Mechanism of inner membrane sigma-regulator function in Gram-negative bacteria

描述(由申请人提供)：疾控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。疾控中心报告中特别强调的是，耐多药革兰氏阴性细菌(MDR-GNB)的流行率增加，需要开发下一代抗生素来对抗它们。所有的革兰氏阴性细菌都依赖一组同源但高度特异的外膜TonB依赖转运蛋白(TBDs)从他们的环境中输入关键营养物质，特别是铁等金属，这些金属被称为铁载体的高亲和力金属螯合化合物结合在一起。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，并且这种传递机制克服了几种关键的抗生素耐药性机制。然而，这种递送系统的一个显著限制是TBDTs的低表达水平。这项建议的长期目标是提供对革兰氏阴性细菌如何转录调节其TBDT的机械性理解，以便操纵这一过程，选择性上调TBDT水平，并加强铁载体-抗生素结合疗法治疗MDR-GNB感染。在这个提案中，我们将阐明蛋白质相互作用事件的结构基础，这些事件负责上调特定TBDTs的转录。作为一个模型系统，我们使用了恶臭假单胞菌的假杆菌蛋白BN7/8转运系统，该系统由TbdT、PupB、内膜σ调节因子PupR和细胞质σ因子Pupi组成。为了实现我们的目标，我们将追求以下两个特定的目标：1)描述内膜细胞质表面的PupR：PupI相互作用被PupI转录激活的机制，以及2)建立PupB和PupR之间相互作用的热力学和原子水平的结构细节。为了成功实现我们的目标，我们将采用多学科方法，包括核磁共振光谱学、X射线结晶学、分子生物学、细胞分析和生物物理技术，如等温滴定量热法。本研究将提供σ-调节子的第一个结构信息，解释σ-因子在内膜上的定位如何限制其活性，以及σ-因子与TbDT-调节子之间的周质相互作用在多大程度上导致构象变化，这可能是控制转录激活的重要因素。

DESCRIPTION (provided by applicant): 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. However, a significant limitation of this delivery system is the low expression levels of the TBDTs. The long-term objective of this proposal is to provide a mechanistic understanding of how Gram-negative bacteria transcriptionally regulate their TBDTs in order to manipulate this process to selectively up-regulate TBDT levels and enhance siderophore-antibiotic conjugate therapy for treatment of MDR-GNB infections. In this proposal we will elucidate the structural basis for protein interaction events that are responsible for up-regulatin the transcription of particular TBDTs. As a model system we are using the pseudobactin BN7/8 transport system from Psuedomonas putida that consists of the TBDT, PupB, the inner membrane σ-regulator, PupR, and the cytoplasmic σ-factor, PupI. To accomplish our objective we will pursue the following two specific aims: 1) delineate the mechanism by which the PupR:PupI interaction at the cytoplasmic face of the inner membrane is altered to allow transcriptional activation by PupI, and 2) establish the thermodynamics and atomic-level structural details of the interaction between the PupB and PupR. For the successful completion of our aims we will employ a multidisciplinary approach including NMR spectroscopy, X-ray crystallography, molecular biology, cellular assays, and biophysical techniques such as isothermal titration calorimetry. This research will provide the first structural information for a σ-regulator, explain how localization of a σ-factor to the inner membrane limits its activity, and th extent to which periplasmic interactions between the TBDT and σ-regulator lead to conformational changes that might be important for controlling transcriptional activation.