Defining the Role of WxL Proteins in Enterococcus faecium

描述(申请人提供)：在过去的25年里，肠球菌已经成为重要的医院病原体，粪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。一些带有锚定LPxTG基序的细胞壁的表面蛋白与肠球菌感染有关。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粪肠球菌引起的腹膜炎感染和Coryniformis乳杆菌的聚集中起重要作用。我的初步数据显示，粪肠球菌的WxL蛋白存在于假定的操纵子中，其中包含一个跨膜蛋白和一个LPxTG蛋白，这表明一种新的表面组装形式的可能性。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粪肠球菌定植和毒力的可能贡献。本研究的目标集中在含有新发现的C-末端区域WxL结构域的蛋白质上。这项研究的具体目的将首先通过观察一些毒力相关功能的体外模型和典型的粪肠球菌感染的体内模型中的突变，来研究WxL蛋白在粪肠球菌致病中的作用。其次，将定义WxL结构域的功能，检查蛋白质的定位，并初步了解假定操纵子内蛋白质之间的相互作用。最后，将分析这些蛋白在体内的表达，确定它们的抗原性，并探索利用WxL蛋白保护粪肠球菌感染的可能性。这项建议包括微生物学和传染病领域，特别是集中在新出现的病原体粪肠球菌上。这项研究将有助于了解这种通常共生的有机体，并探索未来的免疫选择。拟议的研究包括在许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，并应涵盖最后三年的博士培训。 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，但与已知的其他革兰氏阳性病原体相比，缺乏关于这种通常共生的微生物如何引发疾病并与宿主相互作用的研究。评估具有“WxL”基序的表面蛋白在毒力中的重要性、鉴定参与细胞壁结合的结合结构域、同一操纵子编码的蛋白之间可能的蛋白质-蛋白质相互作用、编码WxL蛋白的基因在宿主中的表达以及将WxL蛋白用于粪肠球菌感染免疫的可能性将增强对这种生物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者。

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.