PLoS Pathog. 2012 Feb;8(2):e1002554. doi: 10.1371/journal.ppat.1002554. Epub 2012 Feb 23.

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.

Source

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

Erratum in

PLoS Pathog. 2012 Mar;8(3). doi: 10.1371/annotation/ef70d427-0816-4a63-aeaf-874b734793b0.

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.

PMID:: 22383885; [PubMed - indexed for MEDLINE]
PMCID:: PMC3285604

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Figure 3

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.

A Putative Homologue of CDC20/CDH1 in the Malaria Parasite Is Essential for Male Gamete Development

PLoS Pathog. 2012 February; 2012 March;8(2):e1002554.

Figure 4

Analysis of genome replication in activated male gametocytes by direct (immuno) fluorescence microscopy and FACS analysis.

A. Direct immunolabelling of α-tubulin (red) and DNA (Hoechst – blue) in activated gametocytes fixed at different time points post activation (pa). Representative cells from one of three experiments are shown. The 8 min time point shows characteristic axonemic circling of the nucleus (arrow) whereby each of the 8 axonemes lie completely within the cytoplasm, coiled around the nucleus. The 15 min time point illustrates an exflagellating wild-type microgametocyte with condensed DNA entering the flagella (indicated by *). Δcdc20 and Δmap2 parasites are arrested at the exflagellation stage. Bar = 5 µm. mpa = minutes post-activation. B. Fluorometric anaylsis of DNA content (n) after DAPI nuclear staining. Microgametocytes were at 0 mpa (non-activated), 8 mpa or 15 mpa. The mean DNA content (and standard deviation) of 10 nuclei per sample are shown. Values are expressed relative to the average fluorescence intensity of 10 haploid ring-stage parasites from the same slide [69]. All values were corrected for background fluorescence. C. Determination of DNA content of purified, activated male gametocytes at 8 mpa by FACS analyses of Hoechst-stained parasites [63]. Dot plots show the mean percentage of gametocytes in gate G1 (inactivated and activated female gametocytes) and G2 (activated gametocytes with an 8n DNA content. Wild-type, Δcdc20 and Δmap2 parasites show a high percentage of activated male gametocytes with an 8n DNA content (see D). The previously characterised Δcdpk4 parasites [28] were used as a control as they do not undergo DNA replication upon activation. FI = Fluorescence Intensity. D. Mean Hoechst fluorescence intensity (DNA content) (±SD) of gametocytes in gates G1 and G2 in three independent experiments. The DNA content of Δcdc20 and Δmap2 male gametocytes (gate G2) at 8 mpa is comparable to that of wild-type gametocytes whereas activated, DNA replicating males are absent in Δcdpk4 parasites. FI = Fluorescence Intensity; ND = not determined.

A Putative Homologue of CDC20/CDH1 in the Malaria Parasite Is Essential for Male Gamete Development

PLoS Pathog. 2012 February; 2012 March;8(2):e1002554.