The continued emergence of drug-resistant Plasmodium falciparum parasites hinders global attempts to eradicate malaria, emphasizing the need to identify new antimalarial drugs. Attractive targets for chemotherapeutic intervention are the cytochrome (cyt) bc₁ complex, which is an essential component of the mitochondrial electron transport chain (mtETC) required for ubiquinone recycling and mitochondrially localized dihydroorotate dehydrogenase (DHODH) critical for de novo pyrimidine synthesis. Despite the essentiality of this complex, resistance to a novel acridone class of compounds targeting cyt bc₁ was readily attained, resulting in a parasite strain (SB1-A6) that was panresistant to both mtETC and DHODH inhibitors. Here, we describe the molecular mechanism behind the resistance of the SB1-A6 parasite line, which lacks the common cyt bc₁ point mutations characteristic of resistance to mtETC inhibitors. Using Illumina whole-genome sequencing, we have identified both a copy number variation (∼2×) and a single-nucleotide polymorphism (C276F) associated with pfdhodh in SB1-A6. We have characterized the role of both genetic lesions by mimicking the copy number variation via episomal expression of pfdhodh and introducing the identified single nucleotide polymorphism (SNP) using CRISPR-Cas9 and assessed their contributions to drug resistance. Although both of these genetic polymorphisms have been previously identified as contributing to both DSM-1 and atovaquone resistance, SB1-A6 represents a unique genotype in which both alterations are present in a single line, suggesting that the combination contributes to the panresistant phenotype. This novel mechanism of resistance to mtETC inhibition has critical implications for the development of future drugs targeting the bc₁ complex or de novo pyrimidine synthesis that could help guide future antimalarial combination therapies and reduce the rapid development of drug resistance in the field.

中文翻译：

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恶性疟原虫线粒体功能抑制剂耐药的非典型分子基础

耐药性恶性疟原虫寄生虫的持续出现阻碍了全球根除疟疾的努力，强调需要确定新的抗疟疾药物。化疗干预的有吸引力的目标是细胞色素 (cyt) bc ₁复合物，它是泛醌回收和线粒体定位二氢乳清酸脱氢酶 (DHODH) 所需的线粒体电子传递链 (mtETC) 的重要组成部分，对于从头合成嘧啶至关重要。尽管这种复合物很重要，但对针对 cyt bc _{1的新型吖啶酮类化合物的抗性}很容易获得，从而产生对 mtETC 和 DHODH 抑制剂都具有泛抗性的寄生虫菌株 (SB1-A6)。在这里，我们描述了 SB1-A6 寄生虫系抗性背后的分子机制，它缺乏对 mtETC 抑制剂抗性的常见 cyt bc ₁点突变特征。使用 Illumina 全基因组测序，我们已经确定了与SB1-A6 中pfdhodh相关的拷贝数变异（~2×）和单核苷酸多态性（C276F） 。我们通过pfdhodh的附加型表达模拟拷贝数变异来表征这两种遗传病变的作用并使用 CRISPR-Cas9 引入已鉴定的单核苷酸多态性 (SNP)，并评估它们对耐药性的贡献。尽管这两种遗传多态性先前已被确定为对 DSM-1 和 atovaquone 抗性都有贡献，但 SB1-A6 代表了一种独特的基因型，其中两种改变都存在于一个单一的系中，这表明该组合有助于泛抗性表型。这种对 mtETC 抑制的新耐药机制对未来靶向bc ₁复合物或从头嘧啶合成的药物的开发具有重要意义，这可能有助于指导未来的抗疟联合疗法并减少该领域耐药性的快速发展。