Deciphering of the Toxin-Antitoxin Systems in E. coli

描述(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，这些基因在不同的生长和应激条件下诱导，导致细胞生长停滞，最终导致细胞死亡，其方式类似于高等系统中的细胞凋亡或程序性细胞死亡。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，我们的实验室和其他实验室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，这些毒素基因在医学上的重要性才得到充分认识。细菌的生理受到这些毒素或压力条件下的毒素网络的严格调控。结核分枝杆菌在人体组织中的长时间休眠、生物被膜的形成以及其他各种人类病原体的持续多药耐药被认为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调查。为此，我建议对大肠杆菌中的毒素-抗毒素系统进行彻底的研究，作为模式菌，它的基因组上出人意料地含有多达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，但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，也是研究先前未知的由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。我将探讨如何从小区中的TA网络协调TA系统。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，我们将借助TA缺失菌株，研究不同生长或胁迫条件下单个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系统如何在整个TA网络系统中协调表达。对细胞死亡决定因素的识别也将在这项提案中进行，这将为细菌细胞死亡的机制提供重要的见解。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致病性和耐药性持久性中所起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。与公共卫生相关：细菌，包括大多数人类病原体，含有TA(毒素-抗毒素)系统，该系统调节自己的细胞生长并决定自己的生死。对TA系统的研究表明，TA系统在维持人类病原体的耐药性和致病性方面发挥着重要作用。研究的目的是确定大肠杆菌中所有TA系统的特征，阐明单个毒素的细胞靶标、作用机制以及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的细胞生长或导致它们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.