The molecular circuitry of toxin-antitoxin systems and their contribution to microbial dormancy.

毒素-抗毒素系统的分子回路及其对微生物休眠的贡献。

• 批准号: RGPIN-2020-06636
• 负责人: Ensminger, Alexander
• 金额: $ 3.06万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744129
• 关键词: molecular; circuitry; toxin; antitoxin; systems

In the lab, bacteria are often studied in nutrient-rich environments under maximal growth rates. The reality is that microbial life is typically characterized by punctuated, suboptimal conditions of growth. In order to survive extended periods of time under nutrient-poor, stressful conditions, many bacteria enter a growth-arrested state until there is a return to more favourable conditions. Our research program seeks to define the molecular circuitry behind these transitions, using a highly tractable model bacterium, Legionella pneumophila. L. pneumophila typically replicates inside freshwater amoebae, but can spend months at a time in a non-replicative, extracellular state. One mechanism by which bacteria regulate their growth is through toxin-antitoxin systems. Despite their name, these "toxins" are not delivered to other cells, but rather serve to limit the replication of the cells that encode for them. Toxin-antitoxin systems typically exist as two-gene modules within an operon that encode a protein toxin capable of inhibiting growth along with a corresponding antitoxin. They are ubiquitous in bacteria and have been implicated in phage defense, biofilm formation, and dormancy, yet much of their function in cellular physiology is still unknown. The large number of systems in most species complicates their experimental interrogation: for instance, there are over 30 systems in most Escherichia and Salmonella species, and nearly 90 in Mycobacterium. In contrast, L. pneumophila has only 7 predicted toxin-antitoxin systems yet has a well-defined need to survive for extended periods in a non-replicative state. Put simply, our research program seeks to establish L. pneumophila as a consummate model organism for the systematic study of toxin-antitoxin systems. We will define their molecular targets and their mechanisms of action. We will systematically examine the contributions of these systems to L. pneumophila environmental persistence, intracellular replication, and genome stability. Within this program, our objectives are to: 1. Place each toxin-antitoxin system within the genetic network of the microbial cell. 2. Extensively characterize bacteria devoid of all toxin-antitoxin systems. 3. Determine the contribution of individual toxin-antitoxin systems to specific phenotypes through systematic complementation studies. The field of toxin-antitoxin systems is full of unlocked mysteries, controversies, and promise. Specifically, while there is a growing body of literature on the molecular mechanisms underpinning toxin-antitoxin systems, the underlying biology behind such systems remains poorly defined. Leveraging a uniquely tractable and well-suited model microbe, our program seeks to address this knowledge-gap head-on. With dozens of additional Legionella species recently sequenced - each with a corresponding diverse set of toxin-antitoxin systems to explore - the program we propose has a clear path forward for the conceivable future.

在实验室中，细菌通常在营养丰富的环境中以最大生长速率进行研究。现实情况是，微生物生命的典型特征是生长时断时续、次优。为了在营养贫乏、压力大的条件下长时间生存，许多细菌进入生长停滞状态，直到恢复到更有利的条件。我们的研究项目旨在使用高度易处理的模型细菌嗜肺军团菌来定义这些转变背后的分子电路。嗜肺军团菌通常在淡水变形虫体内复制，但可以一次处于非复制的细胞外状态数月。细菌调节其生长的一种机制是通过毒素-抗毒素系统。尽管有它们的名字，这些“毒素”并不会传递到其他细胞，而是用于限制为其编码的细胞的复制。毒素-抗毒素系统通常作为操纵子内的双基因模块存在，编码能够抑制生长的蛋白质毒素以及相应的抗毒素。它们在细菌中普遍存在，并与噬菌体防御、生物膜形成和休眠有关，但它们在细胞生理学中的大部分功能仍然未知。大多数物种的大量系统使实验研究变得复杂：例如，大多数埃希氏菌和沙门氏菌物种有 30 多个系统，分枝杆菌有近 90 个系统。相比之下，嗜肺军团菌只有 7 个预测的毒素-抗毒素系统，但具有明确的在非复制状态下长时间生存的需求。简而言之，我们的研究计划旨在将嗜肺军团菌建立为系统研究毒素-抗毒素系统的完美模式生物。 我们将定义它们的分子靶点及其作用机制。我们将系统地研究这些系统对嗜肺军团菌环境持久性、细胞内复制和基因组稳定性的贡献。在该计划中，我们的目标是： 1. 将每个毒素-抗毒素系统置于微生物细胞的遗传网络中。 2. 广泛表征缺乏所有毒素-抗毒素系统的细菌。 3. 通过系统互补研究确定个体毒素-抗毒素系统对特定表型的贡献。 毒素-抗毒素系统领域充满了未解之谜、争议和希望。具体来说，虽然关于毒素-抗毒素系统的分子机制的文献越来越多，但此类系统背后的基础生物学仍然不清楚。我们的计划利用独特的易处理且非常适合的模型微生物，力求正面解决这一知识差距。最近对数十种其他军团菌物种进行了测序——每种都有一套相应的不同毒素-抗毒素系统可供探索——我们提出的计划为可想象的未来提供了明确的前进道路。