Glycosylation & Function of an Oral Streptococcal Adhesin

描述（由申请人提供）：本研究基金主要研究我们几年前发现的副血链球菌的一种重要的链球菌粘附素Fap 1。有趣的是，Fap 1被保守的基因簇糖基化，该基因簇也存在于致病性链球菌和葡萄球菌中。提出了两个具体的目标，以表征这种新的糖基化和分泌途径的母基金。目的1是确定Fap 1糖基化的第一步，目的2是表征辅助分泌蛋白SecY 2介导的Fap 1糖基化步骤。在研究过程中，我们发现Fap 1是一种有效的抗原，并通过先天免疫Toll样受体2介导促炎反应。TLR及其相关的天然免疫信号在人类疾病的发病机制中起着重要作用。在本修订中，我们将验证我们的假设，即Fap 1和Fap 1糖基化在S.副豚鼠介导的TLR信号传导和感染性心内膜炎（IE）的发病机制，如口腔链球菌与IE的发展密切相关。我们将通过追求三个子目标来测试我们的假设：A.确定S.副豚鼠介导的TLR信号传导。Fap 1介导先天免疫应答。由于Fap 1是一种糖蛋白，我们将剖析Fap 1的功能结构域（聚糖和肽）在TLR信号传导中的重要作用。此外，我们将确定先天免疫信号传导对Fap 1介导的炎症反应的功能贡献。部分B。探讨Fap 1及其糖基化在IE发病机制中的作用。我们将评估Fap 1和Fap 1糖基化在IE发病机制中的作用，使用Fap 1和Fap 1糖基化缺陷突变体在良好表征的IE兔模型中构建。部分C.建立小鼠IE模型，并确定先天免疫在IE发病机制中的作用。由于转基因动物可用于小鼠，我们将使用TLR 2-/-和MyD 88-/-小鼠及其对照同窝小鼠建立IE小鼠模型，以确定TLR 2及其受体MyD 88在调节疾病发展中的作用。 Fap 1样蛋白在许多链球菌和致病性链球菌中高度保守，并与IE的发病机制有关。Fap 1的糖基化和生物合成是一条尚未被探索的生物合成途径，因此，确定Fap 1的功能及其与先天免疫系统在疾病发病机制中的关系将为设计和开发新的治疗药物提供独特的机会。 我们提出的竞争性修订扩大了母基金的科学范围。支持该提案将使我们能够雇用更多的工作人员和合同额外需要的技能，以刺激经济，复苏法案的目标。 公共卫生相关性：我们提出的竞争性修订很好地符合我们的母基金的范围，并将扩大我们对细菌发病机制的研究，并确定新的治疗设计目标。此外，支持该提案将使我们能够雇用更多的工作人员（一名研究生和一名研究助理），因此，我们的修订不仅将加快科学研究的克里思，而且还将创造就业机会，这是《复苏法》的目标。

DESCRIPTION (provided by applicant): This parent grant studies an important streptococcal adhesin Fap1 of Streptococcus parasanguinis we discovered few years ago. Interestingly Fap1 is glycosylated by a conserved gene cluster that is also present in pathogenic streptococci and staphylococci. Two specific aims were proposed to characterize this new glycosylation and secretion pathway in the parent grant. Aim 1 was to determine the first step of the Fap1 glycosylation and Aim 2 was to characterize accessory secretion protein SecY2-mediated Fap1 glycosylation step. During the study, we have discovered that Fap1 is a potent antigen and mediates proinflammatory responses via innate immune Toll-Like Receptor 2. TLRs and related innate immune signaling play important roles in pathogenesis of human diseases. In this revision, we will test our hypothesis that Fap1 and Fap1 glycosylation play an important role in S. parasanguinis-mediated TLR signaling and in the pathogenesis of infective endocarditis (IE) as oral streptococci have been strongly associated with the development of IE. We will test our hypothesis by pursuing three sub-aims: Part. A. Determine the function of the Fap1 glycosylation in S. parasanguinis-mediated TLR signaling. Fap1 mediates innate immune response. As Fap1 is a glycoprotein, we will dissect functional domains of Fap1 (glycans and peptides) that are important in TLR signaling. In addition, we will determine the functional contribution of innate immune signaling to Fap1-mediated inflammatory responses. Part. B. Determine the function of Fap1 and Fap1 glycosylation in the pathogenesis of IE. We will evaluate the role of Fap1 and Fap1 glycosylation in the pathogenesis of IE using Fap1 and Fap1 glycosylation deficient mutants constructed in the parent grant in a well-characterized rabbit model of IE. Part. C. Develop a mouse model of IE and determine the functional contribution of innate immunity to the pathogenesis of IE. As genetically modified animals are available for mice, we will establish a mouse model of IE to define the role of TLR2 and its receptor MyD88 in regulating the disease development using TLR2-/- and MyD88-/- mice and their control littermates. Fap1-like proteins are highly conserved in many streptococci and pathogenic straphylcocci and have been implicated in the pathogenesis of IE. Glycosylation and biogenesis of Fap1 is a previously unexplored biosynthetic pathway, therefore establishing the function of Fap1 and its relationship with innate immune system in the disease pathogenesis will provide a unique opportunity to design and develop new therapeutics. Our proposed competitive revision expands the scientific scope of the parent grant. Supporting of the proposal will allow us to hire additional staff and contract additional needed skills to stimulate economy, the objectives of the Recovery Act. PUBLIC HEALTH RELEVANCE: Our proposed competitive revision fits nicely with the scope of our parent grant and will expand our study on bacterial pathogenesis and identify new targets for the design of therapeutics. In addition, supporting of the proposal will allow us to hire additional staff (a graduate student and a Research Associate), thus, our revision will not only accelerate the tempo of scientific research but also allow for job creation, the objectives of the Recovery Act.