单细菌水平毒素－抗毒素系统研究新方法

Single-cell analysis is vital for providing insights into the heterogeneity of molecular content and phenotypic characteristics of cells from complex and clonal cell populations. The overall goal of this proposal is to develop a new method for studying toxin-antitoxin (TA) systems within individual bacterial cells by combining ultra-sensitive flow cytometry with a biarsenical-tetracysteine motif. Nearly all bacteria have toxin-antitoxin systems, which are comprised of a stable toxin that arrests cellular processes and a labile antitoxin that prevents toxin activity. A small subpopulation of cells has heightened toxin activation, giving rise to dormant persister cells that tolerate antibiotics by "sleeping" through treatment. Using a laboratory-built high-sensitivity flow cytometer (HSFCM), it is possible to perform a high-throughput and multi-parameter analysis of biarsenical-labeled TA systems within individual bacteria. Statistical distribution of a TA system can be determined quickly by analyzing thousands of individual cells within a couple of minutes, revealing the heterogeneous expression pattern that is otherwise masked by the ensemble analysis. TA systems have an important role in bacterial stress physiology, which might form the basis of multidrug resistance. This high-throughput technique will be used to map TA system stress response detection modules under different types of stress, which will provide a better understanding of the fundamental role of TA systems in persister cell formation.Most bacterial genomes contain multiple TA loci, so it is extremely important to consider cross-talk when studying TA-related signal transduction pathways. In addition to this high-throughput technique, a two-step cascade method would also be utilized to detect cross-activation between different TA systems. The sensitivity and speed of the HSFCM offers great capability in high-throughput analysis of cross-activation between different TA systems. The above mentioned method can also be extended to screen toxin activated molecules. In conclusion, an in vivo, quantitative, high-throughput, and versatile new method is ideal for studying TA systems within individual bacteria. This method will pave the way for the study of signal transduction and molecular mechanisms of TA systems during programmed cell arrest and persister cell formation, which will have a significant impact on strategies for designing antibacterials.

毒素－抗毒素（TA）系统几乎存在于所有致病菌中，它的异质性表达产生多药耐药的休眠存留细胞。对单细菌体内的毒素－抗毒素蛋白进行快速的逐一分析可揭示因集权平均而被掩盖的个体差异，有益于TA系统的异质性考察及亚类表征。本申请拟利用实验室自行研制的超高灵敏流式检测装置的灵敏、快速、多参数分析性能，结合四半胱氨酸－双砷染料体系的蛋白标记优点，通过对单个细菌散射光和荧光信号的同时检测，发展TA系统研究新方法，建立单个细菌体内毒素、抗毒素蛋白定量表征技术并考察各参数之间的相互关系；建立TA系统应激检测模式，确定TA系统在诱发细菌程序性阻滞和程序性死亡中的作用；引入Two-Step Cascade调控机制，考察TA系统间交互作用并发展方便、快捷的毒素蛋白诱导因子筛选法。本研究将创立单细菌水平、原位、高通量、普适性的TA系统研究新方法，对揭示TA系统的作用机理和研究对抗致病菌及细菌耐药性的战略具有重要价值。

毒素－抗毒素（TA）系统几乎存在于所有致病菌中，它的异质性表达产生多药耐药的存留细胞。对单细菌体内的毒素－抗毒素蛋白进行快速的逐一分析可揭示因集权平均而被掩盖的个体差异。本项目通过对单个细菌散射光、毒素和抗毒素蛋白的双荧光信号的同时检测，发展TA系统研究新方法，建立单个细菌体内毒素、抗毒素蛋白定量表征技术并考察T/A比例与各参数之间的相互关系；建立TA系统应激检测模式，确定TA系统在细菌生长中的作用；考察TA系统间交互作用并发展方便、快捷的毒素蛋白诱导因子筛选法。取得的重要进展如下：.1) 构建由天然启动子调控的带标签TA系统。在原计划成功构建携带四半胱氨酸标签的TA系统的基础上分别在毒素和抗毒素蛋白上新增构建His-tag和Flag-tag融合标签。结合双砷染色和免疫荧光染色方法，实现对毒素和抗毒素蛋白的同时检测。 .2) 建立单细菌水平TA系统蛋白表达的超高灵敏流式检测方法。首次在单细菌水平观测到表达量极低的毒素蛋白MqsR的表达。发现在正常生长条件下，细菌存在高表达和低表达抗毒素MqsA两个群体，在抑制细菌生长的条件下，高表达的MqsA被降解。.3) 建立应激条件下单细菌水平毒素、抗毒素蛋白表达的超高灵敏流式分析方法。发现抗毒素MqsA在氧化应激时被降解，在热应激时持续合成，在氨基酸饥饿时被降解后再生（首次发现）。.4) 建立抗毒素MqsA蛋白与细菌生长同时检测的超高灵敏流式分析方法。发现抗毒素MqsA蛋白表达与细菌生长呈正相关，且细菌生长不管是抑制还是促进都比MqsA蛋白反应滞后60 min。.5) 实现了单细菌水平Lon蛋白酶调控MqsR/MqsA TA系统的超高灵敏检测，初步建立了TA系统交互作用的超高灵敏流式研究方法。.6) 建立持留菌形成过程中单细菌水平T/A比例的监测方法，获得T/A比例与细菌耐药相关持留菌形成的关系。.7) 此外，以重组噬菌体PPO1-TC及E. coli O157:H7作为模型，建立痕量致病活菌的灵敏、特异、快速检测体系。 (Biosens Bioelectron., 2016，86, 102-108）.8) 建立了可以对蛋白质相互作用进行检测和强度排序对比的定量蛋白相作用研究新方法。（Anal. Chem., 2017, 89, 2782-2789）