Probing the multifactorial basis of Plasmodium falciparum quinine resistance: Evidence for a strain-specific contribution of the sodium-proton exchanger PfNHE

Quinine (QN) continues to be an important treatment option for severe malaria, however resistance to this drug has emerged in field isolates of the etiologic agent Plasmodium falciparum. Quantitative trait loci investigations of QN resistance have mapped three loci of this complex trait. Two coincide with pfcrt and pfmdr1, involved in resistance to chloroquine (CQ) and other quinoline-based antimalarials. A third locus on chromosome 13 contains the sodium-proton exchanger (pfnhe) gene. Previous studies have associated pfnhe polymorphisms with reduced QN sensitivity in culture-adapted field isolates. Here, we provide direct evidence supporting the hypothesis that pfnhe contributes to QN resistance. Using allelic exchange, we reduced pfnhe expression by introducing a truncated 3′ untranslated region (UTR) from pfcrt into the endogenous pfnhe 3′UTR. Transfections were performed with 1BB5 and 3BA6 (both CQ- and QN-resistant) as well as GC03 (CQ- and QN-sensitive), all progenies of the HB3×Dd2 genetic cross. RNA and protein analyses of the ensuing recombinant clones demonstrated a ∼50% decrease in pfnhe expression levels. A statistically significant 30% decrease in QN IC₅₀ values was associated with these decreased expression levels in 1BB5 and 3BA6 but not in GC03. CQ, mefloquine and lumefantrine IC₅₀ values were unaltered. Cytosolic pH values were similar in all parental lines and recombinant clones. Our observations support a role for pfnhe in QN resistance in a strain-dependent manner, which might be contingent on pre-existing resistance to CQ and/or QN. These data bolster observations that QN resistance is a complex trait requiring the contribution of multiple transporter proteins.