Abstract

Doc number: 184

Abstract

Background: To facilitate development within erythrocytes, malaria parasites increase their host cell uptake of diverse solutes including Ca++ . The mechanism and molecular basis of increased Ca++ permeability remains less well studied than that of other solutes.

Methods: Based on an appropriate Ca++ affinity and its greater brightness than related fluorophores, Fluo-8 was selected and used to develop a robust fluorescence-based assay for Ca++ uptake by human erythrocytes infected with Plasmodium falciparum .

Results: Both uninfected and infected cells exhibited a large Ca++ -dependent fluorescence signal after loading with the Fluo-8 dye. Probenecid, an inhibitor of erythrocyte organic anion transporters, abolished the fluorescence signal in uninfected cells; in infected cells, this agent increased fluorescence via mechanisms that depend on parasite genotype. Kinetic fluorescence measurements in 384-well microplates revealed that the infected cell Ca++ uptake is not mediated by the plasmodial surface anion channel (PSAC), a parasite nutrient channel at the host membrane; it also appears to be distinct from mammalian Ca++ channels. Imaging studies confirmed a low intracellular Ca++ in uninfected cells and higher levels in both the host and parasite compartments of infected cells. Parasite growth inhibition studies revealed a conserved requirement for extracellular Ca++ .

Conclusions: Nondestructive loading of Fluo-8 into human erythrocytes permits measurement of Ca++ uptake kinetics. The greater Ca++ permeability of cells infected with malaria parasites is apparent when probenecid is used to inhibit Fluo-8 efflux at the host membrane. This permeability is mediated by a distinct pathway and may be essential for intracellular parasite development. The miniaturized assay presented here should help clarify the precise transport mechanism and may identify inhibitors suitable for antimalarial drug development.

Details

Title
A kinetic fluorescence assay reveals unusual features of Ca++ uptake in Plasmodium falciparum -infected erythrocytes
Author
Zipprer, Elizabeth M; Neggers, McKinzie; Kushwaha, Ambuj; Rayavara, Kempaiah; Desai, Sanjay A
Pages
184
Publication year
2014
Publication date
2014
Publisher
BioMed Central
e-ISSN
14752875
Source type
Scholarly Journal
Language of publication
English
ProQuest document ID
1537604052
Copyright
© 2014 Zipprer et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.