Structure and Function Studies of LL-37 Binding to CsrS of Group A Streptococcus

 描述（由申请人提供）：A组链球菌（GAS）尽管是研究最多的人类病原体之一，但仍会导致显著的发病率和死亡率。除了更常见的口咽或皮肤感染外，病原体还可导致侵袭性感染，如坏死性筋膜炎或链球菌中毒性休克。GAS的CsrRS双组分信号系统被认为在毒力中起主要作用，并调节病原体基因组的10-15%。最近，它表明，LL-37，一种人类抗菌肽，可以矛盾地导致通过这种双组分系统的毒力因子的表达增加。我们有证据表明LL-37和CsrS受体的细胞外结构域之间存在直接的结合相互作用。我们还证明了受体能够独立于胞内结构域二聚化。本研究的目的是扩展这些观察结果，并确定CsrS上LL-37的结合位点。我们还将确定可能的配体诱导的构象变化和二聚体构象对CSRS信号的作用。我们将确定CsrS胞外结构域的结构，其跨膜结构域嵌入在膜纳米盘中，并将这些技术扩展到另外的双组分受体。这些数据将导致更好地理解LL-37的CsrS信号传导的结构和功能，并将为继续研究GAS信号传导机制和潜在的治疗抑制以及一般双组分受体的信号传导奠定基础。 作为拟议培训计划的一部分，短期目标之一是获得细菌信号系统和膜蛋白生物化学和结构生物学研究方面的技能。该项目将在GAS发病机制领域的领导者和专家Michael Wessels博士和膜蛋白结构生物学专家James Chou博士的指导下开发;由细菌发病机制和结构生物学专家组成的科学咨询委员会将提供项目总体方向和职业指导。培训计划将包括结构生物学和晶体学，膜生物化学和计算生物学的课程;以及当地研讨会以及国家和国际会议的教学学习。这项研究将在波士顿儿童医院的Wessels博士和哈佛医学院的Chou博士的实验室进行。哈佛医学院是一个充满活力的研究环境，在拟议研究的各个方面提供多种专业发展和专业知识的机会。波士顿儿童医院也是一个非常支持的环境，并致力于为这项研究提供85%的保护时间。该项目将建立在候选人过去在微生物发病机理和核磁共振光谱学方面的经验基础上，并使他能够从事医学科学家的职业生涯，目标是在儿科传染病部门从事学术事业。

 DESCRIPTION (provided by applicant): Group A Streptococcus (GAS), despite being among the best-studied human pathogens, continues to cause significant morbidity and mortality. The pathogen can result in invasive infections, such as necrotizing fasciitis or streptococcal toxic shock, in addition to the more common oropharyngeal or skin infections. The CsrRS two-component signaling system of GAS is thought to play a major role in virulence and regulates 10-15% of the pathogen's genome. Recently, it was shown that LL-37, a human antimicrobial peptide, can paradoxically lead to increased expression of virulence factors through this two-component system. We have evidence for a direct binding interaction between LL-37 and the extracellular domain of the CsrS receptor. We have also demonstrated that the receptor is able to dimerize independently of the intracellular domain. The goals of this study are to extend these observations and to determine the binding site for LL-37 on CsrS. We will also determine possible ligand-induced conformational changes and the role of the dimer conformation on CsrS signaling. We will determine the structure of the CsrS extracellular domain with its transmembrane domains embedded in a membrane nanodisc and expand these techniques to an additional two-component receptor. These data will lead to a better understanding of the structure and function of CsrS signaling by LL-37 and will establish the foundation for continued studies of GAS signaling mechanisms and potential therapeutic inhibition, and signaling by two-component receptors in general. As part of the proposed training program, one of the short-term goals is to acquire skills in the study of bacterial signaling systems and the biochemistry and structural biology of membrane proteins. The project will be developed under the mentorship of Dr. Michael Wessels, a leader and expert in the field of GAS pathogenesis, and Dr. James Chou, an expert in the structural biology of membrane proteins; a scientific advisory committee composed of experts in bacterial pathogenesis and structural biology will provide advice on overall direction of the project and career guidance. The training program will include coursework in structural biology and crystallography, membrane biochemistry, and computational biology; and didactic learning from local seminars as well as national and international conferences. The research will take place in the laboratories of Dr. Wessels at Boston Children's Hospital and Dr. Chou at Harvard Medical School. The Harvard medical campus is a dynamic research environment with multiple opportunities for professional development and expertise in all aspects of the proposed research. Boston Children's Hospital is also an extremely supportive environment and is committed to providing 85% protected time for this research. The project will build on the candidate's past experiences in microbial pathogenesis and NMR spectroscopy and allow him to pursue a career as a physician-scientist with the goal of an academic career in a pediatric division of infectious diseases.