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

描述（由申请人提供）：美国疾病控制与预防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。疾病预防控制中心的报告特别强调了耐多药革兰氏阴性菌（MDR- GNB）流行率的增加以及开发下一代抗生素以对抗它们的必要性。所有革兰氏阴性菌都依赖于一组同源的、高度特异性的外膜转运蛋白（TBDTs）从环境中进口关键营养物质，尤其是铁等金属，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。然而，这种递送系统的一个重要限制是TBDTs的低表达水平。本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，以便操纵这一过程选择性上调TBDT水平，并加强铁载体-抗生素结合治疗耐多药gnb感染。在这个建议中，我们将阐明负责上调特定tbdt转录的蛋白质相互作用事件的结构基础。我们使用恶臭假单胞菌的假bactin BN7/8转运系统作为模型系统，该系统由TBDT、pub、细胞膜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目标：1)描述PupR:PupI在细胞膜细胞质表面的相互作用被改变以允许PupI的转录激活的机制；2)建立PupR和PupR之间相互作用的热力学和原子水平结构细节。为了成功完成我们的目标，我们将采用多学科方法，包括核磁共振波谱，x射线晶体学，分子生物学，细胞分析和生物物理技术，如等温滴定量热法。本研究将提供第一个关于σ-调节因子的结构信息，解释σ-因子在细胞膜上的定位如何限制其活性，以及TBDT和σ-调节因子之间的质周相互作用在多大程度上导致构象变化，这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.