Alternative Approaches for E. faecalis Infections

描述（由申请人提供）：肠球菌是社区发病感染性心内膜炎（IE）的第三大最常见原因，也是医疗保健相关感染的第二至第三大最常见原因（但在医疗保健相关IE中排名第一至第二，E.粪（Efs）占主导地位）。抗生素耐药性可能促进了医院环境和住院患者植物群中肠球菌的建立，并且肯定会使治疗更加困难，特别是对于IE。我们最近的工作主要集中在Efs粘附ECM蛋白的发病机制的贡献，通常不会看到标准的体外生长后的表型。我们鉴定了Ace，并证明它是Efs的胶原粘附素（CN），血清、CN和46 ℃生长等因素诱导Ace表达，普遍存在的Ace基因在Efs实验性IE中是重要的，Ace免疫保护EIE，并阐明了Ace与CN结合的分子机制。我们对其他Efs表面蛋白（如Ace，具有异常的Ig样折叠）的研究使我们发现了“Ebp”基因及其在心内膜炎、生物膜和菌毛形成中的作用;后来我们发现Ebp基因是Efs核心基因组的一部分，在小鼠UTI上升和第二个CN粘附表型中也很重要。我们还鉴定了一个纤维蛋白原（FG）结合MSCRA家族，该家族需要Ebp皮利的存在才能赋予FG对Efs细胞的粘附，最近，我们发现血清a）在生长期间促进ECM粘附，并且B）可能通过在ECM蛋白和粘附素之间形成桥梁而充当粘附的激活剂。在这次更新中，Ace的计划是：1）证明（通过向ace中引入降低rec-Ace的体外CN结合的突变），用ace敲除突变体观察到的衰减与Ace的CN结合活性的丧失特异性相关。（相对于由于这种表面蛋白的损失而引起的不相关的影响），2）探讨抗Ace单克隆抗体预防Efs IE的有效性及替代单克隆抗体作用的分子基础; 3）确定血清和CN诱导Ace的机制。与Ebp皮利和FG粘附素相关的目标是：1）将Ebp菌毛蛋白亚基定位于皮利内，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）鉴定负责菌毛相关（非Ace）CN粘附的机制以及单个菌毛蛋白在生物膜、细胞粘附和毒力中的作用，3）确定消除菌毛突变体中FG粘附的基础，即使FG粘附素仍然存在，4）确定针对Ebp亚基的免疫是否防止实验性IE，（5）探讨血清绒毛的形成机制及调节条件和机制。最后，我们将研究ace和ebpABC在EIE中的可能的相加效应，以及针对它们的mAb在抑制Efs细胞的CN粘附中的可能的相加效应。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感染的方法奠定基础，3）更好地了解这种生物体如何与人类宿主相互作用。 公共卫生相关性：该提案旨在了解肠球菌（在医院相关感染中很重要的细菌）如何引起心脏瓣膜感染，这是一种在没有抗生素的情况下一律致命的疾病。我们试图了解这些细菌如何附着在心脏瓣膜和其他人体组织和蛋白质上，并开发预防这些感染的方法。

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.