Chloroquine resistance transporter of Plasmodium falciparum (PfCRT) is a food vacuolar transmembrane protein that mediates susceptibility of the parasite to chloroquine. A mutation at K76T of the Pfcrt gene is a key determinant for chloroquine resistance phenotype. In the absence of drug pressure, in vitro growth rate of chloroquine-resistance parasites was outcompeted by wild-type parasites unless intragenic compensatory mutations occurred. Chloroquine-resistant P. falciparum bearing the Cam734 haplotype known to circulate in endemic areas of Cambodia bordering Thailand contains 9 mutations in Pfcrt and exhibits both chloroquine resistance and comparable growth rate to the chloroquine-sensitive 3D7 strain. To analyze the evolution of the Cam734 haplotype, codon-based analysis was performed by using the mixed effects model of evolution (MEME), branch-site random effects likelihood (BR-REL) and other related methods. Results revealed that the Cam734 haplotype has evolved distinctively from other known mutant haplotypes including the most common Dd2 haplotype in Southeast Asia. Evidence of episodic positive selection was detected at codon 144, characterized by c.[430G>T; 431C>T] (p.A144F), known to be indispensable for both chloroquine resistance and restoration of growth rate of the parasites. To survey the prevalence of mutations at codons 76 and 144 in Pfcrt among Thai isolates, restriction fragment analysis of 548 P. falciparum isolates collected from six endemic provinces of Thailand during 1991 and 2016 was performed. The 144F Pfcrt mutant was detected in 7 (1.28%) isolates. All Thai isolates analyzed herein harbored a mutation at codon 76 whilst the wild-type parasite was not found. The low prevalence of isolates bearing the mutation 144F in PfCRT could imply little or lack of survival advantage of this mutant in endemic areas of Thailand where the wild-type parasites seem to be absent or extremely rare.
Plasmodium knowlesi and P. cynomolgi are simian malaria parasites capable of causing symptomatic human infections. The interaction between the Duffy binding protein alpha on P. knowlesi merozoite and the Duffy-antigen receptor for chemokine (DARC) on human and macaque erythrocyte membrane is prerequisite for establishment of blood stage infection whereas DARC is not required for erythrocyte invasion by P. cynomolgi. To gain insights into the evolution of the PkDBP gene family comprising PkDBPα, PkDBPβ and PkDBPγ, and a member of the DBP gene family of P. cynomolgi (PcyDBP1), the complete coding sequences of these genes were analyzed from Thai field isolates and compared with the publicly available DBP sequences of P. vivax (PvDBP). The complete coding sequences of PkDBPα (n=11), PkDBPβ (n=11), PkDBPγ (n=10) and PcyDBP1 (n=11) were obtained from direct sequencing of the PCR products. Nucleotide diversity of DBP is highly variable across malaria species. PcyDBP1 displayed the greatest level of nucleotide diversity while all PkDBP gene members exhibited comparable levels of diversity. Positive selection occurred in domains I, II and IV of PvDBP and in domain V of PcyDBP1. Although deviation from neutrality was not detected in domain II of PkDBPα, a signature of positive selection was identified in the putative DARC binding site in this domain. The DBP gene families seem to have arisen following the model of concerted evolution because paralogs rather than orthologs are clustered in the phylogenetic tree. The presence of identical or closely related repeats exclusive for the PkDBP gene family suggests that duplication of gene members postdated their divergence from the ancestral PcyDBP and PvDBP lineages. Intragenic recombination was detected in all DBP genes of these malaria species. Despite the limited number of isolates, P. knowlesi from Thailand shared phylogenetically related domain II sequences of both PkDBPα and PkDBPγ with those from Peninsular Malaysia, consistent with their geographic proximity.
The amino acid substitution at residue 76 of the food vacuolar transmembrane protein encoded by the chloroquine resistance transporter gene of Plasmodium falciparum (Pfcrt) is an important, albeit imperfect, determinant of chloroquine susceptibility status of the parasite. Other mutations in Pfcrt can modulate susceptibility of P. falciparum to other antimalarials capable of interfering with heme detoxification process, and may exert compensatory effect on parasite growth rate. To address whether nationwide implementation of artemisinin combination therapy (ACT) in Thailand could affect sequence variation in exon 2 and introns of Pfcrt, we analyzed 136 P. falciparum isolates collected during 1997 and 2016 from endemic areas bordering Myanmar, Cambodia and Malaysia. Results revealed 6 haplotypes in exon 2 of Pfcrt with 2 novel substitutions at c.243A > G (p.R81) and c.251A > T (p.N84I). Positive selection was observed at amino acid residues 75, 76 and 97. Four, 3, and 2 alleles of microsatellite (AT/TA) repeats occurred in introns 1, 2 and 4, respectively, resulting in 7 different 3-locus haplotypes. The number of haplotypes and haplotype diversity of exon 2, and introns 1, 2 and 4 were significantly greater among isolates collected during 2009 and 2016 than those collected during 1997 and 2008 when 3-day ACT and 2-day ACT regimens were implemented nationwide, respectively (p