Laboratory of Microbial Technology, Graduate School of Science and Technology, Kyoto Institute of Technology, Matsugasaki, Kyoto, 606-8585, Japan.
Faculty of Electrical Engineering and Electronics, Kyoto Institute of Technology, Kyoto, Japan.
Appl Microbiol Biotechnol. 2018 Mar;102(5):2279-2288. doi: 10.1007/s00253-018-8758-2. Epub 2018 Jan 22.
Cold atmospheric pressure plasma (CAP) does not cause thermal damage or generate toxic residues; hence, it is projected as an alternative agent for sterilization in food and pharmaceutical industries. The fungicidal effects of CAP have not yet been investigated as extensively as its bactericidal effects. We herein examined the effects of CAP on yeast proteins using a new CAP system with an improved processing capacity. We demonstrated that protein ubiquitination and the formation of protein aggregates were induced in the cytoplasm of yeast cells by the CAP treatment. GFP-tagged Tsa1 and Ssa1, an HO-responsive molecular chaperone and constitutively expressed Hsp70, respectively, formed cytoplasmic foci in CAP-treated cells. Furthermore, Tsa1 was essential for the formation of Ssa1-GFP foci. These results indicate that the denaturation of yeast proteins was caused by CAP, at least partially, in a HO-dependent manner. Furthermore, misfolded protein levels in the endoplasmic reticulum (ER) and the oligomerization of Ire1, a key sensor of ER stress, were enhanced by the treatment with CAP, indicating that CAP causes ER stress in yeast cells as a specific phenomenon to eukaryotic cells. The pretreatment of yeast cells at 37 °C significantly alleviated cell death caused by CAP. Our results strongly suggest that the induction of protein denaturation is a primary mechanism of the fungicidal effects of CAP.
冷等离体大气压等离子体（CAP）不会造成热损伤或产生有毒残留物；因此，它被认为是食品和制药行业中一种替代的灭菌剂。CAP 的杀菌效果尚未像其杀菌效果那样得到广泛研究。我们在此使用具有改进处理能力的新型 CAP 系统，研究了 CAP 对酵母蛋白的影响。我们证明，CAP 处理会诱导酵母细胞细胞质中蛋白质泛素化和蛋白质聚集体的形成。作为 HO 反应性分子伴侣的 GFP 标记的 Tsa1 和组成型表达的 Hsp70 分别在 CAP 处理的细胞中形成细胞质焦点。此外，Tsa1 对于 Ssa1-GFP 焦点的形成是必不可少的。这些结果表明，CAP 至少部分地以 HO 依赖性方式导致酵母蛋白变性。此外，内质网（ER）中错误折叠的蛋白质水平和 ER 应激的关键传感器 Ire1 的寡聚化通过 CAP 处理而增强，表明 CAP 作为真核细胞的一种特有现象引起酵母细胞中的 ER 应激。在 37°C 下对酵母细胞进行预处理可显著减轻 CAP 引起的细胞死亡。我们的结果强烈表明，蛋白质变性的诱导是 CAP 杀菌效果的主要机制。
