Conformation and functional dynamics of a bacterial PASTA kinase

项目总结/摘要 肠球菌，如粪肠球菌，是通常在肠道中共存的数万亿肠道微生物之一。 与宿主的共生关系然而，耐药肠球菌也是三种最常见的肠球菌之一。 这是医院获得性感染的常见原因，因此代表了严重的公共卫生问题。一个 获得医院获得性肠球菌感染的重要危险因素是既往治疗， 抗生素，如广谱头孢菌素，肠球菌对这些抗生素具有内在抗性。因此 肠球菌在肠道定植和抵抗抗微生物剂攻击的固有能力尤其 对肠球菌感染的发病机理至关重要。然而，肠球菌肠道定植和 抗微生物剂耐药性尚未完全了解。这些内在特征的一个关键决定因素是 称为IreK的跨膜Ser/Thr激酶，其属于细菌激酶家族，包含 胞外PASTA结构域。这种“PASTA激酶”控制关键过程，包括抗生素抗性， 几乎所有革兰氏阳性菌的毒素产生、毒力或细胞分裂;在某些细菌中，PASTA激酶 对生存能力至关重要因此，PASTA激酶代表了新疗法的有吸引力的靶标。但 基本了解PASTA激酶在溶液中的结构和动力学，以及结构-功能 缺乏促进环境感知和体内生物反应协调的关系。 这些信息对于开发新的治疗方法至关重要。这里提出的研究 试图通过阐明基本的结构-功能关系来解决这一差距， 代表性的PASTA激酶，来自E.粪便，我们已经证明，这是必要的内在 抗性E.粪便中存在的细胞壁活性抗生素和洗涤剂。这一协作 研究旨在应用新的方法和观点来阐明新的见解的功能， IreK激酶使用体内功能测定和EPR光谱的互补组合 确定PASTA模块在溶液中构象的方法，并了解激酶结构域如何 动力学有助于激酶活化。通过成功完成这项工作，我们将获得重要的新成果。 深入了解一种代表性的细菌PASTA激酶，以帮助定义一种 关键细菌信号蛋白的整个家族，其长期目标是调节这些蛋白的活性， 激酶治疗。

Project Summary/Abstract Enterococci such as Enterococcus faecalis are among the trillions of intestinal microbes that normally coexist in a symbiotic relationship with their host. However, antibiotic-resistant enterococci are also among the three most common causes of hospital-acquired infections and therefore represent a serious public health problem. An important risk factor for the acquisition of hospital-acquired enterococcal infections is prior therapy with antibiotics, such as broad-spectrum cephalosporins, to which enterococci are intrinsically resistant. Thus, the inherent abilities of enterococci to colonize the gut and withstand assault by antimicrobial agents are especially critical for pathogenesis of enterococcal infections. Yet, the mechanisms of enterococcal gut colonization and antimicrobial resistance are not fully understood. One critical determinant of these intrinsic traits is a transmembrane Ser/Thr kinase known as IreK, which belongs to a family of bacterial kinases containing extracellular PASTA domains. Such “PASTA kinases” control critical processes including antibiotic resistance, toxin production, virulence, or cell division in nearly all Gram-positive bacteria; in some bacteria, a PASTA kinase is essential for viability. As such, PASTA kinases represent attractive targets for new therapeutics. However, a basic understanding of the structure and dynamics of PASTA kinases in solution, and the structure-function relationships that promote environmental sensing and coordination of biological responses in vivo, is lacking. Such information is critical to inform development of new therapeutic approaches. The research proposed here seeks to address this gap by elucidating fundamental structure-function relationships in solution for a representative PASTA kinase, the IreK kinase from E. faecalis, which we have shown is required for intrinsic resistance of E. faecalis to cell-wall-active antibiotics and to detergents present in bile. This collaborative research is designed to apply new approaches and perspectives to elucidate novel insights into the function of the IreK kinase using a complementary combination of in vivo functional assays and EPR spectroscopy approaches to determine the PASTA module conformation in solution and understand how kinase domain dynamics contribute to kinase activation. By successfully completing this work, we will obtain important new insights into a representative bacterial PASTA kinase to help define the structure-function relationships for an entire family of critical bacterial signaling proteins with the long-term goal of modulating the activity of these kinases therapeutically.