Alternative Approaches for E. faecalis Infections

描述（由申请人提供）：肠球菌是社区性感染性心内膜炎（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大常见原因（但在医疗保健相关的IE中排名第一至第二位，其中粪肠球菌（Efs）占主导地位）。抗菌素耐药性可能促进肠球菌在医院环境和住院患者菌群中的建立，并且肯定会使治疗更加困难，特别是对于IE。我们最近的工作集中在Efs粘附于ECM蛋白的发病机制上，这是一种在标准体外生长后通常看不到的表型。我们鉴定了Ace，发现它是Efs的胶原黏附素（adhesion in to Collagen， CN），血清、CN和46℃生长等因素诱导Ace表达，Ace基因在Efs实验性IE中起重要作用，Ace免疫可预防EIE，我们描述了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igg样折叠，使我们发现了“Ebp”基因及其在心内膜炎，生物膜和毛菌形成中的作用；后来，我们发现ebp基因是Efs核心基因组的一部分，并且在上升的小鼠uti和第二种cn粘附表型中也很重要。我们还发现了一个纤维蛋白原（FG）结合的msmrms家族，需要Ebp菌毛的存在才能使FG粘附到Efs细胞上，最近，我们发现血清a)在生长过程中诱导ECM粘附，b)作为粘附的激活剂，可能通过在ECM蛋白和粘附素之间形成桥梁。在这次更新中，Ace的计划是1)证明（通过引入Ace中减少rec-Ace的体外CN结合的突变）Ace敲除突变所见的衰减与Ace的CN结合活性的丧失特别相关（相对于由于这种表面蛋白的丧失而产生的无关效应），2)探索抗Ace单克隆抗体在预防Efs IE中的功效以及取代单克隆抗体功能的分子基础。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)在菌毛中定位Ebp菌毛亚基，并确定这些亚基和两种分选酶在菌毛生物发生中的作用；2)确定菌毛相关（非ace） CN粘附的机制，以及单个菌毛在生物膜、细胞粘附和毒力中的作用；3)确定即使FG粘附素仍然存在，菌毛突变体中FG粘附消除的基础。4)确定对Ebp亚基的免疫是否会阻止实验性IE； 5)研究血清诱导毛毛形成的机制以及毛毛调节的条件和机制。最后，我们将研究ace和eebabc在EIE中以及单克隆抗体与它们之间在抑制Efs细胞CN粘附方面可能存在的叠加效应。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为合理设计预防或改善Efs感染的模式奠定基础，3)将更好地了解这种生物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. PUBLIC HEALTH RELEVANCE: This proposal seeks to understand how enterococci, bacteria that are important in hospital-associated infections, cause heart valve infections, a uniformly fatal disease without antibiotics. We seek to understand how these bacteria attach to heart valves and other human tissues and proteins, and to develop ways to prevent these infections.