Understanding molecular strategies of resistance used by the malarial parasite, Plasmodium falciparum 356 views

The malaria parasite, Plasmodium falciparum, is one of the deadliest human protozoan parasites largely due to its rapid adaptation to overcome antimalarial drug treatment. Work from our group and others have shown that resistance can be accomplished by copy number variations (CNVs) and single nucleotide polymorphisms (SNPs) in the malaria genome. Little is known about how these genetic alterations arise and are propagated to confer enhanced parasite drug resistance. In order to address these gaps in research, this thesis is divided in two parts: resistance and transmission. Chapter 2 highlights the novel detection of extra-chromosomal(ec) DNA in highly resistant P. falciparum parasites. Employing specialized PCR (droplet digital(dd) PCR) and Illumina sequencing we observed high copy numbers over genomic copies, uniquely associated with resistant parasites. The innovative detection strategies outlined in this thesis provided the opportunity to detect an otherwise, invisible molecule. We hope that the data presented in this thesis will lead to new discoveries of ecDNA in other well characterized resistance models. Furthermore, ecDNA provides a new, targetable approach to disrupt transmission of antimalarial resistance. Chapter 3 investigates for the first time, extracellular vesicles (EVs) harboring endogenously-derived dhodh amplicons used by resistant Plasmodium parasites. We combined ImageStreamX flow cytometry with cryogenic electron microscopy to characterize vesicle morphology. Also, we used ddPCR to characterize the DNA contents found within EVs. We detected CNVs found in genomic DNA and vesicle-contained DNA. The discovery of genetic material harboring resistance-conferring genes found in vesicles indicates a novel mechanism of genome evolution adapted by P. falciparum. Our results suggest vesicle exchange between a resistant donor and a sensitive recipient parasite may also emerge as a new targetable mechanism to inhibit resistance propagation. Chapter 4 focuses on the surveillance of molecular markers of chloroquine (CQ) resistance in an endemic country to determine whether or not an antiquated antimalarial, CQ, will be effective once again. Returning to an antiquated antimalarial provides an alternative to our current frontline, artemisinin, aimed to dampened resistance. Resistance profiling in Southwest Uganda during 2010 and 2015 revealed a slow reversal to CQ sensitivity via high resolution melt analysis (HRM). We concluded that the significant regional variation in Uganda as well as other areas of the world emphasizes the need to perform local assessment of resistant profiles. Investigating resistance profiles can be used to inform treatment policies and drug implementation. Overall, we hope that the data generated in this thesis can be used to uncover the Achille’s heel of malaria and move the scientific community closer to malaria eradication.

PHD (Doctor of Philosophy)

Malaria ; Drug resistance; Extra-chromosomal DNA

English

2020-04-30