Identification and Characterization of Novel Drug Resistance Loci in Plasmodium falciparum

Abstract

Malaria has plagued mankind for millennia. Antimalarial drug use over the last century has generated highly drug-resistant parasites, which amplify the burden of this disease and pose a serious obstacle to control efforts. This dissertation is motivated by the simple fact that malaria parasites have become resistant to nearly every antimalarial drug that has ever been used, yet the precise genetic mechanisms of parasite drug resistance remain largely unknown. Our work pairs genomics-age technologies with molecular biology, genetics and molecular epidemiology in order to identify and characterize novel genes that contribute to drug resistance in P. falciparum. In the Introduction, we highlight relevant opportunities and challenges in trying to identify and understand the genetic basis of malaria drug resistance as it emerges to currently used therapies. In Chapter One, we demonstrate how genome-wide association studies (GWAS) can be applied to P. falciparum in order to identify novel drug resistance loci. Functional follow-up revealed that overexpression of the novel candidate gene PF10_0355 made parasites more resistant to the drugs halofantrine, lumefantrine and mefloquine. These findings show that PF10_0355 plays a role in parasite drug response, as well as provides validation of our GWAS approach. In Chapter Two, we further characterize PF10_0355 and show that modulation of the gene by either knockout or allelic replacement changes parasite drug sensitivity. Furthermore, we show that moderate changes in drug response measured in the short-term can have dramatic effects when parasites are competed with one another under drug pressure. In Chapter Three, we use an overexpression approach to functionally follow up other novel drug resistance genes generated by GWAS in P. falciparum. We find that overexpression is a useful way to begin to screen candidate drug resistance loci in the malaria parasite. In Chapter Four, we use a DAPI-based ex vivo drug assay to monitor drug resistance among parasites circulating in Thiés, Senegal. In the future, we will look for genetic markers of parasite drug resistance in this population by GWAS. Finally, in the Discussion we present an essay about malaria evolution and eradication written for non-specialists. Our hope is that the work presented in this dissertation furthers understanding of drug resistance in the malaria parasite, both within and beyond the malaria research community.