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Plasmodium P-Type Cyclin CYC3 Modulates Endomitotic Growth during Oocyst Development in Mosquitoes.

Roques M, Wall RJ, Douglass AP, Ramaprasad A, Ferguson DJ, Kaindama ML, Brusini L, Joshi N, Rchiad Z, Brady D, Guttery DS, Wheatley SP, Yamano H, Holder AA, Pain A, Wickstead B, Tewari R - PLoS Pathog. (2015)

Bottom Line: Deletion of cyc3 resulted in defects in size, number and growth of oocysts, with abnormalities in budding and sporozoite formation.Furthermore, global transcript analysis of the cyc3-deleted and wild type parasites at gametocyte and ookinete stages identified differentially expressed genes required for signalling, invasion and oocyst development.Collectively these data suggest that cyc3 modulates oocyst endomitotic development in Plasmodium berghei.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Queens Medical Centre, University of Nottingham, Nottingham, United Kingdom.

ABSTRACT
Cell-cycle progression and cell division in eukaryotes are governed in part by the cyclin family and their regulation of cyclin-dependent kinases (CDKs). Cyclins are very well characterised in model systems such as yeast and human cells, but surprisingly little is known about their number and role in Plasmodium, the unicellular protozoan parasite that causes malaria. Malaria parasite cell division and proliferation differs from that of many eukaryotes. During its life cycle it undergoes two types of mitosis: endomitosis in asexual stages and an extremely rapid mitotic process during male gametogenesis. Both schizogony (producing merozoites) in host liver and red blood cells, and sporogony (producing sporozoites) in the mosquito vector, are endomitotic with repeated nuclear replication, without chromosome condensation, before cell division. The role of specific cyclins during Plasmodium cell proliferation was unknown. We show here that the Plasmodium genome contains only three cyclin genes, representing an unusual repertoire of cyclin classes. Expression and reverse genetic analyses of the single Plant (P)-type cyclin, CYC3, in the rodent malaria parasite, Plasmodium berghei, revealed a cytoplasmic and nuclear location of the GFP-tagged protein throughout the lifecycle. Deletion of cyc3 resulted in defects in size, number and growth of oocysts, with abnormalities in budding and sporozoite formation. Furthermore, global transcript analysis of the cyc3-deleted and wild type parasites at gametocyte and ookinete stages identified differentially expressed genes required for signalling, invasion and oocyst development. Collectively these data suggest that cyc3 modulates oocyst endomitotic development in Plasmodium berghei.

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Related in: MedlinePlus

Transcript analysis of genes involved in parasite development.(A) Ratio-Intensity scatter plots for ∆cyc3 activated gametocytes and ookinetes. The y-axis shows the log2 fold change between wild-type and mutant and the x-axis shows the average of normalised FPKM (See also S2 Table). Significantly up-regulated genes are highlighted in green while down-regulated genes are highlighted in red. (B) Heatmaps for invasion, kinase and phosphatase gene clusters based on their log2 fold change in Δcyc3 activated gametocytes (inner track) and Δcyc3 ookinetes (outer track) relative to WT. Functional groups were inferred from annotations available in GeneDB (http://www.genedb.org/). Genes that were found significantly misregulated are shown in bold and those validated by qRT-PCR are shown in red. Full gene list and functional clusters are shown in S3 Table. (C) Log2 fold transcript change in Δcyc3 at different life-cycle stages of cell cycle, signalling and transcription genes, and invasion and oocyst development genes, studied in ∆cyc3 (compared against WT) using qRT-PCR. Data were normalised against an endogenous control gene, hsp70 (PBANKA_081890). Each bar is the mean of relative expression in comparison to WT from three biological replicates ± SEM. Sch: schizonts; AG: activated gametocytes; Ook: ookinetes; Spor: 14 dpi oocysts/sporozoites. * p ≤0.05; ** p ≤0.01, *** p ≤0.001. (D) Comparison of qRT-PCR and RNA-seq data (Cuffdiff2 analysis) using Log2 values for activated gametocyte and ookinete samples.
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ppat.1005273.g006: Transcript analysis of genes involved in parasite development.(A) Ratio-Intensity scatter plots for ∆cyc3 activated gametocytes and ookinetes. The y-axis shows the log2 fold change between wild-type and mutant and the x-axis shows the average of normalised FPKM (See also S2 Table). Significantly up-regulated genes are highlighted in green while down-regulated genes are highlighted in red. (B) Heatmaps for invasion, kinase and phosphatase gene clusters based on their log2 fold change in Δcyc3 activated gametocytes (inner track) and Δcyc3 ookinetes (outer track) relative to WT. Functional groups were inferred from annotations available in GeneDB (http://www.genedb.org/). Genes that were found significantly misregulated are shown in bold and those validated by qRT-PCR are shown in red. Full gene list and functional clusters are shown in S3 Table. (C) Log2 fold transcript change in Δcyc3 at different life-cycle stages of cell cycle, signalling and transcription genes, and invasion and oocyst development genes, studied in ∆cyc3 (compared against WT) using qRT-PCR. Data were normalised against an endogenous control gene, hsp70 (PBANKA_081890). Each bar is the mean of relative expression in comparison to WT from three biological replicates ± SEM. Sch: schizonts; AG: activated gametocytes; Ook: ookinetes; Spor: 14 dpi oocysts/sporozoites. * p ≤0.05; ** p ≤0.01, *** p ≤0.001. (D) Comparison of qRT-PCR and RNA-seq data (Cuffdiff2 analysis) using Log2 values for activated gametocyte and ookinete samples.

Mentions: The marked changes in Δcyc3 oocyst morphology and growth led us to analyse the regulation of mRNA in Δcyc3 parasites compared with WT. We first used strand-specific RNA sequencing (RNA-seq) to investigate the global transcript levels in Δcyc3 and WT activated gametocytes and ookinetes. The deletion of cyc3 was confirmed by RNAseq, with no reads mapping to the region of the gene targeted for disruption (S6A Fig). Generally, most transcript levels were very similar in activated gametocytes, with strong linkage between levels in Δcyc3 and WT lines. However, the ookinete transcriptome was greatly altered by loss of cyc3, with many genes down-regulated and a smaller number up-regulated. In total, 813 and 2,069 genes showed modulated expression in activated gametocytes and ookinetes, respectively (p <0.05 and fold change >2; 702 and 1,891 genes in activated gametocytes and ookinetes, respectively, with p <0.01 and fold change >2) (Fig 6A, S6B Fig and S2 Table), including those with roles in reversible phosphorylation, transcription, cell signalling and inner membrane complex function (Fig 6B, S6C Fig and S3 Table). We identified several functional clusters that were significantly differentially expressed in Δcyc3 (Fig 6B, S6C Fig and S3 Table) which may be collectively responsible for the observed phenotype. Global Gene Ontology (GO) analysis showed enrichment of genes associated with GO terms ‘kinase activity’, ‘protein phosphorylation’ and’ inner membrane complex’ (S6D Fig).


Plasmodium P-Type Cyclin CYC3 Modulates Endomitotic Growth during Oocyst Development in Mosquitoes.

Roques M, Wall RJ, Douglass AP, Ramaprasad A, Ferguson DJ, Kaindama ML, Brusini L, Joshi N, Rchiad Z, Brady D, Guttery DS, Wheatley SP, Yamano H, Holder AA, Pain A, Wickstead B, Tewari R - PLoS Pathog. (2015)

Transcript analysis of genes involved in parasite development.(A) Ratio-Intensity scatter plots for ∆cyc3 activated gametocytes and ookinetes. The y-axis shows the log2 fold change between wild-type and mutant and the x-axis shows the average of normalised FPKM (See also S2 Table). Significantly up-regulated genes are highlighted in green while down-regulated genes are highlighted in red. (B) Heatmaps for invasion, kinase and phosphatase gene clusters based on their log2 fold change in Δcyc3 activated gametocytes (inner track) and Δcyc3 ookinetes (outer track) relative to WT. Functional groups were inferred from annotations available in GeneDB (http://www.genedb.org/). Genes that were found significantly misregulated are shown in bold and those validated by qRT-PCR are shown in red. Full gene list and functional clusters are shown in S3 Table. (C) Log2 fold transcript change in Δcyc3 at different life-cycle stages of cell cycle, signalling and transcription genes, and invasion and oocyst development genes, studied in ∆cyc3 (compared against WT) using qRT-PCR. Data were normalised against an endogenous control gene, hsp70 (PBANKA_081890). Each bar is the mean of relative expression in comparison to WT from three biological replicates ± SEM. Sch: schizonts; AG: activated gametocytes; Ook: ookinetes; Spor: 14 dpi oocysts/sporozoites. * p ≤0.05; ** p ≤0.01, *** p ≤0.001. (D) Comparison of qRT-PCR and RNA-seq data (Cuffdiff2 analysis) using Log2 values for activated gametocyte and ookinete samples.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4643991&req=5

ppat.1005273.g006: Transcript analysis of genes involved in parasite development.(A) Ratio-Intensity scatter plots for ∆cyc3 activated gametocytes and ookinetes. The y-axis shows the log2 fold change between wild-type and mutant and the x-axis shows the average of normalised FPKM (See also S2 Table). Significantly up-regulated genes are highlighted in green while down-regulated genes are highlighted in red. (B) Heatmaps for invasion, kinase and phosphatase gene clusters based on their log2 fold change in Δcyc3 activated gametocytes (inner track) and Δcyc3 ookinetes (outer track) relative to WT. Functional groups were inferred from annotations available in GeneDB (http://www.genedb.org/). Genes that were found significantly misregulated are shown in bold and those validated by qRT-PCR are shown in red. Full gene list and functional clusters are shown in S3 Table. (C) Log2 fold transcript change in Δcyc3 at different life-cycle stages of cell cycle, signalling and transcription genes, and invasion and oocyst development genes, studied in ∆cyc3 (compared against WT) using qRT-PCR. Data were normalised against an endogenous control gene, hsp70 (PBANKA_081890). Each bar is the mean of relative expression in comparison to WT from three biological replicates ± SEM. Sch: schizonts; AG: activated gametocytes; Ook: ookinetes; Spor: 14 dpi oocysts/sporozoites. * p ≤0.05; ** p ≤0.01, *** p ≤0.001. (D) Comparison of qRT-PCR and RNA-seq data (Cuffdiff2 analysis) using Log2 values for activated gametocyte and ookinete samples.
Mentions: The marked changes in Δcyc3 oocyst morphology and growth led us to analyse the regulation of mRNA in Δcyc3 parasites compared with WT. We first used strand-specific RNA sequencing (RNA-seq) to investigate the global transcript levels in Δcyc3 and WT activated gametocytes and ookinetes. The deletion of cyc3 was confirmed by RNAseq, with no reads mapping to the region of the gene targeted for disruption (S6A Fig). Generally, most transcript levels were very similar in activated gametocytes, with strong linkage between levels in Δcyc3 and WT lines. However, the ookinete transcriptome was greatly altered by loss of cyc3, with many genes down-regulated and a smaller number up-regulated. In total, 813 and 2,069 genes showed modulated expression in activated gametocytes and ookinetes, respectively (p <0.05 and fold change >2; 702 and 1,891 genes in activated gametocytes and ookinetes, respectively, with p <0.01 and fold change >2) (Fig 6A, S6B Fig and S2 Table), including those with roles in reversible phosphorylation, transcription, cell signalling and inner membrane complex function (Fig 6B, S6C Fig and S3 Table). We identified several functional clusters that were significantly differentially expressed in Δcyc3 (Fig 6B, S6C Fig and S3 Table) which may be collectively responsible for the observed phenotype. Global Gene Ontology (GO) analysis showed enrichment of genes associated with GO terms ‘kinase activity’, ‘protein phosphorylation’ and’ inner membrane complex’ (S6D Fig).

Bottom Line: Deletion of cyc3 resulted in defects in size, number and growth of oocysts, with abnormalities in budding and sporozoite formation.Furthermore, global transcript analysis of the cyc3-deleted and wild type parasites at gametocyte and ookinete stages identified differentially expressed genes required for signalling, invasion and oocyst development.Collectively these data suggest that cyc3 modulates oocyst endomitotic development in Plasmodium berghei.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Queens Medical Centre, University of Nottingham, Nottingham, United Kingdom.

ABSTRACT
Cell-cycle progression and cell division in eukaryotes are governed in part by the cyclin family and their regulation of cyclin-dependent kinases (CDKs). Cyclins are very well characterised in model systems such as yeast and human cells, but surprisingly little is known about their number and role in Plasmodium, the unicellular protozoan parasite that causes malaria. Malaria parasite cell division and proliferation differs from that of many eukaryotes. During its life cycle it undergoes two types of mitosis: endomitosis in asexual stages and an extremely rapid mitotic process during male gametogenesis. Both schizogony (producing merozoites) in host liver and red blood cells, and sporogony (producing sporozoites) in the mosquito vector, are endomitotic with repeated nuclear replication, without chromosome condensation, before cell division. The role of specific cyclins during Plasmodium cell proliferation was unknown. We show here that the Plasmodium genome contains only three cyclin genes, representing an unusual repertoire of cyclin classes. Expression and reverse genetic analyses of the single Plant (P)-type cyclin, CYC3, in the rodent malaria parasite, Plasmodium berghei, revealed a cytoplasmic and nuclear location of the GFP-tagged protein throughout the lifecycle. Deletion of cyc3 resulted in defects in size, number and growth of oocysts, with abnormalities in budding and sporozoite formation. Furthermore, global transcript analysis of the cyc3-deleted and wild type parasites at gametocyte and ookinete stages identified differentially expressed genes required for signalling, invasion and oocyst development. Collectively these data suggest that cyc3 modulates oocyst endomitotic development in Plasmodium berghei.

Show MeSH
Related in: MedlinePlus