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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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Expression profile of the Pfge genes through gametocytogenesis.MACS purified late stage asexual parasite cultures were set up at 6% hematocrit and sorbitol synchronized 2 hours later to remove all but the newly invaded ring stage parasites. Parasitemia was monitored by daily Giemsa-stained smear. Ring (blue), schizont stage parasitemias (red), and stage II gametocytemia (green) are plotted (A & B). The relative abundance, (log2) of the indicated gene in relation to the expression level on day 2 was calculated using the 2−ΔΔCT method [43] with seryl tRNA synthetase as the reference and plotted in C and D): Pfaldolase (blue), Pfkahrp (brown), Pfmsp7-5 (purple), Pfs16 (green), Pfgdv1 (bright red); while Pfge1 (orange), Pfge2 (pale pink), Pfge3 (beige), Pfg27 (light blue), Pfge7 (dark pink), Pfge8 (light green), Pfs47(turquoise) and Pfs16 (green) are shown in (E and F). The 3 asexual cycles are indicated by numbers as well as gray dotted lines, and NAG treatment is indicated by the gray box. Representative data from one of three independent experiments is shown. The samples from the different time points were tested in triplicate and the average relative expression is plotted with the error bars representing the range.
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ppat-1002964-g004: Expression profile of the Pfge genes through gametocytogenesis.MACS purified late stage asexual parasite cultures were set up at 6% hematocrit and sorbitol synchronized 2 hours later to remove all but the newly invaded ring stage parasites. Parasitemia was monitored by daily Giemsa-stained smear. Ring (blue), schizont stage parasitemias (red), and stage II gametocytemia (green) are plotted (A & B). The relative abundance, (log2) of the indicated gene in relation to the expression level on day 2 was calculated using the 2−ΔΔCT method [43] with seryl tRNA synthetase as the reference and plotted in C and D): Pfaldolase (blue), Pfkahrp (brown), Pfmsp7-5 (purple), Pfs16 (green), Pfgdv1 (bright red); while Pfge1 (orange), Pfge2 (pale pink), Pfge3 (beige), Pfg27 (light blue), Pfge7 (dark pink), Pfge8 (light green), Pfs47(turquoise) and Pfs16 (green) are shown in (E and F). The 3 asexual cycles are indicated by numbers as well as gray dotted lines, and NAG treatment is indicated by the gray box. Representative data from one of three independent experiments is shown. The samples from the different time points were tested in triplicate and the average relative expression is plotted with the error bars representing the range.

Mentions: Strain 3D7 P. falciparum gametocyte cultures were initiated with 0.1% synchronized ring stage parasites and followed daily for 2 weeks, through 3 asexual cycles (1, 2, 3, Fig. 4A and B). Asexual parasitemia peaked in 6–7 days with a subsequent decline after day 7 and a rise in Giemsa-detectable stage II gametocytes that peaked on day 10. In one set of cultures (Fig. 4A), N-acetyl-D-glucosamine (NAG) was added on day 6–8 when the ring stage parasitemia reached 4% to prevent schizont maturation and merozoite invasion as previously described [41], [42]. In the absence of NAG (Fig. 4B), ring forms observed on day 6 developed into schizonts by day 7, but did not form new ring stage parasites, causing the parasitemia to decline rapidly by day 8. Consequently, the major difference between NAG-treated and untreated cultures was the third schizont peak on day 7. In both cases the asexual parasitemia declined by day 8 and a similar number of ring stage parasites and gametocytes were produced in both conditions. These findings suggest that gametocytogenesis was induced prior to the death of the schizonts on day 7 and was not triggered directly by the conditions that cause the natural decline in asexual growth (crash). The gametocyte conversion rate was estimated to be 13.8% and 13% in NAG treated and untreated cultures, respectively, by using the daily RBC counts and Giemsa-stained culture smears to calculate the total number of gametocytes and ring stage parasites produced over the first 10 days of culture.


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)

Expression profile of the Pfge genes through gametocytogenesis.MACS purified late stage asexual parasite cultures were set up at 6% hematocrit and sorbitol synchronized 2 hours later to remove all but the newly invaded ring stage parasites. Parasitemia was monitored by daily Giemsa-stained smear. Ring (blue), schizont stage parasitemias (red), and stage II gametocytemia (green) are plotted (A & B). The relative abundance, (log2) of the indicated gene in relation to the expression level on day 2 was calculated using the 2−ΔΔCT method [43] with seryl tRNA synthetase as the reference and plotted in C and D): Pfaldolase (blue), Pfkahrp (brown), Pfmsp7-5 (purple), Pfs16 (green), Pfgdv1 (bright red); while Pfge1 (orange), Pfge2 (pale pink), Pfge3 (beige), Pfg27 (light blue), Pfge7 (dark pink), Pfge8 (light green), Pfs47(turquoise) and Pfs16 (green) are shown in (E and F). The 3 asexual cycles are indicated by numbers as well as gray dotted lines, and NAG treatment is indicated by the gray box. Representative data from one of three independent experiments is shown. The samples from the different time points were tested in triplicate and the average relative expression is plotted with the error bars representing the range.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002964-g004: Expression profile of the Pfge genes through gametocytogenesis.MACS purified late stage asexual parasite cultures were set up at 6% hematocrit and sorbitol synchronized 2 hours later to remove all but the newly invaded ring stage parasites. Parasitemia was monitored by daily Giemsa-stained smear. Ring (blue), schizont stage parasitemias (red), and stage II gametocytemia (green) are plotted (A & B). The relative abundance, (log2) of the indicated gene in relation to the expression level on day 2 was calculated using the 2−ΔΔCT method [43] with seryl tRNA synthetase as the reference and plotted in C and D): Pfaldolase (blue), Pfkahrp (brown), Pfmsp7-5 (purple), Pfs16 (green), Pfgdv1 (bright red); while Pfge1 (orange), Pfge2 (pale pink), Pfge3 (beige), Pfg27 (light blue), Pfge7 (dark pink), Pfge8 (light green), Pfs47(turquoise) and Pfs16 (green) are shown in (E and F). The 3 asexual cycles are indicated by numbers as well as gray dotted lines, and NAG treatment is indicated by the gray box. Representative data from one of three independent experiments is shown. The samples from the different time points were tested in triplicate and the average relative expression is plotted with the error bars representing the range.
Mentions: Strain 3D7 P. falciparum gametocyte cultures were initiated with 0.1% synchronized ring stage parasites and followed daily for 2 weeks, through 3 asexual cycles (1, 2, 3, Fig. 4A and B). Asexual parasitemia peaked in 6–7 days with a subsequent decline after day 7 and a rise in Giemsa-detectable stage II gametocytes that peaked on day 10. In one set of cultures (Fig. 4A), N-acetyl-D-glucosamine (NAG) was added on day 6–8 when the ring stage parasitemia reached 4% to prevent schizont maturation and merozoite invasion as previously described [41], [42]. In the absence of NAG (Fig. 4B), ring forms observed on day 6 developed into schizonts by day 7, but did not form new ring stage parasites, causing the parasitemia to decline rapidly by day 8. Consequently, the major difference between NAG-treated and untreated cultures was the third schizont peak on day 7. In both cases the asexual parasitemia declined by day 8 and a similar number of ring stage parasites and gametocytes were produced in both conditions. These findings suggest that gametocytogenesis was induced prior to the death of the schizonts on day 7 and was not triggered directly by the conditions that cause the natural decline in asexual growth (crash). The gametocyte conversion rate was estimated to be 13.8% and 13% in NAG treated and untreated cultures, respectively, by using the daily RBC counts and Giemsa-stained culture smears to calculate the total number of gametocytes and ring stage parasites produced over the first 10 days of culture.

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