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

Abstract

Increases in the copy number of large genomic regions, termed genome amplification, are an important adaptive strategy for malaria parasites. Numerous amplifications across the Plasmodium falciparum genome contribute directly to drug resistance or impact the fitness of this protozoan parasite. During the characterization of parasite lines with amplifications of the dihydroorotate dehydrogenase (DHODH) gene, we detected increased copies of an additional genomic region that encompassed 3 genes (~5 kb) including GTP cyclohydrolase I (GCH1 amplicon). While this gene is reported to increase the fitness of antifolate resistant parasites, GCH1 amplicons had not previously been implicated in any other antimalarial resistance context. Here, we further explored the association between GCH1 and DHODH copy number. Using long read sequencing and single read visualization, we directly observed a higher number of tandem GCH1 amplicons in parasites with increased DHODH copies (up to 9 amplicons) compared to parental parasites (3 amplicons). While all GCH1 amplicons shared a consistent structure, expansions arose in 2-unit steps (from 3 to 5 to 7, etc copies). Adaptive evolution of DHODH and GCH1 loci was further bolstered when we evaluated prior selection experiments; DHODH amplification was only successful in parasite lines with pre-existing GCH1 amplicons. These observations, combined with the direct connection between metabolic pathways that contain these enzymes, lead us to propose that the GCH1 locus is beneficial for the fitness of parasites exposed to DHODH 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.

Fig 1.. GCH1 copy number increase is positively correlated with DHODH copy number in one family of DSM1 selected parasites.

(A) DSM1 selection schematic, as presented previously (11). Blue text: Illumina short read sequenced lines. Underline: modern lines confirmed by ddPCR analysis (Table 1). Asterisk (*): lines subjected to long read sequencing in this study. Wild-type (WT1, Dd2) P. falciparum was selected with DSM1 in two steps; the first step selected for low-level (L) resistance and the second step selected for moderate- (M) or high-level (H) resistance. DSM1 EC50 values are as follows: L1 (1 μM), L2 (0.9 μM), M1 (7.2 μM), H2 (85 μM), H6 (56 μM), H4 (49 μM). All values were previously reported and clone names were adapted as previously (11,36). (B) Relationship between GCH1 and DHODH copy number in DSM1 selected parasites as quantified using short read data from Guler et al. 2013. A trend line was added to show the relationship between GCH1 and DHODH copy numbers but a correlation coefficient could not be calculated due to the small sample size (n=5) and dependence among the lines.

Fig 2.. Long-read visualization shows conserved boundaries and structure of GCH1 amplicon from parental and resistance parasites.

(A-C) Representative images from the Shiny application comparing the GCH1 amplicon in WT1 (A), M1 (B), and H2 (C) spanning reads. Red dashed square: copies of GCH1 amplicon covering 3 genes. Each gene sequence from the 3D7 reference genome (no GCH1 amplicon represented) was split into <=500 bp fragments and blasted against individual Nanopore reads (dark gray: genic regions, light grey: intergenic regions, more details in Materials and Methods). (D) Orientation of GCH1 amplicons from each parasite line. The three genes within the GCH1 amplicon unit include PF3D7_1224000 (GTP cyclohydrolase I, GCH1), PF3D7_1223900 (50S ribosomal protein L24, putative, 50S RPL24), and PF3D7_1223800 (citrate/oxoglutarate carrier protein, putative, YHM2). Note: The gene order (GCH1–50S RPL24-YHM2) is reversed compared to the 3D7 reference genome (YHM2–50S RPL24-GCH1) in order to facilitate comparison with the read images in panels A-C. Red circles: amplicon junction sequences that act as potential secondary recombination sites.

Fig 3.. Quantification of long reads displays an increase in GCH1 amplicon number in DSM1 selected parasites that is not dependent on overall read length.

(A) GCH1 copy number from WT1 and selected (M1 and H2) parasite lines. Spanning and non-spanning reads are included; spanning reads were grouped with their corresponding non-spanning count (e.g. a read showing 2 copies of the GCH1 amplicon is counted as a 2+ read). Bars represent mean, error bars represent standard deviation (dotted lines, values that represent detected spanning reads, see Figure S1). (B) Read length distribution from long reads (>=10kb) covering the GCH1 amplicon. Thick line represents median, thin line represents quartiles.

Fig 4.. Preexisting GCH1 amplicons confer resistance competence.

(A) Relationship between GCH1 and DHODH copy number in parental (black outline) and DHODH inhibitor-selected parasite lines as quantified using short read data (see Table 2 for source data). Each data point represents a copy number from an individual parasite clone or line from a distinct selection (see Tables S3 and S4 for copy number assessment). 3D7, yellow squares (2 selections); Dd2, blue circles (2 selections); K1, red triangle (1 selection, unknown DHODH copy number post-selection); HB3, grey diamonds (2 selections). (B) GCH1 amplicon size and gene number vary between parasite lines used for DHODH inhibitor selections (see Table 2 for selection details). 3D7, 4 copy, single gene (PF3D7_1224000, 2kb, this study); Dd2, 3 copy, three genes (PF3D7_1223800- PF3D7_1224000, 5kb, this study); K1, unknown copy number (grey dash, anticipated 2 copy), 7 genes (PF3D7_1223500 - PF3D7_1224100, 19kb) (39); HB3, 1 copy of region (40).

Fig 5.. The connection between pyrimidine and folate biosynthesis pathways.

Enzymes with gene copy number variations are indicated in blue text (DHODH: dihydroorotate dehydrogenase; GCH1: GTP cyclohydrolase 1) and connections between the two pathways in blue arrows. Gln: Glutamine; DHO: Dihydroorotate; UMP: Uridine monophosphate; dUMP: Deoxyuridine monophosphate; dTMP: deoxythymidine monophosphate; GTP: Guanosine-5’-triphosphate; DHPS: Dihydropteroate synthase; DHFR: Dihydrofolate reductase; DHF: Dihydrofolate; THF: Tetrahydrofolate; HMDP-P2: 6-hydroxymethyl-7, 8-dihydropterin diphosphate; pABA: para-amino-benzoic acid; 5, 10-CH2-THF: 5,10-Methylenetetrahydrofolate. *Dd2 carries 3 mutations in both DHPS and DHFR (6 total) and HB3 and 3D7 have a single mutation each in DHFR and DHPS, respectively (15,48).