Importance of adhesin-like proteins of Enteroccocus faecium

肠球菌是心内膜炎的第三大常见原因， 一百多年了虽然在过去，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，现在它是最困难的之一 来治疗有机体。最新一代的E.屎肠菌心内膜炎分离株TX 16 （=DO），PI、G. Weinstock和JGI让我们确定了Acm， E.屎室尽管acm存在于所有的屎肠分离物中，但胶原粘附， 仅3/60（5%）的非临床分离株对36/63（57%）的临床分离株显示了由ACM编码的P21（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 从健康人或动物的粪便分离物中的假基因;我们有一个\so表明，acm是完整的，在所有 心内膜炎分离株的研究，迄今为止，有证据表明，在人类感染过程中，即使当 不在体外表达。最近的初步数据发现，我们产生的一种非粘附性acm缺失突变体， 从一个人屎心内膜炎分离与一个新的ts载体，我们构建了这个项目，是减毒的， 它能引起大鼠心内膜炎。我们还确定了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加基序（Ig样）的结构特征的粘附素 折叠），其中三个在具有分选酶样基因的基因座中。重组蛋白 其中两种提取物粘附于纤维蛋白原或纤连蛋白（各一种），与纤连蛋白减少一致 在其中一个中观察到破坏突变。 我们工作的长期目标是翻译与这些基因和ECM相关的体外观察结果 坚持对防治肠球菌感染有用的知识，其中，心内膜炎是最重要的 有问题与Acm相关的具体目的是证实其在动物模型中的重要性;表征Acm的生物学特性; Acm的结合区域及其与Acm的胶原粘附素Ace结合差异的机制 faecalis;调查影响Acm产生的遗传和/或环境条件;确定是否抗- Acm抗体可以防止胶原粘附和/或分离与胶原结合的细菌;并探索被动 和主动免疫，用于预防动物的屎心内膜炎和其他感染。目标 与其他（潜在）的粘附素有关的是使用多方面的方法来了解 原因和相关性的粪便粘附到其他ECM蛋白。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，通过将这些基因克隆入 非粘附宿主，通过测试重组蛋白的体外粘附性，以及用感染患者的血清 这些蛋白质在体内表达的证据，并且，如果时间允许，启动实验，将解决这些问题。 这些粘附素在体内的作用。该提案的主要假设是（1）（至少）其中一些 已证实和假定的粘附素有助于使屎成为心内膜炎和其他疾病的成功原因。 感染，（2）有些是负责“untaming”的医院内粪菌分离株看到，在最近几年， 和（3）更好地了解这些已证实和假定的粘附素将有助于预防 和/或逆转其效果。

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 ¿ 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\soshown 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 ¿ 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 fibrinqgen 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 ¿ 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 ¿ faecium endocarditis and other infections in animals. The aims relating to the other (potential) adhesins of ¿ faecium are to use a multi-faceted approach to understand the causes and relevance of ¿ 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 ¿ faecium a successful cause of endocarditis and other infections, (2) that some are responsible for the "untaming" of nosocomial ¿ 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.