The influence of genetic background on drug resistance in the malaria parasite Plasmodium falciparum
Plasmodium falciparum is a parasite that causes the most severe forms of human malaria; with multidrug resistance to modern antimalarials emerging and spreading in certain parasite populations, understanding the mechanisms for acquiring multidrug resistance will be key for predicting which genes may become important for clinical resistance in the future. Transporter proteins have key roles in drug resistance across a variety of organisms — in P. falciparum, the spread of resistance to the antimalarial chloroquine in the 1950s and 60s is largely due to mutations in the Chloroquine Resistance Transporter (PfCRT). Variations in the ABC transporter pfmdr1 modulate sensitivity to multiple antimalarials, including mefloquine and lumefantrine, popular partner drugs in first-line artemisinin combination therapies.
During my PhD, I assessed a panel of parasite lines that had undergone in vitro evolution experiments, identifying polymorphisms in a poorly characterised ABC transporter, ABCI3, that confer resistance to several experimental antimalarial compounds with diverse chemical scaffolds, suggesting that ABCI3 could mediate resistance to next-generation antimalarial drugs. Next, I examined a novel PfCRT mutation currently spreading in Southeast Asia that confers piperaquine resistance when edited into the wild-type pfcrt allele of laboratory lines using CRISPR/Cas9, raising concerns that piperaquine resistance could arise in susceptible populations with a single nucleotide polymorphism. Finally, I introduced unique 11-nucleotide ‘barcodes’ into 37 progeny from the NF54 × Cam3.II genetic cross using CRISPR/Cas9. The African NF54 parasite is considered wild-type and is broadly drug susceptible. The Cambodian Cam3.II parasite is multidrug-resistant owing to numerous mutations, including the R539T mutation in pfkelch13, a gene associated with artemisinin resistance. R539T has been demonstrated to have a high fitness cost, and similar PfKelch13 mutations display fitness costs that are exacerbated when engineered into non-Southeast Asian parasites.
Combination of ‘barcoded’ parasite lines into a single flask, or ‘pool’ creates a highly valuable screening resource, allowing one to observe the change in proportion of each progeny within this pool over time via next-generation sequencing of the barcoded locus; linkage analysis can therefore be performed on data generated within a single experimental run. Using this tool, I note the enrichment of certain haplotypes of interest under various antimalarial pressures, including pfabci3, pfcrt and pfkelch13 variants. I discuss the future avenues of research that will reveal the quantitative trait loci responsible for the differential survival of progeny under these antimalarial pressures, which will shed light on the complex interplay between drug resistance mutations, fitness, and genetic background.