Deciphering of the Toxin-Antitoxin Systems in E. coli

描述（由申请人提供）：包括人类病原体在内的几乎所有细菌都含有由毒素-抗毒素（TA）系统编码的自杀或毒素基因，这些基因在各种生长和应激条件下被诱导，导致细胞生长停滞并最终以类似于高等系统中的细胞凋亡或程序性细胞死亡的方式死亡。令人惊讶的是，许多细菌，特别是人类病原体，如结核分枝杆菌，在它们的基因组上含有大量独立的毒素基因。直到最近，当我们的实验室和其他人开始破译这些毒素的细胞靶标，并证明这些毒素如何抑制细胞生长导致细胞死亡时，这些毒素基因在医学科学中的重要性才得到充分认识。细菌的生理受到这些毒素或应激条件下的毒素网络的严格调节。据推测，结核分枝杆菌在人体组织中的长期休眠、生物膜的形成以及各种其他人类病原体的持续多药耐药与这些病原体中的毒素表达密切相关。此外，利用这些毒素的自杀特性，有可能开发出前所未有的针对人类病原体的新型抗生素。因此，迫切需要对这一新兴领域进行全面的研究。因此，我建议对大肠杆菌的毒素-抗毒素系统进行彻底的研究，大肠杆菌作为一种模式细菌，令人惊讶的是，它的基因组中含有多达22个独立的毒素-抗毒素系统。由于这些毒素在功能上高度多样化，而且在许多其他细菌中都是保守的，因此它们不仅是研究细菌毒素的理想范例，而且是研究以前未知的由TA网络控制的细菌生理的理想范例。我们的目标是破译单个大肠杆菌毒素的细胞靶标及其作用机制。我将探讨阐明细胞中的TA系统是如何与TA网络协调的。在目标#1中，将使用遗传，生化和结构方法的组合来破译新的TA系统，包括识别细胞靶标和毒素的作用机制。在目标#2中，在TA缺失菌株的帮助下，我们将研究不同生长或应激条件下单个TA系统和一组TA系统在细菌生理中的可能作用，以及单个TA系统的表达如何在整个TA网络系统中协调。细胞死亡决定因素的鉴定也将在本提案中进行，将提供对细菌细胞死亡机制的重要见解。与公共卫生的相关性：本研究将为人类细菌病原体中存在的自杀基因在其致病性和耐药持久性中的作用提供新的见解，为开发针对TA系统的新型抗生素提供重要线索。公共卫生相关性：细菌，包括大多数人类病原体，含有TA（毒素-抗毒素）系统，该系统调节其自身的细胞生长并决定其自身的生死。对TA系统的研究揭示了TA系统在人类病原体持续耐药和致病性方面的重要作用。提出的研究是表征大肠杆菌中所有的TA系统，阐明单个毒素的细胞靶点，它们的作用机制和它们在细菌生理学中的作用。这项研究的结果对我们理解细菌生理学的基本原理、TA系统在人类病原体中的作用具有广泛而重要的意义，并为抑制人类病原体细胞生长或导致其死亡的新技术提供了重要的见解。

DESCRIPTION (provided by applicant): Almost all bacteria including human pathogens contain suicide or toxin genes encoded by the toxin-antitoxin (TA) systems, which are induced under various growth and stress conditions leading to cell growth arrest and eventual cell death in a way similar to apoptosis or programmed cell death in higher systems. Surprisingly, many bacteria, particularly human pathogens such as Mycobacterium tuberculosis contain a large number of independent toxin genes on their genomes. The importance of these toxin genes in medical science has not been fully appreciated until most recently when our laboratory and others started to decipher the cellular targets of these toxins and demonstrated how these toxins inhibit cell growth leading to cell death. Bacterial physiology is tightly regulated by these toxins or by the toxin networks under stress conditions. The prolonged dormancy of M. tuberculosis in human tissues, biofilm formation, and persistent multi-drug resistance of various other human pathogens are presumed to be closely associated with toxin expression in these pathogens. Furthermore, taking advantage of suicidal properties of these toxins, it is possible to develop novel unprecedented antibiotics against human pathogens. Therefore, all-out comprehensive investigation in this emerging field is urgently needed. For this reason, I propose to carry out thorough investigation of the toxin-antitoxin systems in Escherichia coli as a model bacterium, which surprisingly contains as many as twenty two independent toxin-antitoxin systems on its genome. As these toxins are functionally highly diversified and yet are conserved in many other bacteria, they are ideal paradigms for the study of not only bacterial toxins, but also previously unknown bacterial physiology governed by the TA network. Our goal is to decipher cellular targets of individual E. coli toxins and their mechanisms of action. I will explore to elucidate how the TA systems are coordinated to from the TA network in the cell. In Aim #1, a combination of genetic, biochemical and structural approaches will be used to decipher new TA systems including identification of cellular targets and mechanism of action of toxins. In Aim #2, with help of TA deletion strains we will investigate possible roles of individual TA systems and a group of the TA systems in bacterial physiology under different growth or stress conditions and how the expression of individual TA systems is coordinated in the overall TA network system. Identification of cell death determinants which will be also carried out in this proposal will provide important insights into the mechanism of bacterial cell death. Relevance in public heath: The present studies will provide new insights into the role of suicide genes present in human bacterial pathogens in their pathogenicity and drug-resistant persistence, providing important clues for development of novel antibiotics targeting the TA systems. PUBLIC HEALTH RELEVANCE: Bacteria, including most of human pathogens, contain the TA (toxin-antitoxin) systems, which regulate their own cell growth and determine their own life and death. Research on the TA Systems has revealed their important roles in persisting drug-resistance and pathogenicity of human pathogens. The proposed search is to characterize all the TA systems in E. coli elucidating cellular targets of individual toxins, their mechanisms of action and their roles in bacterial physiology. The outcome from this research has a wide, important implication to our understanding of the basic principle of bacterial physiology, the roles of the TA systems in human pathogens and provides crucial insights into a novel technology to suppress cell growth of human pathogens or to lead them to death.