Importance of adhesin-like proteins of Enteroccocus faecium

描述（由申请人提供）：肠球菌是心内膜炎的第三大常见原因，已知引起这种感染已有100多年的历史。虽然在过去，屎肠球菌引起的感染并不常见，但这种微生物在医院环境中的频率和重要性显着增加，现在它是最难治疗的微生物之一。最新一代的E.屎肠菌心内膜炎分离株TX 16（=DO），PI，G. Weinstock和JGI，使我们能够鉴定出Acm，即E.屎室尽管acm存在于所有屎肠球菌分离株中，但仅3/60（5%）非临床分离株与36/63（57%）临床分离株显示acm编码的胶原粘附（p < 0.0001）。作为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或动物的粪便分离株中;我们已经证明acm在迄今为止研究的所有心内膜炎分离株中是完整的，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，我们从人屎肠球菌心内膜炎分离株中产生的非粘附acm缺失突变体与我们为此项目构建的新ts载体在其引起大鼠心内膜炎的能力方面减弱。我们还确定了14个其他潜在的细胞外基质（ECM）粘附素，包括一些结构特征的细胞壁锚定蛋白加上其他ECM结合蛋白中看到的基序（Ig样折叠），其中三个是在位点与分选酶样基因。其中两种重组蛋白提取物粘附于纤维蛋白原或纤连蛋白（各一种），这与在其中一种中观察到的破坏突变的纤连蛋白结合减少一致。 我们工作的长期目标是将与这些基因和ECM粘附相关的体外观察转化为对对抗肠球菌感染有用的知识，其中，心内膜炎是最有问题的。与Acm相关的具体目的是确认其在动物模型中的重要性;表征Acm的结合区域及其与粪肠球菌的胶原粘附素Ace结合差异的机制;研究影响Acm产生的遗传和/或环境条件;确定抗Acm抗体是否可以防止胶原粘附和/或分离与胶原结合的细菌;探索预防动物屎肠球菌心内膜炎等感染的被动免疫和主动免疫方法。与屎肠球菌的其他（潜在）粘附素相关的目的是使用多方面的方法来了解屎肠球菌粘附其他ECM蛋白的原因和相关性。这些目标将通过以下方式实现：将突变引入粘附阳性菌株的（推定的）粘附素基因中，将这些基因克隆到非粘附宿主中，测试重组蛋白的体外粘附性，并使用感染患者血清检测这些蛋白的体内表达证据，以及在时间允许的情况下，启动实验以阐明这些粘附素在体内的作用。这一建议的主要假设是：（1）（至少）这些已证实和推定的粘附素中的一些有助于使屎肠球菌成为心内膜炎和其他感染的成功原因，（2）一些粘附素负责近年来所见的医院内屎肠球菌分离株的“未驯服”，以及（3）更好地理解这些已证实和推定的粘附素将有助于导致预防和/或逆转其作用的方式。

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.