Integrating Cell Envelope and Starvation Stress: Regulation of Sigma(E) by ppGpp

描述（由申请人提供）：该？E细胞膜敏感途径是细菌维持细胞膜完整性的关键调控系统。? E是几种重要细菌病原体生存所必需的，也是其他细菌病原体毒力所必需的。? E活性受两个主要信号系统的调节，这两个信号系统整合了不同的信号，细胞被膜应激和营养限制。为了应对信封压力，？E通过其专用抗σ因子的调节性蛋白水解来激活。在营养限制期间？在没有明显的包膜应激的情况下，E被alarmone ppGpp激活，提前为包膜的存活做好准备。激活的程度，由每个系统的服务，以设置的整体活动？在细胞中，使t与细胞的需要相匹配。除了激活？E，ppGpp激活其他替代σ因子和应激途径，同时抑制快速生长所需的基因。实际上，ppGpp介导了从快速生长的转录程序到针对胁迫存活的优化转录程序的转换。ppGpp的调节机制是什么？活动还不太清楚。这些知识对于我们理解这种重要且高度保守的替代性σ因子如何响应变化的环境以维持细胞包膜完整性至关重要。本建议的目的是确定一种或多种机制，E活性由全局调节因子ppGpp调节，从而协调包膜和细胞质对饥饿的反应。拟议的研究的中心假设是，ppGpp，在协同调节DksA和潜在的其他因素，控制？E活动直接激活？E全酶（E？E）和间接地通过增加E的量？E通过其对核心RNA聚合酶（RNAP）的sigma因子之间的竞争的影响。这一假设将通过三个具体目标来解决。(1)定义转录起始的生化特性E？E. ? E是高度保守的、广泛分布的一类替代性σ因子的成员，其缺乏已知对于其他σ因子的转录起始中的特定步骤重要的几个保守区域和氨基酸。这些差异如何改变转录起始尚未研究。转录起始的基本参数E？在评估pGp的影响之前，必须确定E。(2)阐明ppGpp和DksA调节E？E活动。自激活E？70和ppGpp/DksA的替代sigma因子尚未得到很好的理解，来自所提出的工作的结果将对这些全局调节因子的转录激活的生化基础的整体理解产生强烈的积极影响。(3)确定ppGpp如何调节？E活性独立于DksA。初步工作表明，一种新的机制？E by ppGpp的存在无法用当前模型解释，这需要DksA与ppGpp一起作用。将探讨这种新的监管策略。因为这两个？E和ppGpp系统对重要细菌病原体的毒力至关重要，更好地理解这些反应的交叉可能会为药物发现和疫苗设计带来新的途径。 公共卫生相关性：革兰氏阴性菌是非常成功的病原体，并且这些细菌中抗生素耐药性的日益普遍对人类健康构成重大风险。拟议的研究将阐明细菌用于整合对细胞包膜和饥饿应激的两种关键反应的机制，这两种反应对于在环境中、感染期间和抗生素治疗期间的生存至关重要。更好地理解这些反应的交叉点可能会为药物发现和疫苗设计带来新的途径

DESCRIPTION (provided by applicant): The ?E cell envelope-sensing pathway is a key regulatory system used by bacteria to maintain cell envelope integrity. ?E is essential for viabiliy of several important bacterial pathogens and required for virulence of others. ?E activity is regulated by two major signaling systems that integrate distinct signals, cell envelope stress and nutrient limitation. In response to envelope stress, ?E is activated by regulated proteolysis of it dedicated antisigma factor. During nutrient limitation ?E is activated by the alarmone ppGpp, in the absence of apparent envelope stress, preparing the envelope for survival in advance. The extent of activation by each system serves to set the overall activity of ?E in the cell, matching t to cellular needs. In addition to activating ?E, ppGpp activates other alternative sigma factors and stress pathways, while repressing genes required for rapid growth. In effect, ppGpp mediates a switch from a transcriptional program for rapid growth to one optimized for stress survival. The mechanism by which ppGpp regulates ?E activity is not wel understood. Such knowledge is critical for our understanding of how this important and highly conserved alternative sigma factor responds to changing environments to maintain cell envelope integrity. The objective of this proposal is to determine the mechanism(s) by which ?E activity is regulated by the global regulator, ppGpp, thereby coordinating envelope and cytoplasmic responses to starvation. The central hypothesis of the proposed research is that ppGpp, in conjunction with the coregulator DksA and potentialy other factors, controls ?E activity by directly activating ?E holoenzyme (E?E) and indirectly by increasing the amount of E?E through its influence on the competition among sigma factors for core RNA polymerase (RNAP). This hypothesis will be addressed through three specific aims. (1) Define the biochemical properties of transcription initiation by E?E. ?E is a member of a highly conserved, widely distributed class of alternative sigma factors that lack several conserved regions and amino acids known to be important for specific steps in transcription initiation by other sigma factors. How these differences alter transcription initiation has not been investigated. The fundamental parameters of transcription initiation by E?E must be established before the influence of pGp can be assessed. (2) Elucidate the mechanism by which ppGpp and DksA regulate E?E activity. Since activation of E?70 and alternative sigma factors by ppGpp/DksA is not well understood, results from the proposed work will have a strong positive impact on the overall understanding of the biochemical basis of transcriptional activation by these global regulators. (3) Determine how ppGpp regulates ?E activity independently of DksA. Preliminary work suggests that a novel mechanism of regulation of ?E by ppGpp exists that cannot be explained by current models, which require DksA to act with ppGpp. This novel regulatory strategy will be explored. Because both the ?E and ppGpp systems are critical for virulence in important bacterial pathogens, a better understanding of the intersection of these responses may lead to new avenues for drug discovery and vaccine design. PUBLIC HEALTH RELEVANCE: Gram-negative bacteria are remarkably successful pathogens and the increasing prevalence of antibiotic resistance in these bacteria presents a significant risk to human health. The proposed studies will elucidate the mechanism used by bacteria to integrate two key responses to cell envelope and starvation stress that are critical for survival i the environment, during infection, and during antibiotic treatment. A better understanding of the intersection of these responses may lead to new avenues for drug discovery and vaccine design