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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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Ultrastructure analysis of oocyst development in ∆cyc3 mutant.(A) Low power ultrastructural images of oocyst development at 7–10 days (i, iv, vii) and 14–21 days post-infection (dpi) showing normal sporulation of WT (ii, iii) and certain Δcyc3 (v, vi) parasites while other Δcyc3 mutants show evidence of cytoplasmic vacuolation (V) and degeneration (vii, viii, ix). N–nucleus. Bars represent 10 μm. (B) Details showing progressive stages in sporozoite formation in wild type parasites (i-iii) and various abnormal developmental stages of the Δcyc3 parasite (iv-vi). Bars represent 1 μm. i. Initiation of sporozoite formation with formation of the inner membrane complex (I) beneath the plasmalemma and above a peripherally located nucleus (N) with nuclear pole (NP). ii. Early sporozoite (S) with rhoptry anlagen (R) budding from the surface of the sporoblast. N–nucleus. iii. Late stage in sporozoite formation showing the elongated sporozoites (S). R–rhoptry. iv. Detail of the sporoblast cytoplasm showing nuclei (N) enclosed by abnormal membrane whorls v. Part of a nucleus with extensive nuclear spindle microtubule (Mt) not seen in WT parasite. vi. Detail of a late stage in parasite degeneration showing apoptotic-like nuclei (N) and dilated endoplasmic reticulum (ER).
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ppat.1005273.g005: Ultrastructure analysis of oocyst development in ∆cyc3 mutant.(A) Low power ultrastructural images of oocyst development at 7–10 days (i, iv, vii) and 14–21 days post-infection (dpi) showing normal sporulation of WT (ii, iii) and certain Δcyc3 (v, vi) parasites while other Δcyc3 mutants show evidence of cytoplasmic vacuolation (V) and degeneration (vii, viii, ix). N–nucleus. Bars represent 10 μm. (B) Details showing progressive stages in sporozoite formation in wild type parasites (i-iii) and various abnormal developmental stages of the Δcyc3 parasite (iv-vi). Bars represent 1 μm. i. Initiation of sporozoite formation with formation of the inner membrane complex (I) beneath the plasmalemma and above a peripherally located nucleus (N) with nuclear pole (NP). ii. Early sporozoite (S) with rhoptry anlagen (R) budding from the surface of the sporoblast. N–nucleus. iii. Late stage in sporozoite formation showing the elongated sporozoites (S). R–rhoptry. iv. Detail of the sporoblast cytoplasm showing nuclei (N) enclosed by abnormal membrane whorls v. Part of a nucleus with extensive nuclear spindle microtubule (Mt) not seen in WT parasite. vi. Detail of a late stage in parasite degeneration showing apoptotic-like nuclei (N) and dilated endoplasmic reticulum (ER).

Mentions: To define further the defect in oocyst growth during different developmental stages of sporogony, Δcyc3 and WT parasite-infected mosquito midguts at 7, 10, 14, and 21 dpi were examined by transmission electron microscopy. Marked differences were observed at the later time points in the ultrastructure of the majority of Δcyc3 compared to WT oocysts, although some Δcyc3 oocysts appeared similar to WT (Fig 5). At every time point there were significantly fewer oocysts in the guts of mosquitoes infected with Δcyc3 compared to the WT parasites (5 oocysts/gut Δcyc3 compared to 60 oocyst/gut WT at 7 dpi).


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)

Ultrastructure analysis of oocyst development in ∆cyc3 mutant.(A) Low power ultrastructural images of oocyst development at 7–10 days (i, iv, vii) and 14–21 days post-infection (dpi) showing normal sporulation of WT (ii, iii) and certain Δcyc3 (v, vi) parasites while other Δcyc3 mutants show evidence of cytoplasmic vacuolation (V) and degeneration (vii, viii, ix). N–nucleus. Bars represent 10 μm. (B) Details showing progressive stages in sporozoite formation in wild type parasites (i-iii) and various abnormal developmental stages of the Δcyc3 parasite (iv-vi). Bars represent 1 μm. i. Initiation of sporozoite formation with formation of the inner membrane complex (I) beneath the plasmalemma and above a peripherally located nucleus (N) with nuclear pole (NP). ii. Early sporozoite (S) with rhoptry anlagen (R) budding from the surface of the sporoblast. N–nucleus. iii. Late stage in sporozoite formation showing the elongated sporozoites (S). R–rhoptry. iv. Detail of the sporoblast cytoplasm showing nuclei (N) enclosed by abnormal membrane whorls v. Part of a nucleus with extensive nuclear spindle microtubule (Mt) not seen in WT parasite. vi. Detail of a late stage in parasite degeneration showing apoptotic-like nuclei (N) and dilated endoplasmic reticulum (ER).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4643991&req=5

ppat.1005273.g005: Ultrastructure analysis of oocyst development in ∆cyc3 mutant.(A) Low power ultrastructural images of oocyst development at 7–10 days (i, iv, vii) and 14–21 days post-infection (dpi) showing normal sporulation of WT (ii, iii) and certain Δcyc3 (v, vi) parasites while other Δcyc3 mutants show evidence of cytoplasmic vacuolation (V) and degeneration (vii, viii, ix). N–nucleus. Bars represent 10 μm. (B) Details showing progressive stages in sporozoite formation in wild type parasites (i-iii) and various abnormal developmental stages of the Δcyc3 parasite (iv-vi). Bars represent 1 μm. i. Initiation of sporozoite formation with formation of the inner membrane complex (I) beneath the plasmalemma and above a peripherally located nucleus (N) with nuclear pole (NP). ii. Early sporozoite (S) with rhoptry anlagen (R) budding from the surface of the sporoblast. N–nucleus. iii. Late stage in sporozoite formation showing the elongated sporozoites (S). R–rhoptry. iv. Detail of the sporoblast cytoplasm showing nuclei (N) enclosed by abnormal membrane whorls v. Part of a nucleus with extensive nuclear spindle microtubule (Mt) not seen in WT parasite. vi. Detail of a late stage in parasite degeneration showing apoptotic-like nuclei (N) and dilated endoplasmic reticulum (ER).
Mentions: To define further the defect in oocyst growth during different developmental stages of sporogony, Δcyc3 and WT parasite-infected mosquito midguts at 7, 10, 14, and 21 dpi were examined by transmission electron microscopy. Marked differences were observed at the later time points in the ultrastructure of the majority of Δcyc3 compared to WT oocysts, although some Δcyc3 oocysts appeared similar to WT (Fig 5). At every time point there were significantly fewer oocysts in the guts of mosquitoes infected with Δcyc3 compared to the WT parasites (5 oocysts/gut Δcyc3 compared to 60 oocyst/gut WT at 7 dpi).

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