Chloroquine Tolerance in 3D7 Strain of P. falciparum was Associated with Change in Allelic Structure and not Pfcrt and Pfmdr1 Transporter Genes nor DNA Methylation
Development of anti-malarial resistance occurs at a fitness cost to the parasites. It makes sense to assume that P. 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 Pfcrt and Pfmdr1. A CQ 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. The surviving parasite density at each drug level was determined by SYBR Green I fluorassay. Allelic diversity of CQ unexposed and those surviving drug pressure 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. Mutations at Pfcrt and Pfmdr1 genes were assessed by single nucleotide polymorphisms (SNPs). The CQ 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. 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 IC50 could not be determined. At 5.99 ng/mL of CQ, change in parasite structure 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. CQ tolerant populations remained wild type at Pfcrt and Pfmdr1. 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 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.
Plasmodium falciparum, DNA Methylation, Pfcrt, Pfmdr1, Single Nucleotide Polymorphisms, Chloroquine Resistance, Genetic Diversity
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