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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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CYC3 is dispensable in asexual and sexual stages but important for oocyst development.(A) Average number of nuclei per schizont measured using Giemsa stained slides at 100x magnification. Bar is the mean ± SEM. n = 4 independent experiments. (B) Microgametogenesis of ∆cyc3 compared with WT measured as the number of exflagellation centres per field. Means ± SEM are shown. n = 4 independent experiments. (C) Ookinete conversion as a percentage in ∆cyc3 and WT lines. Ookinetes were identified using the marker 13.1 and defined as those cells that successfully differentiated into elongated ‘banana shaped’ ookinetes. Bar is the mean ± SEM. n = 5 independent experiments. (D) Total number of GFP-positive oocysts per infected mosquito, including normal and small oocysts, at 5, 7, 10, 14, 21 dpi for ∆cyc3 and WT lines. Bar is the mean ± SEM. n = 3 independent experiments (20 mosquitoes for each). Example of relative oocyst size and numbers at (E) 10x and (F) 63x magnification in ∆cyc3 and WT lines. Images show DIC and GFP at 5, 7, 10, 14 and 21 dpi. Scale bar = 100 μm for 10x and 20 μm for 63x. (G) Individual ∆cyc3 and WT oocyst diameters in μm at 5, 7, 10, 14 and 21 dpi. Horizontal line indicates the mean from 3 independent experiments (20 mosquitoes for each) of ∆cyc3 and WT. p <0.001 for all time points. (H) Total number of sporozoites per mosquito from 14 and 21 dpi midguts for ∆cyc3 and WT lines. Three independent experiments are described, n = 20 mosquitoes for each replicate. ** p ≤ 0.01, *** p ≤ 0.001. (I) Genetic complementation of ∆cyc3. Mosquitoes were fed with a combination of WT, ∆cyc3 or ∆cyc3 with either male (∆p48/45 and ∆hap2) or female (∆dozi and ∆nek4) mutants. Shown is a representation of one experiment (20 mosquitoes per line).
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ppat.1005273.g003: CYC3 is dispensable in asexual and sexual stages but important for oocyst development.(A) Average number of nuclei per schizont measured using Giemsa stained slides at 100x magnification. Bar is the mean ± SEM. n = 4 independent experiments. (B) Microgametogenesis of ∆cyc3 compared with WT measured as the number of exflagellation centres per field. Means ± SEM are shown. n = 4 independent experiments. (C) Ookinete conversion as a percentage in ∆cyc3 and WT lines. Ookinetes were identified using the marker 13.1 and defined as those cells that successfully differentiated into elongated ‘banana shaped’ ookinetes. Bar is the mean ± SEM. n = 5 independent experiments. (D) Total number of GFP-positive oocysts per infected mosquito, including normal and small oocysts, at 5, 7, 10, 14, 21 dpi for ∆cyc3 and WT lines. Bar is the mean ± SEM. n = 3 independent experiments (20 mosquitoes for each). Example of relative oocyst size and numbers at (E) 10x and (F) 63x magnification in ∆cyc3 and WT lines. Images show DIC and GFP at 5, 7, 10, 14 and 21 dpi. Scale bar = 100 μm for 10x and 20 μm for 63x. (G) Individual ∆cyc3 and WT oocyst diameters in μm at 5, 7, 10, 14 and 21 dpi. Horizontal line indicates the mean from 3 independent experiments (20 mosquitoes for each) of ∆cyc3 and WT. p <0.001 for all time points. (H) Total number of sporozoites per mosquito from 14 and 21 dpi midguts for ∆cyc3 and WT lines. Three independent experiments are described, n = 20 mosquitoes for each replicate. ** p ≤ 0.01, *** p ≤ 0.001. (I) Genetic complementation of ∆cyc3. Mosquitoes were fed with a combination of WT, ∆cyc3 or ∆cyc3 with either male (∆p48/45 and ∆hap2) or female (∆dozi and ∆nek4) mutants. Shown is a representation of one experiment (20 mosquitoes per line).

Mentions: Analysis of two independent cyc3 deletion clones, cyc3 cl1 and cyc3 cl3 (hence forward called Δcyc3) showed no overt phenotype during blood stage asexual proliferation, microgamete exflagellation or ookinete conversion in vitro when compared with control parasites which constitutively express untagged GFP (WTGFPcon 507 cl1 line, henceforth known as WT) [44] (Fig 3A–3C).


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)

CYC3 is dispensable in asexual and sexual stages but important for oocyst development.(A) Average number of nuclei per schizont measured using Giemsa stained slides at 100x magnification. Bar is the mean ± SEM. n = 4 independent experiments. (B) Microgametogenesis of ∆cyc3 compared with WT measured as the number of exflagellation centres per field. Means ± SEM are shown. n = 4 independent experiments. (C) Ookinete conversion as a percentage in ∆cyc3 and WT lines. Ookinetes were identified using the marker 13.1 and defined as those cells that successfully differentiated into elongated ‘banana shaped’ ookinetes. Bar is the mean ± SEM. n = 5 independent experiments. (D) Total number of GFP-positive oocysts per infected mosquito, including normal and small oocysts, at 5, 7, 10, 14, 21 dpi for ∆cyc3 and WT lines. Bar is the mean ± SEM. n = 3 independent experiments (20 mosquitoes for each). Example of relative oocyst size and numbers at (E) 10x and (F) 63x magnification in ∆cyc3 and WT lines. Images show DIC and GFP at 5, 7, 10, 14 and 21 dpi. Scale bar = 100 μm for 10x and 20 μm for 63x. (G) Individual ∆cyc3 and WT oocyst diameters in μm at 5, 7, 10, 14 and 21 dpi. Horizontal line indicates the mean from 3 independent experiments (20 mosquitoes for each) of ∆cyc3 and WT. p <0.001 for all time points. (H) Total number of sporozoites per mosquito from 14 and 21 dpi midguts for ∆cyc3 and WT lines. Three independent experiments are described, n = 20 mosquitoes for each replicate. ** p ≤ 0.01, *** p ≤ 0.001. (I) Genetic complementation of ∆cyc3. Mosquitoes were fed with a combination of WT, ∆cyc3 or ∆cyc3 with either male (∆p48/45 and ∆hap2) or female (∆dozi and ∆nek4) mutants. Shown is a representation of one experiment (20 mosquitoes per line).
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1005273.g003: CYC3 is dispensable in asexual and sexual stages but important for oocyst development.(A) Average number of nuclei per schizont measured using Giemsa stained slides at 100x magnification. Bar is the mean ± SEM. n = 4 independent experiments. (B) Microgametogenesis of ∆cyc3 compared with WT measured as the number of exflagellation centres per field. Means ± SEM are shown. n = 4 independent experiments. (C) Ookinete conversion as a percentage in ∆cyc3 and WT lines. Ookinetes were identified using the marker 13.1 and defined as those cells that successfully differentiated into elongated ‘banana shaped’ ookinetes. Bar is the mean ± SEM. n = 5 independent experiments. (D) Total number of GFP-positive oocysts per infected mosquito, including normal and small oocysts, at 5, 7, 10, 14, 21 dpi for ∆cyc3 and WT lines. Bar is the mean ± SEM. n = 3 independent experiments (20 mosquitoes for each). Example of relative oocyst size and numbers at (E) 10x and (F) 63x magnification in ∆cyc3 and WT lines. Images show DIC and GFP at 5, 7, 10, 14 and 21 dpi. Scale bar = 100 μm for 10x and 20 μm for 63x. (G) Individual ∆cyc3 and WT oocyst diameters in μm at 5, 7, 10, 14 and 21 dpi. Horizontal line indicates the mean from 3 independent experiments (20 mosquitoes for each) of ∆cyc3 and WT. p <0.001 for all time points. (H) Total number of sporozoites per mosquito from 14 and 21 dpi midguts for ∆cyc3 and WT lines. Three independent experiments are described, n = 20 mosquitoes for each replicate. ** p ≤ 0.01, *** p ≤ 0.001. (I) Genetic complementation of ∆cyc3. Mosquitoes were fed with a combination of WT, ∆cyc3 or ∆cyc3 with either male (∆p48/45 and ∆hap2) or female (∆dozi and ∆nek4) mutants. Shown is a representation of one experiment (20 mosquitoes per line).
Mentions: Analysis of two independent cyc3 deletion clones, cyc3 cl1 and cyc3 cl3 (hence forward called Δcyc3) showed no overt phenotype during blood stage asexual proliferation, microgamete exflagellation or ookinete conversion in vitro when compared with control parasites which constitutively express untagged GFP (WTGFPcon 507 cl1 line, henceforth known as WT) [44] (Fig 3A–3C).

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