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

Abstract

AbstractIncreases in the copy number of large genomic regions, termed genome amplification, are an important adaptive strategy for malaria parasites. Numerous amplifications across thePlasmodium falciparumgenome contribute directly to drug resistance or impact the fitness of this protozoan parasite. During the characterization of parasite lines with amplifications of thedihydroorotate dehydrogenase(DHODH) gene, we detected increased copies of an additional genomic region that encompassed 3 genes (~5 kb) includingGTP cyclohydrolase I(GCH1amplicon). While this gene is reported to increase the fitness of antifolate resistant parasites,GCH1amplicons had not previously been implicated in any other antimalarial resistance context. Here, we further explored the association betweenGCH1andDHODHcopy number. Using long read sequencing and single read visualization, we directly observed a higher number of tandemGCH1amplicons in parasites with increasedDHODHcopies (up to 9 amplicons) compared to parental parasites (3 amplicons). While allGCH1amplicons shared a consistent structure, expansions arose in 2-unit steps (from 3 to 5 to 7, etc copies). Adaptive evolution ofDHODHandGCH1loci was further bolstered when we evaluated prior selection experiments;DHODHamplification was only successful in parasite lines with pre-existingGCH1amplicons. These observations, combined with the direct connection between metabolic pathways that contain these enzymes, lead us to propose that theGCH1locus is beneficial for the fitness of parasites exposed toDHODHinhibitors. 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.Author SummaryMalaria is caused by a protozoan parasite that readily evolves resistance to drugs that are used to treat this deadly disease. Changes that arise in the parasite genome, including extra copies of important genes, directly contribute to this resistance or improve how well the resistant parasite competes. In this study, we identified that extra copies of one gene (GTP cyclohydrolaseorGCH1) were more likely to be found in parasites with extra copies of another gene on a different chromosome (dihydroorotate dehydrogenaseorDHODH). A method that allows us to view long pieces of DNA from individual genomes was especially important for this study; we were able to assess gene number, arrangement, and boundary sequences, which provided clues into how extra copies evolved. Additionally, by analyzing previous experiments, we identified that extraGCH1copies improved resistance to drugs that target DHODH. The relationship between these two loci is supported by a direct connection between the folate and pyrimidine biosynthesis pathways that the parasite uses to make DNA. SinceGCH1amplicons are common in clinical parasites worldwide, this finding highlights the need to study metabolic connections to avoid resistance evolution.