Departamento de Bioquímica and Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay.
Front Cell Infect Microbiol. 2022 Feb 4;12:749476. doi: 10.3389/fcimb.2022.749476. eCollection 2022.
is the causative agent of Chagas disease which is currently treated by nifurtimox (NFX) and benznidazole (BZ). Nevertheless, the mechanism of action of NFX is not completely established. Herein, we show the protective effects of mitochondrial peroxiredoxin (MPX) in macrophage infections and in response to NFX toxicity. After a 3-day treatment of epimastigotes with NFX, MPX content increased (2.5-fold) with respect to control, and interestingly, an MPX-overexpressing strain was more resistant to the drug. The generation of mitochondrial reactive species and the redox status of the low molecular weight thiols of the parasite were not affected by NFX treatment indicating the absence of oxidative stress in this condition. Since MPX was shown to be protective and overexpressed in drug-challenged parasites, non-classical peroxiredoxin activity was studied. We found that recombinant MPX exhibits holdase activity independently of its redox state and that its overexpression was also observed in temperature-challenged parasites. Moreover, increased holdase activity (2-fold) together with an augmented protease activity (proteasome-related) and an enhancement in ubiquitinylated proteins was found in NFX-treated parasites. These results suggest a protective role of MPX holdase activity toward NFX toxicity. has a complex life cycle, part of which involves the invasion of mammalian cells, where parasite replication inside the host occurs. In the early stages of the infection, macrophages recognize and engulf with the generation of reactive oxygen and nitrogen species toward the internalized parasite. Parasites overexpressing MPX produced higher macrophage infection yield compared with wild-type parasites. The relevance of peroxidase vs. holdase activity of MPX during macrophage infections was assessed using conoidin A (CA), a covalent, cell-permeable inhibitor of peroxiredoxin peroxidase activity. Covalent adducts of MPX were detected in CA-treated parasites, which proves its action . The pretreatment of parasites with CA led to a reduced infection index in macrophages revealing that the peroxidase activity of peroxiredoxin is crucial during this infection process. Our results confirm the importance of peroxidase activity during macrophage infection and provide insights for the relevance of MPX holdase activity in NFX resistance.
是恰加斯病的病原体，目前用硝呋替莫（NFX）和苯并咪唑（BZ）治疗。然而，NFX 的作用机制尚未完全确定。本文显示了线粒体过氧化物酶（MPX）在巨噬细胞感染和对 NFX 毒性反应中的保护作用。在用 NFX 处理 3 天的前鞭毛体后，MPX 含量增加（增加 2.5 倍）与对照相比，有趣的是，过表达 MPX 的菌株对药物更具抗性。药物处理后，寄生虫的线粒体活性氧生成和低分子量硫醇的氧化还原状态没有受到影响，表明在这种情况下没有氧化应激。由于 MPX 在受到药物挑战的寄生虫中显示出保护作用和过表达，因此研究了非经典过氧化物酶活性。我们发现重组 MPX 独立于其氧化还原状态具有持留酶活性，并且在受到温度挑战的寄生虫中也观察到过表达。此外，在 NFX 处理的寄生虫中发现了 2 倍的持留酶活性（2 倍）增加，蛋白酶活性（与蛋白酶体相关）增加，泛素化蛋白增加。这些结果表明 MPX 持留酶活性对 NFX 毒性具有保护作用。有一个复杂的生命周期，其中一部分涉及哺乳动物细胞的入侵，寄生虫在宿主内部复制。在感染的早期阶段，巨噬细胞识别并吞噬，产生针对内部寄生虫的活性氧和氮物质。与野生型寄生虫相比，过表达 MPX 的寄生虫产生了更高的巨噬细胞感染产量。使用共角素 A（CA）评估了 MPX 的过氧化物酶与持留酶活性在巨噬细胞感染中的相关性，CA 是一种半胱氨酸残基修饰的、细胞通透的过氧化物酶抑制剂。在 CA 处理的寄生虫中检测到 MPX 的共价加合物，证明了它的作用。用 CA 预处理寄生虫可降低巨噬细胞中的感染指数，表明过氧化物酶在感染过程中的重要性。我们的结果证实了过氧化物酶活性在巨噬细胞感染中的重要性，并为 MPX 持留酶活性在 NFX 抗性中的相关性提供了依据。
