Oxidative Stress and DNA Lesions: The Role of 8-Oxoguanine Lesions in Trypanosoma cruzi Cell Viability

The main consequence of oxidative stress is the formation of DNA lesions, which can result in genomic instability and lead to cell death. Guanine is the base that is most susceptible to oxidation, due to its low redox potential, and 8-oxoguanine (8-oxoG) is the most common lesion. These characteristics make 8-oxoG a good cellular biomarker to indicate the extent of oxidative stress. If not repaired, 8-oxoG can pair with adenine and cause a G:C to T:A transversion. When 8-oxoG is inserted during DNA replication, it could generate double-strand breaks, which makes this lesion particularly deleterious. Trypanosoma cruzi needs to address various oxidative stress situations, such as the mammalian intracellular environment and the triatomine insect gut where it replicates. We focused on the MutT enzyme, which is responsible for removing 8-oxoG from the nucleotide pool. To investigate the importance of 8-oxoG during parasite infection of mammalian cells, we characterized the MutT gene in T. cruzi (TcMTH) and generated T. cruzi parasites heterologously expressing Escherichia coli MutT or overexpressing the TcMTH enzyme. In the epimastigote form, the recombinant and wild-type parasites displayed similar growth in normal conditions, but the MutT-expressing cells were more resistant to hydrogen peroxide treatment. The recombinant parasite also displayed significantly increased growth after 48 hours of infection in fibroblasts and macrophages when compared to wild-type cells, as well as increased parasitemia in Swiss mice. In addition, we demonstrated, using western blotting experiments, that MutT heterologous expression can influence the parasite antioxidant enzyme protein levels. These results indicate the importance of the 8-oxoG repair system for cell viability.氧化应激（oxidative stress）的主要后果是DNA损伤（DNA lesions）的形成，该过程可引发基因组不稳定性（genomic instability）并最终导致细胞死亡（cell death）。鸟嘌呤（Guanine）是最易发生氧化的碱基，因其较低的氧化还原电位（redox potential），而8-氧代鸟嘌呤（8-oxoguanine, 8-oxoG）是最为常见的DNA损伤类型。上述特性使得8-oxoG成为反映氧化应激程度的优质细胞生物标志物（cellular biomarker）。 若未被修复，8-oxoG可与腺嘌呤（adenine）配对，引发G:C到T:A的碱基颠换（transversion）。在DNA复制过程中若插入8-oxoG，则可能产生双链断裂（double-strand breaks），令该类损伤具有极强的危害性。 克氏锥虫（Trypanosoma cruzi）需应对多种氧化应激场景，例如其寄生的哺乳动物细胞内环境以及繁殖所处的锥蝽昆虫肠道（triatomine insect gut）。本研究聚焦于负责从核苷酸池（nucleotide pool）中清除8-oxoG的MutT酶（MutT enzyme）。为探究8-oxoG在寄生虫感染哺乳动物细胞过程中的重要性，我们对克氏锥虫中的MutT基因（TcMTH）进行了特征化分析，并构建了异源表达大肠杆菌（Escherichia coli）MutT酶或过表达TcMTH酶的克氏锥虫重组株。 在上鞭毛体（epimastigote）形态下，重组寄生虫与野生型寄生虫在正常培养条件下的生长表现无显著差异，但表达MutT酶的重组株对过氧化氢（hydrogen peroxide）处理具有更强的耐受性。与野生型细胞相比，重组寄生虫在感染成纤维细胞（fibroblasts）和巨噬细胞（macrophages）48小时后，生长水平显著提升，且在瑞士小鼠（Swiss mice）体内的寄生虫血症（parasitemia）水平更高。 此外，我们通过蛋白质免疫印迹实验（Western blotting）证实，异源表达MutT酶可影响寄生虫的抗氧化酶蛋白水平。上述结果表明，8-oxoG修复系统对细胞存活至关重要。