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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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Ca2+/H+ exchange activity of PfCAX.ΔpH-dependent uptake of 10 µM 45Ca2+ into vacuolar-enriched membrane vesicles isolated from K665 (pmc1 vcx1) yeast transformed with empty vector (piHGpd) (A), scrcax1 (B), pfcax (C), and spfcax (D), measured over a 22 min time course. Transport measurements were determined in the presence of 0.1 mM NaN3, 10 mM KCl, 1 mM ATP, 1 mM MgSO4 and 0.2 mM orthovanadate (a Ca2+-ATPase inhibitor). 45Ca2+ uptake in the absence (open symbols) or presence (closed symbols) of 5 µM of the protonophore CCCP is shown. The Ca2+ ionophore A23187 was added at 12 min at a concentration of 5 µM to dissipate any vesicle-loaded 45Ca2+, as indicated by arrows. Points represent the mean ± SEM of 4–5 independent experiments (where not shown errors lie within the symbols).
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ppat-1003191-g003: Ca2+/H+ exchange activity of PfCAX.ΔpH-dependent uptake of 10 µM 45Ca2+ into vacuolar-enriched membrane vesicles isolated from K665 (pmc1 vcx1) yeast transformed with empty vector (piHGpd) (A), scrcax1 (B), pfcax (C), and spfcax (D), measured over a 22 min time course. Transport measurements were determined in the presence of 0.1 mM NaN3, 10 mM KCl, 1 mM ATP, 1 mM MgSO4 and 0.2 mM orthovanadate (a Ca2+-ATPase inhibitor). 45Ca2+ uptake in the absence (open symbols) or presence (closed symbols) of 5 µM of the protonophore CCCP is shown. The Ca2+ ionophore A23187 was added at 12 min at a concentration of 5 µM to dissipate any vesicle-loaded 45Ca2+, as indicated by arrows. Points represent the mean ± SEM of 4–5 independent experiments (where not shown errors lie within the symbols).

Mentions: To confirm that growth of PfCAX-expressing yeast on Ca2+-containing media was due to enhanced vacuolar Ca2+/H+ exchange activity, ΔpH-dependent 45Ca2+ uptake in the presence of the Ca2+-ATPase inhibitor vanadate was examined in vacuolar membrane vesicles isolated from K665 yeast expressing each of the cax plasmids. ΔpH across the vacuolar membrane vesicles was established by activation of endogenous H+-ATPase activity by the addition of Mg2+-ATP. Significant Ca2+/H+ exchange activity, which could be inhibited by the protonophore carbonyl cyanide 3-chlorophenyl hydrazone, CCCP, was measured for each CAX variant (Figure 3). However, sPfCAX activity was reduced compared with that of sCrCAX1 (by 42% at the 12 min time point; 0.662±0.032 versus 1.142±0.076 nmol mg protein-1, after subtraction of basal accumulation; p = 0.0004, unpaired, two-tailed Student's t-test; n = 5). Ca2+/H+ exchange activity mediated by full-length PfCAX was significantly reduced compared with that of sPfCAX (36% reduced Ca2+ uptake at 12 min time point; 0.425±0.063 versus 0.662±0.032 nmol mg protein−1, after subtraction of basal accumulation; p = 0.01, unpaired, two-tailed Student's t-test; n = 5) but was detectable over basal (empty vector) levels (p = 0.0007, unpaired, two-tailed Student's t-test; n≥4).


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)

Ca2+/H+ exchange activity of PfCAX.ΔpH-dependent uptake of 10 µM 45Ca2+ into vacuolar-enriched membrane vesicles isolated from K665 (pmc1 vcx1) yeast transformed with empty vector (piHGpd) (A), scrcax1 (B), pfcax (C), and spfcax (D), measured over a 22 min time course. Transport measurements were determined in the presence of 0.1 mM NaN3, 10 mM KCl, 1 mM ATP, 1 mM MgSO4 and 0.2 mM orthovanadate (a Ca2+-ATPase inhibitor). 45Ca2+ uptake in the absence (open symbols) or presence (closed symbols) of 5 µM of the protonophore CCCP is shown. The Ca2+ ionophore A23187 was added at 12 min at a concentration of 5 µM to dissipate any vesicle-loaded 45Ca2+, as indicated by arrows. Points represent the mean ± SEM of 4–5 independent experiments (where not shown errors lie within the symbols).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585132&req=5

ppat-1003191-g003: Ca2+/H+ exchange activity of PfCAX.ΔpH-dependent uptake of 10 µM 45Ca2+ into vacuolar-enriched membrane vesicles isolated from K665 (pmc1 vcx1) yeast transformed with empty vector (piHGpd) (A), scrcax1 (B), pfcax (C), and spfcax (D), measured over a 22 min time course. Transport measurements were determined in the presence of 0.1 mM NaN3, 10 mM KCl, 1 mM ATP, 1 mM MgSO4 and 0.2 mM orthovanadate (a Ca2+-ATPase inhibitor). 45Ca2+ uptake in the absence (open symbols) or presence (closed symbols) of 5 µM of the protonophore CCCP is shown. The Ca2+ ionophore A23187 was added at 12 min at a concentration of 5 µM to dissipate any vesicle-loaded 45Ca2+, as indicated by arrows. Points represent the mean ± SEM of 4–5 independent experiments (where not shown errors lie within the symbols).
Mentions: To confirm that growth of PfCAX-expressing yeast on Ca2+-containing media was due to enhanced vacuolar Ca2+/H+ exchange activity, ΔpH-dependent 45Ca2+ uptake in the presence of the Ca2+-ATPase inhibitor vanadate was examined in vacuolar membrane vesicles isolated from K665 yeast expressing each of the cax plasmids. ΔpH across the vacuolar membrane vesicles was established by activation of endogenous H+-ATPase activity by the addition of Mg2+-ATP. Significant Ca2+/H+ exchange activity, which could be inhibited by the protonophore carbonyl cyanide 3-chlorophenyl hydrazone, CCCP, was measured for each CAX variant (Figure 3). However, sPfCAX activity was reduced compared with that of sCrCAX1 (by 42% at the 12 min time point; 0.662±0.032 versus 1.142±0.076 nmol mg protein-1, after subtraction of basal accumulation; p = 0.0004, unpaired, two-tailed Student's t-test; n = 5). Ca2+/H+ exchange activity mediated by full-length PfCAX was significantly reduced compared with that of sPfCAX (36% reduced Ca2+ uptake at 12 min time point; 0.425±0.063 versus 0.662±0.032 nmol mg protein−1, after subtraction of basal accumulation; p = 0.01, unpaired, two-tailed Student's t-test; n = 5) but was detectable over basal (empty vector) levels (p = 0.0007, unpaired, two-tailed Student's t-test; n≥4).

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