Surface protein complexes of oral treponemes: assembly and host cell interactions

描述（由申请人提供）：本研究的目的是表征介导与宿主组织相互作用的齿垢密螺旋体（Td）表面蛋白的表达和组装，从而深入了解牙周病的机制。我们专注于分析两个TD蛋白复合物，直接影响宿主细胞：寡聚Msp蛋白和PrtP脂蛋白蛋白酶复合物。总体假设是Msp和PrtP是牙周病中Td细胞病变行为的主要贡献者。Msp和PrtP复合物的表达是相互依赖的。为了了解它们在微生物-宿主相互作用中的作用，我们必须了解这些分子如何在螺旋体中表达和相互作用。我们提出遗传分析的结构特征和这些复合物及其组分的相互作用。我们的方法是定义Msp和PrtP复合体之间的相互依赖机制，以表征它们在微生物-宿主相互作用中的作用。目的1：确定PrtP复合物的组装和活性要求。我们将在蛋白酶位点构建一系列特定的突变，这将使我们能够表征PrcA和PrtP的翻译后加工，PrcB的表达，蛋白酶复合物蛋白的定位和活性蛋白酶复合物的组装。目的2：确定Msp在蛋白酶复合物蛋白合成中的作用，以及蛋白酶在Msp寡聚化中的作用。我们将描述Msp和蛋白酶操纵子蛋白之间的相互作用，影响每个外膜复合物的表达和活性。目的3：表征Td外膜组分与上皮细胞的相互作用。为了进一步研究宿主细胞对Td攻击的反应，我们将表征Msp和上皮细胞上鉴定的推定受体之间的相互作用，并表征上皮细胞对PrtP蛋白酶复合物和Msp的先天反应中的早期事件，其目标是区分Msp和蛋白酶复合物在细胞对Td的反应中的作用。该项目涉及Td外膜复合物表达的遗传和生化分析以及Td对宿主细胞的影响分析，将有助于口腔环境中微生物-宿主相互作用的分子分析。我们的实验室是进行这些研究的唯一场所。这些目标的完成将有助于螺旋体分子生物学的基础知识和慢性感染，如牙周病的微生物-宿主相互作用的理解。公共卫生意义：这个项目将提供有关如何启动牙周病的关键细菌与人类细胞相互作用的信息。这些研究将首先描述蛋白质复合物在细菌表面组装的过程。然后，我们将确定这些复合物与人类细胞上的哪些特定蛋白质结合，然后测定细菌蛋白质复合物与细胞表面受体结合的细胞反应。

DESCRIPTION (provided by applicant): The goal of this research is to characterize expression and assembly of Treponema denticola (Td) surface proteins that mediate interactions with host tissue, thereby gaining insight into mechanisms of periodontal disease. We focus on analysis of two Td protein complexes that directly affect host cells: the oligomeric Msp protein and the PrtP lipoprotein protease complex. The overall hypothesis is that Msp and PrtP are major contributors to Td cytopathic behavior in periodontal disease. Expression of Msp and the PrtP complex are interdependent. To understand their roles in microbe-host interactions, we must understand how these molecules are expressed and interact in the spirochete. We propose genetic analysis of structural features and interactions of these complexes and their components. Our approach is to define the mechanisms of interdependence between Msp and the PrtP complex in order to characterize their roles in microbe-host interactions. Aim 1: Determine requirements for assembly and activity of the PrtP complex. We will construct a series of specific mutations in the protease locus that will enable us to characterize posttranslational processing of PrcA and PrtP, expression of PrcB, localization of protease complex proteins and assembly of the active protease complex. Aim 2: Determine the role of Msp in biosynthesis of protease complex proteins, and the role of the protease in oligomerization of Msp. We will characterize interactions between Msp and protease operon proteins that influence expression and activity of each outer membrane complex. Aim 3: Characterize interactions of Td outer membrane components with epithelial cells. To further studies of host cell responses to Td challenge, we will characterize interactions between Msp and a putative receptor identified on epithelial cells and characterize early events in epithelial cell innate responses to the PrtP protease complex and Msp, with the goal of differentiating between roles of Msp and the protease complex in cellular responses to Td. This project, involving genetic and biochemical analyses of Td outer membrane complex expression and analysis of Td effects on host cells, will contribute to molecular analysis of microbe-host interactions in the oral environment. Our laboratory is uniquely positioned to conduct these studies. Completion of these Aims will contribute to both basic knowledge of spirochete molecular biology and to understanding of microbe-host interactions in chronic infections such as periodontal diseases. Public Health Significance: This project will provide information on how a key bacterium involved in initiation periodontal disease interacts with human cells. The studies will first characterize the process of protein complex assembly on the bacterial surface. Then we will determine which specific proteins on human cells these complexes bind to, and then assay cellular responses to bacterial protein complexes binding to cell surface receptors.