Genomic and Genetic Approaches to Studying Antimalarial Drug Resistance and Plasmodium Biology

Plasmodium falciparum resistance to artemisinin derivatives in southeast Asia threatens malaria control and elimination activities worldwide. To monitor the spread of artemisinin resistance, a molecular marker is urgently needed. Here, using whole-genome sequencing of an artemisinin-resistant parasite line from Africa and clinical parasite isolates from Cambodia, we associate mutations in the PF3D7_1343700 kelch propeller domain ('K13-propeller') with artemisinin resistance in vitro and in vivo. Mutant K13-propeller alleles cluster in Cambodian provinces where resistance is prevalent, and the increasing frequency of a dominant mutant K13-propeller allele correlates with the recent spread of resistance in western Cambodia. Strong correlations between the presence of a mutant allele, in vitro parasite survival rates and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance. K13-propeller polymorphism constitutes a useful molecular marker for large-scale surveillance efforts to contain artemisinin resistance in the Greater Mekong Subregion and prevent its global spread.

Severe malaria is caused by the apicomplexan parasite Plasmodium falciparum. Despite decades of research the unique biology of these parasites has made it challenging to establish high throughput genetic approaches for identification of therapeutic targets. Using transposon mutagenesis of P. falciparum in an approach that exploited its AT-rich genome we generated >38,000 mutants, saturating the genome and defining fitness costs for 95% of genes. Of 5,399 genes we found ~3,000 genes are essential for optimal growth of asexual blood-stages in vitro . Our study defines ∼1000 essential genes, including genes associated with drug resistance, vaccine candidates, and conserved proteins of unknown function. We validated this approach by testing proteasome pathways for individual mutants associated with artemisinin sensitivity. Transposon mutagenesis of Plasmodium falciparum was used to generate >38,000 mutants, saturating the genome and defining fitness costs for 95% of genes. We functionally define the relative fitness cost of disruption for 5,399 genes, and find that ~3,000 genes, ~62% of the genome, are essential for optimal asexual blood-stage in vitro growth. Our study defines ∼1000 essential genes, including genes associated with drug resistance, leading vaccine candidates, and hundreds of Plasmodium- conserved proteins of unknown function that are now potential therapeutic intervention targets. We experimentally validated the essentiality of proteasome pathways with drug studies of individual mutants associated with artemisinin sensitivity. This study defines high-priority targets and pathways and points the way for the future of P. falciparum high throughput genetics. Saturation-scale mutagenesis of Plasmodium falciparum reveals a core set of genes essential for asexual blood-stage growth in vitro . INTRODUCTION: Malaria remains a devastating global parasitic disease, with the majority of malaria deaths caused by the highly virulent Plasmodium falciparum . The extreme AT-bias of the P. falciparum genome has hampered genetic studies through targeted approaches such as homologous recombination or CRISPR-Cas9, and only a few hundred P. falciparum mutants have been experimentally generated in the past decades. In this study, we have used high throughput piggyBac transposon insertional mutagenesis and Quantitative Insertion Site Sequencing (QIseq) to reach saturation-level mutagenesis of this parasite. RATIONALE: Our study exploits the AT-richness of P. falciparum genome, which provides numerous piggyBac transposon insertion targets within both gene coding and non-coding flanking sequences, to generate over 38,000 P. falciparum mutants. At this level of mutagenesis, we could distinguish essential genes as non-mutable and dispensable genes as mutable. Subsequently, we identified 3,357 genes essential for in vitro asexual blood-stage growth. RESULTS: We calculated Mutagenesis Index Scores (MIS) and Mutagenesis Fitness Scores (MFS) to functionally define the relative fitness cost of disruption for 5,399 genes. A competitive growth phenotype screen confirmed that MIS and MFS were predictive of the fitness cost for in vitro asexual growth. Genes predicted to be essential included genes implicated in drug resistance, such as the “ K13 ” Kelch propeller, mdr and dhfr-ts , as well as targets considered to be high-value for drugs development such as pkg , and cdpk5 . The screen revealed essential genes that are specific to human Plasmodium parasites but absent from rodent-infective species, such as lipid metabolic genes that may be crucial to transmission commitment in human infections. MIS and MFS profiling provides a clear ranking of the relative essentiality of gene ontology (GO) functions in P. falciparum . GO pathways associated with translation, RNA metabolism, and cell cycle control are more essential, whereas genes associated with protein phosphorylation, virulence factors, and transcription are more likely to be dispensable. Finally, we confirm that the proteasome-degradation pathway is a high-value druggable target based on its high ratio of essential:dispensable genes, and by functionally confirming its link to the mode of action of artemisinin, the current front-line antimalarial. CONCLUSION: Saturation-scale mutagenesis allows prioritization of intervention targets in the genome of the most important cause of malaria. The identification of the essential genome, consisting of over 3000 genes, will be valuable for antimalarial therapeutic research.

Artemisinin resistance (delayed P. falciparum clearance following artemisinin-based combination therapy), is widespread across Southeast Asia but to date has not been reported in Africa 1–4 . Here we genotyped the P. falciparum K13 (Pfkelch13) propeller domain, mutations in which can mediate artemisinin resistance 5,6 , in pretreatment samples collected from recent dihydroarteminisin-piperaquine and artemether-lumefantrine efficacy trials in Rwanda 7 . While cure rates were >95% in both treatment arms, the Pfkelch13 R561H mutation was identified in 19 of 257 (7.4%) patients at Masaka. Phylogenetic analysis revealed the expansion of an indigenous R561H lineage. Gene editing confirmed that this mutation can drive artemisinin resistance in vitro. This study provides evidence for the de novo emergence of Pfkelch13-mediated artemisinin resistance in Rwanda, potentially compromising the continued success of antimalarial chemotherapy in Africa.