Mind the Traffic: Validation and characterisation of artemisinin resistance pathways in Plasmodium falciparum by genome editing

Artemisinin susceptibility in Plasmodium falciparum is modulated by mutations in the gene pfk13, which encodes a kelch propeller domain protein of unknown function. Reduced susceptibility is demonstrated in vitro by elevated parasite survival after short exposures to physiologic concentrations of drug in the early ring stage. Using CRISPR-Cas9 genome editing, we provide the first evidence of a similar but K13-independent in vitro artemisinin resistance caused by single base changes in the genes encoding the AP-2 adaptor complex μ- subunit (pfap2mu) and the deubiquitinase Ubp1. Through extensive fluorescence and electron microscopy and proteomics, functional characterisation of PfAP2μ shows that gene as encoding a non-canonical AP-2 trafficking factor that may interact with K13 and other important factors in the cytosol and near the ER and is essential for asexual parasite survival. This thesis provides evidence that disruption of trafficking in early rings initiates an ER-based stress response that underlies artemisinin resistance and induced dormancy. A model depicting a role for ER trafficking components in ring-stage artemisinin action is proposed. Brief characterisations of Ubp1 and clathrin heavy chain are also provided, showing for the first time that clathrin may support multiple key secretory organelles and that malarial deubiquitinases are attractive drug targets.