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Conformation and functional dynamics of a bacterial PASTA kinase

细菌 PASTA 激酶的构象和功能动力学

基本信息

批准号：
10526288
负责人：
CHRISTOPHER J KRISTICH
金额：
$ 33.88万
依托单位：
MEDICAL COLLEGE OF WISCONSIN
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-01 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10526288
关键词：
Address Adopted Antibiotic Resistance Antibiotics Antimicrobial Resistance Bacteria Bile fluid Binding Biological Biological Adaptation Biological Assay Biology Cell Wall Cell division Cephalosporins Cytoplasm Cytoplasmic Tail Detergents Development Dimerization Disulfides Electron Spin Resonance Spectroscopy Enterococcus Enterococcus faecalis Family Foundations Goals Gram-Positive Bacteria Hospitals Impairment In Vitro Individual Infection Knowledge Length Mediating Membrane Molecular Molecular Conformation Mutagenesis Mutation NMR Spectroscopy Nature Nosocomial Infections Pathogenesis Pathway interactions Phosphorylation Phosphotransferases Positioning Attribute Prior Therapy Process Production Protein Family Public Health Regulation Reporter Research Resistance Risk Factors Role Scheme Signaling Protein Site Staphylococcus aureus Stimulus Structure Structure-Activity Relationship Therapeutic Toxin Transmembrane Domain Virulence Work X-Ray Crystallography acquired factor antimicrobial antimicrobial drug assault colonization resistance crosslink design extracellular genetic selection gut colonization gut microbiota in vivo innovation insight member mutant new therapeutic target novel novel strategies novel therapeutic intervention pathogen pathogenic bacteria response trait

项目摘要

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.

项目摘要/摘要粪肠球菌等肠球菌是数万亿种肠道微生物中通常共存于与寄主的共生关系。然而，对抗生素具有抗药性的肠球菌也是最多的三种这是医院获得性感染的常见原因，因此是一个严重的公共卫生问题。一个医院获得性肠球菌感染的重要危险因素是事先接受过肠球菌天生耐药的抗生素，如广谱头孢菌素。因此，肠球菌固有的定植肠道和抵抗抗菌剂攻击的能力尤为突出。对肠球菌感染的发病机制至关重要。然而，肠球菌肠道定植的机制和对抗菌素的耐药性还没有完全了解。这些内在特征的一个关键决定因素是跨膜型丝氨酸/苏氨酸蛋白激酶，又称Irek，属于细菌蛋白激酶家族，含有细胞外的意大利面域。这种“面食蛋白激酶”控制着包括抗生素耐药性在内的关键过程，几乎所有革兰氏阳性细菌中的毒素产生、毒力或细胞分裂；在某些细菌中，是一种面食酵素对于生存来说是必不可少的。因此，意大利面激酶是新疗法的诱人靶点。然而，a 基本了解意大利面条蛋白在溶液中的结构和动力学，以及其结构和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。在这里提出的这项研究寻求通过阐明解决方案中的基本结构-功能关系来解决这一差距具有代表性的意大利面激酶，来自粪肠球菌的Irek激酶，我们已经证明了它是内在的粪肠球菌对细胞壁活性抗生素和胆汁中存在的洗涤剂的耐药性。这种协作性研究旨在应用新的方法和视角来阐明对体内功能分析和EPR波谱互补结合的Irek激酶确定溶液中的意大利面模块构象和了解激活域的方法动力学有助于激活剂的激活。通过圆满完成这项工作，我们将获得重要的新的对具有代表性的细菌面蛋白激酶的洞察，以帮助确定其结构-功能关系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治疗上的激活酶。