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

2017; Elsevier BV; Volume: 16; Issue: 4 Linguagem: Inglês

10.1074/mcp.m116.061804

ISSN

1535-9484

Autores

Jun Miao, Zhao Chen, Zenglei Wang, Sony Shrestha, Xiaolian Li, Runze Li, Liwang Cui,

Tópico(s)

Invertebrate Immune Response Mechanisms

Resumo

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 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) 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. 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 (1.Alonso P.L. Brown G. Arevalo-Herrera M. Binka F. Chitnis C. Collins F. Doumbo O.K. Greenwood B. Hall B.F. Levine M.M. Mendis K. Newman R.D. Plowe C.V. Rodriguez M.H. Sinden R. Slutsker L. Tanner M. 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Malaria parasites: genomes and molecular biology. The Netherlands Caister Academic Press, 2004: 445-474Google Scholar). Consistent with this functional division, the MG- and FG-specific proteomes of the rodent parasite P. berghei revealed distinctive sex-specific biology (49.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.Cell. 2005; 121: 675-687Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). 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 (36.Sinden R.E. The cell biology of malaria infection of mosquito: advances and opportunities.Cell Microbiol. 2015; 17: 451-466Crossref PubMed Scopus (44) Google Scholar, 50.Sinden R.E. Carter R. Drakeley C. Leroy D. The biology of sexual development of Plasmodium: the design and implementation of transmission-blocking strategies.Malar. J. 2012; 11: 70Crossref PubMed Scopus (55) Google Scholar). 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 (51.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.Mol. Cell Proteomics. 2014; 13: 2705-2724Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), 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 (52.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.Nucleic Acids Res. 2016; 44: 6087-6101Crossref PubMed Scopus (140) Google Scholar). 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. Cultures of P. falciparum parasite lines were maintained in complete RPMI 1640 medium supplemented with 10% human serum and hypoxanthine as described previously (53.Trager W. Jensen J.B. Human malaria parasites in continuous culture.Science. 1976; 193: 673-675Crossref PubMed Scopus (6158) Google Scholar, 54.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.Trans. R Soc. Trop. Med. Hyg. 1982; 76: 242-250Abstract Full Text PDF PubMed Scopus (144) Google Scholar). A modified induction scheme for gametocytogenesis was used to obtain highly synchronous gametocyte cultures (55.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.PLoS ONE. 2014; 9: e93825Crossref PubMed Scopus (21) Google Scholar, 56.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.Mol. Biochem. Parasitol. 2007; 154: 119-123Crossref PubMed Scopus (156) Google Scholar). 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 par

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