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

  1. Rowena E. Martina,2,3
  1. aResearch School of Biology, The Australian National University, Canberra, ACT 0200, Australia;
  2. bDepartment of Infectious Diseases, Parasitology, Universitätsklinikum Heidelberg, 69120 Heidelberg, Germany; and
  3. cDepartment of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel

  1. Edited* by Thomas E. Wellems, National Institutes of Health, Bethesda, MD, and approved March 17, 2014 (received for review December 17, 2013)

Significance

This study provides detailed insights into the workings of a protein that is a key determinant of drug resistance in the malaria parasite. We found that two main lineages of mutational routes lead to chloroquine transport via the chloroquine resistance transporter (PfCRT) and that a low level of chloroquine transport is conferred by as few as two mutations. However, the attainment of full transport activity is a rigid process that requires the mutations be added in a specific order to avoid decreases in chloroquine transport. Our finding that diverse forms of mutant PfCRT are all limited in their capacity to transport chloroquine indicates that resistance should be overcome by reoptimizing the chloroquine dosage.

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.

Footnotes

  • ↵1R. L. Summers and A.D. contributed equally to this work.

  • ↵2M.L. and R.E.M. contributed equally to this work.

  • ↵3To whom correspondence may be addressed. E-mail: michael.lanzer{at}med.uni-heidelberg.de or rowena.martin{at}anu.edu.au.

  • Author contributions: M.L. and R.E.M. conceived the study; M.L. and R.E.M. designed research; R. L. Summers, A.D., T.J.D., S.B., R.V.M., M.N.N., S.N.R., V.G., R. L. Schenk, C.P.S., M.L., and R.E.M. performed research; R. L. Summers, R.V.M., S.N.R., and R.E.M. contributed new reagents/analytic tools; R. L. Summers, A.D., S.B., M.N.N., W.D.S., K.K., C.P.S., M.L., and R.E.M. analyzed data; and R. L. Summers and R.E.M. wrote the paper.

  • The authors declare no conflict of interest.

  • ↵*This Direct Submission article had a prearranged editor.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1322965111/-/DCSupplemental.

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