Determination of Epigenetic and Genetic Mechanisms involved in Generation of Chloroquine Tolerant Phenotype Clones of Plasmodium falciparum

Abstract

Development of anti-malarial resistance occurs at a fitness cost to the parasites. It makes sense to assume that Plasmodium falciparum would first use alternative mechanisms to survive transient drug pressure and only resort to genetic fixation if the pressure is sustained. In this study, development of chloroquine (CQ) tolerance was assessed by DNA methylation, allelic diversity and genetic changes at P. falciparum chloroquine resistance transporter (PfCRT), and P. falciparum multidrug resistance 1 (PfMDR1) genes. A chloroquine sensitive 3D7 strain of P. falciparum was cloned by limiting dilution and the derived population exposed to increasing CQ concentrations of 4.51 ng/mL, 5.99 ng/mL and 7.15 ng/mL corresponding to 10%, 30% and 50% inhibitory concentrations (IC) of the parental population. Chemo-sensitivity to CQ of the surviving parasite densities at the initial and at each drug level was determined by SYBR Green I fluorassay. Allelic diversity of CQ unexposed and drug pressure surviving parasite strains were assessed by nested PCR that targeted the polymorphic regions of MSP1, MSP2 and GLURP. Global DNA methylation at 5-methylCytosine (5-mC) was assessed by ELISA. Single nucleotide polymorphisms (SNPs) at PfCRT and PfMDR1 genes were assessed by restriction fragment length polymorphism (RFLP), sequencing and probe based quantitative PCR. The chloroquine unexposed population had an IC50 of 7.03±1.37 ng/mL, one K1 allele (248 bp), two IC3D7 (482 bp and 596 bp) and one 800 bp GLURP. PfCRT and PfMDR1 were wild type. Global 5-mC DNA methylation was not detectable. Post CQ exposure at 4.51 ng/mL and 5.99 ng/mL, IC50 increased to 10.5 ng/mL and 15.05 ng/mL respectively. Parasite growth at 7.15 ng/mL of CQ was minimal and IC50 could not be determined. At 5.99 ng/mL of CQ, evidence of clonal selection was marked by allele reduction in parasites carrying the K1 and the 596 bp IC3D7 alleles. At 7.15 ng/mL of CQ, parasites with these two alleles were lost, but the 482 bp IC3D7 and 800 bp GLURP clones survived. Chloroquine tolerant populations remained wild type at PfCRT and PfMDR1. Global 5-mC DNA methylation was not observed in any of the derived parasite populations. These data suggest that, development of CQ tolerance starts by clonal selection. In absence of obvious selection advantages of genetic or epigenetic changes to the surviving clones, further studies are needed to elucidate how CQ induced changes at PfCRT and PfMDR1 genes eventually occur.