Expression of parasite transporters in Xenopus oocytes

Abstract

The Xenopus laevis oocyte heterologous expression system is particularly useful for the study of transporter proteins. We demonstrated the utility of this expression system for studies on Plasmodium falciparum transporters by inducing increased uptake of metabolites or their analogues (nucleosides, nucleobases, lactate and glucose) into oocytes after microinjection of mRNA obtained from asexual stages of P. falciparum. We identified a hexose transporter of P. falciparum (PfHT1) and studied its function. Higher levels of functional activity are obtained when 5' and 3' untranslated Xenopus globin gene sequences and a strong Kozak consensus are included in RNA used for microinjection studies. PfHT1 is a saturable, sodium-independent and stereospecific transporter with a relatively high affinity for glucose (K(m) = 0.48 mM). Competition experiments with glucose analogues show that hydroxyl groups at positions C3 and C4 are important for ligand binding. mRNA levels for PfHT1 are highest during the small ring stages of infection and lowest in gametocytes. Confocal immunofluorescence microscopy localizes PfHT1 to the region of the parasite plasma membrane and not to host structures. When hypoglycaemia complicates cerebral malaria, modelling studies using data obtained from oocyte experiments suggest that the high affinity of PfHT1 may increase the proportion of glucose taken up by parasites compared with that transported across the blood-brain barrier.