Mutations in the P. falciparum Digestive Vacuole Transmembrane Protein PfCRT and Evidence for Their Role in Chloroquine Resistance

Abstract

SummaryThe determinant of verapamil-reversible chloroquine resistance (CQR) in a Plasmodium falciparum genetic cross maps to a 36 kb segment of chromosome 7. This segment harbors a 13-exon gene, pfcrt, having point mutations that associate completely with CQR in parasite lines from Asia, Africa, and South America. These data, transfection results, and selection of a CQR line harboring a novel K76I mutation point to a central role for the PfCRT protein in CQR. This transmembrane protein localizes to the parasite digestive vacuole (DV), the site of CQ action, where increased compartment acidification associates with PfCRT point mutations. Mutations in PfCRT may result in altered chloroquine flux or reduced drug binding to hematin through an effect on DV pH.

Polymorphic patterns of pfcrt and pfmdr1 in Plasmodium falciparum isolates along the Thai-Myanmar border

“…PCR-RFLP was employed to detect pfcrt mutation at codons 76, 220, 271, 326, 356 and 371 [12] and pfmdr1 mutation at codon 86 [13] . DNA of P. falciparum laboratory clones G112 and K1 served as control for chloroquine sensitive and chloroquineresistant genotype, respectively.…”

Chloroquine-resistant isoforms of the Plasmodium falciparum chloroquine resistance transporter acidify lysosomal pH in HEK293 cells more than chloroquine-sensitive isoforms

“…The biotin acceptor domain (bad) sequences were excised by simultaneous NotI and HindIII digestion and re-ligated to form the HB3 and Dd2 alleles. Both constructs were sequenced in their entirety to ensure that their inferred amino acid sequences corresponded to the published HB3 and Dd2 sequences [5]. To generate monomeric Red Fluorescent Protein (RFP) fusion constructs with PfCRT, the HB3-RFP and Dd2-RFP fusions, an AgeI site was introduced before the stop codon of HB3 and Dd2 using a Quikchange kit (Stratagene, CA).…”

“…The development of affordable alternatives to CQ has been slow [4]. The analysis of a genetic cross facilitated the identification of a molecular determinant of P. falciparum CQ resistance [5]. Mutations in this gene, named the P. falciparum chloroquine resistance transporter (pfcrt), are sufficient to confer CQ resistance [6].…”

“…PfCRT displays weak primary sequence similarity to bacterial drug metabolite transporters [7,8], and has been implicated in the development of resistance to other antimalarial agents, including halofantrine and amantidine [9]. CQ-resistant (CQR) patient isolates contain a K76T mutation in a background that contains several other mutations [5]. Expression of the "back mutation" of T76K in a CQ-resistant allele increases the CQ susceptibility [10].…”

“…For example, while verapamil, a calcium channel antagonist, reverses CQ resistance [21], the mechanism of reversal is uncertain, despite evidence that PfCRT is involved [5,10,20]. Physiological and biochemical studies of Plasmodium proteins in situ are time consuming and subject to technical barriers [22].…”