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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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Related in: MedlinePlus

Identification of Plasmodium falciparum gametocyte development 1 gene (Pfgdv1).A) Schematic of chromosome 9 (1–1,541,723 bp) showing the segment deleted (arrows) in the 3D7.Gdef clone. For orientation, the putative centromere is indicated by an O and the 1,500,000 bp position is marked (1500 k). Colored boxes indicate the location of annotated genes: blue, genes transcribed toward the telomere; red, transcribed toward the centromere; horizontal stripes, Pfgdv1; diagonal stripes, var and rifins. The 70-mer oligonucleotide that identified the chromosome 9 deletion in 3D7.Gdef parasites is indicated by an asterisk (*). The numbered gray bars below the line indicate the positions of the eight PCR products used to map the chromosome 9 deletion. B) Amplification products from chromosome 9 using Indochina, 3D7.Gdef, FCR3.Gdef, or HB3.Gdef gDNA as a template. Peak gametocytemias attained in two independent experiments are indicated on the right of the ethidium bromide-stained agarose gel of the PCR products generated using the eight primer pairs (1–8). C) Southern blot of BsaBI-digested gDNA from 3D7.G+ (+), 3D7.Gdef (d), 3D7.Gdef +Pfgdv1 (a), 3D7.Gdef +HA.Pfgdv1 (b) and the parental 3D7 parasites (WT). Digested gDNA was probed with Pfgdv1 (bp 1423–1800) or Pfg27 (bp 1–654). D–E) Gametocyte production in WT (black square), 3D7.Gdef (black triangle), complemented line a, 3D7.Gdef +Pfgdv1 (Panel D, black inverted triangle) and line b, 3D7.Gdef +HA.Pfgdv1 (Panel E, unfilled inverted triangle). Cultures set up at 0.1% asexual parasitemia on day 0 were followed for gametocyte production by Giemsa-stained smears for the next 16 days. Mean gametocytemia and standard deviation of two (D) or three (E) independent experiments are shown (P<0.003 by linear regression analysis). F) Giemsa-stained smear of parasitized erythrocytes purified on a 16% Nycodenz cushion from day 16 gametocyte cultures of WT, 3D7.Gdef +HA.Pfgdv1 (HA.Pfgdv1), and 3D7.Gdef (Gdef) lines. G) Northern blots of RNA harvested from WT 3D7 (w), 3D7.Gdef (d), 3D7.Gdef+Pfgdv1 (a), and 3D7.Gdef +HA.Pfgdv1 (b) complemented lines were hybridized with probes corresponding to Pfgdv1 (gdv1), Pfge genes (ge1–3, 5–11), and merozoite surface protein-1 (msp1) as an asexual parasite control. Autoradiographs are shown with the corresponding ethidium bromide-stained gel. H) Expression of gametocyte specific antigen Pfs48/45. Methanol-fixed WT, 3D7.Gdef+Pfgdv1 (a) and 3D7.Gdef+HA.Pfgdv1 (b) mature gametocyte cultures were incubated with Pfs48/45 mAb IIC5B10 (1∶250 dilution) and labeled secondary antibodies (1∶500 dilution). I) The average gametocytemia of 4 independent cultures of WT 3D7 (WT), WT 3D7 transformed with a Pfgdv1 episomal expression plasmid (WT+HA.Pfgdv1) and the Gdef (Gdef) line is graphed. The error bars represent the SEM and a significant difference from WT and Gdef is indicated by an asterisk (p<0.05 ANOVA followed by Tukey multiple comparison test).
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ppat-1002964-g002: Identification of Plasmodium falciparum gametocyte development 1 gene (Pfgdv1).A) Schematic of chromosome 9 (1–1,541,723 bp) showing the segment deleted (arrows) in the 3D7.Gdef clone. For orientation, the putative centromere is indicated by an O and the 1,500,000 bp position is marked (1500 k). Colored boxes indicate the location of annotated genes: blue, genes transcribed toward the telomere; red, transcribed toward the centromere; horizontal stripes, Pfgdv1; diagonal stripes, var and rifins. The 70-mer oligonucleotide that identified the chromosome 9 deletion in 3D7.Gdef parasites is indicated by an asterisk (*). The numbered gray bars below the line indicate the positions of the eight PCR products used to map the chromosome 9 deletion. B) Amplification products from chromosome 9 using Indochina, 3D7.Gdef, FCR3.Gdef, or HB3.Gdef gDNA as a template. Peak gametocytemias attained in two independent experiments are indicated on the right of the ethidium bromide-stained agarose gel of the PCR products generated using the eight primer pairs (1–8). C) Southern blot of BsaBI-digested gDNA from 3D7.G+ (+), 3D7.Gdef (d), 3D7.Gdef +Pfgdv1 (a), 3D7.Gdef +HA.Pfgdv1 (b) and the parental 3D7 parasites (WT). Digested gDNA was probed with Pfgdv1 (bp 1423–1800) or Pfg27 (bp 1–654). D–E) Gametocyte production in WT (black square), 3D7.Gdef (black triangle), complemented line a, 3D7.Gdef +Pfgdv1 (Panel D, black inverted triangle) and line b, 3D7.Gdef +HA.Pfgdv1 (Panel E, unfilled inverted triangle). Cultures set up at 0.1% asexual parasitemia on day 0 were followed for gametocyte production by Giemsa-stained smears for the next 16 days. Mean gametocytemia and standard deviation of two (D) or three (E) independent experiments are shown (P<0.003 by linear regression analysis). F) Giemsa-stained smear of parasitized erythrocytes purified on a 16% Nycodenz cushion from day 16 gametocyte cultures of WT, 3D7.Gdef +HA.Pfgdv1 (HA.Pfgdv1), and 3D7.Gdef (Gdef) lines. G) Northern blots of RNA harvested from WT 3D7 (w), 3D7.Gdef (d), 3D7.Gdef+Pfgdv1 (a), and 3D7.Gdef +HA.Pfgdv1 (b) complemented lines were hybridized with probes corresponding to Pfgdv1 (gdv1), Pfge genes (ge1–3, 5–11), and merozoite surface protein-1 (msp1) as an asexual parasite control. Autoradiographs are shown with the corresponding ethidium bromide-stained gel. H) Expression of gametocyte specific antigen Pfs48/45. Methanol-fixed WT, 3D7.Gdef+Pfgdv1 (a) and 3D7.Gdef+HA.Pfgdv1 (b) mature gametocyte cultures were incubated with Pfs48/45 mAb IIC5B10 (1∶250 dilution) and labeled secondary antibodies (1∶500 dilution). I) The average gametocytemia of 4 independent cultures of WT 3D7 (WT), WT 3D7 transformed with a Pfgdv1 episomal expression plasmid (WT+HA.Pfgdv1) and the Gdef (Gdef) line is graphed. The error bars represent the SEM and a significant difference from WT and Gdef is indicated by an asterisk (p<0.05 ANOVA followed by Tukey multiple comparison test).

Mentions: To study the genetic basis for the inability of the Gdef line to make gametocytes, we used two P. falciparum microarrays, one with ∼7000 70-mer oligonucleotides and the other with 2.5 million 25-mer tiling oligonucleotides, to compare genomic DNA (gDNA) from the 3D7.G+ and 3D7.Gdef lines [16], [25], [26]. A single oligonucleotide, i13417_1 (MAL9: 1,379,276–1,379,345) designated with an asterisk in Fig. 2A, had a significantly lower signal when the 70-mer oligo array was hybridized with gDNA from the 3D7.Gdef clone than with that from the 3D7.G+ clone, suggesting substitution(s) or a deletion in the 3D7.Gdef genome sequence. Two oligonucleotides (i14759_1 and i12812_3) flanking the i13417_1 probe hybridized to gDNA from both parasite clones with similar intensities. This hybridization data defined a DNA segment of ∼30 kb on chromosome 9 that was likely deleted in the 3D7.Gdef clone (Fig. 2A). To confirm this observation, we designed eight PCR primer pairs to amplify the region between the two positive oligonucleotides (Fig. 2A). PCR products were obtained for all eight primer-pairs using gDNA from 3D7.G+ and wild type (WT) 3D7 lines, but only primer pairs 1, 7, and 8 amplified products from 3D7.Gdef gDNA (Fig. 2B). DNA sequencing of a PCR product spanning the 3D7.Gdef deletion defined the breakpoints to an 18,920 bp region (1,374,582 and 1,393,502 bp) on chromosome 9. The deletion is flanked by polyA tracts of >18 bp that have been reported to be sites of frequent recombination and deletion [27].


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)

Identification of Plasmodium falciparum gametocyte development 1 gene (Pfgdv1).A) Schematic of chromosome 9 (1–1,541,723 bp) showing the segment deleted (arrows) in the 3D7.Gdef clone. For orientation, the putative centromere is indicated by an O and the 1,500,000 bp position is marked (1500 k). Colored boxes indicate the location of annotated genes: blue, genes transcribed toward the telomere; red, transcribed toward the centromere; horizontal stripes, Pfgdv1; diagonal stripes, var and rifins. The 70-mer oligonucleotide that identified the chromosome 9 deletion in 3D7.Gdef parasites is indicated by an asterisk (*). The numbered gray bars below the line indicate the positions of the eight PCR products used to map the chromosome 9 deletion. B) Amplification products from chromosome 9 using Indochina, 3D7.Gdef, FCR3.Gdef, or HB3.Gdef gDNA as a template. Peak gametocytemias attained in two independent experiments are indicated on the right of the ethidium bromide-stained agarose gel of the PCR products generated using the eight primer pairs (1–8). C) Southern blot of BsaBI-digested gDNA from 3D7.G+ (+), 3D7.Gdef (d), 3D7.Gdef +Pfgdv1 (a), 3D7.Gdef +HA.Pfgdv1 (b) and the parental 3D7 parasites (WT). Digested gDNA was probed with Pfgdv1 (bp 1423–1800) or Pfg27 (bp 1–654). D–E) Gametocyte production in WT (black square), 3D7.Gdef (black triangle), complemented line a, 3D7.Gdef +Pfgdv1 (Panel D, black inverted triangle) and line b, 3D7.Gdef +HA.Pfgdv1 (Panel E, unfilled inverted triangle). Cultures set up at 0.1% asexual parasitemia on day 0 were followed for gametocyte production by Giemsa-stained smears for the next 16 days. Mean gametocytemia and standard deviation of two (D) or three (E) independent experiments are shown (P<0.003 by linear regression analysis). F) Giemsa-stained smear of parasitized erythrocytes purified on a 16% Nycodenz cushion from day 16 gametocyte cultures of WT, 3D7.Gdef +HA.Pfgdv1 (HA.Pfgdv1), and 3D7.Gdef (Gdef) lines. G) Northern blots of RNA harvested from WT 3D7 (w), 3D7.Gdef (d), 3D7.Gdef+Pfgdv1 (a), and 3D7.Gdef +HA.Pfgdv1 (b) complemented lines were hybridized with probes corresponding to Pfgdv1 (gdv1), Pfge genes (ge1–3, 5–11), and merozoite surface protein-1 (msp1) as an asexual parasite control. Autoradiographs are shown with the corresponding ethidium bromide-stained gel. H) Expression of gametocyte specific antigen Pfs48/45. Methanol-fixed WT, 3D7.Gdef+Pfgdv1 (a) and 3D7.Gdef+HA.Pfgdv1 (b) mature gametocyte cultures were incubated with Pfs48/45 mAb IIC5B10 (1∶250 dilution) and labeled secondary antibodies (1∶500 dilution). I) The average gametocytemia of 4 independent cultures of WT 3D7 (WT), WT 3D7 transformed with a Pfgdv1 episomal expression plasmid (WT+HA.Pfgdv1) and the Gdef (Gdef) line is graphed. The error bars represent the SEM and a significant difference from WT and Gdef is indicated by an asterisk (p<0.05 ANOVA followed by Tukey multiple comparison test).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475683&req=5

ppat-1002964-g002: Identification of Plasmodium falciparum gametocyte development 1 gene (Pfgdv1).A) Schematic of chromosome 9 (1–1,541,723 bp) showing the segment deleted (arrows) in the 3D7.Gdef clone. For orientation, the putative centromere is indicated by an O and the 1,500,000 bp position is marked (1500 k). Colored boxes indicate the location of annotated genes: blue, genes transcribed toward the telomere; red, transcribed toward the centromere; horizontal stripes, Pfgdv1; diagonal stripes, var and rifins. The 70-mer oligonucleotide that identified the chromosome 9 deletion in 3D7.Gdef parasites is indicated by an asterisk (*). The numbered gray bars below the line indicate the positions of the eight PCR products used to map the chromosome 9 deletion. B) Amplification products from chromosome 9 using Indochina, 3D7.Gdef, FCR3.Gdef, or HB3.Gdef gDNA as a template. Peak gametocytemias attained in two independent experiments are indicated on the right of the ethidium bromide-stained agarose gel of the PCR products generated using the eight primer pairs (1–8). C) Southern blot of BsaBI-digested gDNA from 3D7.G+ (+), 3D7.Gdef (d), 3D7.Gdef +Pfgdv1 (a), 3D7.Gdef +HA.Pfgdv1 (b) and the parental 3D7 parasites (WT). Digested gDNA was probed with Pfgdv1 (bp 1423–1800) or Pfg27 (bp 1–654). D–E) Gametocyte production in WT (black square), 3D7.Gdef (black triangle), complemented line a, 3D7.Gdef +Pfgdv1 (Panel D, black inverted triangle) and line b, 3D7.Gdef +HA.Pfgdv1 (Panel E, unfilled inverted triangle). Cultures set up at 0.1% asexual parasitemia on day 0 were followed for gametocyte production by Giemsa-stained smears for the next 16 days. Mean gametocytemia and standard deviation of two (D) or three (E) independent experiments are shown (P<0.003 by linear regression analysis). F) Giemsa-stained smear of parasitized erythrocytes purified on a 16% Nycodenz cushion from day 16 gametocyte cultures of WT, 3D7.Gdef +HA.Pfgdv1 (HA.Pfgdv1), and 3D7.Gdef (Gdef) lines. G) Northern blots of RNA harvested from WT 3D7 (w), 3D7.Gdef (d), 3D7.Gdef+Pfgdv1 (a), and 3D7.Gdef +HA.Pfgdv1 (b) complemented lines were hybridized with probes corresponding to Pfgdv1 (gdv1), Pfge genes (ge1–3, 5–11), and merozoite surface protein-1 (msp1) as an asexual parasite control. Autoradiographs are shown with the corresponding ethidium bromide-stained gel. H) Expression of gametocyte specific antigen Pfs48/45. Methanol-fixed WT, 3D7.Gdef+Pfgdv1 (a) and 3D7.Gdef+HA.Pfgdv1 (b) mature gametocyte cultures were incubated with Pfs48/45 mAb IIC5B10 (1∶250 dilution) and labeled secondary antibodies (1∶500 dilution). I) The average gametocytemia of 4 independent cultures of WT 3D7 (WT), WT 3D7 transformed with a Pfgdv1 episomal expression plasmid (WT+HA.Pfgdv1) and the Gdef (Gdef) line is graphed. The error bars represent the SEM and a significant difference from WT and Gdef is indicated by an asterisk (p<0.05 ANOVA followed by Tukey multiple comparison test).
Mentions: To study the genetic basis for the inability of the Gdef line to make gametocytes, we used two P. falciparum microarrays, one with ∼7000 70-mer oligonucleotides and the other with 2.5 million 25-mer tiling oligonucleotides, to compare genomic DNA (gDNA) from the 3D7.G+ and 3D7.Gdef lines [16], [25], [26]. A single oligonucleotide, i13417_1 (MAL9: 1,379,276–1,379,345) designated with an asterisk in Fig. 2A, had a significantly lower signal when the 70-mer oligo array was hybridized with gDNA from the 3D7.Gdef clone than with that from the 3D7.G+ clone, suggesting substitution(s) or a deletion in the 3D7.Gdef genome sequence. Two oligonucleotides (i14759_1 and i12812_3) flanking the i13417_1 probe hybridized to gDNA from both parasite clones with similar intensities. This hybridization data defined a DNA segment of ∼30 kb on chromosome 9 that was likely deleted in the 3D7.Gdef clone (Fig. 2A). To confirm this observation, we designed eight PCR primer pairs to amplify the region between the two positive oligonucleotides (Fig. 2A). PCR products were obtained for all eight primer-pairs using gDNA from 3D7.G+ and wild type (WT) 3D7 lines, but only primer pairs 1, 7, and 8 amplified products from 3D7.Gdef gDNA (Fig. 2B). DNA sequencing of a PCR product spanning the 3D7.Gdef deletion defined the breakpoints to an 18,920 bp region (1,374,582 and 1,393,502 bp) on chromosome 9. The deletion is flanked by polyA tracts of >18 bp that have been reported to be sites of frequent recombination and deletion [27].

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