부산대학교 도서관

Antimalarial drug resistance is an unrelenting obstacle to malaria control programs. In Southeast Asia (SEA), parasites have developed some degree of resistance to nearly every malaria drug currently available, with the most recent emergence to artemisinin combination therapies (ACTs). ACTs are the recommended front-line treatments for Plasmodium falciparum malaria worldwide and decreased susceptibility of parasites to both artemisinin and one of the widely used partner drugs, piperaquine, have been reported in multiple locations in SEA. It is therefore necessary to have reliable methods for detecting and evaluating resistant phenotypes. The purpose of this study was to combine clinical data from Cambodia with findings from genomic studies to evaluate putative markers of piperaquine resistance. The study first developed high-throughput assays to reliably detect one of these markers, a copy number variation (CNV) in the plasmepsin 2 and plasmepsin 3 (PM2-PM3) genes, in parasites likely to be PPQ-resistant. In addition to assay development, this study used gene overexpression techniques and CRISPR-Cas9 gene editing to examine the functional role of molecular markers of piperaquine resistance, including the PM2-3 CNV, and two gene candidates with nonsynonymous single nucleotide polymorphisms (SNPs): a putative exonuclease protein (exo-E415G) and a putative mitochondrial carrier protein (mcp-N252D). This research fills a knowledge gap in the lack of functional data for molecular markers of piperaquine resistance by examining the phenotypic relevance of the genotypes observed in contemporary isolates. To complement these functional studies, this doctoral work has also used a P. falciparum hypermutator parasite line to select for a piperaquine-resistant phenotype in vitro. Whole genome sequencing analysis (WGS) of the piperaquine-resistant lines obtained through these experiments has identified nonsynonymous SNPs in gene candidates that have been reported to play a role in antimalarial drug resistance, including SNPs in the chloroquine resistance transporter gene (pfcrt) and the multidrug resistant protein 1 (pfmdr1) transporter. SNPs in pfcrt have been reported to confer piperaquine resistance in the field and in vitro and our recent drug-pressure experiments provide additional evidence to support these findings. The WGS analysis also discovered novel SNPs in gene candidates not previously reported to modulate the piperaquine-resistant phenotype that will require further evaluation. Such work has enabled the possibility of examining whether genetic changes observed in patient isolates can also be investigated and observed in vitro. By combining functional molecular approaches with genomic analyses, this study provides new insights into the mechanisms of piperaquine resistance.