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性机制。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- 本提案的长期目标是了解CSS调控过程并操纵TBDT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。的结果 该提案将有助于阐明革兰氏阴性细菌σ调节剂CSS的结构基础。作为 模型系统来自恶臭假单胞菌的假杆菌素BN 7/8转运系统，其由TBDT组成， 使用PupB、内膜σ-调节因子PupR和细胞质σ-因子PupI。到 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， 抗σ-因子结构域二聚化通过PupI影响转录激活，2）鉴定结构 决定因素和描绘的作用PupR：PupB周质相互作用的PupR的稳定性 周质C末端CSS结构域（CCSSD），和3）确定全长PupB：PupR CCSSD的变化 在存在和不存在其同源铁载体，假杆菌素BN 7/8的情况下的复合物。为成功 完成这些目标需要多学科的方法;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，如等温滴定 量热法和圆二色光谱法;将采用。这项研究将提供关键的结构 关于σ-调节因子的信息;解释它如何与内膜上的σ-因子相互作用，以及 TBDT和σ-调节子之间的周质构象变化导致蛋白水解， 降解，这是重要的控制转录激活。

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 proposal is to understand the CSS regulatory process and manipulate TBDT expression to enhance siderophore-antibiotic conjugate therapy for treatment of MDR-GNB infections. The results of this proposal will help elucidate the structural basis for CSS by a Gram-negative bacteria sigma-regulator. As a model system 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, 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. For the successful completion of these aims 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; will be employed. 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.