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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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Sequence alignments.Amino acid sequence alignment of PfCAX with PbCAX. The Clustal W program was used to generate the alignment. The residues highlighted by a bold black line above correspond to transmembrane segment predictions determined with the TMHMM program (http://www.cbs.dtu.dk/services/TMHMM/). The residues highlighted by a bold green line below correspond to the conserved CAX regions, c-1 and c-2. Green shading denotes residues shown to be essential for Ca2+ transport in AtCAX1 and OsCAX1a [15], [16]. Yellow shading denotes the putative mitochondrial targeting motif [7]. Grey shading denotes cleaved sequences for mitochondrially imported proteins predicted by MitoProt II – v1.101 (http://ihg.gsf.de/ihg/mitoprot.html). Red shading denotes phospo-acceptor sites (GeneDB and [17]). CAX sequences are from (accession no.): Pf, Plasmodium falciparum (XP_966025.1) and Pb, Plasmodium berghei (XP_678577.1). Red letters, identical or conserved residues in all sequences; green letters, conserved substitutions; blue letters, semi-conserved substitutions.
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ppat-1003191-g001: Sequence alignments.Amino acid sequence alignment of PfCAX with PbCAX. The Clustal W program was used to generate the alignment. The residues highlighted by a bold black line above correspond to transmembrane segment predictions determined with the TMHMM program (http://www.cbs.dtu.dk/services/TMHMM/). The residues highlighted by a bold green line below correspond to the conserved CAX regions, c-1 and c-2. Green shading denotes residues shown to be essential for Ca2+ transport in AtCAX1 and OsCAX1a [15], [16]. Yellow shading denotes the putative mitochondrial targeting motif [7]. Grey shading denotes cleaved sequences for mitochondrially imported proteins predicted by MitoProt II – v1.101 (http://ihg.gsf.de/ihg/mitoprot.html). Red shading denotes phospo-acceptor sites (GeneDB and [17]). CAX sequences are from (accession no.): Pf, Plasmodium falciparum (XP_966025.1) and Pb, Plasmodium berghei (XP_678577.1). Red letters, identical or conserved residues in all sequences; green letters, conserved substitutions; blue letters, semi-conserved substitutions.

Mentions: The P. falciparum, P. berghei and T. gondii CAX genes, pfcax (PFF0170w), pbcax (PBANKA_010230) and TgCAX (TGME49_007910), have 1326, 1323 and 1506 base pair open reading frames, respectively, with only the latter having (12) introns. They are located on chromosomes 6, 1 and 1b in their respective genomes and encode polypeptides of 441, 440 and 501 amino acids, with estimated sizes of 48, 49 and 53 kDa, respectively (Figure 1 and S1). All the apicomplexan cax genes identified are single copy genes with no close paralogues. PfCAX has greater than 80%, approximately 50% and 39% amino acid sequence identity compared with other Plasmodium spp., Coccidia (Toxoplasma, Cryptosporidium and Eimeria) and C. reinhardtii CAX sequences, respectively. The phylogenetic relationship between the apicomplexan putative CAX transporters is shown in Figure S2, in which CrCAX1 has been added as the first functionally characterised Type 1-C CAX [10]. Interestingly, BLAST searches, using the PfCAX amino acid sequence, did not reveal cax genes in the genomes of the Piroplasmida, Babesia bovis and bigemina or Theileria annulata, even though they are closely related to Plasmodium parasites.


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)

Sequence alignments.Amino acid sequence alignment of PfCAX with PbCAX. The Clustal W program was used to generate the alignment. The residues highlighted by a bold black line above correspond to transmembrane segment predictions determined with the TMHMM program (http://www.cbs.dtu.dk/services/TMHMM/). The residues highlighted by a bold green line below correspond to the conserved CAX regions, c-1 and c-2. Green shading denotes residues shown to be essential for Ca2+ transport in AtCAX1 and OsCAX1a [15], [16]. Yellow shading denotes the putative mitochondrial targeting motif [7]. Grey shading denotes cleaved sequences for mitochondrially imported proteins predicted by MitoProt II – v1.101 (http://ihg.gsf.de/ihg/mitoprot.html). Red shading denotes phospo-acceptor sites (GeneDB and [17]). CAX sequences are from (accession no.): Pf, Plasmodium falciparum (XP_966025.1) and Pb, Plasmodium berghei (XP_678577.1). Red letters, identical or conserved residues in all sequences; green letters, conserved substitutions; blue letters, semi-conserved substitutions.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585132&req=5

ppat-1003191-g001: Sequence alignments.Amino acid sequence alignment of PfCAX with PbCAX. The Clustal W program was used to generate the alignment. The residues highlighted by a bold black line above correspond to transmembrane segment predictions determined with the TMHMM program (http://www.cbs.dtu.dk/services/TMHMM/). The residues highlighted by a bold green line below correspond to the conserved CAX regions, c-1 and c-2. Green shading denotes residues shown to be essential for Ca2+ transport in AtCAX1 and OsCAX1a [15], [16]. Yellow shading denotes the putative mitochondrial targeting motif [7]. Grey shading denotes cleaved sequences for mitochondrially imported proteins predicted by MitoProt II – v1.101 (http://ihg.gsf.de/ihg/mitoprot.html). Red shading denotes phospo-acceptor sites (GeneDB and [17]). CAX sequences are from (accession no.): Pf, Plasmodium falciparum (XP_966025.1) and Pb, Plasmodium berghei (XP_678577.1). Red letters, identical or conserved residues in all sequences; green letters, conserved substitutions; blue letters, semi-conserved substitutions.
Mentions: The P. falciparum, P. berghei and T. gondii CAX genes, pfcax (PFF0170w), pbcax (PBANKA_010230) and TgCAX (TGME49_007910), have 1326, 1323 and 1506 base pair open reading frames, respectively, with only the latter having (12) introns. They are located on chromosomes 6, 1 and 1b in their respective genomes and encode polypeptides of 441, 440 and 501 amino acids, with estimated sizes of 48, 49 and 53 kDa, respectively (Figure 1 and S1). All the apicomplexan cax genes identified are single copy genes with no close paralogues. PfCAX has greater than 80%, approximately 50% and 39% amino acid sequence identity compared with other Plasmodium spp., Coccidia (Toxoplasma, Cryptosporidium and Eimeria) and C. reinhardtii CAX sequences, respectively. The phylogenetic relationship between the apicomplexan putative CAX transporters is shown in Figure S2, in which CrCAX1 has been added as the first functionally characterised Type 1-C CAX [10]. Interestingly, BLAST searches, using the PfCAX amino acid sequence, did not reveal cax genes in the genomes of the Piroplasmida, Babesia bovis and bigemina or Theileria annulata, even though they are closely related to Plasmodium parasites.

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