Maggi Stefano, Ferrari Alberto, Yabre Korotoum, Bonini Aleksandra Anna, Rivetti Claudio, Folli Claudia
Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, 43124 Parma, Italy.
Department of Food and Drug, University of Parma, 43124 Parma, Italy.
Methods Protoc. 2021 Oct 8;4(4):71. doi: 10.3390/mps4040071.
A large number of bacterial toxin-antitoxin (TA) systems have been identified so far and different experimental approaches have been explored to investigate their activity and regulation both in vivo and in vitro. Nonetheless, a common feature of these methods is represented by the difficulty in cell transformation, culturing, and stability of the transformants, due to the expression of highly toxic proteins. Recently, in dealing with the type I Lpt/RNAII and the type II YafQ/DinJ TA systems, we encountered several of these problems that urged us to optimize methodological strategies to study the phenotype of recombinant host cells. In particular, we have found conditions to tightly repress toxin expression by combining the pET expression system with the C41(DE3) pLysS strain. To monitor the RNase activity of the YafQ toxin, we developed a fluorescence approach based on Thioflavin-T which fluoresces brightly when complexed with bacterial RNA. Fluorescence microscopy was also applied to reveal loss of membrane integrity associated with the activity of the type I toxin Lpt, by using DAPI and ethidium bromide to selectively stain cells with impaired membrane permeability. We further found that atomic force microscopy can readily be employed to characterize toxin-induced membrane damages.
到目前为止，已经鉴定出大量细菌毒素 - 抗毒素（TA）系统，并且已经探索了不同的实验方法来研究它们在体内和体外的活性及调控。然而，由于高毒性蛋白的表达，这些方法的一个共同特点是细胞转化、培养以及转化子稳定性方面存在困难。最近，在处理I型Lpt/RNAII和II型YafQ/DinJ TA系统时，我们遇到了其中的几个问题，这促使我们优化方法策略来研究重组宿主细胞的表型。特别是，我们通过将pET表达系统与C41(DE3)pLysS菌株相结合，找到了紧密抑制毒素表达的条件。为了监测YafQ毒素的核糖核酸酶活性，我们开发了一种基于硫黄素 - T的荧光方法，当与细菌RNA结合时，硫黄素 - T会发出明亮的荧光。还应用荧光显微镜，通过使用DAPI和溴化乙锭选择性地对膜通透性受损的细胞进行染色，来揭示与I型毒素Lpt活性相关的膜完整性丧失。我们进一步发现，原子力显微镜可以很容易地用于表征毒素诱导的膜损伤。
