Importance of adhesin-like proteins of Enteroccocus faecium

屎肠球菌粘附素样蛋白的重要性

• 批准号: 7022475
• 负责人: BARBARA E MURRAY
• 金额: $ 37.13万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7022475
• 关键词: Enterococcus; adhesin; bacterial endocarditis; bacterial genetics; bacterial proteins; extracellular matrix; laboratory mouse; laboratory rat; pathologic process; peritonitis; recombinant proteins; virulence

DESCRIPTION (provided by applicant): Enterococci are the third most common cause of endocarditis and have been known to cause this infection for over 100 years. While, in the past, infections due to Enterococcus faecium were infrequent, this organism has increased markedly in frequency and in importance in the hospital setting where it is now one of the most difficult to treat organisms. The recent generation of the genome sequence of our E. faecium endocarditis isolate TX16 (=DO), part of a collaboration between the PI, G. Weinstock and the JGI, allowed us to identify Acm, the primary adhesin to collagen of E. faecium. Although acm is present in all E faecium isolates, collagen adherence, encoded by acm, is displayed by only 3/60 (5%) non-clinical isolates vs. 36/63 (57%) clinical isolates (p < 0.0001). As a partial explanation for these phenotypic differences, we have shown that acm is frequently present as a pseudogene in fecal isolates from healthy humans or animals; we have a\so shown that acm is intact in all endocarditis isolates studied to date, and there is evidence of Acm expression during infections in man, even when not expressed in vitro. Recent preliminary data found that a non-adhering acm deletion mutant that we generated from a human E faecium endocarditis isolate with a new ts vector we constructed for this project, is attenuated in its ability to cause endocarditis in rats. We have also identified 14 other potential extracellular matrix (ECM) adhesins including some with structural features characteristic of cell-wall anchored proteins plus motifs (Ig-like folds) seen in other ECM binding proteins, three of which are in loci with sortase-like genes. Recombinant protein extracts of two of these adhere to fibrinogen or fibronectin (one each), consistent with decreased fibronectin binding seen with a disruption mutation in one. The long term goals of our work are to translate in vitro observations relating to these genes and ECM adherence into knowledge useful for combating enterococcal infections, among which, endocarditis is the most problematic. Specific Aims related to Acm are to confirm its importance in animal models; to characterize the binding region(s) of Acm and the mechanism for its differences in binding vs. the collagen adhesin Ace of E faecalis; to investigate genetic and/or environmental conditions that influence Acm production; to determine if anti-Acm antibodies can prevent collagen adherence and/or detach bacteria bound to collagen; and to explore passive and active immunization for prevention of E faecium endocarditis and other infections in animals. The aims relating to the other (potential) adhesins of E faecium are to use a multi-faceted approach to understand the causes and relevance of E faecium adherence to other ECM proteins. These aims will be addressed by introducing mutations into the (putative) adhesin genes of adherence-positive strain(s), by cloning these genes into non-adherent host(s), by testing recombinant proteins for in vitro adherence and with infected patients' sera for evidence of in vivo expression of these proteins and, time permitting, initiating experiments that would address the role of these adhesins in vivo. The broad hypotheses of this proposal are (1) that (at least) some of these confirmed and putative adhesins contribute to making E faecium a successful cause of endocarditis and other infections, (2) that some are responsible for the "untaming" of nosocomial E faecium isolates seen in recent years, and (3) that a better understanding of these confirmed and putative adhesins will help lead to modalities to prevent and/or reverse their effects.

描述（由申请人提供）：肠球菌是心内膜炎的第三大常见原因，并且已经知道引起这种感染已有100多年的历史。虽然在过去，由粪肠球菌引起的感染并不常见，但这种生物在医院环境中的频率和重要性显著增加，现在它是最难治疗的生物之一。我们的粪肠心内膜炎分离物TX16 （=DO）的最新一代基因组序列，是PI， G. Weinstock和JGI合作的一部分，使我们能够鉴定出粪肠胶原蛋白的主要粘附素Acm。虽然acm存在于所有粪肠杆菌分离株中，但由acm编码的胶原粘附性仅在3/60（5%）非临床分离株中表现出来，而在36/63（57%）临床分离株中表现出来（p < 0.0001）。作为对这些表型差异的部分解释，我们已经表明，acm经常作为假基因存在于健康人类或动物的粪便分离物中；我们的研究表明，acm在迄今为止研究的所有心内膜炎分离株中都是完整的，并且有证据表明，acm在人类感染期间表达，即使在体外不表达。最近的初步数据发现，我们用为本项目构建的新ts载体从人粪肠菌心内膜炎分离物中产生的非粘附性acm缺失突变体在大鼠中引起心内膜炎的能力减弱。我们还发现了14种其他潜在的细胞外基质（ECM）粘附素，包括一些具有细胞壁锚定蛋白和其他ECM结合蛋白中所见的基序（igg样折叠）的结构特征，其中3种位于排序蛋白样基因的位点。其中两个的重组蛋白提取物粘附在纤维蛋白原或纤维连接蛋白上（每个一个），与纤维连接蛋白结合减少一致，其中一个出现断裂突变。