and confirmed that Pfs25 protein is enriched in FG (Fig. 4), whereas re-analysis of the P. berghei sex-specific proteomes identified one peptide of P25 in FG. Collectively, GFP-tagging of endogenous genes confirmed the accuracy and specificity of our P. falciparum gametocyte-specific proteomes.
      Figure thumbnail gr4
      Fig. 4Experimental evaluation of sex-specific/-biased protein expression of nine selected genes in mature MG and FG. Three MG-specific or biased proteins and six FG-biased proteins were tagged at their C termini with GFP. Numbers are the spectral counts of the respective proteins identified in P. falciparum (left) and P. berghei (right) re-analyzed MG and FG proteomes (Khan et al. 2005, ref.
      • Khan S.M.
      • Franke-Fayard B.
      • Mair G.R.
      • Lasonder E.
      • Janse C.J.
      • Mann M.
      • Waters A.P.
      Proteome analysis of separated male and female gametocytes reveals novel sex-specific Plasmodium biology.
      ; Tao et al., 2014, ref.
      • Tao D.
      • Ubaida-Mohien C.
      • Mathias D.K.
      • King J.G.
      • Pastrana-Mena R.
      • Tripathi A.
      • Goldowitz I.
      • Graham D.R.
      • Moss E.
      • Marti M.
      • Dinglasan R.R.
      Sex-partitioning of the Plasmodium falciparum stage V gametocyte proteome provides insight into falciparum-specific cell biology.
      ). BF, bright field.

       Species-specific Features of Gametocyte Proteomes

      A previous study has determined the sex-specific proteomes from P. berghei (
      • Khan S.M.
      • Franke-Fayard B.
      • Mair G.R.
      • Lasonder E.
      • Janse C.J.
      • Mann M.
      • Waters A.P.
      Proteome analysis of separated male and female gametocytes reveals novel sex-specific Plasmodium biology.
      ). Re-analysis of the P. berghei sex-specific proteomes using updated genome annotation and an advanced MS search engine has expanded the list of identified proteins from 779 to 1546, of which 1180 and 1128 proteins were from FG and MG, respectively (
      • Tao D.
      • Ubaida-Mohien C.
      • Mathias D.K.
      • King J.G.
      • Pastrana-Mena R.
      • Tripathi A.
      • Goldowitz I.
      • Graham D.R.
      • Moss E.
      • Marti M.
      • Dinglasan R.R.
      Sex-partitioning of the Plasmodium falciparum stage V gametocyte proteome provides insight into falciparum-specific cell biology.
      ). Surprisingly, this re-analysis resulted in the assignments of 366 and 418 proteins as MG- and FG-specific, respectively, with the switching of 100 and 85 originally identified MG- and FG-specific proteins to shared proteins, respectively (
      • Tao D.
      • Ubaida-Mohien C.
      • Mathias D.K.
      • King J.G.
      • Pastrana-Mena R.
      • Tripathi A.
      • Goldowitz I.
      • Graham D.R.
      • Moss E.
      • Marti M.
      • Dinglasan R.R.
      Sex-partitioning of the Plasmodium falciparum stage V gametocyte proteome provides insight into falciparum-specific cell biology.
      ) (Fig. 5A). In addition, 762 (49.3%) were shared proteins between the two sexes in P. berghei as compared with 1125 (74.7%) in P. falciparum. As such, many original sex-specific proteins were changed to sex-enriched. Of the 10 sex-specific proteins originally defined as male-specific (six) and female-specific (four), re-analysis of the P. berghei data classified them as male- and female-enriched, respectively (
      • Tao D.
      • Ubaida-Mohien C.
      • Mathias D.K.
      • King J.G.
      • Pastrana-Mena R.
      • Tripathi A.
      • Goldowitz I.
      • Graham D.R.
      • Moss E.
      • Marti M.
      • Dinglasan R.R.
      Sex-partitioning of the Plasmodium falciparum stage V gametocyte proteome provides insight into falciparum-specific cell biology.
      ). Interestingly, our P. falciparum proteomes revealed that four proteins are in agreement with the re-analyzed P. berghei data as shared proteins and four proteins are consistent with the original assignment as sex-specific in the P. berghei data (supplemental Table S6A). Re-analysis of the P. berghei sex-specific proteomes also drastically changed the assignment of nine sex-specific proteins from one sex to the opposite sex (
      • Tao D.
      • Ubaida-Mohien C.
      • Mathias D.K.
      • King J.G.
      • Pastrana-Mena R.
      • Tripathi A.
      • Goldowitz I.
      • Graham D.R.
      • Moss E.
      • Marti M.
      • Dinglasan R.R.
      Sex-partitioning of the Plasmodium falciparum stage V gametocyte proteome provides insight into falciparum-specific cell biology.
      ). The assignment of seven of these proteins is consistent with that in our P. falciparum proteomes (supplemental Table S6B).
      Figure thumbnail gr5
      Fig. 5Comparison of MG and FG proteomes between P. falciparum and P. berghei. A, Comparison of the MG and FG proteomes between P. falciparum and P. berghei. B, Partitioning of the 611 proteins present in both species and in both sexes. Proteins showing opposite sex-partitioning in P. falciparum and P. berghei are labeled as “inconsistent”. C, Sex-specific proteins in one species but shared proteins in the other species. Proteins were grouped as MG- and FG-specific in P. falciparum but shared in both sexes of P. berghei (Pf-M/Pb-Co and Pf-F/Pb-Co, respectively), as well as MG- and FG-specific in P. berghei but shared in both sexes of P. falciparum (Pb-M/Pf-Co and Pb-F/Pf-Co, respectively). Different colors indicate relative protein abundance in different sexes of P. berghei (Pb-Co) or P. falciparum (Pf-Co). D, Comparison of gametocyte proteomes between P. falciparum and P. berghei.
      To identify the functional similarity as well as evolutionary divergence between the two species in their sex-specific proteomes, we performed a comprehensive comparison of the MG and FG proteomes between P. berghei and P. falciparum (Fig. 5A). MG-specific proteins in P. falciparum (119 proteins) and P. berghei (366 proteins) can be divided into three groups: shared (51 proteins), species-specific (55 in P. falciparum and 252 in P. berghei) and MG-specific in one species but not in the other (13 from P. falciparum and 63 from P. berghei) (Fig. 5A, supplemental Table S7A–S7E). Likewise, FG-specific proteins in P. falciparum (262 proteins) and P. berghei (418 proteins) can be divided into three groups: shared (34 proteins), specie-specific (199 in P. falciparum and 179 in P. berghei), and FG-specific in one species but not in the other (29 from P. falciparum and 205 from P. berghei) (Fig. 5A, supplemental Table S7F–S7J). Comparison of the shared proteins of both sexes between P. falciparum (1125) and P. berghei (762) identified 611 common proteins present in both species and in both sexes, as well as 246 and 109 species-specific proteins shared between the two sexes in P. falciparum and P. berghei, respectively (Fig. 5A, supplemental Table S7K–S7M). Further details of the analysis are as follows.

       I. Core Sex-Specific Proteins in Both Species

      As shown in Fig. 5A, the two species share only 51 and 34 proteins as MG- and FG-specific proteins. The shared MG-specific proteins contain a number of proteins known as male-specific such as P230p and mitogen-activated protein kinase 2 (MAPK2), and those functioning in motility (six dyneins, six myosins, one kinesin, and one actin) and DNA replication (supplemental Table S7A). The shared FG-specific proteins contain two ribosome-associated proteins, two transcription-associated proteins, three cytoskeleton-associated proteins (two dyneins and one myosin), and six mitochondrion/aplicoplast associated proteins (supplemental Table S7F). The rest of the core sex-specific proteins largely contain hypothetical proteins with unknown function, which may play important roles in sex-specific biology. These shared MG- and FG-specific proteins should represent the core sex-specific proteins in both species.

       II. Shared Proteins between the Two Sexes and Also in Both Species

      Totally 611 proteins, probably with house-keeping importance, are shared between both sexes and are also common in both species (Fig. 5A, supplemental Table S7K). Using spectral counts as the proxy for protein abundance, we further divided them into three groups as MG-enriched, FG-enriched and equally shared proteins. Of these proteins, 445 (72.8%) show the same sex partitioning in both species (Fig. 5B, supplemental Table S7K), among which 32 (7.2%), 13 (2.9%) and 400 (89.9%) are MG-enriched, equally shared and FG-enriched proteins, respectively. In addition, 21 of the 32 MG-enriched proteins in both species are associated with DNA replication and chromosome regulation, whereas 276 from 400 FG-enriched proteins in both species are associated with translation, transcription, proteasome, redox system, and enzymes in metabolic pathways. The osmiophilic body protein (G377), protein arginine methyltransferase 1 (PRMT1) and 8 of the 11 components of DOZI complex are also in this FG-enriched proteins list (supplemental Table S7K). Such sex-enrichment reflects a similar sex partition in both species.

       III. Sex-Specific Proteins Present Only in One Species

      13 proteins from P. falciparum and 63 proteins from P. berghei are MG-specific only in one species but are shared proteins in the other (Fig. 5A, supplemental Table S7D and E). Likewise, 29 and 205 proteins are FG-specific only in one species (Fig. 5A, supplemental Table S7I and J). Detailed analysis revealed that 11 of 13 MG-specific proteins in P. falciparum are MG-enriched proteins in P. berghei. Likewise, 21 of 63 MG-specific proteins in P. berghei are MG-enriched proteins in P. falciparum, whereas 21 of 29 FG-specific proteins in P. falciparum are FG-enriched proteins in P. berghei, and 172 of 205 FG-specific proteins in P. berghei are FG-enriched proteins in P. falciparum (Fig. 5C).

       IV. Species-specific Proteins

      Despite the high degree of conservation in sex-specific proteomes shown above, comparison between the re-analyzed P. berghei gametocyte proteomes of 1546 proteins and the P. falciparum mature gametocyte proteomes of 1506 proteins revealed 480 proteins from P. falciparum and 520 proteins from P. berghei as species-specific proteins (Fig. 5D, supplemental Table S8). Seventy eight from 480 in P. falciparum and 64 from 520 in P. berghei were identified as unique proteins in one species with no syntenic orthologs in the other species (Fig. 5D, supplemental Table S8A and S8B). Many of these unique proteins were exported proteins such as the nine PfGXPs (P. falciparum gametocyte-exported proteins), eight PHIST in P. falciparum, and four BIR proteins and seven Plasmodium exported proteins in P. berghei. Thirty one of 64 P. berghei- and 37 of 78 P. falciparum-unique proteins are encoded by genes located in the subtelomeric regions, consistent with the fact that the majority of the differences between the two species lies in the subtelomeric regions (
      • Hall N.
      • Karras M.
      • Raine J.D.
      • Carlton J.M.
      • Kooij T.W.
      • Berriman M.
      • Florens L.
      • Janssen C.S.
      • Pain A.
      • Christophides G.K.
      • James K.
      • Rutherford K.
      • Harris B.
      • Harris D.
      • Churcher C.
      • Quail M.A.
      • Ormond D.
      • Doggett J.
      • Trueman H.E.
      • Mendoza J.
      • Bidwell S.L.
      • Rajandream M.A.
      • Carucci D.J.
      • Yates 3rd, J.R.
      • Kafatos F.C.
      • Janse C.J.
      • Barrell B.
      • Turner C.M.
      • Waters A.P.
      • Sinden R.E.
      A comprehensive survey of the Plasmodium life cycle by genomic, transcriptomic, and proteomic analyses.
      ). Other unique proteins include Pfs27, Pfs16, Pf11–1in P. falciparum, and intra-erythrocytic stage-induced structure protein 1, tryptophan-rich antigen in P. berghei.
      Classification of the above species-specific proteins by sex specificity resulted in different proportions of proteins in each species that are classified as MG- and FG-specific, suggesting that the two species have considerable divergence in the sex-specific proteomes (Fig. 5A). A detailed comparison identified several biological groups representing clear divergence between these two species. A striking difference between the two species lies in the detection of 13 cytoskeletal proteins (myosins and inner membrane complex proteins) in mature P. falciparum gametocytes but not in P. berghei gametocytes, which likely represent the building components of the subpellicular membrane complex responsible for the unique crescent shape of the P. falciparum gametocyte (Table II) (
      • Dixon M.W.
      • Dearnley M.K.
      • Hanssen E.
      • Gilberger T.
      • Tilley L.
      Shape-shifting gametocytes: how and why does P. falciparum go banana-shaped?.
      ,
      • Dearnley M.K.
      • Yeoman J.A.
      • Hanssen E.
      • Kenny S.
      • Turnbull L.
      • Whitchurch C.B.
      • Tilley L.
      • Dixon M.W.
      Origin, composition, organization and function of the inner membrane complex of Plasmodium falciparum gametocytes.
      ,
      • Kono M.
      • Herrmann S.
      • Loughran N.B.
      • Cabrera A.
      • Engelberg K.
      • Lehmann C.
      • Sinha D.
      • Prinz B.
      • Ruch U.
      • Heussler V.
      • Spielmann T.
      • Parkinson J.
      • Gilberger T.W.
      Evolution and architecture of the inner membrane complex in asexual and sexual stages of the malaria parasite.
      ,
      • Hliscs M.
      • Millet C.
      • Dixon M.W.
      • Siden-Kiamos I.
      • McMillan P.
      • Tilley L.
      Organization and function of an actin cytoskeleton in Plasmodium falciparum gametocytes.
      ). Other substantial differences in species-specific proteomes between these two species were identified in biological processes such as protein degradation, lipid metabolism and transcriptional regulators (supplemental Table S8D). P. falciparum species-specific proteomes contains a greater number (twelve) of unique proteins associated with ubiquitin-related degradation as compared with three in P. berghei (supplemental Table S8D). Interestingly, the P. falciparum species-specific proteome identified plasmepsin I-III, FP2a and FP3, subtilisin-like protease 2 (SUB2) and dipeptidyl aminopeptidase 2 (DPAP2), whereas the P. berghei species-specific proteomes revealed SUB1 and SERA1–4 (supplemental Table S8D). Previous studies showed that SUB1, SERAs, plasmepsin II, DPAP 3 and FP2 were involved in parasite egress at the schizont stage (
      • Wirth C.C.
      • Pradel G.
      Molecular mechanisms of host cell egress by malaria parasites.
      ), and exflagellation of activated P. berghei and P. falciparum gametocytes can be blocked by the cysteine/serine protease inhibitors (
      • Sologub L.
      • Kuehn A.
      • Kern S.
      • Przyborski J.
      • Schillig R.
      • Pradel G.
      Malaria proteases mediate inside-out egress of gametocytes from red blood cells following parasite transmission to the mosquito.
      ). These different proteases in both species suggest that there might be different degradation pathways in mature gametocytes for egress. P. falciparum species-specific proteome contains a significantly greater number (twenty-four) of proteins associated with lipid metabolism than in P. berghei (seven) (supplemental Table S8D). These include PfABCG2, two acyl-CoA synthetases, proteins associated with de novo fatty acid synthesis, and enzymes associated with signaling lipid metabolism, which is in agreement with the finding that in P. falciparum there is a high demand for lipids at the gametocyte stage (
      • Tran P.N.
      • Brown S.H.
      • Rug M.
      • Ridgway M.C.
      • Mitchell T.W.
      • Maier A.G.
      Changes in lipid composition during sexual development of the malaria parasite Plasmodium falciparum.
      ,
      • Gulati S.
      • Ekland E.H.
      • Ruggles K.V.
      • Chan R.B.
      • Jayabalasingham B.
      • Zhou B.
      • Mantel P.Y.
      • Lee M.C.
      • Spottiswoode N.
      • Coburn-Flynn O.
      • Hjelmqvist D.
      • Worgall T.S.
      • Marti M.
      • Di Paolo G.
      • Fidock D.A.
      Profiling the Essential Nature of Lipid Metabolism in Asexual Blood and Gametocyte Stages of Plasmodium falciparum.
      ) and a need of de novo fatty acid synthesis for subsequent development in the free-living environment in a mosquito (
      • van Schaijk B.C.
      • Kumar T.R.
      • Vos M.W.
      • Richman A.
      • van Gemert G.J.
      • Li T.
      • Eappen A.G.
      • Williamson K.C.
      • Morahan B.J.
      • Fishbaugher M.
      • Kennedy M.
      • Camargo N.
      • Khan S.M.
      • Janse C.J.
      • Sim K.L.
      • Hoffman S.L.
      • Kappe S.H.
      • Sauerwein R.W.
      • Fidock D.A.
      • Vaughan A.M.
      Type II fatty acid biosynthesis is essential for Plasmodium falciparum sporozoite development in the midgut of Anopheles mosquitoes.
      ). Comparison with the recent P. falciparum sex-specific/biased proteomes largely confirmed these species-specific differences (supplemental Table S8D) (
      • Lasonder E.
      • Rijpma S.R.
      • van Schaijk B.C.
      • Hoeijmakers W.A.
      • Kensche P.R.
      • Gresnigt M.S.
      • Italiaander A.
      • Vos M.W.
      • Woestenenk R.
      • Bousema T.
      • Mair G.R.
      • Khan S.M.
      • Janse C.J.
      • Bartfai R.
      • Sauerwein R.W.
      Integrated transcriptomic and proteomic analyses of P. falciparum gametocytes: molecular insight into sex-specific processes and translational repression.
      ).
      Table IIUnique cytoskeleton-associated proteins in P. falciparum gametocyte proteome
      Gene IDMale SC
      SC: spectral count.
      Female SCProtein Name
      PF3D7_13426001814myosin A (MyoA)
      PF3D7_132910033136myosin C (MyoC)
      PF3D7_11107002547actin-like protein, putative (ALP1)
      PF3D7_052260052inner membrane complex protein
      PF3D7_13237002359glideosome associated protein with multiple membrane spans 1 (GAPM1)
      PF3D7_146060025inner membrane complex sub-compartment protein 3, putative (ISP3)
      PF3D7_12227004960glideosome-associated protein 45 (GAP45)
      PF3D7_07080001638cytoskeleton associated protein, putative
      PF3D7_14531005dynactin subunit 4, putative
      PF3D7_03194002kinesin-8, putative
      PF3D7_051400010tubulin–tyrosine ligase, putative (TTL)
      PF3D7_09069103tubulin-specific chaperone, putative
      PF3D7_11159002palmitoyltransferase, putative (DHHC9)
      a SC: spectral count.
      Although both parasite species contain a similar number of species-specific transcriptional regulators, their expression profiles differ significantly (supplemental Table S8D). Specifically, P. falciparum-specific proteome contains three AP2 proteins in relative higher abundance than the only one AP2 in P. berghei. Of the six LCCL domain-containing proteins, three (PbCCp2, PbCCp4 and PbFNPA) were not identified in P. berghei, whereas all PfCCps except PfCCp4 were identified in P. falciparum (supplemental Table S8D). This is in agreement with previous studies, which showed translational repression of PbCCps in P. berghei but not in P. falciaprum (
      • Saeed S.
      • Carter V.
      • Tremp A.Z.
      • Dessens J.T.
      Translational repression controls temporal expression of the Plasmodium berghei LCCL protein complex.
      ,
      • Simon N.
      • Scholz S.M.
      • Moreira C.K.
      • Templeton T.J.
      • Kuehn A.
      • Dude M.A.
      • Pradel G.
      Sexual stage adhesion proteins form multi-protein complexes in the malaria parasite Plasmodium falciparum.
      ). Taken together, these results highlighted considerable differences in the gametocyte proteomes between these two parasite species.
      An earlier effort, based on subtraction between the proteomes of normal and male-defective P. falciparum gametocytes, and P. berghei sex-specific proteomes under the assumption of a high-degree conservation between the P. falciparum and P. berghei sex-specific proteomes, predicted 258 FG- and 174 MG-specific proteins in P. falciparum gametocytes (supplemental Fig. S4A and S4B) (
      • Tao D.
      • Ubaida-Mohien C.
      • Mathias D.K.
      • King J.G.
      • Pastrana-Mena R.
      • Tripathi A.
      • Goldowitz I.
      • Graham D.R.
      • Moss E.
      • Marti M.
      • Dinglasan R.R.
      Sex-partitioning of the Plasmodium falciparum stage V gametocyte proteome provides insight into falciparum-specific cell biology.
      ). However, of these proteins, only 21 and 38 are found in the 262 FG- and 119 MG-specific proteins in P. falciparum, respectively (supplemental Fig. S4A and S4B, supplemental Table S9A and S9B). Interestingly, these common proteins are all found in the 51 and 34 shared sex-specific proteins between P. berghei and P. falciparum (Fig. 5A), indicating that the earlier deduction-based approach only captured the conserved, putative core sex-specific proteins in the malaria parasites. However, 189 FG- and 56 MG-specific proteins deduced in the earlier approach are found in the shared proteins between MG and FG proteomes in our analysis (supplemental Fig. S4C). Additionally, 41 FG- and 39 MG-specific proteins from this earlier prediction are in different sex partitions in our proteomes, whereas 148 FG- and 17 MG-specific proteins are in the same sex-specific partitions (supplemental Fig. S4D, supplemental Table S9A and S9B).

       Sex-specific/biased Proteins in Plasmodium - Lasonder et al

      recently separated mature MG and FG based on marker expression under the male-specific protein dynein heavy chain (PF3D7_1023100) and the female-specific protein Pfs47 and identified 1960 proteins (at least two unique peptides per protein) (
      • Lasonder E.
      • Rijpma S.R.
      • van Schaijk B.C.
      • Hoeijmakers W.A.
      • Kensche P.R.
      • Gresnigt M.S.
      • Italiaander A.
      • Vos M.W.
      • Woestenenk R.
      • Bousema T.
      • Mair G.R.
      • Khan S.M.
      • Janse C.J.
      • Bartfai R.
      • Sauerwein R.W.
      Integrated transcriptomic and proteomic analyses of P. falciparum gametocytes: molecular insight into sex-specific processes and translational repression.
      ). This combined FG and MG proteome data set showed a strong correlation (r = 0.77) with our combined proteome. The FG proteome showed even stronger correlation with our FG proteome (r = 0.81), however, correlation was relatively low between these two MG proteomes (r = 0.56). This protein data set and our present MG and FG data set shared 1338 proteins (supplemental Fig. S5A). Together with the re-analyzed P. berghei gametocyte proteomes (
      • Tao D.
      • Ubaida-Mohien C.
      • Mathias D.K.
      • King J.G.
      • Pastrana-Mena R.
      • Tripathi A.
      • Goldowitz I.
      • Graham D.R.
      • Moss E.
      • Marti M.
      • Dinglasan R.R.
      Sex-partitioning of the Plasmodium falciparum stage V gametocyte proteome provides insight into falciparum-specific cell biology.
      ), the three data sets shared 986 proteins. Here we used these three gametocyte proteome data sets based on sorted MG and FG to derive high-confidence lists of sex-specific/biased proteins. Among these shared proteins, our data set and the P. berghei data set had 99 and 219 proteins showing ≥ 2-fold higher expression in MG, and 321 and 577 proteins showing ≥ 2-fold higher expression in FG. Using proteins showing biased expression in the three data sets, we obtained a high-confidence list of 73 MG-specific/biased and 89 FG-specific/biased proteins, which represent the core MG- and FG-specific/biased proteins in Plasmodium (supplemental Fig. S5B and C, supplemental Table S9C and S9D).
      These core MG- and FG-biased protein lists clearly show the sex-specific biology of Plasmodium. In the 73 core MG-specific/biased proteome, 33 and 26% of proteins are involved in DNA replication and motility/cytoskeleton, respectively, whereas in the 89 core FG-specific/biased proteome, there is a significant enrichment of proteins associated with translation, proteasome, mitochondrion/apicoplast, transport and redox (supplemental Fig. S5D and S5E).

      DISCUSSION

      Malaria parasites with the same set of haploid genome can differentiate into two drastically different sexual forms that are needed for continued transmission through mosquitoes. A thorough understanding of the differences between the two sexes in the human malaria parasite P. falciparum is hindered by the difficulties in accurately separating the two sexes and thus relied heavily on the only known sex-specific proteomes of P. berghei gametocytes. Here, by using a parasite line 3D7α−tubII/GFP that produces two distinct populations of gametocytes differing in GFP fluorescence intensity, we were able to clearly separate P. falciparum mature MG and FG by flow cytometry. With three highly reproducible replicates of ∼3.5 × 106 sorted MG and FG each and using highly stringent protein identification criteria after LC-MS/MS analysis, we identified 1506 proteins from 178,812 spectra, which account for ∼28% of the predicted proteins encoded by the parasite genome and constitute the first comprehensive sex-specific proteomes of accurately separated mature gametocytes in P. falciparum. This sex-specific P. falciparum gametocyte proteome, with about 2-fold higher total spectral counts and unique peptides than the re-analyzed P. berghei sex-specific proteomes (
      • Tao D.
      • Ubaida-Mohien C.
      • Mathias D.K.
      • King J.G.
      • Pastrana-Mena R.
      • Tripathi A.
      • Goldowitz I.
      • Graham D.R.
      • Moss E.
      • Marti M.
      • Dinglasan R.R.
      Sex-partitioning of the Plasmodium falciparum stage V gametocyte proteome provides insight into falciparum-specific cell biology.
      ), provides much increased depth and accuracy of protein identification and a better understanding of the sex-specific biology in this parasite. Whereas this analysis identified significant differences between the two sexes that are consistent with their functional divisions, it also identified substantial sharing of proteins in both sexes with 1125 proteins (74.7% of all P. falciparum gametocyte proteins) being identified in the 1244 MG proteome and the 1387 FG proteome. By excluding proteins that are also expressed in asexual stages, 91, 171, and 349 gametocyte proteins are MG-specific, FG-specific and shared.
      It is intriguing that we detected proteins such as liver-specific protein 2, merozoite surface protein 1, and cytoadherence linked asexual protein 8 in our sex-specific proteomes, which are unlikely expressed in gametocytes. Similarly, such type of proteins were also identified in almost all earlier gametocyte proteome work (
      • Lasonder E.
      • Ishihama Y.
      • Andersen J.S.
      • Vermunt A.M.
      • Pain A.
      • Sauerwein R.W.
      • Eling W.M.
      • Hall N.
      • Waters A.P.
      • Stunnenberg H.G.
      • Mann M.
      Analysis of the Plasmodium falciparum proteome by high-accuracy mass spectrometry.
      ,
      • Florens L.
      • Washburn M.P.
      • Raine J.D.
      • Anthony R.M.
      • Grainger M.
      • Haynes J.D.
      • Moch J.K.
      • Muster N.
      • Sacci J.B.
      • Tabb D.L.
      • Witney A.A.
      • Wolters D.
      • Wu Y.
      • Gardner M.J.
      • Holder A.A.
      • Sinden R.E.
      • Yates J.R.
      • Carucci D.J.
      A proteomic view of the Plasmodium falciparum life cycle.
      ,
      • Silvestrini F.
      • Lasonder E.
      • Olivieri A.
      • Camarda G.
      • van Schaijk B.
      • Sanchez M.
      • Younis Younis S.
      • Sauerwein R.
      • Alano P.
      Protein export marks the early phase of gametocytogenesis of the human malaria parasite Plasmodium falciparum.
      ), including the recent P. falciparum sex-specific proteome identified by Lasonder et al. (
      • Lasonder E.
      • Rijpma S.R.
      • van Schaijk B.C.
      • Hoeijmakers W.A.
      • Kensche P.R.
      • Gresnigt M.S.
      • Italiaander A.
      • Vos M.W.
      • Woestenenk R.
      • Bousema T.
      • Mair G.R.
      • Khan S.M.
      • Janse C.J.
      • Bartfai R.
      • Sauerwein R.W.
      Integrated transcriptomic and proteomic analyses of P. falciparum gametocytes: molecular insight into sex-specific processes and translational repression.
      ). Whereas these proteins might be resulted from contaminating asexual stages, it should be rare or impossible in the present and recent studies (
      • Lasonder E.
      • Rijpma S.R.
      • van Schaijk B.C.
      • Hoeijmakers W.A.
      • Kensche P.R.
      • Gresnigt M.S.
      • Italiaander A.
      • Vos M.W.
      • Woestenenk R.
      • Bousema T.
      • Mair G.R.
      • Khan S.M.
      • Janse C.J.
      • Bartfai R.
      • Sauerwein R.W.
      Integrated transcriptomic and proteomic analyses of P. falciparum gametocytes: molecular insight into sex-specific processes and translational repression.
      ) because both studies used sorted MG and FG that were cultured for an extended period and were supposedly devoid of asexual stages. Thus, future work is needed to verify whether these proteins are also expressed at very low levels in gametocytes.
      Like in P. berghei, the sex-specific proteomes are consistent with the major morphological distinctions of MG and FG in the malaria parasites, highlighting conceivable conservation among different parasite species. As MG are prepared for three rounds of rapid DNA replication to produce eight motile flagella-like gametes during gametogenesis, the MG proteome is specially enriched in proteins involved in these processes. In fact, 9/119 MG-specific proteins are involved DNA replication, whereas 10/119 are predicted to be associated with flagella formation. Intriguingly, a specific dynein (PF3D7_0729900) was extremely enriched in the FG-specific proteome. Because FG have been shown to also produce long nanotubes during gametogenesis (
      • Rupp I.
      • Sologub L.
      • Williamson K.C.
      • Scheuermayer M.
      • Reininger L.
      • Doerig C.
      • Eksi S.
      • Kombila D.U.
      • Frank M.
      • Pradel G.
      Malaria parasites form filamentous cell-to-cell connections during reproduction in the mosquito midgut.
      ), it would be interesting to determine whether this dynein is involved in the formation of the nanotubes or in other transport processes. Ultrastructurally, FG are highly enriched in ribosomes, indicating that FG are prepared for subsequent protein synthesis. Consistently, FG are enriched in proteins involved in translation, metabolic pathways, energy generation and utility, protein degradation and redox system. In addition, the distributions of proteins in the two organelles mitochondrion and apicoplast are also FG-biased. A similar sex-partition was found in P. berghei for mitochondrion only, whereas apicoplast-associated proteins are equally present in both sexes (
      • Khan S.M.
      • Franke-Fayard B.
      • Mair G.R.
      • Lasonder E.
      • Janse C.J.
      • Mann M.
      • Waters A.P.
      Proteome analysis of separated male and female gametocytes reveals novel sex-specific Plasmodium biology.
      ). The facts that apicoplast and mitochondrion are closely associated with critical functions in gametocytes and apicoplast is lost only during microgametogenesis but not macrogametogenesis indicate that the apicoplast presumably plays a more pivotal role in FG (
      • van Schaijk B.C.
      • Kumar T.R.
      • Vos M.W.
      • Richman A.
      • van Gemert G.J.
      • Li T.
      • Eappen A.G.
      • Williamson K.C.
      • Morahan B.J.
      • Fishbaugher M.
      • Kennedy M.
      • Camargo N.
      • Khan S.M.
      • Janse C.J.
      • Sim K.L.
      • Hoffman S.L.
      • Kappe S.H.
      • Sauerwein R.W.
      • Fidock D.A.
      • Vaughan A.M.
      Type II fatty acid biosynthesis is essential for Plasmodium falciparum sporozoite development in the midgut of Anopheles mosquitoes.
      ,
      • Okamoto N.
      • Spurck T.P.
      • Goodman C.D.
      • McFadden G.I.
      Apicoplast and mitochondrion in gametocytogenesis of Plasmodium falciparum.
      ).
      The original study of the P. berghei gametocyte proteomes identified very limited sharing of proteins between the two sexes (
      • Khan S.M.
      • Franke-Fayard B.
      • Mair G.R.
      • Lasonder E.
      • Janse C.J.
      • Mann M.
      • Waters A.P.
      Proteome analysis of separated male and female gametocytes reveals novel sex-specific Plasmodium biology.
      ), whereas reanalysis of the spectral data increased the number of shared proteins from 371 to 762, making up 67% of the 1128 MG proteome and 64% of the 1180 FG proteome, respectively (
      • Tao D.
      • Ubaida-Mohien C.
      • Mathias D.K.
      • King J.G.
      • Pastrana-Mena R.
      • Tripathi A.
      • Goldowitz I.
      • Graham D.R.
      • Moss E.
      • Marti M.
      • Dinglasan R.R.
      Sex-partitioning of the Plasmodium falciparum stage V gametocyte proteome provides insight into falciparum-specific cell biology.
      ). This reanalysis changed 185 originally defined MG- or FG-specific proteins to the shared category. The present study, with dramatically increased depth of protein discovery, revealed more extensive sharing of proteins between the MG (91%) and FG (80%) proteomes. We speculate that many proteins that are very differentially expressed in the two sexes were identified previously in one sex only, and with enhanced sensitivity of protein identification, they were identified in both sexes albeit with a strong sex bias. As such, a large number of FG-biased proteins function in translation and a variety of metabolic pathways such as TCA, glycolysis, protein degradation, energy and redox processes. In contrast, a number of MG-enriched proteins function in motility and DNA replication. Such sex-biased proteomes were further corroborated by the recent analysis of sorted P. falciparum MG and FG proteomes (
      • Lasonder E.
      • Rijpma S.R.
      • van Schaijk B.C.
      • Hoeijmakers W.A.
      • Kensche P.R.
      • Gresnigt M.S.
      • Italiaander A.
      • Vos M.W.
      • Woestenenk R.
      • Bousema T.
      • Mair G.R.
      • Khan S.M.
      • Janse C.J.
      • Bartfai R.
      • Sauerwein R.W.
      Integrated transcriptomic and proteomic analyses of P. falciparum gametocytes: molecular insight into sex-specific processes and translational repression.
      ). Furthermore, we confirmed several of these new predictions of sex partitioning by tagging the endogenous genes with GFP. Nevertheless, several sex-specific proteins have been identified and confirmed in this study, which may serve as markers for quantification of MG or FG in samples. In addition, comparison of the three sex-specific proteome studies led to the identification of 73 core MG-specific/biased and 89 core FG-specific/biased proteins, which may play conserved roles in sex-specific biology in all Plasmodium species. Among them, new candidates can be identified for future functional studies of sexual differentiation in malaria parasites.
      This study identified a Puf-family RNA-binding protein PfPuf1 and all 11 components of the DOZI complex (
      • Cui L.
      • Lindner S.
      • Miao J.
      Translational regulation during stage transitions in malaria parasites.
      ) as FG-enriched proteins. In the P. berghei gametocyte proteome, PbPuf1 was a FG-specific protein and 9 of DOZI complex as FG-enriched (8 proteins) or FG-specific (1 protein), suggesting that RNA-binding proteins may play conserved roles in translational regulation between these parasite species.
      The comparison of sex-specific proteomes on P. falciparum mature gametocytes between this study and the recent study by Lasonder et al. revealed a core MG- and FG-specific/biased proteomes shared by the two data sets, although some discrepancies existed between the two studies, possibly because of different markers used to separate the two sexes and study methods (
      • Lasonder E.
      • Rijpma S.R.
      • van Schaijk B.C.
      • Hoeijmakers W.A.
      • Kensche P.R.
      • Gresnigt M.S.
      • Italiaander A.
      • Vos M.W.
      • Woestenenk R.
      • Bousema T.
      • Mair G.R.
      • Khan S.M.
      • Janse C.J.
      • Bartfai R.
      • Sauerwein R.W.
      Integrated transcriptomic and proteomic analyses of P. falciparum gametocytes: molecular insight into sex-specific processes and translational repression.
      ). The predicted P. falciparum sex-specific proteomes based on a subtraction method (
      • Khan S.M.
      • Franke-Fayard B.
      • Mair G.R.
      • Lasonder E.
      • Janse C.J.
      • Mann M.
      • Waters A.P.
      Proteome analysis of separated male and female gametocytes reveals novel sex-specific Plasmodium biology.
      ), while differing vastly from those in the present study, contain such core sex-specific proteomes. Further comparison with the sex-specific proteomes of gametocytes between P. berghei and P. falciparum reveals core sex-specific/biased proteomes that are likely conserved among most malaria species. In addition to the overall conservation in sex-specific proteomes between the two species, the present study also identified considerable divergence between the two species, which could underpin their differences in gametocyte biology (
      • Sinden R.E.
      The cell biology of malaria infection of mosquito: advances and opportunities.
      ,
      • Janse C.
      • Waters A.P.
      Sexual development of malaria Parasites.
      ,
      • Sinden R.E.
      • Carter R.
      • Drakeley C.
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      ). Whereas MG-specific proteins such as those involved in motility and genome replication as well as FG-specific proteins in translation, mitochondrion and many metabolic pathways are agreeable between the two species (
      • Khan S.M.
      • Franke-Fayard B.
      • Mair G.R.
      • Lasonder E.
      • Janse C.J.
      • Mann M.
      • Waters A.P.
      Proteome analysis of separated male and female gametocytes reveals novel sex-specific Plasmodium biology.
      ,
      • Tao D.
      • Ubaida-Mohien C.
      • Mathias D.K.
      • King J.G.
      • Pastrana-Mena R.
      • Tripathi A.
      • Goldowitz I.
      • Graham D.R.
      • Moss E.
      • Marti M.
      • Dinglasan R.R.
      Sex-partitioning of the Plasmodium falciparum stage V gametocyte proteome provides insight into falciparum-specific cell biology.
      ), species-specific proteomes that diverged substantially are involved in cytoskeleton, lipid metabolism and signal transduction, protein degradation, DNA replication and transcription. More cytoskeleton proteins discovered in this study is in concordance with the presence of the subpellicular membrane structure needed for the transformation of P. falciparum gametocytes to a crescent shape (
      • Dixon M.W.
      • Dearnley M.K.
      • Hanssen E.
      • Gilberger T.
      • Tilley L.
      Shape-shifting gametocytes: how and why does P. falciparum go banana-shaped?.
      ,
      • Dearnley M.K.
      • Yeoman J.A.
      • Hanssen E.
      • Kenny S.
      • Turnbull L.
      • Whitchurch C.B.
      • Tilley L.
      • Dixon M.W.
      Origin, composition, organization and function of the inner membrane complex of Plasmodium falciparum gametocytes.
      ,
      • Kono M.
      • Herrmann S.
      • Loughran N.B.
      • Cabrera A.
      • Engelberg K.
      • Lehmann C.
      • Sinha D.
      • Prinz B.
      • Ruch U.
      • Heussler V.
      • Spielmann T.
      • Parkinson J.
      • Gilberger T.W.
      Evolution and architecture of the inner membrane complex in asexual and sexual stages of the malaria parasite.
      ,
      • Hliscs M.
      • Millet C.
      • Dixon M.W.
      • Siden-Kiamos I.
      • McMillan P.
      • Tilley L.
      Organization and function of an actin cytoskeleton in Plasmodium falciparum gametocytes.
      ). More proteins associated lipid metabolism is in agreement with a significant increase and compositional change of lipids in gametocytes (
      • Tran P.N.
      • Brown S.H.
      • Rug M.
      • Ridgway M.C.
      • Mitchell T.W.
      • Maier A.G.
      Changes in lipid composition during sexual development of the malaria parasite Plasmodium falciparum.
      ,
      • Gulati S.
      • Ekland E.H.
      • Ruggles K.V.
      • Chan R.B.
      • Jayabalasingham B.
      • Zhou B.
      • Mantel P.Y.
      • Lee M.C.
      • Spottiswoode N.
      • Coburn-Flynn O.
      • Hjelmqvist D.
      • Worgall T.S.
      • Marti M.
      • Di Paolo G.
      • Fidock D.A.
      Profiling the Essential Nature of Lipid Metabolism in Asexual Blood and Gametocyte Stages of Plasmodium falciparum.
      ). Of them, three signal lipid-associated proteins (phosphoethanolamine methyltransferase, cytidine diphosphate-diacylglycerol synthase and calponin homology domain-containing protein) have been studied, showing that the disruption of these genes resulted in the arrest of gametocyte development (
      • Bobenchik A.M.
      • Witola W.H.
      • Augagneur Y.
      • Nic Lochlainn L.
      • Garg A.
      • Pachikara N.
      • Choi J.Y.
      • Zhao Y.O.
      • Usmani-Brown S.
      • Lee A.
      • Adjalley S.H.
      • Samanta S.
      • Fidock D.A.
      • Voelker D.R.
      • Fikrig E.
      • Ben Mamoun C.
      Plasmodium falciparum phosphoethanolamine methyltransferase is essential for malaria transmission.
      ,
      • Ikadai H.
      • Shaw Saliba K.
      • Kanzok S.M.
      • McLean K.J.
      • Tanaka T.Q.
      • Cao J.
      • Williamson K.C.
      • Jacobs-Lorena M.
      Transposon mutagenesis identifies genes essential for Plasmodium falciparum gametocytogenesis.
      ). The mechanisms underlining these biological disparities between these Plasmodium species remain to be further elucidated.

      Supplementary Material

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