Structural basis for cell surface siganling by a Gram-negative bacteria sigma-regulator

革兰氏阴性菌西格玛调节器细胞表面信号的结构基础

• 批准号: 10240569
• 负责人: Christopher L Colbert
• 金额: $ 29万
• 依托单位: NORTH DAKOTA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-20 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10240569
• 关键词: Adjuvant; Affinity; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Antibiotic Resistance; Bacterial Infections; Bacteriophages; Biological Models; C-terminal; Calorimetry; Cause of Death; Cell surface; Cells; Cellular Assay; Centers for Disease Control and Prevention (U.S.); Cessation of life; Circular Dichroism Spectroscopy; Complex; Cyclic AMP Receptor Protein; Development; Diarrhea; Dimerization; Disease; Drug Delivery Systems; Environment; Equilibrium; Equus caballus; Escherichia coli; Future; Gastrointestinal tract structure; Genetic Transcription; Gram-Negative Bacteria; Health; Health Care Costs; Heavy Metals; Hospitals; Human; Incidence; Iron; Lead; Length; Length of Stay; Livestock; Membrane; Metals; Microbial Biofilms; Modernization; Molecular Biology; Molecular Conformation; Multi-Drug Resistance; Natural Products; Nutrient; Pathogenicity; Pharmacology; Population; Prevalence; Primary Infection; Process; Pseudomonas aeruginosa; Pump; Recovery; Regulation; Reporting; Research; Resistance; Resources; Roentgen Rays; Role; Siderophores; Sigma Factor; Signal Transduction; Specificity; Stimulus; Stomach; Structure; System; Therapeutic; Time; Titrations; Toxic effect; Transcriptional Activation; Transcriptional Regulation; Up-Regulation; Vancomycin; X-Ray Crystallography; antimicrobial; bacteriocin; beta-Lactams; biophysical techniques; carbapenem-resistant Enterobacteriaceae; combat; cystic fibrosis infection; cystic fibrosis patients; design; disability; efflux pump; experimental study; gastrointestinal; health care settings; improved; interdisciplinary approach; interest; metal chelator; microorganism; multidrug-resistant Pseudomonas aeruginosa; next generation; novel; novel therapeutics; pathogen; pathogenic bacteria; periplasm; pressure; prevent; receptor; resistance mechanism; secondary infection; side effect; targeted delivery; uptake

PROJECT SUMMARY/ABSTRACT The CDC recently released a report detailing antibiotic resistant threats in the US. Of particular emphasis in the CDC report is the increased prevalence of multidrug-resistant, Gram-negative bacteria (MDR- GNB) and the need to develop the next generation of antibiotics to combat them. All Gram-negative bacteria rely on a set of homologous, yet highly-specific, outer membrane TonB-dependent transporters (TBDTs) to import critical nutrients from their environment, especially metals like iron, which are bound by high-affinity, metal chelating compounds called siderophores. Recent antibiotic developments have shown that siderophore-antibiotic conjugates can be selectively targeted to specific bacteria, and that this delivery mechanism overcomes several key antibiotic resistance mechanisms. A significant limitation of this delivery system is the low expression levels of the TBDTs. However, a subset of these TBDTs controls their own expression through a cell-surface signaling (CSS) process that up-regulates their own expression. The long- term objective of this research is to understand the CSS regulatory process and manipulate TBDT expression to enhance siderophore-antibiotic conjugate therapy for treatment of MDR-GNB infections. Research outlined in this proposal will help elucidate the structural basis for CSS by a sigma-regulator. As a model system, the pseudobactin BN7/8 transport system of Psuedomonas putida, which consists of the TBDT, PupB, the inner membrane σ-regulator, PupR, and the cytoplasmic σ-factor, PupI, is being used. To accomplish this proposal's objective the following three specific aims will be pursued: 1) establish that PupR anti-σ-factor domain dimerization influences transcriptional activation by PupI, 2) identify the structural determinants and delineate the role of the PupR:PupB periplasmic interactions on the stability of the PupR periplasmic C- terminal CSS domain (CCSSD), and 3) determine changes in the full-length PupB:PupR CCSSD complex in the presence and absence of its cognate siderophore, pseudobactin BN7/8. These aims will be accomplished using a multidisciplinary approach; including X-ray crystallography, small-angle X-ray scattering, molecular biology, cellular assays, and biophysical techniques such as isothermal titration calorimetry and circular dichroism spectroscopy. This research will provide critical structural information about a σ-regulator; explain how it interacts with a σ-factor at the inner membrane, and the extent to which periplasmic conformational changes between the TBDT and σ-regulator lead to proteolytic degradation that is important for controlling transcriptional activation.

项目概要/摘要 疾病预防控制中心最近发布了一份报告，详细介绍了美国的抗生素耐药性威胁。特别是 CDC 报告强调的是多重耐药革兰氏阴性菌（MDR- GNB）以及开发下一代抗生素来对抗它们的需要。所有革兰氏阴性细菌 依赖于一组同源但高度特异性的外膜 TonB 依赖性转运蛋白 (TBDT) 从环境中输入关键的营养物质，特别是像铁这样的金属，它们通过高亲和力结合在一起， 称为铁载体的金属螯合化合物。最近抗生素的发展表明 铁载体-抗生素缀合物可以选择性地靶向特定细菌，并且这种递送 机制克服了几个关键的抗生素耐药机制。这种交付的一个重大限制 系统中TBDTs的表达水平较低。然而，这些 TBDT 的一个子集控制着它们自己的 通过细胞表面信号传导（CSS）过程上调其自身表达。长- 本研究的长期目标是了解 CSS 调控过程并操纵 TBDT 表达 增强铁载体-抗生素结合疗法治疗 MDR-GNB 感染。研究概述 该提案中的内容将有助于通过 sigma 调节器阐明 CSS 的结构基础。作为一个模型系统， 恶臭假单胞菌的假杆菌素BN7/8转运系统，由TBDT、PupB、内 使用膜 σ 调节因子 PupR 和细胞质 σ 因子 PupI。为了实现这一点 提案的目标 将追求以下三个具体目标： 1) 确定 PupR 抗 σ 因子 结构域二聚化影响 PupI 的转录激活，2) 识别结构决定因素和 描述 PupR:PupB 周质相互作用对 PupR 周质 C-稳定性的作用 终端 CSS 域 (CCSSD)，以及 3) 确定全长 PupB:PupR CCSSD 复合体的变化 其同源铁载体假杆菌素 BN7/8 的存在和不存在。这些目标将会实现 采用多学科方法；包括X射线晶体学、小角X射线散射、分子 生物学、细胞测定和生物物理技术，例如等温滴定量热法和循环法 二色性光谱。这项研究将提供有关 σ 调节器的关键结构信息；解释 它如何与内膜的 σ 因子相互作用，以及周质构象的程度 TBDT 和 σ 调节剂之间的变化会导致蛋白水解降解，这对于控制蛋白水解很重要 转录激活。