Defining the Role of WxL Proteins in Enterococcus faecium

描述（由申请人提供）：肠球菌在过去25年中已成为重要的医院病原体，屎肠球菌感染最近从不到临床分离株的10%增加到引起所有肠球菌感染的三分之一以上。具有细胞壁锚定LPxTG基序的几种表面蛋白与肠球菌感染相关。最近，研究人员指出了一个新的细胞壁结合基序的可能性，命名为WxL结构域。含有该结构域的蛋白质在由粪肠球菌引起的腹膜炎感染和棒状乳杆菌中的聚集中具有重要意义。我的初步数据表明，E.屎存在于假定的操纵子含有跨膜蛋白和LPxTG蛋白，表明一种新形式的表面组装的可能性。这项建议的长期目标是更好地了解细胞壁相关蛋白及其对屎肠球菌定殖和毒力的可能贡献。本研究的目标集中在蛋白质含有新确定的WXL结构域的C-末端区域。本研究的具体目标是首先研究WxL蛋白在大肠杆菌中的作用。通过观察一些体外毒力相关功能模型和屎肠球菌引起的典型感染的体内模型中的突变体，研究屎肠球菌的致病机制。其次，将定义WxL结构域的功能，检查蛋白质的定位，并初步了解推定的操纵子内的蛋白质之间的相互作用。最后，分析这些蛋白在体内的表达，确定其抗原性，并探讨其抗大肠杆菌的可能性。将探索使用WxL蛋白的屎肠菌感染。该提案包括微生物学和传染病领域，特别是集中在新兴的病原体E。屎室这项研究将有助于了解这种正常的微生物，以及探索未来的免疫选择。拟议的研究包括在众多领域使用的各种技术，包括分子生物学，遗传学，生物化学，免疫学和结构生物学，并应涵盖博士学位的最后三年的持续时间。训练 公共卫生相关性：虽然屎肠球菌是一种重要的医院相关病原体，但与其他革兰氏阳性病原体的已知信息相比，缺乏关于这种正常肠道微生物如何引发疾病并与宿主相互作用的研究。本文对具有WxL基序的表面蛋白在大肠杆菌毒力中的重要性、参与细胞壁结合的结合结构域的鉴定、同一操纵子编码的蛋白质之间可能的相互作用、WxL蛋白基因在宿主体内的表达以及WxL蛋白用于大肠杆菌免疫的可能性进行了探讨。屎菌感染将增强人们对这种生物体致病机制的非常有限的了解，并可能为预防肠球菌感染提供可能的免疫候选物。

DESCRIPTION (provided by applicant): Enterococci have become important nosocomial pathogens over the past 25 years, with Enterococcus faecium infections recently increasing from less than ten percent of clinical isolates to causing over one-third of all enterococcal infections. Several surface proteins with a cell wall anchoring LPxTG motif are associated with enterococcal infection. Recently, researchers have indicated the probability of a new cell wall binding motif, designated the WxL domain. Proteins containing this domain have been implicated as important in peritonitis infections caused by Enterococcus faecalis and aggregation in Lactobacillus coryniformis. My preliminary data have shown that the WxL proteins of E. faecium exist in putative operons containing a transmembrane protein and an LPxTG protein, indicating the possibility of a novel form of surface assembly. The long term goal of this proposal is to better understand cell wall associated proteins and their possible contribution to colonization and virulence in Enterococcus faecium. The goals of this study concentrate on proteins containing the newly identified WxL domain at the C-terminal region. The specific aims of the proposed study will first investigate the role of WxL proteins in E. faecium pathogenesis by looking at mutants in a number of in vitro models of virulence related functions and in vivo models of typical infections caused by Enterococcus faecium. Second, the function of the WxL domain will be defined, the localization of the proteins examined, and an initial understanding of the interactions between the proteins within the putative operons will be gained. Lastly, the in vivo expression of these proteins will be analyzed, their antigenicity determined, and the possibility of protecting against E. faecium infection using WxL proteins will be explored. This proposal encompasses the microbiology and infectious disease fields, specifically concentrating on the emerging pathogen E. faecium. This study will contribute to the knowledge of this normally commensal organism, as well as explore future options for immunization. The proposed study includes a variety of techniques used in numerous fields including molecular biology, genetics, biochemistry, immunology, and structural biology and should cover the duration of the last three years of Ph.D. training. PUBLIC HEALTH RELEVANCE: Although Enterococcus faecium is an important hospital-associated pathogen, research on how this normally commensal organism initiates disease and interacts with the host is lacking when compared to the information known about other gram positive pathogens. Assessment of the importance of putative surface proteins with the "WxL" motif in virulence, identification of the binding domains involved in cell wall association, possible protein-protein interactions between proteins encoded in the same operon, expression of the genes encoding WxL proteins within the host, and the possibility of using the WxL proteins in immunization for E. faecium infections will enhance the very limited knowledge of the mechanisms for pathogenicity of this organism, and may provide for possible immunization candidates for the prevention of enterococcal infections.