Table_1_Auranofin Resistance in Toxoplasma gondii Decreases the Accumulation of Reactive Oxygen Species but Does Not Target Parasite Thioredoxin Reductase.pdf

Auranofin, a reprofiled FDA-approved drug originally designed to treat rheumatoid arthritis, has emerged as a promising anti-parasitic drug. It induces the accumulation of reactive oxygen species (ROS) in parasites, including Toxoplasma gondii. We generated auranofin resistant T. gondii lines through chemical mutagenesis to identify the molecular target of this drug. Resistant clones were confirmed with a competition assay using wild-type T. gondii expressing yellow fluorescence protein (YFP) as a reference strain. The predicted auranofin target, thioredoxin reductase, was not mutated in any of our resistant lines. Subsequent whole genomic sequencing analysis (WGS) did not reveal a consensus resistance locus, although many have point mutations in genes encoding redox-relevant proteins such as superoxide dismutase (TgSOD2) and ribonucleotide reductase. We investigated the SOD2 L201P mutation and found that it was not sufficient to confer resistance when introduced into wild-type parasites. Resistant clones accumulated less ROS than their wild type counterparts. Our results demonstrate that resistance to auranofin in T. gondii enhances its ability to abate oxidative stress through diverse mechanisms. This evidence supports a hypothesized mechanism of auranofin anti-parasitic activity as disruption of redox homeostasis.

金诺芬（Auranofin）是一款经老药新用开发的美国食品药品监督管理局（FDA）获批药物，最初用于治疗类风湿关节炎，现已成为颇具潜力的抗寄生虫药物。该药物可诱导包括刚地弓形虫（Toxoplasma gondii）在内的寄生虫体内活性氧（reactive oxygen species, ROS）积累。为鉴定该药物的分子作用靶点，我们通过化学诱变构建了耐金诺芬的刚地弓形虫菌株。以表达黄色荧光蛋白（yellow fluorescence protein, YFP）的野生型刚地弓形虫作为参考菌株，通过竞争实验验证了所获得的耐药克隆。
此前预测的金诺芬靶点硫氧还蛋白还原酶（thioredoxin reductase）在所有耐药株中均未发生突变。后续全基因组测序分析（whole genomic sequencing analysis, WGS）未发现一致的耐药基因座，不过诸多耐药株在编码氧化还原相关蛋白的基因中存在点突变，例如超氧化物歧化酶（TgSOD2）与核糖核苷酸还原酶。
我们针对SOD2 L201P突变展开研究，发现将该突变引入野生型弓形虫中并不足以赋予其耐药性。耐药克隆体内积累的ROS水平低于野生型对应菌株。
本研究结果表明，刚地弓形虫对金诺芬的耐药性可通过多种机制增强其抵御氧化应激的能力。该发现支持了金诺芬抗寄生虫活性的假说机制：通过破坏氧化还原稳态发挥作用。