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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+ tolerance of yeast mediated by PfCAX.(A) RT-PCR analysis of pfcax and spfcax expression in yeast compared with yeast transformed with the empty vector control. (B) Saturated liquid cultures of K665 (pmc1 vcx1) yeast transformed with pfcax in piHGpd, N-terminally truncated spfcax in piHGpd, scrcax1 in piHGpd and empty vector alone were serially diluted to the cell densities as indicated, then spotted onto selection medium lacking histidine (SD –His) and YPD medium containing 50 mM CaCl2. Yeast growth at 30°C is shown after 4 days. A representative experiment is shown. (C) K665 yeast transformed with the various plasmids described in (B) were diluted to a cell density of 0.5 A600 nm and inoculated into YPD medium containing concentrations of CaCl2 from 25 to 150 mM. Yeast cell density was determined by absorbance measurements at 600 nm following growth shaking at 30°C for 16 h. Bars represent the mean ± SEM of 4 independent experiments (each with 8–12 replicates). Vector only, open bars; sCrCAX, closed bars; PfCAX, horizontally lined bars; sPfCAX, diagonally lined bars.
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ppat-1003191-g002: Ca2+ tolerance of yeast mediated by PfCAX.(A) RT-PCR analysis of pfcax and spfcax expression in yeast compared with yeast transformed with the empty vector control. (B) Saturated liquid cultures of K665 (pmc1 vcx1) yeast transformed with pfcax in piHGpd, N-terminally truncated spfcax in piHGpd, scrcax1 in piHGpd and empty vector alone were serially diluted to the cell densities as indicated, then spotted onto selection medium lacking histidine (SD –His) and YPD medium containing 50 mM CaCl2. Yeast growth at 30°C is shown after 4 days. A representative experiment is shown. (C) K665 yeast transformed with the various plasmids described in (B) were diluted to a cell density of 0.5 A600 nm and inoculated into YPD medium containing concentrations of CaCl2 from 25 to 150 mM. Yeast cell density was determined by absorbance measurements at 600 nm following growth shaking at 30°C for 16 h. Bars represent the mean ± SEM of 4 independent experiments (each with 8–12 replicates). Vector only, open bars; sCrCAX, closed bars; PfCAX, horizontally lined bars; sPfCAX, diagonally lined bars.

Mentions: Expression of pfcax and spfcax in yeast was detectable by RT-PCR (Figure 2A). Comparison of PfCAX, sPfCAX, sCrCAX1 and empty vector expressed in yeast grown on YPD (yeast-peptone-dextrose) media supplemented with 50 mM CaCl2 (Figure 2B) found that sPfCAX could suppress the Ca2+ hypersensitivity of the yeast mutant as efficiently as sCrCAX1, while full-length PfCAX-expressing yeast growth was slightly reduced (as determined by the non-uniform growth of yeast expressing PfCAX at a starting cell density of 0.04 absorbance units at A600 nm). The Ca2+ tolerance of yeast expressing PfCAX and sPfCAX was demonstrated further by cell growth in liquid YPD media supplemented with various concentrations of CaCl2 (Figure 2C). sPfCAX provided K665 yeast with tolerance to high concentrations of CaCl2. sPfCAX-expressing yeast growth was significantly greater than PfCAX-expressing yeast at each CaCl2 concentration (p<0.02 unpaired, two-tailed Student's t-test; n = 4), while the cell growth of PfCAX-expressing yeast was significantly greater than that of the empty vector control yeast at all CaCl2 concentrations, including at 150 mM CaCl2 (p<0.02, unpaired, two-tailed Student's t-test; n = 4). A truncated version of TgCAX (sTgCAX), in which translation was initiated by introducing an AUG prior to nucleotide position 295 (encoding Ala-99), was also generated and expressed in K665 yeast. The transformation was assessed to have worked by PCR (Figure S4A) and, as with PfCAX and sPfCAX, sTgCAX suppressed the Ca2+ hypersensitivity of the yeast (Figure S4B). These data demonstrate that apicomplexan CAXs and truncated variants are functional in yeast, and in this heterologous system, can function in providing Ca2+ tolerance.


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+ tolerance of yeast mediated by PfCAX.(A) RT-PCR analysis of pfcax and spfcax expression in yeast compared with yeast transformed with the empty vector control. (B) Saturated liquid cultures of K665 (pmc1 vcx1) yeast transformed with pfcax in piHGpd, N-terminally truncated spfcax in piHGpd, scrcax1 in piHGpd and empty vector alone were serially diluted to the cell densities as indicated, then spotted onto selection medium lacking histidine (SD –His) and YPD medium containing 50 mM CaCl2. Yeast growth at 30°C is shown after 4 days. A representative experiment is shown. (C) K665 yeast transformed with the various plasmids described in (B) were diluted to a cell density of 0.5 A600 nm and inoculated into YPD medium containing concentrations of CaCl2 from 25 to 150 mM. Yeast cell density was determined by absorbance measurements at 600 nm following growth shaking at 30°C for 16 h. Bars represent the mean ± SEM of 4 independent experiments (each with 8–12 replicates). Vector only, open bars; sCrCAX, closed bars; PfCAX, horizontally lined bars; sPfCAX, diagonally lined bars.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585132&req=5

ppat-1003191-g002: Ca2+ tolerance of yeast mediated by PfCAX.(A) RT-PCR analysis of pfcax and spfcax expression in yeast compared with yeast transformed with the empty vector control. (B) Saturated liquid cultures of K665 (pmc1 vcx1) yeast transformed with pfcax in piHGpd, N-terminally truncated spfcax in piHGpd, scrcax1 in piHGpd and empty vector alone were serially diluted to the cell densities as indicated, then spotted onto selection medium lacking histidine (SD –His) and YPD medium containing 50 mM CaCl2. Yeast growth at 30°C is shown after 4 days. A representative experiment is shown. (C) K665 yeast transformed with the various plasmids described in (B) were diluted to a cell density of 0.5 A600 nm and inoculated into YPD medium containing concentrations of CaCl2 from 25 to 150 mM. Yeast cell density was determined by absorbance measurements at 600 nm following growth shaking at 30°C for 16 h. Bars represent the mean ± SEM of 4 independent experiments (each with 8–12 replicates). Vector only, open bars; sCrCAX, closed bars; PfCAX, horizontally lined bars; sPfCAX, diagonally lined bars.
Mentions: Expression of pfcax and spfcax in yeast was detectable by RT-PCR (Figure 2A). Comparison of PfCAX, sPfCAX, sCrCAX1 and empty vector expressed in yeast grown on YPD (yeast-peptone-dextrose) media supplemented with 50 mM CaCl2 (Figure 2B) found that sPfCAX could suppress the Ca2+ hypersensitivity of the yeast mutant as efficiently as sCrCAX1, while full-length PfCAX-expressing yeast growth was slightly reduced (as determined by the non-uniform growth of yeast expressing PfCAX at a starting cell density of 0.04 absorbance units at A600 nm). The Ca2+ tolerance of yeast expressing PfCAX and sPfCAX was demonstrated further by cell growth in liquid YPD media supplemented with various concentrations of CaCl2 (Figure 2C). sPfCAX provided K665 yeast with tolerance to high concentrations of CaCl2. sPfCAX-expressing yeast growth was significantly greater than PfCAX-expressing yeast at each CaCl2 concentration (p<0.02 unpaired, two-tailed Student's t-test; n = 4), while the cell growth of PfCAX-expressing yeast was significantly greater than that of the empty vector control yeast at all CaCl2 concentrations, including at 150 mM CaCl2 (p<0.02, unpaired, two-tailed Student's t-test; n = 4). A truncated version of TgCAX (sTgCAX), in which translation was initiated by introducing an AUG prior to nucleotide position 295 (encoding Ala-99), was also generated and expressed in K665 yeast. The transformation was assessed to have worked by PCR (Figure S4A) and, as with PfCAX and sPfCAX, sTgCAX suppressed the Ca2+ hypersensitivity of the yeast (Figure S4B). These data demonstrate that apicomplexan CAXs and truncated variants are functional in yeast, and in this heterologous system, can function in providing Ca2+ tolerance.

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