A yeast expression system for functional and pharmacological studies of the malaria parasite Ca2+/H+ antiporter.

Salcedo, Enrique, Ward, Stephen ORCID: https://orcid.org/0000-0003-2331-3192 and Biagini, Giancarlo ORCID: https://orcid.org/0000-0001-6356-6595 (2012) 'A yeast expression system for functional and pharmacological studies of the malaria parasite Ca2+/H+ antiporter.'. Malaria Journal, Vol 11, p. 254.

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Abstract

ABSTRACT: BACKGROUND: Calcium (Ca2+) signalling is fundamental for host cell invasion, motility, in vivo synchronicity and sexual differentiation of the malaria parasite. Consequently, cytoplasmic free Ca2+ is tightly regulated through the co-ordinated action of primary and secondary Ca2+ transporters. Identifying selective inhibitors of Ca2+ transporters is key towards understanding their physiological role as well as having therapeutic potential, therefore screening systems to facilitate the search for potential inhibitors are a priority. Here, the methodology for the expression of a Calcium membrane transporter that can be scaled to high throughputs in yeast is presented. METHODS: The Plasmodium falciparum Ca2+/H+ antiporter (PfCHA) was expressed in the yeast Saccharomyces cerevisiae and its activity monitored by the bioluminescence from apoaequorin triggered by divalent cations, such as calcium, magnesium and manganese. RESULTS: Bioluminescence assays demonstrated that PfCHA effectively suppressed induced cytoplasmic peaks of Ca2+, Mg2+ and Mn2+ in yeast mutants lacking the homologue yeast antiporter Vcx1p. In the scalable format of 96-well culture plates pharmacological assays with a cation antiporter inhibitor allowed the measurement of inhibition of the Ca2+ transport activity of PfCHA conveniently translated to the familiar concept of fractional inhibitory concentrations. Furthermore, the cytolocalization of this antiporter in the yeast cells showed that whilst PfCHA seems to locate to the mitochondrion of P. falciparum, in yeast PfCHA is sorted to the vacuole. This facilitates the real-time Ca2+-loading assays for further functional and pharmacological studies. DISCUSSION: The functional expression of PfCHA in S. cerevisiae and luminescence-based detection of cytoplasmic cations as presented here offer a tractable system that facilitates functional and pharmacological studies in a high-throughput format. PfCHA is shown to behave as a divalent cation/H+ antiporter susceptible to the effects of cation/H+ inhibitors such as KBR7943. This type of gene expression systems should advance the efforts for the screening of potential inhibitors of this type of divalent cation transporters as part of the malaria drug discovery initiatives and for functional studies in general. CONCLUSION: The expression and activity of the PfCHA detected in yeast by a bioluminescence assay that follows the levels of cytoplasmic Ca2+ as well as Mg2+ and Mn2+ lend itself to highthroughput and quantitative settings for pharmacological screening and functional studies.

Item Type:	Article
Uncontrolled Keywords:	Calcium, Magnesium, Manganese, Malaria, Yeast, Plasmodium, Ca2+/H+ antiporter, Saccharomyces cerevisiae, Vacuole
Subjects:	QU Biochemistry > Proteins. Amino Acids. Peptides > QU 58.7 RNA QX Parasitology > Protozoa > QX 135 Plasmodia WC Communicable Diseases > Tropical and Parasitic Diseases > WC 750 Malaria
Faculty: Department:	Groups (2002 - 2012) > Molecular & Biochemical Parasitology Group
Digital Object Identifer (DOI):	https://doi.org/10.1186/1475-2875-11-254
Depositing User:	Mary Creegan
Date Deposited:	06 Nov 2012 15:58
Last Modified:	17 Jul 2020 10:58
URI:	https://archive.lstmed.ac.uk/id/eprint/3048

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