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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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PbCAX-GFP localisation.High resolution deconvolution microscopy images of a live female gametocyte shortly after activation and a female gamete/zygote and an ookinete 24 h post activation expressing PbCAX-GFP and co-stained with MitoTracker Red CMXRos (10 ng/ml) and Hoechst 33342. Scale bar: 5 µm.
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ppat-1003191-g004: PbCAX-GFP localisation.High resolution deconvolution microscopy images of a live female gametocyte shortly after activation and a female gamete/zygote and an ookinete 24 h post activation expressing PbCAX-GFP and co-stained with MitoTracker Red CMXRos (10 ng/ml) and Hoechst 33342. Scale bar: 5 µm.

Mentions: To improve resolution of live parasite fluorescence images, deconvolution microscopy was used. In activated gametocytes, GFP signal was confined to membranous regions surrounding the parasite nucleus and a mass to the side (possibly the endoplasmic reticulum). Little signal colocalised with MitoTracker, used as a marker for parasite mitochondrion (Figure 4). Non-converting female gametes/zygotes and ookinetes 24 h after activation had more dispersed GFP signal and only very partial colocalisation with the mitochondrion could be observed (Figure 4). Additional images of the latter stages co-stained with a parasite surface marker rather than MitoTracker can be seen in Figure S6B.


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)

PbCAX-GFP localisation.High resolution deconvolution microscopy images of a live female gametocyte shortly after activation and a female gamete/zygote and an ookinete 24 h post activation expressing PbCAX-GFP and co-stained with MitoTracker Red CMXRos (10 ng/ml) and Hoechst 33342. Scale bar: 5 µm.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003191-g004: PbCAX-GFP localisation.High resolution deconvolution microscopy images of a live female gametocyte shortly after activation and a female gamete/zygote and an ookinete 24 h post activation expressing PbCAX-GFP and co-stained with MitoTracker Red CMXRos (10 ng/ml) and Hoechst 33342. Scale bar: 5 µm.
Mentions: To improve resolution of live parasite fluorescence images, deconvolution microscopy was used. In activated gametocytes, GFP signal was confined to membranous regions surrounding the parasite nucleus and a mass to the side (possibly the endoplasmic reticulum). Little signal colocalised with MitoTracker, used as a marker for parasite mitochondrion (Figure 4). Non-converting female gametes/zygotes and ookinetes 24 h after activation had more dispersed GFP signal and only very partial colocalisation with the mitochondrion could be observed (Figure 4). Additional images of the latter stages co-stained with a parasite surface marker rather than MitoTracker can be seen in Figure S6B.

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