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Abstract
BackgroundRecent work has perfected yeast-based methods for measuring drug transport by the Plasmodium falciparum chloroquine (CQ) resistance transporter (PfCRT).MethodsThe approach relies on inducible heterologous expression of PfCRT in Saccharomyces cerevisiae yeast. In these experiments selecting drug concentrations are not toxic to the yeast, nor is expression of PfCRT alone toxic. Only when PfCRT is expressed in the presence of CQ is the growth of yeast impaired, due to inward transport of chloroquine (CQ) via the transporter.ResultsDuring analysis of all 53 known naturally occurring PfCRT isoforms, two isoforms (PH1 and PH2 PfCRT) were found to be intrinsically toxic to yeast, even in the absence of CQ. Additional analysis of six very recently identified PfCRT isoforms from Malaysia also showed some toxicity. In this paper the nature of this yeast toxicity is examined. Data also show that PH1 and PH2 isoforms of PfCRT transport CQ with an efficiency intermediate to that catalyzed by previously studied CQR conferring isoforms. Mutation of PfCRT at position 160 is found to perturb vacuolar physiology, suggesting a fitness cost to position 160 amino acid substitutions.ConclusionThese data further define the wide range of activities that exist for PfCRT isoforms found in P. falciparum isolates from around the globe.Graphical abstractV5 Western blot quantifying relative PfCRT expression in total (CM) vs vacuolar (VAC) yeast membranes for unmodified HB3 PfCRT vs HB3 and PH1 PMA–PfCRT chimeras.
Highlights
Recent work has perfected yeast-based methods for measuring drug transport by the Plasmodium falciparum chloroquine (CQ) resistance transporter (PfCRT)
Growth delay depends on the expression of PfCRT, the concentration of extracelluar CQ, and the magnitude of membrane potential across the yeast plasma membrane (PM) [3, 4, 17]
Recently, CQ transport for 45 of the 53 naturally occurring isoforms of PfCRT has been measured and found to have a wide range of activities that predict a range of CQ sensitivities for P. falciparum isolates expressing these isoforms [3, 4, 17]
Summary
Recent work has perfected yeast-based methods for measuring drug transport by the Plasmodium falciparum chloroquine (CQ) resistance transporter (PfCRT). Since the discovery of Plasmodium falciparum chloroquine (CQ) resistance transporter PfCRT and its role as the primary genetic determinant of chloroquine resistance (CQR) in P. falciparum, 53 distinct isoforms of this 424 amino acid protein have been found to be expressed in parasite isolates from around the globe [1,2,3,4] These have descended from at least five independent ‘founder events’ in four regions: Southeast Asia (SEA), South America (SA) (two events), Papua New Guinea (PNG), and the Philippines [5, 6], presumably via variable drug selection pressure in these regions. Direct analysis of the function of these isoforms is required to test this conclusion, since possible exceptions to CQR status for parasites expressing K76T mutant PfCRTs have been noted [10,11,12]
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