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

整合细胞包膜和饥饿应激：ppGpp 对 Sigma(E) 的调节

• 批准号: 8895984
• 负责人: SARAH E ADES
• 金额: $ 26.8万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8895984
• 关键词: Address; Affect; Amino Acids; Antibiotic Resistance; Antibiotic Therapy; Bacteria; Biochemical; Cell physiology; Cells; Cellular Structures; Characteristics; DNA-Directed RNA Polymerase; Environment; Escherichia coli; Face; Family; Generations; Genes; Genetic; Genetic Transcription; Goals; Gram-Negative Bacteria; Health; Holoenzymes; Human; Immune; In Vitro; Infection; Kinetics; Knowledge; Laboratories; Lead; Life Style; Mediating; Methods; Modeling; Molecular Genetics; Nutrient; Organism; Pathogenesis; Pathway interactions; Play; Prevalence; Process; Property; Proteolysis; Regulation; Research; Risk; Role; Series; Sigma Factor; Signal Pathway; Signal Transduction; Site; Starvation; Stress; Structure; System; Thermodynamics; Transcription Initiation; Transcriptional Activation; Vaccine Design; Virulence; Work; base; biological adaptation to stress; cell envelope; combat; drug discovery; genetic approach; in vivo; inorganic phosphate; insight; member; novel; pathogen; programs; promoter; rapid growth; response; uptake

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.

描述（由申请人提供）： ?E 细胞包膜感应途径是细菌用来维持细胞包膜完整性的关键调节系统。 ?E 对于几种重要的细菌病原体的生存至关重要，并且是其他细菌病原体的毒力所必需的。 ?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：细胞包膜应激和营养限制。为了响应包膜应激，?E 通过其专用抗西格玛因子的调节蛋白水解而被激活。在营养限制期间，在没有明显的包膜应激的情况下，ΔE 被报警素 ppGpp 激活，提前为包膜的生存做好准备。每个系统的激活程度用于设定细胞中 ΔE 的总体活性，使 t 与细胞的需求相匹配。除了激活 ?E 之外，ppGpp 还激活其他替代 sigma 因子和应激途径，同时抑制快速生长所需的基因。实际上，ppGpp 介导从用于快速生长的转录程序向针对应激生存而优化的转录程序的转变。 ppGpp 调节 ?E 活性的机制尚不清楚。这些知识对于我们理解这一重要且高度保守的替代西格玛因子如何响应不断变化的环境以维持细胞包膜完整性至关重要。该提案的目的是确定全局调节器 ppGpp 调节 ?E 活性的机制，从而协调包膜和细胞质对饥饿的反应。本研究的中心假设是，ppGpp 与辅助调节剂 DksA 和其他潜在因子结合，通过直接激活 ?E 全酶 (E?E) 来控制 ?E 活性，并通过影响 sigma 因子之间对核心 RNA 聚合酶 (RNAP) 的竞争来间接增加 E?E 的量，从而控制 ?E 活性。这一假设将通过三个具体目标来解决。 (1) 定义E?E转录起始的生化特性。 ?E 是高度保守、分布广泛的替代 sigma 因子类别的成员，其缺乏已知对其他 sigma 因子转录起始中的特定步骤很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得到研究。在评估 pGp 的影响之前，必须先确定 E?E 转录起始的基本参数。 (2)阐明ppGpp和DksA调节E?E活性的机制。由于 ppGpp/DksA 对 E?70 和替代 sigma 因子的激活尚不清楚，因此拟议工作的结果将对这些全球调节因子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 如何独立于 DksA 调节 ?E 活性。初步工作表明，存在一种 ppGpp 调节 ?E 的新机制，目前的模型无法解释该机制，该机制需要 DksA 与 ppGpp 共同作用。将探索这种新颖的监管策略。由于 ?E 和 ppGpp 系统对于重要细菌病原体的毒力至关重要，因此更好地了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。