Instituto de Parasitología y Biomedicina "López-Neyra". Consejo Superior de Investigaciones Científicas. Parque Tecnológico de Ciencias de la Salud, Avenida del Conocimiento, 17, 18016 Armilla, Granada, Spain.
Instituto de Parasitología y Biomedicina "López-Neyra". Consejo Superior de Investigaciones Científicas. Parque Tecnológico de Ciencias de la Salud, Avenida del Conocimiento, 17, 18016 Armilla, Granada, Spain.
Free Radic Biol Med. 2019 Feb 1;131:59-71. doi: 10.1016/j.freeradbiomed.2018.11.025. Epub 2018 Nov 22.
Uracil-DNA glycosylase (UNG) initiates the base excision repair pathway by excising uracil from DNA. We have previously shown that Trypanosoma brucei cells defective in UNG exhibit reduced infectivity thus demonstrating the relevance of this glycosylase for survival within the mammalian host. In the early steps of the immune response, nitric oxide (NO) is released by phagocytes, which in combination with oxygen radicals produce reactive nitrogen species (RNS). These species can react with DNA generating strand breaks and base modifications including deaminations. Since deaminated cytosines are the main substrate for UNG, we hypothesized that the glycosylase might confer protection towards nitrosative stress. Our work establishes the occurrence of genotoxic damage in Trypanosoma brucei upon exposure to NO in vitro and shows that deficient base excision repair results in increased levels of damage in DNA and a hypermutator phenotype. We also evaluate the incidence of DNA damage during infection in vivo and show that parasites recovered from mice exhibit higher levels of DNA strand breaks, base deamination and repair foci compared to cells cultured in vitro. Notably, the absence of UNG leads to reduced infectivity and enhanced DNA damage also in animal infections. By analysing mRNA and protein levels, we found that surviving UNG-KO trypanosomes highly express tryparedoxin peroxidase involved in trypanothione/tryparedoxin metabolism. These observations suggest that the immune response developed by the host enhances the activation of genes required to counteract oxidative stress and emphasize the importance of DNA repair pathways in the protection to genotoxic and oxidative stress in trypanosomes.
尿嘧啶-DNA 糖基化酶（UNG）通过从 DNA 中切除尿嘧啶来启动碱基切除修复途径。我们之前已经表明，UNG 缺陷的非洲锥虫细胞的感染性降低，这表明这种糖苷酶对于在哺乳动物宿主中生存是相关的。在免疫反应的早期阶段，吞噬细胞释放一氧化氮（NO），与氧自由基结合产生活性氮物种（RNS）。这些物质可以与 DNA 反应生成链断裂和碱基修饰，包括脱氨。由于脱氨的胞嘧啶是 UNG 的主要底物，我们假设糖苷酶可能对硝化应激提供保护。我们的工作确立了在体外暴露于 NO 时，在 Trypanosoma brucei 中发生遗传毒性损伤，并且表明缺乏碱基切除修复会导致 DNA 损伤水平增加和超突变表型。我们还评估了体内感染过程中 DNA 损伤的发生率，并表明与体外培养的细胞相比，从小鼠中回收的寄生虫表现出更高水平的 DNA 链断裂、碱基脱氨和修复焦点。值得注意的是，UNG 的缺失也会导致感染动物中的感染性降低和 DNA 损伤增加。通过分析 mRNA 和蛋白质水平，我们发现存活的 UNG-KO 锥虫高度表达参与 trypanothione/tryparedoxin 代谢的 tryparedoxin 过氧化物酶。这些观察结果表明，宿主产生的免疫反应增强了激活基因的激活，这些基因需要对抗氧化应激，强调了 DNA 修复途径在保护锥虫免受遗传毒性和氧化应激方面的重要性。
