Diverse mutational pathways converge on saturable chloroquine transport via the malaria parasite’s chloroquine resistance transporter

Abstract

Mutations in the chloroquine resistance transporter (PfCRT) are the primary determinant of chloroquine (CQ) resistance in the malaria parasite Plasmodium falciparum. A number of distinct PfCRT haplotypes, containing between 4 and 10 mutations, have given rise to CQ resistance in different parts of the world. Here we present a detailed molecular analysis of the number of mutations (and the order of addition) required to confer CQ transport activity upon the PfCRT as well as a kinetic characterization of diverse forms of PfCRT. We measured the ability of more than 100 variants of PfCRT to transport CQ when expressed at the surface of Xenopus laevis oocytes. Multiple mutational pathways led to saturable CQ transport via PfCRT, but these could be separated into two main lineages. Moreover, the attainment of full activity followed a rigid process in which mutations had to be added in a specific order to avoid reductions in CQ transport activity. A minimum of two mutations sufficed for (low) CQ transport activity, and as few as four conferred full activity. The finding that diverse PfCRT variants are all limited in their capacity to transport CQ suggests that resistance could be overcome by reoptimizing the CQ dosage.drug resistance | evolutionary biochemistry | Xenopus oocytes

Functional Comparison of 45 Naturally Occurring Isoforms of the Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT)

“…In contrast, another recent study reports good correlation between CQ transport for six PfCRT isoforms and the CQ IC 50 found for the cognate strains expressing those isoforms. 41 Our data for these isoforms show some degree of correlation; however, the correlation does not hold when the data are included within larger data sets (Figure 9). …”

“…7 Data in this paper further highlight this ΔΨ dependence and show that transport measurements taken at lower ΔΨ values do not necessarily reveal the complete CQ transport potential for some PfCRT isoforms. As previously discussed, 1 this concept is important when comparing data obtained via oocyte expression models 36,41 versus these yeast models or versus proteoliposome preparations. 7,9,18,25 For yeast and proteoliposomes, ΔΨ can be manipulated to much higher values whereas ΔΨ is quite low for the oocyte plasma membrane.…”

Characterization of the Chloroquine Resistance Transporter Homologue in Toxoplasma gondii

“…TgCRT, however, resembles wild-type PfCRT at several other positions with a known role in conferring chloroquine transport activity, such as N76 and N326 (see Fig. S1B) (23). TgCRT contains numerous other regions that are conserved in CRT homologues from other organisms, including a cysteine-rich motif in the loop between transmembrane domains 7 and 8 (see Fig.…”

“…S1C). A key mutation that confers chloroquine resistance in PfCRT is the conversion of lysine 76 to a threonine (K76T) (5,23). Intriguingly, TgCRT and other coccidian CRTs harbor a threonine in this position (see Fig.…”

Temporal and Seasonal Changes of Genetic Polymorphisms Associated with Altered Drug Susceptibility to Chloroquine, Lumefantrine, and Quinine in Guinea-Bissau between 2003 and 2012

“…pfcrt 76T is essential for resistance to the standard dose of CQ, and additional SNPs in pfcrt and pfmdr1 86Y appear to compensate for fitness loss and to modulate CQ susceptibility (11). In Guinea-Bissau, CQ resistance was linked to the pfcrt haplotype 72-76 CVIET ϩ 271E ϩ 326S, whereas the haplotype 72-76 CVMNK ϩ Q271 ϩ N326 was linked to CQ sensitivity (12,13).…”