Extrachromosomal DNA amplicons in antimalarial‐resistant <i>Plasmodium falciparum</i>

McDaniels, Jennifer; Huckaby, Adam C; Carter, Sabrina A.; Lingeman, Sabrina; Francis, Audrey; Congdon, Molly D.; Santos, Webster L.; Rathod, Pradipsinh K.; Guler, Jennifer L.

doi:10.1111/mmi.14624

Cited by 6 publications

(6 citation statements)

References 87 publications

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“…Furthermore, allele frequencies from the two parts of the locus were uncorrelated across MAL (mean pairwise R 2 adj=-0.01, linear models; In light of these patterns, we hypothesized that common var polymorphisms were not generated de novo during the MA experiment, but instead maintained on extrachromosomal DNA (ecDNA) inherited from ANC with stochastic loss (Fig 2C). ecDNA is ubiquitous in eukaryotes 12 and has been reported in one P. falciparum study to date, where it was implicated in selective amplification of a drug-resistance gene 13 . We performed molecular experiments to quantify var polymorphism and test the ecDNA hypothesis, again focusing on the most polymorphic internal locus on chr12.…”

Section: D7 Reference Chromosomesmentioning

confidence: 99%

Antigenic diversity in malaria parasites is maintained on extrachromosomal DNA

Ebel

Kim

McDew-White

et al. 2023

Preprint

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Sequence variation among antigenic var genes enables Plasmodium falciparum malaria parasites to evade host immunity. Using long sequence reads from haploid clones from a mutation accumulation experiment, we detect var diversity inconsistent with simple chromosomal inheritance. We discover putatively circular DNA that is strongly enriched for var genes, which exist in multiple alleles per locus separated by recombination and indel events. Extrachromosomal DNA likely contributes to rapid antigenic diversification in P. falciparum.

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Section: D7 Reference Chromosomesmentioning

confidence: 99%

Antigenic diversity in malaria parasites is maintained on extrachromosomal DNA

Ebel

Kim

McDew-White

et al. 2023

Preprint

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“…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.…”

Section: Resultsmentioning

confidence: 99%

“…We diluted the digested DNA for ddPCR reactions. We performed ddPCR using ddPCR Supermix for Probe (no dUTP, Bio-Rad Laboratories, California, USA) with DNA input 0.1 ng (in duplicate per sample), 0.025 ng (in duplicate per sample) as previously described (McDaniels et al, 2021). The primers and probes used in reactions are included in Supplementary Table 3 .…”

Section: Methodsmentioning

confidence: 99%

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

Liu

Ebel

Kim

et al. 2023

Preprint

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Changes in the copy number of large genomic regions, termed copy number variations or CNVs, are an important adaptive strategy for malaria parasites. Numerous CNVs across the Plasmodium falciparum genome contribute directly to drug resistance or impact fitness of this protozoan parasite. CNVs that encompass the dihydroorotate dehydrogenase (DHODH) gene confer resistance to antimalarials that target this enzyme in the pyrimidine biosynthesis pathway (i.e. DSM1). During the characterization of DSM1 resistant parasite lines with DHODH CNVs, we detected selection of an additional CNV that encompasses 3 genes (~5 kb) including GTP cyclohydrolase I (GCH1 amplicon). While this locus has been implicated in increased fitness of antifolate resistant parasites, GCH1 CNVs had not previously been reported to contribute to resistance to other antimalarials. Here, we further explored the association between GCH1 and DHODH copy number. We visualized single long reads and directly quantified the number of tandem GCH1 amplicons in a parental line versus a DSM1-selected line. We found that the GCH1 amplicons share a consistent structure in all lines. However, we detected more reads that encompassed a higher number of amplicons in the resistant (up to 7 amplicons) compared to the parental line (3 amplicons). To better understand the implications of this result, we evaluated variation at this locus across multiple short- and long-read data sets collected from various parasite lines. Based on our analysis of parasites resistant to other DHODH inhibitors (DSM265, DSM267, and DSM705), GCH1 is not likely contributing directly to resistance; however, higher numbers of the GCH1 amplicon are associated with increased DHODH copies and may compensate for changes in metabolism of parasites. This is supported by the direct connection between folate and pyrimidine metabolism, which together contribute to nucleic acid biosynthesis. This study highlights the importance of studying clonal variation and potential biochemical connections as novel antimalarials move closer to clinical approval.

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“…Because prior P. falciparum CNV analyses were confined to bulk DNA sequencing, our view of minor variants in parasite populations is limited. The recent discovery of P. falciparum extrachromosomal DNA that is derived from regions of the genome that harbor CNVs [125] suggests that there are cellular pathways that could contribute to cell-to-cell variations in CNV boundaries and dynamics (i.e., perhaps through the excision and reintegration of extrachromosomal DNA). While the differences in start position of true CNVs from single-cell genomes (Tables 4 and 5) could represent true minor variants, they could also be due to analysis artifacts that contribute to excess CNV calls and inaccuracies in estimating boundaries.…”

Section: Cnv Analysis and Related Considerationsmentioning

confidence: 99%

Single-cell sequencing of the small and AT-skewed genome of malaria parasites

et al. 2021

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Single-cell genomics is a rapidly advancing field; however, most techniques are designed for mammalian cells. We present a single-cell sequencing pipeline for an intracellular parasite, Plasmodium falciparum, with a small genome of extreme base content. Through optimization of a quasi-linear amplification method, we target the parasite genome over contaminants and generate coverage levels allowing detection of minor genetic variants. This work, as well as efforts that build on these findings, will enable detection of parasite heterogeneity contributing to P. falciparum adaptation. Furthermore, this study provides a framework for optimizing single-cell amplification and variant analysis in challenging genomes.

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Extrachromosomal DNA amplicons in antimalarial‐resistant Plasmodium falciparum

Abstract: This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Cited by 6 publications

References 87 publications

Antigenic diversity in malaria parasites is maintained on extrachromosomal DNA

Antigenic diversity in malaria parasites is maintained on extrachromosomal DNA

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

Single-cell sequencing of the small and AT-skewed genome of malaria parasites

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