Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance

Abstract

The 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 K761 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.

Figure 1. The Dd2 pfcrt Gene Predicts a 424 Amino Acid Transmembrane Protein

The ten predicted transmembrane segments are shaded, and positions of eight amino acid substitutions are indicated that distinguish CQR and CQS parasites in the HB3 × Dd2 genetic cross. Inverted arrows indicate the intron splice sites in the corresponding nucleotide sequence. A map illustrating the exon-intron organization of pfcrt and its position relative to neighboring genes of chromosome 7 is available as supplemental data at www.molecule.org/cgi/content/full/6/4/861/DC1.

Figure 2. The Mutant CQR 106/1 Line 34-1/E Does Not Show Size Modifications in the pfcrt or pfmdr1 Genes but Instead Has Acquired a Novel K76I Codon in the pfcrt Sequence

(A) Southern blot analysis of BamHI-digested P. falciparum genomic DNA (from CQR lines 34-1/F and FCB and the CQS line 34-1/A_rm) or pNHSC plasmid DNA hybridized with sequences from pfcrt (left) or pfmdr1 (right). The pfcrt probe can hybridize to both an 11.8 kb BamHI chromosomal fragment including all the pfcrt coding regions and the pfcrt cDNA sequence present in linearized 6.5 kb pNHSC. The pfmdr1 probe is a mixture of 5′ and 3′ sequences that hybridize to 11.3 kb and 3.4 kb BamHI chromosomal fragments. The pfcrt and pfmdr1 hybridization results were the same for FCB and 106/1 as well as for 34-1/F and 34-1/E (data not shown). (B) The mutant CQR 106/1 line 34-1/E acquired a novel pfcrt mutation encoding K76I. Electropherograms show pfcrt sequences spanning codon 76 in lines 106/1, 34-1/B, 34-1/D, and 34-1/E (DNA prepared on days 74, 112, and 111 post-electroporation, respectively). The 34-1/E, 34-1/E_rm, and 34-1/F lines carried only the I76 codon (34-1/E panel; other data not shown). This mutation represented about 65% of the mixed signal from line 34-1/D. Only the original K76 codon was found in the episomally transformed 34-1/B line as well as the CQS lines 34-1/A_rm and 106/1.

Figure 3. The Transmembrane Protein PfCRT Migrates as an M_r 45K Band and Localizes to the Digestive Vacuole

(A) FLAG-specific M2 mAb recognizes epitope-tagged PfCRT as a M_r 45K protein in transformed 33-1/FLAG trophozoites. Rabbit IgG specific to the PfCRT-K peptide also recognize a protein of the same M_r in trophozoites from the 34-1/E_rm line and in DV from the 34-1/E_rm and 33-1/FLAG lines. (B–D) Rabbit anti-PfCRT-K IgG specifically stain the DV in both 34-1/E_rm (B and C) and 33-1/FLAG (D) parasites. Parasite location within the erythrocytes is evident in the differential interference contrast images (upper panels). Arrows indicate margins of the imaged intraerythrocytic parasites. Green fluorescence localizes PfCRT to the DV around the hemozoin pigment.