New approaches for measuring fitness of Plasmodium falciparum mutations implicated in drug resistance
The repeated emergence of drug resistance in Plasmodium falciparum underscores the importance of understanding the genetic architecture of current resistance pathways, as well as any associated fitness costs. Why resistance emerges in particular regions of the world has been linked to particular genetic backgrounds that better tolerate resistance-associated polymorphisms; this is likely to play a key role in driving the epidemiology of drug resistance, however is infrequently studied at a large scale in a laboratory setting. The first results chapter establishes a barcoding approach for P. falciparum with the aim of tracking parasite growth in vitro. The strategy used was adapted for P. falciparum by using a pseudogene (PfRh3) as a safe harbour to insert unique molecular barcodes. These libraries of barcoded P. falciparum vectors were also used as a readout of transfection efficiency. The second chapter establishes a proof of principle for phenotyping by barcode sequencing, using a panel of barcoded parasites generated in different genetic backgrounds that comprise sufficient genetic diversity to pilot the method. These were grown in the presence and absence of antimalarial compounds, and growth phenotypes were measured in parallel using BarSeq. The third results chapter studies the contribution of mutations in Pfkelch13, a molecular marker of artemisinin resistance, to parasite fitness. Combining CRISPR/Cas9-based genome editing and high throughput sequencing, the impact of Pfkelch13 alleles on fitness in the context of particular strain backgrounds is revealed. In particular, the impact of genetic background in the emergence and spread of drug-resistant lineages (referred to as KEL1) in Southeast Asia carrying a Y580 Pfkelch13 allele. Overall, given the current pace of genome sequencing of pathogenic organisms such as P. falciparum, it will be important to increase the scale of experimental genetics, in order to tackle in real-time natural variation that might be under constant selection from drugs, thus anticipating the emergence of drug resistance in changing parasite populations. Through this work, tools were developed to facilitate parallel phenotyping by measuring in vitro growth using high-throughput sequencing. The work also develops novel approaches to address the importance of genetic background and a potential role for positive epistasis in a lineage responsible for the recent outbreak of drug-resistant malaria in Southeast Asia.