Key Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China.
Key Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China.
Ticks Tick Borne Dis. 2019 Aug;10(5):1018-1027. doi: 10.1016/j.ttbdis.2019.05.004. Epub 2019 May 22.
Babesia microti is an emerging zoonotic pathogen that is transmitted by ticks and parasites and propagates in mammalian erythrocytes. Thioredoxin reductase (TrxR) plays a crucial role in B. microti survival by maintaining cellular redox homeostasis. In the present study, 4-nitro-2,1,3-benzothiadiazole (4-NBT) was selected as a specific B. microti TrxR inhibitor by comparing rat and parasite TrxR inhibition levels. Reactive oxygen species (ROS) levels were evaluated using flow cytometry, and in B. microti treated with 4-NBT, ROS levels increased with increasing inhibitor concentration. Furthermore, the inhibitor treatment increased lipid peroxidation and protein carbonyl levels, thus indicating a state of oxidative stress. While B. microti treated with 4-NBT appeared to lose the ability to multiply in mice, the fastigium of parasitemia between the treated and control groups was comparable. Furthermore, a TUNEL assay showed that 4-NBT induces apoptosis in B. microti. Proteomic analysis of B. microti treated with 4-NBT detected 960 proteins. Label-free quantitative proteomic analysis identified 118 proteins that were significantly up-regulated and 37 that were significantly down-regulated in the treatment group relative to the control. Of the differential proteins, proteasome and ribosomal subunit expression was up-regulated, thus suggesting that redundant proteins may be damaged by oxidation and waiting for degradation, while proteins for subsistence are waiting for de novo synthesis. Moreover, the findings obtained herein suggest that the DNA and lipids were also damaged and awaiting synthesis or repair. In conclusion, TrxR dysfunction in B. microti results in the breakdown of redox homeostasis and promotes apoptosis.
微小巴贝斯虫是一种新兴的动物源性病原体，通过蜱虫和寄生虫传播，并在哺乳动物的红细胞中繁殖。硫氧还蛋白还原酶（TrxR）通过维持细胞氧化还原稳态在微小巴贝斯虫的存活中起着至关重要的作用。在本研究中，通过比较大鼠和寄生虫 TrxR 的抑制水平，选择 4-硝基-2,1,3-苯并噻二唑（4-NBT）作为微小巴贝斯虫 TrxR 的特异性抑制剂。使用流式细胞术评估活性氧（ROS）水平，并且在用 4-NBT 处理的微小巴贝斯虫中，ROS 水平随抑制剂浓度的增加而增加。此外，抑制剂处理增加了脂质过氧化和蛋白质羰基水平，从而表明存在氧化应激状态。虽然用 4-NBT 处理的微小巴贝斯虫似乎失去了在小鼠中繁殖的能力，但处理组和对照组之间的疟原虫血症尖峰相似。此外，TUNEL 测定表明 4-NBT 诱导微小巴贝斯虫凋亡。用 4-NBT 处理的微小巴贝斯虫的蛋白质组分析检测到 960 种蛋白质。无标记定量蛋白质组学分析鉴定出 118 种在处理组中显著上调的蛋白质和 37 种在处理组中显著下调的蛋白质。在差异蛋白中，蛋白酶体和核糖体亚基的表达上调，这表明氧化损伤的冗余蛋白可能在等待降解，而生存所需的蛋白则在等待从头合成。此外，本文的研究结果表明，DNA 和脂质也受到损伤，正在等待合成或修复。总之，微小巴贝斯虫 TrxR 功能障碍导致氧化还原稳态崩溃并促进凋亡。
