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. 2008 Jul;69(1):110-8.
doi: 10.1111/j.1365-2958.2008.06267.x. Epub 2008 Apr 28.

Disruption of a Plasmodium falciparum cyclic nucleotide phosphodiesterase gene causes aberrant gametogenesis

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Disruption of a Plasmodium falciparum cyclic nucleotide phosphodiesterase gene causes aberrant gametogenesis

Cathy J Taylor et al. Mol Microbiol. 2008 Jul.

Abstract

Phosphodiesterase (PDE) and guanylyl cyclase (GC) enzymes are key components of the cGMP signalling pathway and are encoded in the genome of Plasmodium falciparum. Here we investigate the role of specific GC and PDE isoforms in gamete formation--a process that is essential for malaria transmission and occurs in the Anopheles mosquito midgut following feeding on an infected individual. Details of the intracellular signalling events controlling development of the male and female gametes from their precursors (gametocytes) remain sparse in P. falciparum. Previous work involving the addition of pharmacological agents to gametocytes implicated cGMP in exflagellation--the emergence of highly motile, flagellated male gametes from the host red blood cell. In this study we show that decreased GC activity in parasites having undergone disruption of the PfGCbeta gene had no significant effect on gametogenesis. By contrast, decreased cGMP-PDE activity during gametocyte development owing to disruption of the PfPDEdelta gene, led to a severely reduced ability to undergo gametogenesis. This suggests that the concentration of cGMP must be maintained below a threshold in the developing gametocyte to allow subsequent differentiation to proceed normally. The data indicate that PfPDEdelta plays a crucial role in regulating cGMP levels during sexual development.

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Figures

Fig. 1
Fig. 1
Phenotypic analysis of the PfGCβ parasites. A. Stage V gametocyte particulate fractions from WT and the two PfGCβ mutant clones (formula image and formula image) were assayed for GC activity. Uninfected RBC and WT asexual blood stage particulate fractions were included as controls. The amount of cGMP produced by 104 cells was measured using a 96-well plate cGMP competitive enzyme immunoassay system. The data shown are the mean of five individual assays from multiple parasite cultures carried out in triplicate, with error bars showing the standard error of the mean. B. Rounding up of WT (black bars), formula image (grey bars) and formula image (white bars) gametocytes was measured after addition of either XA (100 μM) or zaprinast (400 μM). Ten minutes post stimulation, a minimum of 200 live cells were counted by light microscopy and scored as either round or gametocyte-shaped. Results show mean counts for a minimum of four experiments counted in duplicate, with error bars showing the standard error of the mean. The asterisk represents a statistically significant result. C. Exflagellation of WT (black bars), formula image (grey bars) and formula image (white bars) male gametocytes was measured after addition of either XA (100 μM) or zaprinast (400 μM). Ten minutes post stimulation, cells were observed with a light microscope for 10 min and the number of centres of exflagellation scored per 10 000 cells. Results show mean counts for a minimum of three experiments counted in duplicate, with error bars showing the standard error of the mean.
Fig. 2
Fig. 2
cGMP-PDE activity in PfPDEδ parasites. A. A northern blot containing total RNA (4 μg in each lane) from PfPDEδ, PfPDEγ clones and control Pfrh3 parasites. The blot was probed with the catalytic domain of PfPDEδ (upper panel) and the membrane was stripped and reprobed with a region of the P. falciparum glycerol kinase gene (PlasmoDB identifier PF13_0269) (lower panel) to demonstrate equal loading. Lane 1, Pfrh3 RNA; lanes 2 and 3, PfPDEδ clones 4 and 14 respectively; lanes 4 and 5, PfPDEγ clones A3 and A4 respectively. Sizes in kilobases are indicated to the left. B. The total intracellular cGMP concentration was measured in stages III to V gametocytes in PfPDEδ clones and controls using an enzyme immunoassay based on a competition assay. Black bars denote control (Pfrh3) cells, PfPDEδclones are represented by dark grey (clone 4) and light grey (clone 14) bars. The white bar represents the cGMP content of uninfected erythrocytes. Gametocytes were harvested using Nycodenz and aliquots of 107 cells were assayed in triplicate wells. The assay was carried out three times and a representative result is shown. Error bars denote the standard error of the mean. C. cGMP-PDE activity was measured in particulate fractions of stage V gametocytes from PfPDEδ clones (PDEd4 and PDEd14) and Pfrh3 controls. Fractions were assayed in the absence (black bars) and presence (grey bars) of 400 μM zaprinast. Gametocytes were harvested using Nycodenz and aliquots of 107 cells were assayed in triplicate wells. The results are the mean of three independent experiments. RBC, uninfected red blood cells. Error bars represent the standard error of the mean.
Fig. 3
Fig. 3
Rounding up and emergence levels in PfPDEδ parasites. A. The percentage of rounded up gametocytes in three independent PfPDEδ clones (PfPDE4, 7 and 14) and Pfrh3 controls (Pfrh3) was evaluated 10 min after the addition of 20 μM XA by counting 200 live gametocytes and scoring them as round or gametocyte-shaped. Results are based on three independent counts of each clone on three consecutive days (days 10–12). Error bars represent the standard error of the mean. B. The number of centres of exflagellation in the three independent PfPDEδ clones and Pfrh3 controls was evaluated under the same conditions. Results are based on three independent counts of each clone on three consecutive days. Error bars represent the standard error of the mean. C. Merged confocal immunofluorescence images of Pfrh3 control stage V gametocytes before (pre-XA, left panel) and after (post-XA, centre panel) addition of 20 μM XA and PfPDEδ stage V gametocytes (PDEdelta post-XA) after (right panel) addition of 20 μM XA. Parasites were fixed with paraformaldehyde and stained with a human Band 3 antibody (red) to determine whether parasites had emerged from their host RBC. Blue shows DAPI stain. D. The percentage of emergence was evaluated by counting reactivity of cells with a Band 3 antibody in two independent PfPDEδ clones and Pfrh3 controls before and after addition of 20 μM XA. Black bars denote control (Pfrh3) cells, PfPDEδclones are represented by dark grey (clone 4) and light grey (clone 14) bars. Results are based on duplicate independent counts from the same flask of gametocytes on a single day. Error bars represent the standard error of the mean.

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References

    1. Alano P, Billker O. Gametocytes and gametes. In: Sherman IW, editor. Molecular Approaches to Malaria. Washington, DC: American Society for Microbiology Press; 2005. pp. 191–219.
    1. Billker O, Lindo V, Panico M, Etienne AE, Paxton T, Dell A, et al. Identification of xanthurenic acid as the putative inducer of malaria development in the mosquito. Nature. 1998;392:289–292. - PubMed
    1. Billker O, Shaw MK, Jones IW, Ley SV, Mordue AJ, Sinden RE. Azadirachtin disrupts formation of organised microtubule arrays during microgametogenesis of Plasmodium berghei. J Eukaryot Microbiol. 2002;49:489–497. - PubMed
    1. Billker O, Dechamps S, Tewari R, Wenig G, Franke-Fayard B, Brinkmann V. Calcium and a calcium-dependent protein kinase regulate gamete formation and mosquito transmission in a malaria parasite. Cell. 2004;117:503–514. - PubMed
    1. Carucci DJ, Witney AA, Muhia DK, Warhurst DC, Schaap P, Meima M, et al. Guanylyl cyclase activity associated with putative bifunctional integral membrane proteins in Plasmodium falciparum. J Biol Chem. 2000;275:22147–22156. - PubMed

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