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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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TgCAX localisation.(A) Immunofluorescence assay of intracellular Toxoplasma tachyzoites transiently transfected with TgCAX-Ty expressed under the control of the tubulin promotor (upper panels) and when expressed stably (lower panels). Parasites are immunostained with the surface marker GAP45 (red). (B) TgCAX transiently transfected tachyzoites detected by anti-TgCAX antibodies and co-stained with anti-Ty antibodies and the nuclear marker DAPI (upper panels) or with GAP45 antibodies (lower panels). (C) TgCAX transiently expressed in tachyzoites co-localised with 2 mitochondrial markers, anti-HSP70 antibodies [26] (upper panels) or transiently co-transfected SPTPSOD2GFP (SP: signal peptide, TP: transit peptide, [27]) (lower panels). Scale bars: 2 µm.
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ppat-1003191-g008: TgCAX localisation.(A) Immunofluorescence assay of intracellular Toxoplasma tachyzoites transiently transfected with TgCAX-Ty expressed under the control of the tubulin promotor (upper panels) and when expressed stably (lower panels). Parasites are immunostained with the surface marker GAP45 (red). (B) TgCAX transiently transfected tachyzoites detected by anti-TgCAX antibodies and co-stained with anti-Ty antibodies and the nuclear marker DAPI (upper panels) or with GAP45 antibodies (lower panels). (C) TgCAX transiently expressed in tachyzoites co-localised with 2 mitochondrial markers, anti-HSP70 antibodies [26] (upper panels) or transiently co-transfected SPTPSOD2GFP (SP: signal peptide, TP: transit peptide, [27]) (lower panels). Scale bars: 2 µm.

Mentions: In a highly complementary approach to allow comparative tagging and knock-out studies with P. berghei, additional experiments were performed using the genetically amenable apicomplexan, T. gondii, at the tachyzoite stage. When TgCAX was expressed transiently under the control of the tubulin promoter, as a second copy detectable by a C-terminal Ty-tag (Figure S9A), the protein was found predominantly in a large vesicular-like compartment located in the apical end of the intracellular parasite, as well as in much smaller vesicle-like structures dispersed throughout the parasite cytosol (Figure 8A). This compartment is reminiscent of the plant-like vacuole (PLV) described recently in extracellular tachyzoites [23]. However, there was no co-localization with antibodies directed against a marker for the PLV and acidocalcisomes, the vacuolar proton pyrophosphatase, VP1 (Figure S9B). When TgCAX-Ty was stably expressed in a pool of intracellular parasites, only the dispersed signal was observed (Figure 8A), similar but seemingly not identical to that reported for VP1 [23].


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)

TgCAX localisation.(A) Immunofluorescence assay of intracellular Toxoplasma tachyzoites transiently transfected with TgCAX-Ty expressed under the control of the tubulin promotor (upper panels) and when expressed stably (lower panels). Parasites are immunostained with the surface marker GAP45 (red). (B) TgCAX transiently transfected tachyzoites detected by anti-TgCAX antibodies and co-stained with anti-Ty antibodies and the nuclear marker DAPI (upper panels) or with GAP45 antibodies (lower panels). (C) TgCAX transiently expressed in tachyzoites co-localised with 2 mitochondrial markers, anti-HSP70 antibodies [26] (upper panels) or transiently co-transfected SPTPSOD2GFP (SP: signal peptide, TP: transit peptide, [27]) (lower panels). Scale bars: 2 µm.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003191-g008: TgCAX localisation.(A) Immunofluorescence assay of intracellular Toxoplasma tachyzoites transiently transfected with TgCAX-Ty expressed under the control of the tubulin promotor (upper panels) and when expressed stably (lower panels). Parasites are immunostained with the surface marker GAP45 (red). (B) TgCAX transiently transfected tachyzoites detected by anti-TgCAX antibodies and co-stained with anti-Ty antibodies and the nuclear marker DAPI (upper panels) or with GAP45 antibodies (lower panels). (C) TgCAX transiently expressed in tachyzoites co-localised with 2 mitochondrial markers, anti-HSP70 antibodies [26] (upper panels) or transiently co-transfected SPTPSOD2GFP (SP: signal peptide, TP: transit peptide, [27]) (lower panels). Scale bars: 2 µm.
Mentions: In a highly complementary approach to allow comparative tagging and knock-out studies with P. berghei, additional experiments were performed using the genetically amenable apicomplexan, T. gondii, at the tachyzoite stage. When TgCAX was expressed transiently under the control of the tubulin promoter, as a second copy detectable by a C-terminal Ty-tag (Figure S9A), the protein was found predominantly in a large vesicular-like compartment located in the apical end of the intracellular parasite, as well as in much smaller vesicle-like structures dispersed throughout the parasite cytosol (Figure 8A). This compartment is reminiscent of the plant-like vacuole (PLV) described recently in extracellular tachyzoites [23]. However, there was no co-localization with antibodies directed against a marker for the PLV and acidocalcisomes, the vacuolar proton pyrophosphatase, VP1 (Figure S9B). When TgCAX-Ty was stably expressed in a pool of intracellular parasites, only the dispersed signal was observed (Figure 8A), similar but seemingly not identical to that reported for VP1 [23].

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