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The Plasmodium berghei Ca(2+)/H(+) exchanger, PbCAX, is essential for tolerance to environmental Ca(2+) during sexual development.

Guttery DS, Pittman JK, Frénal K, Poulin B, McFarlane LR, Slavic K, Wheatley SP, Soldati-Favre D, Krishna S, Tewari R, Staines HM - PLoS Pathog. (2013)

Bottom Line: Furthermore, genetically disrupted parasites failed to develop further from "round" form zygotes, suggesting that PbCAX is essential for ookinete development and differentiation.Therefore, PbCAX provides a mechanism for free living parasites to multiply within the ionic microenvironment of the mosquito midgut.Ca(2+) homeostasis mediated by PbCAX is critical and suggests plasmodial CAXs may be targeted in approaches designed to block parasite transmission.

View Article: PubMed Central - PubMed

Affiliation: Centre for Genetics and Genomics, School of Biology, Queen's Medical Centre, University of Nottingham, Nottingham, United Kingdom.

ABSTRACT
Ca(2+) contributes to a myriad of important cellular processes in all organisms, including the apicomplexans, Plasmodium and Toxoplasma. Due to its varied and essential roles, free Ca(2+) is tightly regulated by complex mechanisms. These mechanisms are therefore of interest as putative drug targets. One pathway in Ca(2+) homeostatic control in apicomplexans uses a Ca(2+)/H(+) exchanger (a member of the cation exchanger family, CAX). The P. falciparum CAX (PfCAX) has recently been characterised in asexual blood stage parasites. To determine the physiological importance of apicomplexan CAXs, tagging and knock-out strategies were undertaken in the genetically tractable T. gondii and P. berghei parasites. In addition, a yeast heterologous expression system was used to study the function of apicomplexan CAXs. Tagging of T. gondii and P. berghei CAXs (TgCAX and PbCAX) under control of their endogenous promoters could not demonstrate measureable expression of either CAX in tachyzoites and asexual blood stages, respectively. These results were consistent with the ability of parasites to tolerate knock-outs of the genes for TgCAX and PbCAX at these developmental stages. In contrast, PbCAX expression was detectable during sexual stages of development in female gametocytes/gametes, zygotes and ookinetes, where it was dispersed in membranous networks within the cytosol (with minimal mitochondrial localisation). Furthermore, genetically disrupted parasites failed to develop further from "round" form zygotes, suggesting that PbCAX is essential for ookinete development and differentiation. This impeded phenotype could be rescued by removal of extracellular Ca(2+). Therefore, PbCAX provides a mechanism for free living parasites to multiply within the ionic microenvironment of the mosquito midgut. Ca(2+) homeostasis mediated by PbCAX is critical and suggests plasmodial CAXs may be targeted in approaches designed to block parasite transmission.

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Δpbcax parasite ookinete conversion images.Immunofluorescence images of in vitro wild-type (WT) and Δpbcax cl9 P. berghei ookinete cultures, 8 and 24 h after gametocyte activation and in the presence of EGTA (10 mM; added directly prior to gametocyte activation). Parasites are immunostained for the female gamete/zygote/ookinete marker P28 (red) and co-stained with the nuclear marker Hoechst 33342 (blue). Development of elongated ookinetes was completely ablated in the Δpbcax cl9 line, a phenotype which could be reversed by the removal of extracellular Ca2+ using EGTA. The arrow indicates the diffuse DNA staining observed in Δpbcax cl9 parasites 24 h after gametocyte activation. Scale bar: 5 µm.
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ppat-1003191-g006: Δpbcax parasite ookinete conversion images.Immunofluorescence images of in vitro wild-type (WT) and Δpbcax cl9 P. berghei ookinete cultures, 8 and 24 h after gametocyte activation and in the presence of EGTA (10 mM; added directly prior to gametocyte activation). Parasites are immunostained for the female gamete/zygote/ookinete marker P28 (red) and co-stained with the nuclear marker Hoechst 33342 (blue). Development of elongated ookinetes was completely ablated in the Δpbcax cl9 line, a phenotype which could be reversed by the removal of extracellular Ca2+ using EGTA. The arrow indicates the diffuse DNA staining observed in Δpbcax cl9 parasites 24 h after gametocyte activation. Scale bar: 5 µm.

Mentions: Figure 6 shows examples of the typical phenotype that was observed for the mutant parasites. At 8 h post-activation, wild-type parasites were predominantly “retort” forms (a round parasite that contains the nucleus, with an apical protrusion), whereas Δpbcax parasites remained round. At 24 h post-activation, wild-type parasites were predominantly fully converted into ookinetes (elongated forms with the nucleus in the centre). At this time, Δpbcax parasites were still round in form, fewer in number and were often smaller in size and had degenerated membranes (as judged from discontinuous P28 staining). Furthermore, they often had diffuse nuclei, suggestive of possible necrosis or late stage apoptosis. In a single semi-quantitative experiment with three repeats, the number of “round” form PbCAX knock-out parasites per field of view was counted in ookinete development cultures at 2, 6 and 24 h after activation (Figure S8). An approximate 70% reduction in the number of parasites present after 24 h was observed, suggesting that the parasites are degrading rather than arrested or slow growing.


The Plasmodium berghei Ca(2+)/H(+) exchanger, PbCAX, is essential for tolerance to environmental Ca(2+) during sexual development.

Guttery DS, Pittman JK, Frénal K, Poulin B, McFarlane LR, Slavic K, Wheatley SP, Soldati-Favre D, Krishna S, Tewari R, Staines HM - PLoS Pathog. (2013)

Δpbcax parasite ookinete conversion images.Immunofluorescence images of in vitro wild-type (WT) and Δpbcax cl9 P. berghei ookinete cultures, 8 and 24 h after gametocyte activation and in the presence of EGTA (10 mM; added directly prior to gametocyte activation). Parasites are immunostained for the female gamete/zygote/ookinete marker P28 (red) and co-stained with the nuclear marker Hoechst 33342 (blue). Development of elongated ookinetes was completely ablated in the Δpbcax cl9 line, a phenotype which could be reversed by the removal of extracellular Ca2+ using EGTA. The arrow indicates the diffuse DNA staining observed in Δpbcax cl9 parasites 24 h after gametocyte activation. Scale bar: 5 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585132&req=5

ppat-1003191-g006: Δpbcax parasite ookinete conversion images.Immunofluorescence images of in vitro wild-type (WT) and Δpbcax cl9 P. berghei ookinete cultures, 8 and 24 h after gametocyte activation and in the presence of EGTA (10 mM; added directly prior to gametocyte activation). Parasites are immunostained for the female gamete/zygote/ookinete marker P28 (red) and co-stained with the nuclear marker Hoechst 33342 (blue). Development of elongated ookinetes was completely ablated in the Δpbcax cl9 line, a phenotype which could be reversed by the removal of extracellular Ca2+ using EGTA. The arrow indicates the diffuse DNA staining observed in Δpbcax cl9 parasites 24 h after gametocyte activation. Scale bar: 5 µm.
Mentions: Figure 6 shows examples of the typical phenotype that was observed for the mutant parasites. At 8 h post-activation, wild-type parasites were predominantly “retort” forms (a round parasite that contains the nucleus, with an apical protrusion), whereas Δpbcax parasites remained round. At 24 h post-activation, wild-type parasites were predominantly fully converted into ookinetes (elongated forms with the nucleus in the centre). At this time, Δpbcax parasites were still round in form, fewer in number and were often smaller in size and had degenerated membranes (as judged from discontinuous P28 staining). Furthermore, they often had diffuse nuclei, suggestive of possible necrosis or late stage apoptosis. In a single semi-quantitative experiment with three repeats, the number of “round” form PbCAX knock-out parasites per field of view was counted in ookinete development cultures at 2, 6 and 24 h after activation (Figure S8). An approximate 70% reduction in the number of parasites present after 24 h was observed, suggesting that the parasites are degrading rather than arrested or slow growing.

Bottom Line: Furthermore, genetically disrupted parasites failed to develop further from "round" form zygotes, suggesting that PbCAX is essential for ookinete development and differentiation.Therefore, PbCAX provides a mechanism for free living parasites to multiply within the ionic microenvironment of the mosquito midgut.Ca(2+) homeostasis mediated by PbCAX is critical and suggests plasmodial CAXs may be targeted in approaches designed to block parasite transmission.

View Article: PubMed Central - PubMed

Affiliation: Centre for Genetics and Genomics, School of Biology, Queen's Medical Centre, University of Nottingham, Nottingham, United Kingdom.

ABSTRACT
Ca(2+) contributes to a myriad of important cellular processes in all organisms, including the apicomplexans, Plasmodium and Toxoplasma. Due to its varied and essential roles, free Ca(2+) is tightly regulated by complex mechanisms. These mechanisms are therefore of interest as putative drug targets. One pathway in Ca(2+) homeostatic control in apicomplexans uses a Ca(2+)/H(+) exchanger (a member of the cation exchanger family, CAX). The P. falciparum CAX (PfCAX) has recently been characterised in asexual blood stage parasites. To determine the physiological importance of apicomplexan CAXs, tagging and knock-out strategies were undertaken in the genetically tractable T. gondii and P. berghei parasites. In addition, a yeast heterologous expression system was used to study the function of apicomplexan CAXs. Tagging of T. gondii and P. berghei CAXs (TgCAX and PbCAX) under control of their endogenous promoters could not demonstrate measureable expression of either CAX in tachyzoites and asexual blood stages, respectively. These results were consistent with the ability of parasites to tolerate knock-outs of the genes for TgCAX and PbCAX at these developmental stages. In contrast, PbCAX expression was detectable during sexual stages of development in female gametocytes/gametes, zygotes and ookinetes, where it was dispersed in membranous networks within the cytosol (with minimal mitochondrial localisation). Furthermore, genetically disrupted parasites failed to develop further from "round" form zygotes, suggesting that PbCAX is essential for ookinete development and differentiation. This impeded phenotype could be rescued by removal of extracellular Ca(2+). Therefore, PbCAX provides a mechanism for free living parasites to multiply within the ionic microenvironment of the mosquito midgut. Ca(2+) homeostasis mediated by PbCAX is critical and suggests plasmodial CAXs may be targeted in approaches designed to block parasite transmission.

Show MeSH
Related in: MedlinePlus