Direct long read visualization reveals metabolic interplay between two antimalarial drug targets.

Increases in the copy number of large genomic regions, termed genome amplification, are an important adaptive strategy for malaria parasites. Numerous amplifications across the genome contribute directly to drug resistance or impact the fitness of this protozoan parasite. During the characterization of parasite lines with amplifications of the () gene, we detected increased copies of an additional genomic region that encompassed 3 genes (~5 kb) including ( amplicon). While this gene is reported to increase the fitness of antifolate resistant parasites, amplicons had not previously been implicated in any other antimalarial resistance context. Here, we further explored the association between and copy number. Using long read sequencing and single read visualization, we directly observed a higher number of tandem amplicons in parasites with increased copies (up to 9 amplicons) compared to parental parasites (3 amplicons). While all amplicons shared a consistent structure, expansions arose in 2-unit steps (from 3 to 5 to 7, etc copies). Adaptive evolution of and loci was further bolstered when we evaluated prior selection experiments; amplification was only successful in parasite lines with pre-existing amplicons. These observations, combined with the direct connection between metabolic pathways that contain these enzymes, lead us to propose that the locus is beneficial for the fitness of parasites exposed to inhibitors. This finding highlights the importance of studying variation within individual parasite genomes as well as biochemical connections of drug targets as novel antimalarials move towards clinical approval.