Studies on the Artemisinin Resistome of Plasmodium Falciparum and the Role of Extracellular Vesicles in the Development of Antimalarial Drug Resistance

Abstract

The development of antimalarial drug resistance by Plasmodium falciparum has historically derailed efforts at effective control of malaria especially in sub-Saharan Africa (SSA). Presently, this risk has resurfaced following the emergence of artemisinin (ART) resistant (ARTr) P. falciparum parasites in the Greater Mekong subregion (GMS) of Southeast Asia (SEA). To mitigate the risk of the spread of ARTr P. falciparum parasites to SSA, molecular surveillance is indispensable. This need has partly been met by the identification of P. falciparum kelch 13 (PfK13) variants associated with ART resistance. However, the genomic architecture of ART resistance is complex and multiple layers of evidence have shown that PfK13-driven ARTr is not the universally exclusive molecular marker(s) of ARTr P. falciparum parasites. This fluidity of the molecular marker associated with ARTr parasites is linked to the genetic background of the regional P. falciparum parasite population and the promiscuous mechanism of action of ART that makes possible a plurality of molecular mechanisms to drive the evolution of ART resistance. Therefore, in regions at risk of ARTr P. falciparum parasites emerging, interrogating the artemisinin resistome (all resistance genes including the precursor genes capable of contributing to ART resistance) may shed light on the topology of ART resistance evolution - PfK13 dependent or not. This paradigm has the long term potential to be retrofitted into a molecular surveillance forecasting tool for ARTr P. falciparum parasites. Furthermore, the impact of ARTr P. falciparum parasites emerging in SSA can be mitigated by deepening our understanding of the molecular mechanisms undergirding ART resistance development and translating that knowledge into the discovery of novel antimalarial drug targets. With mounting evidence implicating PfK13 gene variants in the emergence of the ARTr phenotype in P. falciparum parasites, efforts to understand its underlying molecular mechanisms have progressed swiftly. Presently, two mechanistic propositions capture our understanding of the molecular mechanisms of ART resistance in P. falciparum. These are reduced hemoglobin endocytosis into the digestive food vacuole and increased tolerance to ART-induced oxidative stress. This model of ART resistance may also accommodate a role for extracellular vesicles (EVs), which are ubiquitously expressed in the parasite and have been already reported to play a role in the transfer of the ARTr phenotype in P. falciparum parasites. However, a role for EVs in developing ART resistance outside this motif of parasite-parasite communication is yet to be explored. To approach these problems, it was hypothesized that PfK13-dependent ARTr parasites will evolve in Ghana if there are signatures of balancing selection at genomic loci that have been reported to serve as genetic background on which the ART resistance-conferring PfK13 variant(s) emerges. Diversity at these loci will suggest that the population of P. falciparum parasites were primed for a fast selection sweep under ART pressure. Secondly, it was hypothesized that extracellular vesicles (EVs) are implicated in the development of ART resistance as actively secreted organelles involved in the export of toxic proteinopathic molecules, to maintain cellular homeostasis under ART pressure. This is the EVs export hypothesis. Therefore, the transcriptome of ARTr parasites under ART pressure will be enriched with genes known to be involved in P. falciparum EVs biogenesis. Finally, the EVs export hypothesis predicts an alteration in EVs abundance under ART exposure. In this thesis, the power of population genomic tools has been leveraged to show that two genes, previously reported as background loci for ART resistance, were under balancing selection in clinical isolates of P. falciparum from Ghana in 2010. This finding demonstrates the potential utility of looking for signatures of selection in ART resistance background genes as an early warning molecular surveillance tool and argues for a further exploration of this approach as an addendum to existing tools for molecular surveillance of ARTr P. falciparum parasites. This finding also provides empirical evidence for malaria molecular surveillance in Ghana to continue to use PfK13 as the marker for ARTr despite the possibility of PfK13 independent emergence of ARTr parasites in Ghana. Secondly, an EVs module (set of genes with correlated expression) associated with the transcriptome of ARTr P. falciparum parasites was reported. This implicates EVs in the molecular mechanisms undergirding ART resistance and warrants further exploration of the EVs export hypothesis. Finally, the EVs export hypothesis was tested, and it was observed that a positive correlation between DHA exposure and EVs abundance which suggests that EVs biogenesis is one of the P. falciparum’s response pathways to ART and implicates EVs in the ARTr phenotype.