Liu Shiwei, Zulawinska Julia, Ebel Emily R, Luniewski Aleksander, Danis Charles, Simpson Mary Lewis, Kim Jane, Ene Nnenna, Braukmann Thomas Werner Anthony, Congdon Molly, Santos Webster, Yeh Ellen, Guler Jennifer L
Department of Biology, University of Virginia, Charlottesville, VA, USA.
Indiana University School of Medicine, Indianapolis, IN, USA.
BMC Genomics. 2025 Jul 17;26(1):671. doi: 10.1186/s12864-025-11859-5.
Increases in the copy number of large genomic regions, termed amplifications, are an important adaptive strategy for many organisms. Numerous amplifications across the AT-rich Plasmodium falciparum genome contribute directly to drug resistance or impact the fitness of this protozoan parasite. During the characterization of malaria parasites selected with a dihydroorotate dehydrogenase (DHODH) inhibitor that targets pyrimidine biosynthesis, we detected increased copies of a genomic region that encompassed 3 genes (~ 5 kb) including GTP cyclohydrolase I (GCH1 amplicon). While amplification of this gene is reported in antifolate-resistant parasites, GCH1 amplicons had not previously been implicated in DHODH inhibitor resistance.
Here, we explored the expansion of the GCH1 locus in this family of parasite lines using long-read sequencing and single-read visualization. We directly quantified higher numbers of tandem GCH1 amplicons in selected parasite lines (up to 9 GCH1 amplicons) compared to parental P. falciparum parasites (strictly 3 GCH1 amplicons). Because each read represents DNA from an individual genome, we were able to appreciate hidden variation within a single parasite line (3, to 5, to 7 amplicons) that was not reflected in other DNA-based analysis methods. While all GCH1 amplicons shared a consistent structure, expansions arose in precise 2-unit steps within selected lines. We found conserved AT-rich sequences at amplicon boundaries, which is consistent with the Plasmodium model of CNV formation. Parasite lines with expanded GCH1 also had DHODH amplicons on a separate chromosome. When we evaluated prior DHODH inhibitor selections, we observed that GCH1 amplification was not required for resistance; however, selection outcomes suggest that pre-existing GCH1 amplicons may support amplification at the DHODH locus.
We identified previously undetected heterogeneity in gene copy number by viewing long pieces of DNA from individual genomes. This approach was possible due to the amplicon's tandem orientation and relatively small size that can be spanned by a single long ONT read. The positive association between DHODH and GCH1 copy number, combined with the metabolic connection between P. falciparum pyrimidine and folate biosynthesis, justifies further investigation into the adaptive evolution of these two genomic loci.
大基因组区域拷贝数的增加，即扩增，是许多生物体的一种重要适应性策略。恶性疟原虫富含AT的基因组中的大量扩增直接导致耐药性，或影响这种原生动物寄生虫的适应性。在用靶向嘧啶生物合成的二氢乳清酸脱氢酶（DHODH）抑制剂筛选疟原虫的过程中，我们检测到一个包含3个基因（约5 kb）的基因组区域的拷贝数增加，该区域包括GTP环化水解酶I（GCH1扩增子）。虽然在抗叶酸寄生虫中报道了该基因的扩增，但GCH1扩增子此前并未被认为与DHODH抑制剂耐药性有关。
在这里，我们使用长读长测序和单读长可视化技术，探索了该寄生虫系家族中GCH1基因座的扩增情况。与亲本恶性疟原虫相比，我们直接定量了选定寄生虫系中更高数量的串联GCH1扩增子（多达9个GCH1扩增子）（亲本严格为3个GCH1扩增子）。由于每个读段代表单个基因组的DNA，我们能够识别单个寄生虫系内隐藏的变异（3个、5个、7个扩增子），而这在其他基于DNA的分析方法中并未体现。虽然所有GCH1扩增子具有一致的结构，但在选定的系中，扩增以精确的2单位步长出现。我们在扩增子边界发现了保守的富含AT的序列，这与疟原虫CNV形成模型一致。GCH1扩增的寄生虫系在另一条染色体上也有DHODH扩增子。当我们评估先前的DHODH抑制剂筛选时，我们观察到耐药性并不需要GCH1扩增；然而，筛选结果表明，预先存在的GCH1扩增子可能支持DHODH基因座的扩增。
通过查看单个基因组的长片段DNA，我们发现了以前未检测到的基因拷贝数异质性。由于扩增子的串联方向和相对较小的大小可以被单个长ONT读段跨越，这种方法才得以实现。DHODH和GCH1拷贝数之间的正相关，以及恶性疟原虫嘧啶和叶酸生物合成之间的代谢联系，证明有必要进一步研究这两个基因组位点的适应性进化。
