Journal of Biological Chemistry
Volume 278, Issue 35, 29 August 2003, Pages 33593-33601
Journal home page for Journal of Biological Chemistry

Genes: Structure and Regulation
Chloroquine Resistance Modulated in Vitro by Expression Levels of the Plasmodium falciparum Chloroquine Resistance Transporter*

https://doi.org/10.1074/jbc.M302215200Get rights and content
Under a Creative Commons license
open access

Plasmodium falciparum malaria is increasingly difficult to treat and control due to the emergence of parasite resistance to the major antimalarials, notably chloroquine. Recent work has shown that the chloroquine resistance phenotype can be conferred by multiple amino acid mutations in the parasite digestive vacuole transmembrane protein PfCRT. Here, we have addressed whether chloroquine resistance can also be affected by changes in expression levels of this protein. Transient transfection reporter assays revealed that truncation of the pfcrt 3′-untranslated region just prior to putative polyadenylation sites resulted in a 10-fold decrease in luciferase expression levels. Using allelic exchange on a chloroquine-resistant line (7G8 from Brazil), this truncated 3′-untranslated region was inserted downstream of the pfcrt coding sequence, in the place of the endogenous 3′-untranslated region. The resulting pfcrt-modified “knockdown” clones displayed a marked decrease in pfcrt transcription and an estimated 30–40% decrease in PfCRT protein expression levels. [3H]hypoxanthine incorporation assays demonstrated up to a 40% decrease in chloroquine with or without verapamil IC50 levels of pfcrt knockdown clones, relative to the 7G8 parent. Single-cell photometric analyses were consistent with an altered intracellular pH in the knockdown clones, providing further evidence for a relationship between PfCRT, pH regulation, and chloroquine resistance. Genetic truncation of 3′-untranslated regions provides a useful approach for assessing the impact of candidate genes on drug resistance or other quantifiable phenotypes in P. falciparum.

Cited by (0)

*

This work was supported in part by National Institutes of Health Grants R01 AI50234 (to D. A. F.) and R01 AI45957 (to P. D. R.), The Ellison Medical Foundation (New Scholar Award in Global Infectious Disease to D. A. F.), U.S. Agency for International Development Grant DPE-963-6001-29 (to H. F.), and the Wellcome Trust (to P. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Present address: The Dept. of Cardiology, Albert Einstein College of Medicine, Bronx, NY 10461.