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

项目摘要/摘要 美国疾病控制与预防中心最近发布了一份报告，详细描述了美国的抗生素耐药性威胁。特别的 疾控中心报告中的重点是多重耐药的革兰氏阴性细菌(MDR-细菌)的流行增加。 (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 依赖于一组同源但高度特异的外膜TonB依赖转运蛋白(TBDTs)来 从它们的环境中进口关键的营养物质，特别是铁等金属，它们被高亲和力结合在一起， 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-抗生素结合物可以选择性地针对特定的细菌，这种传递 该机制克服了几个关键的抗生素耐药机制。这次交付的一个重要限制是 TbDts的低表达水平是该系统的一个重要特征。然而，这些TBDT的子集控制着它们自己的 通过细胞表面信号传递(CSS)过程上调自身的表达。长的- 本研究的术语目标是了解css的调控过程并操纵tbdt的表达。 加强铁载体-抗生素联合治疗MDR-GNB感染。研究概述 这一建议将有助于阐明西格玛调节器对css的结构基础。作为一个模范系统， 恶臭假单胞菌的假细菌蛋白BN7/8转运系统，由TBDT、PupB、内 正在使用膜σ调节因子PupR和细胞质σ因子Pupi。要做到这一点 提案的目标将追求以下三个具体目标：1)建立PupR抗σ-因子 结构域二聚化影响PUPI的转录激活，2)确定结构决定因素和 PupR：PupB周质相互作用对PupR周质C-稳定性的影响 终端CSS域(CCSSD)，以及3)确定全长PupB：PupR CCSSD复合体的变化 它的同源铁载体BN7/8的存在和不存在。这些目标将被实现 使用多学科方法；包括X射线结晶学、小角X射线散射、分子 生物学、细胞分析和生物物理技术，如等温滴定量热法和循环 二向色性光谱。这项研究将提供有关σ调节器的关键结构信息；解释 它是如何与内膜上的σ因子相互作用的，以及周质构象的程度 Tbdt和σ-调节子之间的变化导致蛋白质降解，这对控制是重要的 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.