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: 10004679
• 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 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10004679
• 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.

项目摘要/摘要 疾病预防控制中心最近发布了一份报告，详细介绍了美国抗生素抗性威胁。特别 CDC报告中的重点是抗多药，革兰氏阴性细菌的患病率增加（MDR- GNB）以及开发下一代抗生素以对抗它们的需求。所有革兰氏阴性细菌 依靠一组同源但高度特异性的外膜TONB依赖性转运蛋白（TBDT） 从其环境中进口关键营养物质，尤其是由高亲和力约束的铁等金属， 金属螯合化合物称为铁载体。最近的抗生素发展表明 可以选择性地靶向特定细菌，而这种递送可以选择性地靶向侧虫 - 抗生素结合物 机制克服了几种关键的抗生素抗性机制。此交付的重大限制 系统是TBDT的低表达水平。但是，这些TBDT的子集控制着自己的 通过细胞表面信号传导（CSS）进行表达，从而在上调了自己的表达。长期 这项研究的术语目标是了解CSS调节过程并操纵TBDT表达 为了增强铁载体 - 抗生素结合疗法，以治疗MDR-GNB感染。研究概述了 在此提案中，将有助于阐明Sigma-Regulator的CSS结构基础。作为模型系统， pseudobactin bn7/8 putida的传输系统，由tbdt，pupb组成 正在使用膜σ调节剂，PUPR和细胞质σ因子Pupi。实现这一目标 提案的目标将实现以下三个特定目标：1）确定PUPR抗σ因子 域二聚化影响pupi的转录激活，2）确定结构确定器和 描述了pUPR：pupb的周围相互作用对pupr阴性c-稳定性的作用 端子CSS域（CCSSD），3）确定全长PUPB的变化：PUPR CCSSD复合物 其同源辅助载体的存在和不存在Pseudobactin BN7/8。这些目标将实现 使用多学科的方法；包括X射线晶体学，小角度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圆形 二分色谱法。这项研究将提供有关σ调节器的关键结构信息；解释 它如何与内膜处的σ因子相互作用，以及周围构象的程度 TBDT和σ调节剂之间的变化导致蛋白水解降解，这对于控制很重要 转录激活。