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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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Related in: MedlinePlus

Δpbcax parasite rescue with EGTA.Line graph illustrating ookinete conversion, measured at 24 h post-gametocyte activation, in wild-type (WT, open circles: WT GFP, open triangles) and Δpbcax cl9 (closed circles) and cl5 gfp (closed triangles) parasites in the presence of 10 mM EGTA added at 0, 0.5, 2 and 3 h post-gametocyte activation. The conversion rate is the percentage of P28-positive parasites that had successfully differentiated into elongated ‘banana-shaped’ ookinetes. Data are expressed as the percentage of wild-type controls. Points represent the mean ± SEM (n = 3) except for WT, where the points represent the mean ± range (n = 2).
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ppat-1003191-g007: Δpbcax parasite rescue with EGTA.Line graph illustrating ookinete conversion, measured at 24 h post-gametocyte activation, in wild-type (WT, open circles: WT GFP, open triangles) and Δpbcax cl9 (closed circles) and cl5 gfp (closed triangles) parasites in the presence of 10 mM EGTA added at 0, 0.5, 2 and 3 h post-gametocyte activation. The conversion rate is the percentage of P28-positive parasites that had successfully differentiated into elongated ‘banana-shaped’ ookinetes. Data are expressed as the percentage of wild-type controls. Points represent the mean ± SEM (n = 3) except for WT, where the points represent the mean ± range (n = 2).

Mentions: Given that transmission stages of parasite development are extracellular (the in vitro culture medium used here contained 0.42 mM Ca2+) and that CAX activity often provides a mechanism to enable organisms to survive in the presence of extracellular Ca2+[14], we tested the ability of the Ca2+ chelator EGTA (ethylene glycol tetraacetic acid) to restore ookinete conversion of both Δpbcax cl9 and cl5 gfp parasites (Figure 7). EGTA (10 mM) had no significant effect on the normal ookinete conversion efficiency of wild-type parasites, which constitutively expressed GFP (control parasites used for comparison with Δpbcax cl5 gfp; open triangles in Figure 7), measured at 24 h post-activation when added immediately prior to gametogenesis (t = 0 h), 30 min post-activation (at which point exflagellation has occurred), 2 h post-activation (at which point zygote formation will be complete, predominantly) or 3 h post-activation (p>0.05, ANOVA with Dunnett's post test; n = 3). Apparently similar results were found for the development of non-GFP expressing wild-type parasites (control parasites used for comparison with Δpbcax cl9; open circles in Figure 7) grown in the presence of EGTA, although this could not be tested statistically as only 2 control experiments were performed. The effect of EGTA on Δpbcax cl9 and cl5 gfp parasites was to restore ookinete conversion, when added 3 h post-activation or prior to this point, although the ability to restore ookinete conversion reduced the longer after activation EGTA was added. In the case of all Δpbcax cl9 experiments and one of the three using Δpbcax cl5 gfp parasites, addition of EGTA at 0 and 0.5 h led seemingly to complete restoration of ookinete conversion. Images of restored Δpbcax cl9 ookinetes in the presence of EGTA added at t = 0 h can be seen in the lower panels of Figure 6. Addition of EGTA at 3 h post-activation still enabled ookinete conversion of Δpbcax cl9 and cl5 gfp parasites but at significantly lower levels than the GFP-expressing wild-type control (p<0.05, ANOVA with Dunnett's post test; n = 3). In case the rescuing effect of EGTA was osmotic rather than due to its Ca2+ chelation properties, additional controls were performed in the presence of 20 mM NaCl added to the culture medium immediately prior to gametogenesis. This had no effect on the ability of wild-type parasites to develop into ookinetes (p = 0.6, unpaired, two-tailed Student's t-test; n = 3) or the inability of mutant parasite to develop (data not shown).


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 rescue with EGTA.Line graph illustrating ookinete conversion, measured at 24 h post-gametocyte activation, in wild-type (WT, open circles: WT GFP, open triangles) and Δpbcax cl9 (closed circles) and cl5 gfp (closed triangles) parasites in the presence of 10 mM EGTA added at 0, 0.5, 2 and 3 h post-gametocyte activation. The conversion rate is the percentage of P28-positive parasites that had successfully differentiated into elongated ‘banana-shaped’ ookinetes. Data are expressed as the percentage of wild-type controls. Points represent the mean ± SEM (n = 3) except for WT, where the points represent the mean ± range (n = 2).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003191-g007: Δpbcax parasite rescue with EGTA.Line graph illustrating ookinete conversion, measured at 24 h post-gametocyte activation, in wild-type (WT, open circles: WT GFP, open triangles) and Δpbcax cl9 (closed circles) and cl5 gfp (closed triangles) parasites in the presence of 10 mM EGTA added at 0, 0.5, 2 and 3 h post-gametocyte activation. The conversion rate is the percentage of P28-positive parasites that had successfully differentiated into elongated ‘banana-shaped’ ookinetes. Data are expressed as the percentage of wild-type controls. Points represent the mean ± SEM (n = 3) except for WT, where the points represent the mean ± range (n = 2).
Mentions: Given that transmission stages of parasite development are extracellular (the in vitro culture medium used here contained 0.42 mM Ca2+) and that CAX activity often provides a mechanism to enable organisms to survive in the presence of extracellular Ca2+[14], we tested the ability of the Ca2+ chelator EGTA (ethylene glycol tetraacetic acid) to restore ookinete conversion of both Δpbcax cl9 and cl5 gfp parasites (Figure 7). EGTA (10 mM) had no significant effect on the normal ookinete conversion efficiency of wild-type parasites, which constitutively expressed GFP (control parasites used for comparison with Δpbcax cl5 gfp; open triangles in Figure 7), measured at 24 h post-activation when added immediately prior to gametogenesis (t = 0 h), 30 min post-activation (at which point exflagellation has occurred), 2 h post-activation (at which point zygote formation will be complete, predominantly) or 3 h post-activation (p>0.05, ANOVA with Dunnett's post test; n = 3). Apparently similar results were found for the development of non-GFP expressing wild-type parasites (control parasites used for comparison with Δpbcax cl9; open circles in Figure 7) grown in the presence of EGTA, although this could not be tested statistically as only 2 control experiments were performed. The effect of EGTA on Δpbcax cl9 and cl5 gfp parasites was to restore ookinete conversion, when added 3 h post-activation or prior to this point, although the ability to restore ookinete conversion reduced the longer after activation EGTA was added. In the case of all Δpbcax cl9 experiments and one of the three using Δpbcax cl5 gfp parasites, addition of EGTA at 0 and 0.5 h led seemingly to complete restoration of ookinete conversion. Images of restored Δpbcax cl9 ookinetes in the presence of EGTA added at t = 0 h can be seen in the lower panels of Figure 6. Addition of EGTA at 3 h post-activation still enabled ookinete conversion of Δpbcax cl9 and cl5 gfp parasites but at significantly lower levels than the GFP-expressing wild-type control (p<0.05, ANOVA with Dunnett's post test; n = 3). In case the rescuing effect of EGTA was osmotic rather than due to its Ca2+ chelation properties, additional controls were performed in the presence of 20 mM NaCl added to the culture medium immediately prior to gametogenesis. This had no effect on the ability of wild-type parasites to develop into ookinetes (p = 0.6, unpaired, two-tailed Student's t-test; n = 3) or the inability of mutant parasite to develop (data not shown).

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