Analysis of Plasmodium vivax hexose transporters and effects of a parasitocidal inhibitor

Abstract

Plasmodium vivax is the second most common species of malaria parasite and causes up to 80 million episodes of infection each year. New drug targets are urgently needed because of emerging resistance to current treatments. To study new potential targets, we have functionally characterized two natural variants of the hexose transporter of P. vivax (PvHT) after heterologous expression in Xenopus oocytes. We show that PvHT transports both glucose and fructose. Differences in the affinity for fructose between the two variants of PvHT establishes that sequence variation is associated with phenotypic plasticity. Mutation of a single glutamine residue, Gln(167), predicted to lie in transmembrane helix 5, abolishes fructose transport by PvHT, although glucose uptake is preserved. In contrast, the exofacial site located between predicted helices 5 and 6 of PvHT is not an important determinant of substrate specificity, despite exhibiting sequence polymorphisms between hexose transporters of different Plasmodium spp. Indeed, replacement of twelve residues located within this region of PvHT by those found in the orthologous Plasmodium falciparum sequence (PfHT) is functionally silent with respect to affinity for hexoses. All PvHT variants are inhibited by compound 3361, a long-chain O-3 derivative of D-glucose effective against PfHT. Furthermore, compound 3361 kills short term cultures of P. vivax isolated from patients. These data provide unique insights into the function of hexose transporters of Plasmodium spp. as well as further evidence that they could be targeted by drugs.

Figure 1. Sequence alignment of predicted helix 5 region from different P. vivax hexose transporters

Alignment in the predicted helix 5 region of amino acid sequences from different natural variants of PvHT was generated by the ClustalW program. Non-conserved residues are highlighted in black boxes. Assignment of helix 5 is based on predictions determined with the Tmpred program. PvHT_belem, PvHT_sal and PvHT_ind were identified from the Belem strain, Salvador 1 strain and from an Indian isolate respectively. Site-directed mutants generated in this study are also shown. PvHT Q167N corresponds to the replacement of the glutamine by an asparagine in position 167. PvHT_chim corresponds to a PvHT chimera in which amino acids located at the exofacial site between predicted helices 5 and 6 have been replaced by those of PfHT (see Materials and methods).

Figure 2. Transport properties of PvHT_sal, PvHT_ind and Q167N mutant PvHT_ind in Xenopus laevis oocytes

(A) Initial mean uptake rates (eight oocytes per concentration; mean±S.E.M.) of D-glucose are shown against concentration of substrate (representative of three experiments; see Table 1). (B) D-fructose used to compete for D-glucose transport. Uptake assays (mean±S.E.M. of 8 oocytes per condition) were carried out with D-[¹⁴C]glucose. Percentage of D-glucose uptake at various fructose concentration was compared with uptake in uncompeted oocytes (control). PvHT_ind (▪); PvHT_ind Q167N (▾); PvHT_sal (○).

Figure 3. Active analogue studies on PvHT_Ind in Xenopus laevis oocytes

Uptake assays (mean±S.E.M. of 8 oocytes per condition) were carried out with D-[¹⁴C]glucose. Percentage of D-glucose uptake for each condition was compared with uptake in uncompeted oocytes (control). All compounds were used at 10 mM. Grey bars, PfHT; black bars, PvHT_ind; hatched bars, PkHT.