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A putative homologue of CDC20/CDH1 in the malaria parasite is essential for male gamete development.

Guttery DS, Ferguson DJ, Poulin B, Xu Z, Straschil U, Klop O, Solyakov L, Sandrini SM, Brady D, Nieduszynski CA, Janse CJ, Holder AA, Tobin AB, Tewari R - PLoS Pathog. (2012)

Bottom Line: Furthermore, qRT-PCR analysis in parasite lines with deletions of two kinase genes involved in male sexual development (map2 and cdpk4), showed a significant increase in cdc20 transcription in activated gametocytes.Changes in global protein phosphorylation patterns in the Δcdc20 mutant parasites were largely different from those observed in the Δmap2 mutant.This suggests that CDC20 and MAP2 are both likely to play independent but vital roles in male gametogenesis.

View Article: PubMed Central - PubMed

Affiliation: Centre for Genetics and Genomics, School of Biology Queens Medical Centre, University of Nottingham, Nottingham, UK.

ABSTRACT
Cell-cycle progression is governed by a series of essential regulatory proteins. Two major regulators are cell-division cycle protein 20 (CDC20) and its homologue, CDC20 homologue 1 (CDH1), which activate the anaphase-promoting complex/cyclosome (APC/C) in mitosis, and facilitate degradation of mitotic APC/C substrates. The malaria parasite, Plasmodium, is a haploid organism which, during its life-cycle undergoes two stages of mitosis; one associated with asexual multiplication and the other with male gametogenesis. Cell-cycle regulation and DNA replication in Plasmodium was recently shown to be dependent on the activity of a number of protein kinases. However, the function of cell division cycle proteins that are also involved in this process, such as CDC20 and CDH1 is totally unknown. Here we examine the role of a putative CDC20/CDH1 in the rodent malaria Plasmodium berghei (Pb) using reverse genetics. Phylogenetic analysis identified a single putative Plasmodium CDC20/CDH1 homologue (termed CDC20 for simplicity) suggesting that Plasmodium APC/C has only one regulator. In our genetic approach to delete the endogenous cdc20 gene of P. berghei, we demonstrate that PbCDC20 plays a vital role in male gametogenesis, but is not essential for mitosis in the asexual blood stage. Furthermore, qRT-PCR analysis in parasite lines with deletions of two kinase genes involved in male sexual development (map2 and cdpk4), showed a significant increase in cdc20 transcription in activated gametocytes. DNA replication and ultra structural analyses of cdc20 and map2 mutants showed similar blockage of nuclear division at the nuclear spindle/kinetochore stage. CDC20 was phosphorylated in asexual and sexual stages, but the level of modification was higher in activated gametocytes and ookinetes. Changes in global protein phosphorylation patterns in the Δcdc20 mutant parasites were largely different from those observed in the Δmap2 mutant. This suggests that CDC20 and MAP2 are both likely to play independent but vital roles in male gametogenesis.

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Phenotypic analysis of Δcdc20.A. Immunofluorescence images of Plasmodium cultures after 24 hr in vitro immunostained for the female gamete/zygote/ookinete marker P28 (red) and counterstained with the nuclear marker Hoechst (blue). Development of elongated ookinetes was completely ablated in Δcdc20 lines, which produced only round female gametes. Bar = 5 µm. B. Bar graph illustrating ookinete conversion in wild-type and Δcdc20 parasites. The conversion rate is the percentage of P28-positive parasites that had successfully differentiated into elongated ‘banana-shaped’ ookinetes (error bar = arithmetic mean ±SD; n = 3). C. Ookinete conversion after crossing Δcdc20 parasites with a female-defective nek4 mutant (Δnek4) and a male-defective cdpk4 mutant (Δcdpk4). Wild-type parasites were used as a control. Bar graph represents the percentage of round P28-positive parasites that had converted into elongated ookinetes (arithmetic mean ±SD; n = 3). D. Bar graph showing average numbers of oocysts per gut (error bar indicates ±SEM; n = 60 of wild-type or Δcdc20 infected mosquitoes from three independent experiments). Overall infection prevalence was 80% for wild-type and 0% for Δcdc20. E. Wild-type mRNA expression of cdc20, cdpk4 and map2 relative to hsp70 and arginyl-tRNA synthetase as endogenous controls (ΔΔCt method). Error bars represent ±SEM, n = 3 from three independent experiments. The key to the shading of bars is indicated in F. F. Relative expression of cdc20, cdpk4 and map2 in Δcdc20, Δcdpk4 and Δmap2 parasites compared to wild-type parasites (Pfaffl method). Error bars represent ±SEM, n = 3 from three independent experiments. ASB  =  Asexual blood; Sch  =  Schizont; IG  =  Inactivated gametocytes; AG  =  Activated gametocytes; RQ  =  relative quantification.
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ppat-1002554-g003: Phenotypic analysis of Δcdc20.A. Immunofluorescence images of Plasmodium cultures after 24 hr in vitro immunostained for the female gamete/zygote/ookinete marker P28 (red) and counterstained with the nuclear marker Hoechst (blue). Development of elongated ookinetes was completely ablated in Δcdc20 lines, which produced only round female gametes. Bar = 5 µm. B. Bar graph illustrating ookinete conversion in wild-type and Δcdc20 parasites. The conversion rate is the percentage of P28-positive parasites that had successfully differentiated into elongated ‘banana-shaped’ ookinetes (error bar = arithmetic mean ±SD; n = 3). C. Ookinete conversion after crossing Δcdc20 parasites with a female-defective nek4 mutant (Δnek4) and a male-defective cdpk4 mutant (Δcdpk4). Wild-type parasites were used as a control. Bar graph represents the percentage of round P28-positive parasites that had converted into elongated ookinetes (arithmetic mean ±SD; n = 3). D. Bar graph showing average numbers of oocysts per gut (error bar indicates ±SEM; n = 60 of wild-type or Δcdc20 infected mosquitoes from three independent experiments). Overall infection prevalence was 80% for wild-type and 0% for Δcdc20. E. Wild-type mRNA expression of cdc20, cdpk4 and map2 relative to hsp70 and arginyl-tRNA synthetase as endogenous controls (ΔΔCt method). Error bars represent ±SEM, n = 3 from three independent experiments. The key to the shading of bars is indicated in F. F. Relative expression of cdc20, cdpk4 and map2 in Δcdc20, Δcdpk4 and Δmap2 parasites compared to wild-type parasites (Pfaffl method). Error bars represent ±SEM, n = 3 from three independent experiments. ASB  =  Asexual blood; Sch  =  Schizont; IG  =  Inactivated gametocytes; AG  =  Activated gametocytes; RQ  =  relative quantification.

Mentions: Analysis of two cdc20 deletion mutant clones, N10 cl7 and N10 cl9 (henceforward called Δcdc20), showed no developmental abnormalities during asexual proliferation or gametocyte formation, as assessed on blood smears (data not shown). However, in vitro cultures analysed for differentiation into ookinete stages [20], [37] showed complete ablation of ookinete development (Figure 3A, B). To ascertain whether the block in ookinete formation was a defect along the male or female line, we performed genetic crosses as previously described [37], [38]. Crossing of Δcdc20 with a cdpk4 deletion mutant (a previously characterised male mutant [28], henceforward called Δcdpk4) showed no rescue of the phenotype. Conversely, crossing with a nek4 deletion mutant (a previously characterised female mutant [38], henceforward called Δnek4) resulted in 36% ookinete formation (Figure 3C). These data prove that Δcdc20 parasites are defective along the male line. As a result of this observation, we analysed exflagellation of the activated male gametocytes [26], which was completely blocked in Δcdc20 parasites. To substantiate the in vitro findings, we fed mosquitoes on mice infected with either wild-type or Δcdc20 parasites and analysed oocyst development. Wild type parasites developed normally and oocysts were detected in the mosquito gut, whereas no oocysts were found in the guts of mosquitoes fed on Δcdc20 parasites and analysed 14 or 21 days after feeding (Figure 3D). This result confirms that CDC20 is vital to male gamete development and that fertilization/zygote formation/ookinete development is completely blocked in the Δcdc20 parasites, preventing oocyst formation.


A putative homologue of CDC20/CDH1 in the malaria parasite is essential for male gamete development.

Guttery DS, Ferguson DJ, Poulin B, Xu Z, Straschil U, Klop O, Solyakov L, Sandrini SM, Brady D, Nieduszynski CA, Janse CJ, Holder AA, Tobin AB, Tewari R - PLoS Pathog. (2012)

Phenotypic analysis of Δcdc20.A. Immunofluorescence images of Plasmodium cultures after 24 hr in vitro immunostained for the female gamete/zygote/ookinete marker P28 (red) and counterstained with the nuclear marker Hoechst (blue). Development of elongated ookinetes was completely ablated in Δcdc20 lines, which produced only round female gametes. Bar = 5 µm. B. Bar graph illustrating ookinete conversion in wild-type and Δcdc20 parasites. The conversion rate is the percentage of P28-positive parasites that had successfully differentiated into elongated ‘banana-shaped’ ookinetes (error bar = arithmetic mean ±SD; n = 3). C. Ookinete conversion after crossing Δcdc20 parasites with a female-defective nek4 mutant (Δnek4) and a male-defective cdpk4 mutant (Δcdpk4). Wild-type parasites were used as a control. Bar graph represents the percentage of round P28-positive parasites that had converted into elongated ookinetes (arithmetic mean ±SD; n = 3). D. Bar graph showing average numbers of oocysts per gut (error bar indicates ±SEM; n = 60 of wild-type or Δcdc20 infected mosquitoes from three independent experiments). Overall infection prevalence was 80% for wild-type and 0% for Δcdc20. E. Wild-type mRNA expression of cdc20, cdpk4 and map2 relative to hsp70 and arginyl-tRNA synthetase as endogenous controls (ΔΔCt method). Error bars represent ±SEM, n = 3 from three independent experiments. The key to the shading of bars is indicated in F. F. Relative expression of cdc20, cdpk4 and map2 in Δcdc20, Δcdpk4 and Δmap2 parasites compared to wild-type parasites (Pfaffl method). Error bars represent ±SEM, n = 3 from three independent experiments. ASB  =  Asexual blood; Sch  =  Schizont; IG  =  Inactivated gametocytes; AG  =  Activated gametocytes; RQ  =  relative quantification.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285604&req=5

ppat-1002554-g003: Phenotypic analysis of Δcdc20.A. Immunofluorescence images of Plasmodium cultures after 24 hr in vitro immunostained for the female gamete/zygote/ookinete marker P28 (red) and counterstained with the nuclear marker Hoechst (blue). Development of elongated ookinetes was completely ablated in Δcdc20 lines, which produced only round female gametes. Bar = 5 µm. B. Bar graph illustrating ookinete conversion in wild-type and Δcdc20 parasites. The conversion rate is the percentage of P28-positive parasites that had successfully differentiated into elongated ‘banana-shaped’ ookinetes (error bar = arithmetic mean ±SD; n = 3). C. Ookinete conversion after crossing Δcdc20 parasites with a female-defective nek4 mutant (Δnek4) and a male-defective cdpk4 mutant (Δcdpk4). Wild-type parasites were used as a control. Bar graph represents the percentage of round P28-positive parasites that had converted into elongated ookinetes (arithmetic mean ±SD; n = 3). D. Bar graph showing average numbers of oocysts per gut (error bar indicates ±SEM; n = 60 of wild-type or Δcdc20 infected mosquitoes from three independent experiments). Overall infection prevalence was 80% for wild-type and 0% for Δcdc20. E. Wild-type mRNA expression of cdc20, cdpk4 and map2 relative to hsp70 and arginyl-tRNA synthetase as endogenous controls (ΔΔCt method). Error bars represent ±SEM, n = 3 from three independent experiments. The key to the shading of bars is indicated in F. F. Relative expression of cdc20, cdpk4 and map2 in Δcdc20, Δcdpk4 and Δmap2 parasites compared to wild-type parasites (Pfaffl method). Error bars represent ±SEM, n = 3 from three independent experiments. ASB  =  Asexual blood; Sch  =  Schizont; IG  =  Inactivated gametocytes; AG  =  Activated gametocytes; RQ  =  relative quantification.
Mentions: Analysis of two cdc20 deletion mutant clones, N10 cl7 and N10 cl9 (henceforward called Δcdc20), showed no developmental abnormalities during asexual proliferation or gametocyte formation, as assessed on blood smears (data not shown). However, in vitro cultures analysed for differentiation into ookinete stages [20], [37] showed complete ablation of ookinete development (Figure 3A, B). To ascertain whether the block in ookinete formation was a defect along the male or female line, we performed genetic crosses as previously described [37], [38]. Crossing of Δcdc20 with a cdpk4 deletion mutant (a previously characterised male mutant [28], henceforward called Δcdpk4) showed no rescue of the phenotype. Conversely, crossing with a nek4 deletion mutant (a previously characterised female mutant [38], henceforward called Δnek4) resulted in 36% ookinete formation (Figure 3C). These data prove that Δcdc20 parasites are defective along the male line. As a result of this observation, we analysed exflagellation of the activated male gametocytes [26], which was completely blocked in Δcdc20 parasites. To substantiate the in vitro findings, we fed mosquitoes on mice infected with either wild-type or Δcdc20 parasites and analysed oocyst development. Wild type parasites developed normally and oocysts were detected in the mosquito gut, whereas no oocysts were found in the guts of mosquitoes fed on Δcdc20 parasites and analysed 14 or 21 days after feeding (Figure 3D). This result confirms that CDC20 is vital to male gamete development and that fertilization/zygote formation/ookinete development is completely blocked in the Δcdc20 parasites, preventing oocyst formation.

Bottom Line: Furthermore, qRT-PCR analysis in parasite lines with deletions of two kinase genes involved in male sexual development (map2 and cdpk4), showed a significant increase in cdc20 transcription in activated gametocytes.Changes in global protein phosphorylation patterns in the Δcdc20 mutant parasites were largely different from those observed in the Δmap2 mutant.This suggests that CDC20 and MAP2 are both likely to play independent but vital roles in male gametogenesis.

View Article: PubMed Central - PubMed

Affiliation: Centre for Genetics and Genomics, School of Biology Queens Medical Centre, University of Nottingham, Nottingham, UK.

ABSTRACT
Cell-cycle progression is governed by a series of essential regulatory proteins. Two major regulators are cell-division cycle protein 20 (CDC20) and its homologue, CDC20 homologue 1 (CDH1), which activate the anaphase-promoting complex/cyclosome (APC/C) in mitosis, and facilitate degradation of mitotic APC/C substrates. The malaria parasite, Plasmodium, is a haploid organism which, during its life-cycle undergoes two stages of mitosis; one associated with asexual multiplication and the other with male gametogenesis. Cell-cycle regulation and DNA replication in Plasmodium was recently shown to be dependent on the activity of a number of protein kinases. However, the function of cell division cycle proteins that are also involved in this process, such as CDC20 and CDH1 is totally unknown. Here we examine the role of a putative CDC20/CDH1 in the rodent malaria Plasmodium berghei (Pb) using reverse genetics. Phylogenetic analysis identified a single putative Plasmodium CDC20/CDH1 homologue (termed CDC20 for simplicity) suggesting that Plasmodium APC/C has only one regulator. In our genetic approach to delete the endogenous cdc20 gene of P. berghei, we demonstrate that PbCDC20 plays a vital role in male gametogenesis, but is not essential for mitosis in the asexual blood stage. Furthermore, qRT-PCR analysis in parasite lines with deletions of two kinase genes involved in male sexual development (map2 and cdpk4), showed a significant increase in cdc20 transcription in activated gametocytes. DNA replication and ultra structural analyses of cdc20 and map2 mutants showed similar blockage of nuclear division at the nuclear spindle/kinetochore stage. CDC20 was phosphorylated in asexual and sexual stages, but the level of modification was higher in activated gametocytes and ookinetes. Changes in global protein phosphorylation patterns in the Δcdc20 mutant parasites were largely different from those observed in the Δmap2 mutant. This suggests that CDC20 and MAP2 are both likely to play independent but vital roles in male gametogenesis.

Show MeSH
Related in: MedlinePlus