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Plasmodium falciparum gametocyte development 1 (Pfgdv1) and gametocytogenesis early gene identification and commitment to sexual development.

Eksi S, Morahan BJ, Haile Y, Furuya T, Jiang H, Ali O, Xu H, Kiattibutr K, Suri A, Czesny B, Adeyemo A, Myers TG, Sattabongkot J, Su XZ, Williamson KC - PLoS Pathog. (2012)

Bottom Line: Malaria transmission requires the production of male and female gametocytes in the human host followed by fertilization and sporogonic development in the mosquito midgut.Using a gametocyte-defective parasite line and genetic complementation, we show that Plasmodium falciparumgametocyte development 1 gene (Pfgdv1), encoding a peri-nuclear protein, is critical for early sexual differentiation.The expression profile of one of the early gametocyte specific genes, Pfge1, correlated significantly with asexual parasitemia, which is consistent with the ongoing induction of gametocytogenesis during asexual growth observed in vitro and reinforces the need for sustained transmission-blocking strategies to eliminate malaria.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Loyola University Chicago, Chicago, Illinois, USA.

ABSTRACT
Malaria transmission requires the production of male and female gametocytes in the human host followed by fertilization and sporogonic development in the mosquito midgut. Although essential for the spread of malaria through the population, little is known about the initiation of gametocytogenesis in vitro or in vivo. Using a gametocyte-defective parasite line and genetic complementation, we show that Plasmodium falciparumgametocyte development 1 gene (Pfgdv1), encoding a peri-nuclear protein, is critical for early sexual differentiation. Transcriptional analysis of Pfgdv1 negative and positive parasite lines identified a set of gametocytogenesis early genes (Pfge) that were significantly down-regulated (>10 fold) in the absence of Pfgdv1 and expression was restored after Pfgdv1 complementation. Progressive accumulation of Pfge transcripts during successive rounds of asexual replication in synchronized cultures suggests that gametocytes are induced continuously during asexual growth. Comparison of Pfge gene transcriptional profiles in patient samples divided the genes into two groups differing in their expression in mature circulating gametocytes and providing candidates to evaluate gametocyte induction and maturation separately in vivo. The expression profile of one of the early gametocyte specific genes, Pfge1, correlated significantly with asexual parasitemia, which is consistent with the ongoing induction of gametocytogenesis during asexual growth observed in vitro and reinforces the need for sustained transmission-blocking strategies to eliminate malaria.

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Subcellular localization of PfGDV1.Parasites transformed with GFP- or HA-tagged PfGDV1 were stained with DAPI (DNA stain) and the indicated anti-sera, and then examined by fluorescence microscopy (Zeiss Axiovert 200, 1000× magnification). Images are shown of the DAPI stain (DNA), GFP-tagged PfGDV1 epifluorescence (GDV1), and antibodies specific for HA (αHA), Pfs16 (αPfs16), PfGE3 (αPfGE3), PfMCM2 (αMCM2), and PfSir2 (αSir2). The corresponding merged and bright field (BF) images are included on the right. PfGDV1 expression is indicated with an arrow; locations of parasites in the BF image are indicated with a P for parasite or S for schizont. A) A schizont (S) (Upper) expressing GDV1 and a doubly infected erythrocyte (Lower) with one parasite (P1) expressing HA-tagged PfGDV1 (αHA) and another negative (P2) for anti-HA antibodies. B) Co-staining of parasites expressing GDV1 with early gametocytogenesis markers. A doubly infected erythrocyte (Upper) with one parasite (P1) positive for GDV1 and αPfs16 and the other (P2) negative for both. An erythrocyte (Lower) infected with a parasite (P) positive for GDV1 and αPfGE3. C) Co-localization of PfGDV1 with nuclear proteins. A doubly infected erythrocyte (Upper) with one parasite in the plane of the image (P1) and the other below (P2). Both P1 and P2 are positive for GDV1 and αMCM2. A schizont (S) (Lower) expressing GDV1 stained with αSir2.
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ppat-1002964-g003: Subcellular localization of PfGDV1.Parasites transformed with GFP- or HA-tagged PfGDV1 were stained with DAPI (DNA stain) and the indicated anti-sera, and then examined by fluorescence microscopy (Zeiss Axiovert 200, 1000× magnification). Images are shown of the DAPI stain (DNA), GFP-tagged PfGDV1 epifluorescence (GDV1), and antibodies specific for HA (αHA), Pfs16 (αPfs16), PfGE3 (αPfGE3), PfMCM2 (αMCM2), and PfSir2 (αSir2). The corresponding merged and bright field (BF) images are included on the right. PfGDV1 expression is indicated with an arrow; locations of parasites in the BF image are indicated with a P for parasite or S for schizont. A) A schizont (S) (Upper) expressing GDV1 and a doubly infected erythrocyte (Lower) with one parasite (P1) expressing HA-tagged PfGDV1 (αHA) and another negative (P2) for anti-HA antibodies. B) Co-staining of parasites expressing GDV1 with early gametocytogenesis markers. A doubly infected erythrocyte (Upper) with one parasite (P1) positive for GDV1 and αPfs16 and the other (P2) negative for both. An erythrocyte (Lower) infected with a parasite (P) positive for GDV1 and αPfGE3. C) Co-localization of PfGDV1 with nuclear proteins. A doubly infected erythrocyte (Upper) with one parasite in the plane of the image (P1) and the other below (P2). Both P1 and P2 are positive for GDV1 and αMCM2. A schizont (S) (Lower) expressing GDV1 stained with αSir2.

Mentions: The subcellular location of PfGDV1 was evaluated by tagging the gene with green fluorescent protein (GFP) or a HA epitope. Consistent with the nuclear localization predicted by PSORT, both tagged proteins had a punctate expression pattern around the nuclear periphery in a subpopulation (∼0.2–0.4%) of trophozoites and schizonts (Fig. 3A). Parasites expressing GFP-tagged PfGDV1 also stained with anti-sera against early gametocyte markers Pfs16 (PfGE11) and Pfg14.748 (PfGE3), suggesting that these parasites were committed to gametocytogenesis (Fig. 3B) [19], [38]. The nuclear location of PfGDV1 was further evaluated using antibodies against nuclear protein minichromosome maintenance protein 2 (MCM2) [39] or silencing information regulator 2 (Sir2) (Fig. 3C). PfMCM2, which is part of the replication origin, co-localizes with the DAPI stained nucleus in trophozoites and is internal to the PfGDV1-GFP fluorescence (Fig. 3C, upper row). PfSir2 binds to Rep20 telomere sequences and has been implicated in the silencing of var genes in peri-nuclear repressive centers [39], [40]. Anti-sera against PfSir2 also localized to the nuclear periphery adjacent to PfGDV1-GFP, but the two patterns did not overlap (Fig. 3C, lower row). This is the first report of a gametocyte-associated gene that has been localized to the nucleus raising the possibility that Pfgdv1 could have a role regulating gametocyte production pathways.


Plasmodium falciparum gametocyte development 1 (Pfgdv1) and gametocytogenesis early gene identification and commitment to sexual development.

Eksi S, Morahan BJ, Haile Y, Furuya T, Jiang H, Ali O, Xu H, Kiattibutr K, Suri A, Czesny B, Adeyemo A, Myers TG, Sattabongkot J, Su XZ, Williamson KC - PLoS Pathog. (2012)

Subcellular localization of PfGDV1.Parasites transformed with GFP- or HA-tagged PfGDV1 were stained with DAPI (DNA stain) and the indicated anti-sera, and then examined by fluorescence microscopy (Zeiss Axiovert 200, 1000× magnification). Images are shown of the DAPI stain (DNA), GFP-tagged PfGDV1 epifluorescence (GDV1), and antibodies specific for HA (αHA), Pfs16 (αPfs16), PfGE3 (αPfGE3), PfMCM2 (αMCM2), and PfSir2 (αSir2). The corresponding merged and bright field (BF) images are included on the right. PfGDV1 expression is indicated with an arrow; locations of parasites in the BF image are indicated with a P for parasite or S for schizont. A) A schizont (S) (Upper) expressing GDV1 and a doubly infected erythrocyte (Lower) with one parasite (P1) expressing HA-tagged PfGDV1 (αHA) and another negative (P2) for anti-HA antibodies. B) Co-staining of parasites expressing GDV1 with early gametocytogenesis markers. A doubly infected erythrocyte (Upper) with one parasite (P1) positive for GDV1 and αPfs16 and the other (P2) negative for both. An erythrocyte (Lower) infected with a parasite (P) positive for GDV1 and αPfGE3. C) Co-localization of PfGDV1 with nuclear proteins. A doubly infected erythrocyte (Upper) with one parasite in the plane of the image (P1) and the other below (P2). Both P1 and P2 are positive for GDV1 and αMCM2. A schizont (S) (Lower) expressing GDV1 stained with αSir2.
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getmorefigures.php?uid=PMC3475683&req=5

ppat-1002964-g003: Subcellular localization of PfGDV1.Parasites transformed with GFP- or HA-tagged PfGDV1 were stained with DAPI (DNA stain) and the indicated anti-sera, and then examined by fluorescence microscopy (Zeiss Axiovert 200, 1000× magnification). Images are shown of the DAPI stain (DNA), GFP-tagged PfGDV1 epifluorescence (GDV1), and antibodies specific for HA (αHA), Pfs16 (αPfs16), PfGE3 (αPfGE3), PfMCM2 (αMCM2), and PfSir2 (αSir2). The corresponding merged and bright field (BF) images are included on the right. PfGDV1 expression is indicated with an arrow; locations of parasites in the BF image are indicated with a P for parasite or S for schizont. A) A schizont (S) (Upper) expressing GDV1 and a doubly infected erythrocyte (Lower) with one parasite (P1) expressing HA-tagged PfGDV1 (αHA) and another negative (P2) for anti-HA antibodies. B) Co-staining of parasites expressing GDV1 with early gametocytogenesis markers. A doubly infected erythrocyte (Upper) with one parasite (P1) positive for GDV1 and αPfs16 and the other (P2) negative for both. An erythrocyte (Lower) infected with a parasite (P) positive for GDV1 and αPfGE3. C) Co-localization of PfGDV1 with nuclear proteins. A doubly infected erythrocyte (Upper) with one parasite in the plane of the image (P1) and the other below (P2). Both P1 and P2 are positive for GDV1 and αMCM2. A schizont (S) (Lower) expressing GDV1 stained with αSir2.
Mentions: The subcellular location of PfGDV1 was evaluated by tagging the gene with green fluorescent protein (GFP) or a HA epitope. Consistent with the nuclear localization predicted by PSORT, both tagged proteins had a punctate expression pattern around the nuclear periphery in a subpopulation (∼0.2–0.4%) of trophozoites and schizonts (Fig. 3A). Parasites expressing GFP-tagged PfGDV1 also stained with anti-sera against early gametocyte markers Pfs16 (PfGE11) and Pfg14.748 (PfGE3), suggesting that these parasites were committed to gametocytogenesis (Fig. 3B) [19], [38]. The nuclear location of PfGDV1 was further evaluated using antibodies against nuclear protein minichromosome maintenance protein 2 (MCM2) [39] or silencing information regulator 2 (Sir2) (Fig. 3C). PfMCM2, which is part of the replication origin, co-localizes with the DAPI stained nucleus in trophozoites and is internal to the PfGDV1-GFP fluorescence (Fig. 3C, upper row). PfSir2 binds to Rep20 telomere sequences and has been implicated in the silencing of var genes in peri-nuclear repressive centers [39], [40]. Anti-sera against PfSir2 also localized to the nuclear periphery adjacent to PfGDV1-GFP, but the two patterns did not overlap (Fig. 3C, lower row). This is the first report of a gametocyte-associated gene that has been localized to the nucleus raising the possibility that Pfgdv1 could have a role regulating gametocyte production pathways.

Bottom Line: Malaria transmission requires the production of male and female gametocytes in the human host followed by fertilization and sporogonic development in the mosquito midgut.Using a gametocyte-defective parasite line and genetic complementation, we show that Plasmodium falciparumgametocyte development 1 gene (Pfgdv1), encoding a peri-nuclear protein, is critical for early sexual differentiation.The expression profile of one of the early gametocyte specific genes, Pfge1, correlated significantly with asexual parasitemia, which is consistent with the ongoing induction of gametocytogenesis during asexual growth observed in vitro and reinforces the need for sustained transmission-blocking strategies to eliminate malaria.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Loyola University Chicago, Chicago, Illinois, USA.

ABSTRACT
Malaria transmission requires the production of male and female gametocytes in the human host followed by fertilization and sporogonic development in the mosquito midgut. Although essential for the spread of malaria through the population, little is known about the initiation of gametocytogenesis in vitro or in vivo. Using a gametocyte-defective parasite line and genetic complementation, we show that Plasmodium falciparumgametocyte development 1 gene (Pfgdv1), encoding a peri-nuclear protein, is critical for early sexual differentiation. Transcriptional analysis of Pfgdv1 negative and positive parasite lines identified a set of gametocytogenesis early genes (Pfge) that were significantly down-regulated (>10 fold) in the absence of Pfgdv1 and expression was restored after Pfgdv1 complementation. Progressive accumulation of Pfge transcripts during successive rounds of asexual replication in synchronized cultures suggests that gametocytes are induced continuously during asexual growth. Comparison of Pfge gene transcriptional profiles in patient samples divided the genes into two groups differing in their expression in mature circulating gametocytes and providing candidates to evaluate gametocyte induction and maturation separately in vivo. The expression profile of one of the early gametocyte specific genes, Pfge1, correlated significantly with asexual parasitemia, which is consistent with the ongoing induction of gametocytogenesis during asexual growth observed in vitro and reinforces the need for sustained transmission-blocking strategies to eliminate malaria.

Show MeSH
Related in: MedlinePlus