Limits...
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.

Show MeSH

Related in: MedlinePlus

Essentiality of TgCAX in T. gondii tachyzoites.(A) Plaque assays were performed by incubating host cells with ΔKU80 and ΔCAX parasite strains for 7 days. (B) For the intracellular growth assay, ΔKU80 (open bars) and ΔCAX (closed bars) parasites were grown in host cells for 24 h and then the number of parasites per vacuole was determined. Bars represent the mean ± SEM of 3 independent experiments (each with 2 replicates). (C) For the ionophore-induced egress assay, parasites were cultured for 30 h before treatment with DMSO (open bars) or Ca2+-ionophore A23187 (3 µM; closed bars) for 5 min. The results are expressed as a percentage of ruptured vacuoles. Bars represent the mean ± SEM of at least 2 independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585132&req=5

ppat-1003191-g009: Essentiality of TgCAX in T. gondii tachyzoites.(A) Plaque assays were performed by incubating host cells with ΔKU80 and ΔCAX parasite strains for 7 days. (B) For the intracellular growth assay, ΔKU80 (open bars) and ΔCAX (closed bars) parasites were grown in host cells for 24 h and then the number of parasites per vacuole was determined. Bars represent the mean ± SEM of 3 independent experiments (each with 2 replicates). (C) For the ionophore-induced egress assay, parasites were cultured for 30 h before treatment with DMSO (open bars) or Ca2+-ionophore A23187 (3 µM; closed bars) for 5 min. The results are expressed as a percentage of ruptured vacuoles. Bars represent the mean ± SEM of at least 2 independent experiments.

Mentions: To determine the physiological importance of Ca2+/H+ exchange activity during the tachyzoite stage of T. gondii, a similar approach to that used for the study P. berghei was taken (Figure S9E and S9F). Plaque assay experiments were performed and revealed no obvious defect in the lytic cycle of the ΔTgCAX strain compared with wild-type parasites after 7 days (Figure 9A). To confirm this observation, intracellular growth and egress were assessed specifically. In the case of intracellular growth, the distributions of parasites per vacuole were essentially identical for both the ΔTgCAX and wild-type strains (Figure 9B) and parasite egress induced by the Ca2+ ionophore A23187 was also similar between strains (Figure 9C). These data suggest that TgCAX is dispensable during the tachyzoite stage.


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)

Essentiality of TgCAX in T. gondii tachyzoites.(A) Plaque assays were performed by incubating host cells with ΔKU80 and ΔCAX parasite strains for 7 days. (B) For the intracellular growth assay, ΔKU80 (open bars) and ΔCAX (closed bars) parasites were grown in host cells for 24 h and then the number of parasites per vacuole was determined. Bars represent the mean ± SEM of 3 independent experiments (each with 2 replicates). (C) For the ionophore-induced egress assay, parasites were cultured for 30 h before treatment with DMSO (open bars) or Ca2+-ionophore A23187 (3 µM; closed bars) for 5 min. The results are expressed as a percentage of ruptured vacuoles. Bars represent the mean ± SEM of at least 2 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003191-g009: Essentiality of TgCAX in T. gondii tachyzoites.(A) Plaque assays were performed by incubating host cells with ΔKU80 and ΔCAX parasite strains for 7 days. (B) For the intracellular growth assay, ΔKU80 (open bars) and ΔCAX (closed bars) parasites were grown in host cells for 24 h and then the number of parasites per vacuole was determined. Bars represent the mean ± SEM of 3 independent experiments (each with 2 replicates). (C) For the ionophore-induced egress assay, parasites were cultured for 30 h before treatment with DMSO (open bars) or Ca2+-ionophore A23187 (3 µM; closed bars) for 5 min. The results are expressed as a percentage of ruptured vacuoles. Bars represent the mean ± SEM of at least 2 independent experiments.
Mentions: To determine the physiological importance of Ca2+/H+ exchange activity during the tachyzoite stage of T. gondii, a similar approach to that used for the study P. berghei was taken (Figure S9E and S9F). Plaque assay experiments were performed and revealed no obvious defect in the lytic cycle of the ΔTgCAX strain compared with wild-type parasites after 7 days (Figure 9A). To confirm this observation, intracellular growth and egress were assessed specifically. In the case of intracellular growth, the distributions of parasites per vacuole were essentially identical for both the ΔTgCAX and wild-type strains (Figure 9B) and parasite egress induced by the Ca2+ ionophore A23187 was also similar between strains (Figure 9C). These data suggest that TgCAX is dispensable during the tachyzoite stage.

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