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

描述（由申请人提供）：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。CDC报告中特别强调的是多重耐药革兰氏阴性细菌（MDR-GNB）的流行率增加，以及开发下一代抗生素来对抗它们的必要性。所有革兰氏阴性细菌都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）从其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。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性机制。然而，该递送系统的显著限制是TBDT的低表达水平。该提案的长期目标是提供革兰氏阴性菌如何转录调节其TBDT的机制理解，以便操纵该过程以选择性地上调TBDT水平并增强用于治疗MDR-GNB感染的铁载体-抗生素缀合物疗法。在这个提议中，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TBDT的转录。作为模型系统，我们使用来自恶臭假单胞菌的假杆菌素BN 7/8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具体的目标：1）描绘的机制，通过该机制PupR：PupI在内膜的细胞质面的相互作用被改变，以允许转录激活PupI，和2）建立热力学和原子水平的结构细节的PupB和PupR之间的相互作用。为了成功完成我们的目标，我们将采用多学科的方法，包括NMR光谱学，X射线晶体学，分子生物学，细胞测定和生物物理技术，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-因子在内膜的定位如何限制其活性，以及TBDT和σ-调节子之间的周质相互作用导致构象变化的程度，这些构象变化可能对控制转录激活很重要。

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.