Importance of adhesin-like proteins of Enteroccocus faecium

肠球菌是心内膜炎的第三大常见原因，已知引起这种感染的原因是 一百多年了。虽然在过去，由粪肠球菌引起的感染并不常见，但这种细菌已经 在医院环境中的频率和重要性显著增加，而现在这是最困难的之一 用来治疗有机体。粪便棘球菌心内膜炎分离株TX16基因组序列的新一代 (=DO)，这是PI、G.Weinstock和JGI之间合作的一部分，使我们能够识别主要的ACM 粘附素与粪肠球菌胶原蛋白的结合。尽管所有的粪便分离株都存在ACM，但胶原蛋白的粘附性， 由ACM编码，只有3/60(5%)的非临床分离株与36/63(57%)的临床分离株(p&lt；0.0001)显示。 作为对这些表型差异的部分解释，我们已经表明ACM经常作为一种 在健康人或动物的粪便分离中发现假基因；我们已经证明ACM在所有 到目前为止研究的心内膜炎分离株，有证据表明在人类感染期间ACM表达，即使在 不能在体外表达。最近的初步数据发现，我们产生的一个非粘附性ACM缺失突变体 从我们为本项目构建的新ts载体的人粪便心内膜炎分离株中分离出的 它能引起大鼠的心内膜炎。我们还鉴定了其他14种潜在的细胞外基质(ECM) 粘附素，包括一些具有细胞壁锚定蛋白加模体(类Ig)结构特征的粘附素 折叠)在其他ECM结合蛋白中可见，其中三个位于类似于排序酶基因的区域。重组蛋白 其中的两种提取物附着于纤维蛋白原或纤维连接蛋白(各一种)，与减少的纤维连接蛋白一致。 结合可见与其中一种突变的断裂。 我们工作的长期目标是将与这些基因和细胞外基质相关的体外观察转化为 坚持有助于抗击肠球菌感染的知识，其中心内膜炎是最重要的 这是个问题。与ACM相关的具体目标是确认其在动物模型中的重要性； 酸性粒细胞黏附素结合区(S)及其与胶原粘附素结合差异的机制 粪便；调查影响ACM生产的遗传和/或环境条件；确定抗 ACM抗体可以防止胶原蛋白黏附和/或分离结合在胶原蛋白上的细菌；并探索被动 主动免疫，预防动物的粪便心内膜炎和其他感染。目标 与粪便杆菌的其他(潜在)粘附素相关的是使用多方面的方法来理解 粪便杆菌与其他ECM蛋白粘连的原因及相关性。这些目标将通过以下方式实现 将黏附阳性株(S)的(推定)黏附素基因克隆到 非粘附性宿主(S)，通过检测重组蛋白的体外粘附性和与感染患者血清的粘附性 这些蛋白质在体内表达的证据，以及在时间允许的情况下，启动实验以解决 这些粘附素在体内的作用。这项提议的广泛假设是(1)(至少)其中一些 已证实和推定的粘附素有助于使粪便成为心内膜炎和其他疾病的成功原因 感染，(2)一些人对近年来出现的院内粪便分离菌的“不驯服”负有责任， 以及(3)更好地了解这些已确认的和推定的粘附素将有助于找到预防 和/或逆转它们的影响。

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.