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

项目总结/摘要 CDC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 CDC报告中强调的是多重耐药革兰氏阴性菌（MDR-1）的患病率增加。 GNB）以及开发下一代抗生素来对抗它们的必要性。所有革兰氏阴性菌 依赖于一组同源的，但高度特异性的外膜TonB依赖性转运蛋白（TBDT）， 从环境中输入重要的营养物质，特别是铁等金属，它们被高亲和力结合， 金属螯合化合物称为铁载体。最近的抗生素发展表明， 铁载体-抗生素缀合物可以选择性地靶向特定的细菌，并且这种递送 机制克服了几种关键的抗生素耐药机制。这种传递的一个重要限制是 系统是TBDTs的低表达水平。然而，这些TBDT的一个子集控制着它们自己的 通过上调其自身表达的细胞表面信号传导（CSS）过程表达。很长的- 本研究的长期目标是了解CSS的调控过程，并操纵TBDT的表达 以增强用于治疗MDR-GNB感染的铁载体-抗生素缀合物疗法。研究概述 在这一建议将有助于阐明结构基础CSS的西格玛监管机构。作为一个模型系统， Psuedomonas putida的pseudobactin BN 7/8转运系统，由TBDT、PupB、内毒素、内毒素和外毒素组成， 使用膜σ调节因子PupR和细胞质σ因子PupI。为了实现这一 建议的目标将追求以下三个具体目标：1）建立PupR反σ因子， 结构域二聚化通过PupI影响转录激活，2）鉴定结构决定簇， 描述PupR：PupB周质相互作用对PupR周质C- 末端CSS结构域（CCSSD），和3）确定在全长PupB：PupR CCSSD复合物中的变化， 其同源铁载体假杆菌素BN 7/8的存在和不存在。这些目标将会实现 使用多学科方法;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伏安法。 二色性光谱学这项研究将提供关于σ调节器的关键结构信息;解释 它如何与内膜上的σ因子相互作用，以及周质构象在多大程度上 TBDT和σ-调节因子之间的变化导致蛋白水解降解，这对于控制 转录激活

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.