Glucan Binding Proteins of Oral Streptococci

描述：变形链球菌具有三种不同的葡糖转移酶(GTF)和至少三种不同的非酶葡聚糖结合蛋白(GBP)。每种GTF和GBP对斑块形成的确切贡献尚不清楚。最近我们发现，通过等位基因替换而完成的葡聚糖结合蛋白(GbpA)的丢失，极大地改变了变形链球菌培养物形成的生物膜的结构。基于这些数据，我们设计了编码其他胞外蛋白基因失活的菌株。失去gbpc、FruA或P1也会导致生物膜结构的变化。为了解释这些观察结果，我们假设细胞外蛋白的丢失可能导致有机体的特定物理和/或生化变化，或其直接环境影响该有机体形成的成熟生物膜的结构。此应用程序的特定目标旨在测试这些可能性。由于与GbpA的合作取得了最大进展，对GbpA特定物业的调查也包括在目标中。目的1)检测基因敲除菌株的物理性质，包括疏水性、表面电荷，以及它们与钙等阳离子的相互作用，这些阳离子对生物膜的形成至关重要。目的2)使用流动室检查与敲除菌株最初附着在基质上有关的事件，并将结果与成熟生物膜的结构相关联。目的3)研究GbpA与葡聚糖合成的比例与生物膜结构的关系。目的4)删除或替换GbpA的氨基末端结构域(非葡聚糖结合结构域)，并与野生型和GbpA基因敲除菌株比较，研究这一改变对变形链球菌表型特性的影响。目的5)表达地层的表型特征。目的6)利用异源宿主戈登链球菌中的结构基因gbpA，在依赖蔗糖的黏附和生物膜微阵列的背景下检测该生物体，以确定胞外蛋白的丢失和生物膜的表达。

DESCRIPTION: Streptococcus mutans possesses three distinct glucosyltransferases (GTFs) and at least three different nonenzymatic glucan binding proteins (GBPs). The precise contributions of each GTF and GBP to plaque development are unknown. Recently we showed that the loss of a glucan-binding protein (GbpA), accomplished through allelic replacement, dramatically altered the architecture of the biofilm formed by cultures of S. mutans. Based on these data we engineered strains with inactivation in genes encoding other extracellular proteins. The loss of GbpC, FruA, or P1 also resulted in changes in the biofilm architecture. To explain these observations we posit that the loss of an extracellular protein may result in specific physical and/or biochemical changes to the organism, or to its immediate environment that affect the structure of the mature biofilm formed by that organism. The Specific Aims of this application are designed to test these possibilities. Since work with the GbpA has progressed the furthest, investigations of specific GbpA properties are also included in the Aims. Aim 1) Examine the physical properties of the knockout strains including hydrophobicity, surface charge, and their interactions with cations such as calcium which are important in the development of a biofilm. Aim 2) Use a flow chamber to examine the events associated with the initial attachment of the knockout strains to the substratum and correlate the results with the structure of the mature biofilms. Aim 3) Examine how the ratio of GbpA to glucan synthesis correlates with the structure of the biofilm. Aim 4) Delete or replace the amino terminal domain (non glucan-binding domain) of GbpA and examine how this change affects the phenotypic properties of Streptococcus mutans compared to the wild-type and GbpA knockout strains. Aim 5) Express phenotypic properties of formation. Aim 6) Utilize architecture influence gene GbpA in the heterologous host Streptococcus gordonii and examine the organism in the context of sucrose-dependent adhesion and biofilm microarrays to determine how the loss of extracellular proteins and biofilm expression.