Alternative Approaches for E. faecalis Infections

说明(申请人提供)：肠球菌是社区感染性心内膜炎(IE)的第三大常见原因，也是医疗保健相关感染的第二至第三常见原因(但在与医疗保健相关的IE中排名第一至第二，其中粪肠球菌(EFS)占主导地位)。抗菌素耐药性可能有助于在医院环境和住院患者的菌群中建立肠球菌，并肯定会使治疗变得更加困难，特别是对IE。我们最近的工作集中在EFS与ECM蛋白的黏附在发病机制中的贡献，这种表型在标准的体外生长后通常不会出现。我们鉴定了Ace，证明它是EFS胶原(CN)的粘附素，血清、CN和46℃的生长等因素诱导了Ace的表达，普遍存在的Ace基因在EFS实验性IE中起重要作用，Ace免疫对EIE具有保护作用，并阐明了Ace与Cn结合的分子机制。我们对其他EFS表面蛋白的研究，如Ace，具有异常的Ig样折叠，使我们找到了“EBP”基因及其在心内膜炎、生物被膜和毛孔形成中的作用；后来我们发现EBP基因是EFS核心基因组的一部分，在上升的小鼠尿路感染和第二个CN-黏附表型中也是重要的。我们还鉴定了一类与纤维蛋白原(FG)结合的MSCRAMM家族，它们需要EBP菌毛的存在才能使FG黏附到EFS细胞上，最近，我们发现血清a)在生长过程中诱导ECM黏附，b)作为黏附的激活剂，可能是通过在ECM蛋白和粘附素之间建立桥梁。在这一更新中，Ace的计划是：1)证明(通过在Ace中引入减少REC-Ace体外CN结合的突变)，Ace基因敲除突变体的衰减与Ace的CN结合活性的丧失(而不是由于失去这种表面蛋白而产生的无关效应)具体相关；2)探索抗Ace单抗预防EFS IE的有效性和取代mAbs功能的分子基础，以及3)确定血清和CN诱导Ace的机制。与EBP菌毛和FG粘附素相关的目标是1)定位EBP菌毛亚基并确定这些亚基和两种分解酶在菌毛生物发生中的作用，2)确定菌毛相关(非Ace)CN黏附的负责机制以及单个菌毛在生物膜、细胞黏附和毒力中的作用，3)确定即使FG-黏附素仍然存在也消除菌毛突变体中FG黏附的基础，4)确定针对EBP亚单位的免疫是否可以阻止实验性IE，以及5)研究血清诱导菌毛形成的机制以及调节菌毛的条件和机制。最后，我们将探讨EIE中ACE和ebpABC之间以及它们之间的单抗在抑制EFS细胞CN黏附中可能的相加作用。我们的长期目标是1)了解这些粘附素在发病机制中的作用机制，2)希望我们的研究结果将成为合理设计预防或改善EFS感染的方式的基础，以及3)将提供更好的了解这种有机体如何与人类宿主相互作用。

DESCRIPTION (provided by applicant): Enterococci are the 3rd most common cause of community-onset infective endocarditis (IE) and the 2nd to 3rd most common cause of healthcare-associated infections overall (but are 1st-2nd in healthcare-associated IE, with E. faecalis (Efs) predominating). Antimicrobial resistance likely facilitates establishment of enterococci in the hospital setting and in the flora of hospitalized patients, and certainly makes therapy more difficult, particularly for IE. Our recent work has focused on the contributions to pathogenesis of Efs adherence to ECM proteins, a phenotype not usually seen after standard in vitro growth. We identified Ace and showed that it is an Adhesin to Collagen (CN) of Efs, that factors like serum, CN and growth at 46 C induce ace expression, that the ubiquitous ace gene is important in Efs experimental IE, that immunization with Ace protects against EIE, and we delineated the molecular mechanism of Ace's binding to CN. Our work on other Efs surface proteins which, like Ace, have deviant Ig-like folds, led us to the "Ebp" genes and their role in Endocarditis, Biofilm and Pilus formation; later we showed that the ebp genes are part of the Efs core genome and are also important in ascending murine UTIs and for a second CN-adherence phenotype. We also identified a family of fibrinogen (FG)-binding MSCRAMMs that require the presence of Ebp pili to confer FG adherence to Efs cells and, recently, we showed that serum a) elicits, during growth, ECM adherence, and also b) serves as an activator of adherence, perhaps by forming bridges between ECM proteins and adhesins. In this renewal, plans for Ace are to 1) demonstrate (by introducing, into ace, the mutations that decrease in vitro CN-binding of rec-Ace) that the attenuation seen with ace knock-out mutants is specifically associated with loss of Ace's CN- binding activity (vs. an unrelated effect due to loss of this surface protein), 2) explore the efficacy of anti-Ace mAbs in preventing Efs IE and the molecular basis by which displacing mAbs function, and 3) determine the mechanism of ace induction by serum and CN. Goals relating to Ebp pili and FG adhesins are to 1) localize Ebp pilin subunits within pili and define the role of these subunits and two sortases in pilus biogenesis, 2) identify the mechanism responsible for pilus-associated (non-Ace) CN adherence and the role of individual pilins in biofilm, cell adherence, and virulence, 3) determine the basis for elimination of FG adherence in pilus mutants even though FG-adhesins are still present, 4) determine if immunity against Ebp subunits prevents experimental IE, and 5) investigate the mechanism by which serum elicits pilus formation and conditions and mechanisms for pilus regulation. Finally, we will investigate a possible additive effect between ace and ebpABC in EIE and between mAbs to them in inhibition of CN adherence of Efs cells. Our long-range goals are 1) to understand the mechanism of action of these adhesins in pathogenesis, with 2) the hope that the results of our studies will form the basis for rational design of modalities to prevent or ameliorate Efs infections, and 3) will provide a better understanding of how this organism interacts with the human host.