Laboratório de Interação de Tripanossomatídeos e Vetores - Departamento de Bioquímica, IBRAG - UERJ, Rio de Janeiro, Brazil.
Laboratório de Toxicologia e Biologia Molecular, Departamento de Bioquímica, IBRAG - UERJ, Rio de Janeiro, Brazil.
Biochim Biophys Acta Mol Basis Dis. 2020 Dec 1;1866(12):165951. doi: 10.1016/j.bbadis.2020.165951. Epub 2020 Aug 27.
Trypanosoma cruzi has a complex life cycle involving four life stages: the replicative epimastigotes and metacyclic trypomastigotes in the invertebrate host digestive tract, and intracellular amastigotes and bloodstream trypomastigotes in the mammalian host. Trypomastigotes can invade any nucleated cell, including macrophages, which produce ROS that enhance intracellular infection. However, how ROS modulate T. cruzi infection in the mammalian cell remains unclear. Therefore, the present work aimed to investigate the role of ROS during the stimulation of amastigogenesis in vitro. Our results showed that HO improves the differentiation process in vitro and that it was impaired by Peg-Catalase. However, the antioxidants GSH and NAC had no influence on induced amastigogenesis, which suggests the specificity of HO to increase intracellular differentiation. Amastigogenesis physiologically occurs in low pH, thus we investigated whether parasites are able to produce ROS during amastigogenesis. Interestingly, after 60 min of differentiation induction in vitro, we observed an increase in HO production, which was inhibited by the mitochondrial-targeted antioxidant, mitoTEMPO and Cyclosporine A (a mitochondrial permeability transition pore -mPTP- inhibitor), suggesting mitochondrion as a HO source. Indeed, quantitative real time (qPCR) showed an increase of the mitochondrial superoxide dismutase (FeSODA) gene expression after 60 min of induced amastigogenesis, reinforcing the hypothesis of mitochondrial ROS induction during intracellular differentiation of T. cruzi. The reduction of cellular respiration and the decreased ΔΨm observed during amastigogenesis can explain the increased mitochondrial ROS through mPTP opening. In conclusion, our results suggest that HO is involved in the amastigogenesis of T. cruzi.
克氏锥虫具有复杂的生命周期，包括四个生命阶段：无脊椎动物宿主消化道中的复制性前鞭毛体和循环型无鞭毛体，以及哺乳动物宿主体内的内阿米巴型和血液型无鞭毛体。无鞭毛体能侵入任何有核细胞，包括巨噬细胞，巨噬细胞会产生 ROS，从而增强细胞内感染。然而，ROS 如何调节哺乳动物细胞中的克氏锥虫感染尚不清楚。因此，本研究旨在探讨 ROS 在体外诱导无鞭毛体形成过程中的作用。我们的结果表明，HO 可改善体外分化过程，而过氧化氢酶抑制剂 Peg-Catalase 可损害这一过程。然而，抗氧化剂 GSH 和 NAC 对诱导的无鞭毛体形成没有影响，这表明 HO 特异性地增加细胞内分化。无鞭毛体在生理条件下于低 pH 值下发生，因此我们研究了寄生虫在无鞭毛体形成过程中是否能够产生 ROS。有趣的是，在体外分化诱导 60 分钟后，我们观察到 HO 产生增加，这一增加被线粒体靶向抗氧化剂 mitoTEMPO 和环孢菌素 A（线粒体通透性转换孔-mPTP-抑制剂）抑制，提示线粒体是 HO 的来源。事实上，定量实时（qPCR）显示，在诱导无鞭毛体形成 60 分钟后，线粒体超氧化物歧化酶（FeSODA）基因表达增加，这加强了克氏锥虫细胞内分化过程中诱导线粒体 ROS 的假说。在无鞭毛体形成过程中观察到的细胞呼吸减少和 ΔΨm 降低，可以解释 mPTP 开放导致的线粒体 ROS 增加。总之，我们的结果表明，HO 参与了克氏锥虫的无鞭毛体形成。
