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，100多年来一直被认为是导致这种感染的原因。虽然在过去，由粪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已显著增加，现在它是最难治疗的微生物之一。最近产生的粪肠球菌心内膜炎分离株TX16(=DO)的基因组序列是PI，G.Weinstock和JGI合作的一部分，使我们能够鉴定出ACM，它是屎肠球菌胶原蛋白的主要粘附素。虽然在所有的粪肠球菌分离株中都存在acm，但只有3/60(5%)的非临床分离株与36/63(57%)的临床分离株表现出由acm编码的胶原粘附性(p&lt；0.0001)。作为这些表型差异的部分解释，我们已经表明ACM经常以假基因的形式存在于健康人或动物的粪便分离中；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，即使在体外没有表达。最近的初步数据发现，我们用为本项目构建的新的ts载体从人粪便埃希菌心内膜炎分离株中产生的一个非粘附性ACM缺失突变体，其引起大鼠心内膜炎的能力减弱。我们还鉴定了其他14个潜在的细胞外基质(ECM)粘附素，其中一些具有细胞壁锚定蛋白的结构特征加上其他ECM结合蛋白中的基序(Ig样折叠)，其中三个位于索尔特酶样基因的基因座上。其中两个的重组蛋白提取物附着于纤维蛋白原或纤维连接蛋白(各一个)，与纤维连接蛋白结合减少一致，其中一个出现中断突变。 我们工作的长期目标是将与这些基因和ECM黏附相关的体外观察转化为有助于对抗肠球菌感染的知识，其中心内膜炎是最有问题的。与ACM相关的具体目的是确定其在动物模型中的重要性；确定ACM的结合区(S)及其与粪肠球菌的胶原粘附素A的结合差异的机制；调查影响ACM产生的遗传和/或环境条件；确定抗ACM抗体是否可以阻止胶原粘连和/或分离与胶原结合的细菌；以及探索被动和主动免疫以预防动物的粪肠球菌心内膜炎和其他感染。与其他(潜在的)粪肠球菌粘附素相关的目的是使用多方面的方法来了解粪肠球菌与其他ECM蛋白黏附的原因和相关性。为了实现这些目标，将在黏附阳性菌株(S)的(推定)黏附素基因中引入突变，通过将这些基因克隆到非黏附宿主(S)中，通过测试重组蛋白的体外黏附性能并与感染患者的血清一起寻找这些蛋白质在体内表达的证据，以及在时间允许的情况下，启动研究这些黏附素在体内的作用的实验。这一建议的广泛假设是(1)这些已确认和推定的粘附素中的一部分至少有助于使粪肠球菌成为心内膜炎和其他感染的成功原因，(2)一些粘附素是导致近年来出现的医院内粪肠球菌分离株“不驯服”的原因，以及(3)对这些已确认和推定的粘附素的更好理解将有助于找到预防和/或逆转其影响的方法。

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.