The critical roles of (p)ppGpp in homeostasis and antibiotic tolerance in Gram positive bacteria

项目摘要 细菌经常遇到压力，包括营养匮乏、温度变化和 抗生素攻击，这很容易使他们的细胞内环境陷入混乱。为了生存和 为了适应，细菌产生了不同的应激反应来调节细胞内的过程。 相应地。虽然控制应激反应的转录网络已经广泛存在 在特征上，除了转录调控之外，我们的知识还有很大的差距。的主题 我的研究是为了阐明压力信号传递机制，这些机制是通过 丙酮-信号核苷酸的浓度，它有助于向细胞发出警报 及时强调。我的实验室在刻画保守的 警报(P)ppGpp。(P)ppGpp是由压力诱导的，并介导着深刻的、多效性的 几乎所有细菌的生理变化都能使其适应、生存和进化。我们确认了 多个嘌呤合成酶、复制酶和转录抑制物，它们是 在革兰氏阳性芽孢杆菌中由(P)ppGpp直接调节。这些规定进一步 在许多病原体中被发现是保守的，对动态平衡、抗饥饿性、 抗生素的持久性和基因组的完整性。目前，我们还在调查(P)ppGpp如何 调节不同细菌生活方式之间的转换：浮游生长和生物膜形成。 此外，我们还检测到了其他核苷酸丙酮类化合物，包括AppppA、pGpp、ppApp和c-di- AMP，由包括温度和细胞壁应力在内的不同应力诱导，形成一个 强大的保护网络。我们未来的研究将回答以下基本问题：如何 不同的Alarone是由不同的压力触发的吗？细菌是如何合成它们的？ 不同丙酮的直接相互作用靶点是什么？它们如何促进细菌 是否适合并影响细菌的发育，如生物膜的形成和孢子的形成？做什么 细菌整合了来自不同丙酮的多个线索，以快速和适当地适应 不同的环境？我们将代谢组学、转录组学和蛋白质组学与 生物化学和细胞生物学方法来回答这些问题。我们得到了一份名单 利用病原菌芽孢杆菌的蛋白质组进行系统筛选的警报靶标 炭疽病。我们将在相关的非致病细菌枯草杆菌中研究这些过程。 在这方面我们有丰富的经验。枯草杆菌生长迅速，对基因高度敏感 操纵。我们在芽孢杆菌中描述的核苷酸信号机制适用于 其他较难驯化的致病菌，可用于开发抗菌策略 通过瞄准他们的应激反应。

Project Summary Bacteria frequently encounter stresses including nutrient starvation, temperature changes, and antibiotic assault, which could easily throw their intracellular environment into chaos. To survive and to adapt, bacteria developed diverse stress responses to regulate intracellular processes accordingly. While the transcriptional networks governing stress responses have been extensively characterized, there are major gaps in our knowledge beyond transcription regulation. The theme of my research is to elucidate stress signaling mechanisms that are transmitted by rapid changes in concentration of ‘alarmones’ – signaling nucleotides which are instrumental for alerting cells about stresses in a timely manner. My laboratory has extensive experience in characterizing the conserved alarmone (p)ppGpp. (p)ppGpp is induced by stresses and mediates profound, pleiotropic physiological changes in almost all bacteria to allow fitness, survival, and evolution. We identified multiple purine synthesis enzymes, a replication enzyme and a transcription repressor that are directly regulated by (p)ppGpp in Gram-positive Bacillus species. These regulations were further found to be conserved in many pathogens and are critical for homeostasis, starvation resistance, antibiotic persistence, and genome integrity. Currently, we are also investigating how (p)ppGpp regulates the switch between distinct bacterial lifestyles: planktonic growth and biofilm formation. Additionally, we detected other nucleotide alarmones including AppppA, pGpp, ppApp, and c-di- AMP, which are induced by different stresses including temperature and cell wall stress, to form a robust protective network. Our future research will answer the following fundamental questions: How are the different alarmones triggered by different stresses, and how do bacteria synthesize them? What are the direct interaction targets of different alarmones, and how do they promote bacterial fitness and influence bacterial development such as biofilm formation and sporulation? How do bacteria integrate multiple cues from different alarmones for rapid and appropriate adaptation to diverse environments? We combine metabolomics, transcriptomics, and proteomics with biochemical and cell biological approaches to answer these questions. We obtained a list of alarmone targets from systematic screens performed with the proteome of the pathogen Bacillus anthracis. We will study these processes in the related non-pathogenic bacterium Bacillus subtilis for which we have extensive experience. B. subtilis grows fast and is highly amenable to genetic manipulation. The nucleotide signaling mechanisms we characterize in Bacillus are applicable to other, less tractable, pathogenic bacteria, and can be used for developing antimicrobial strategies by targeting their stress responses.