Genetic Analysis-Adhesion of S Sanguis to S Pellicle

描述：先前的资助支持导致了一种新的副猪链球菌菌毛相关粘连的发现，Fapl。参与fap糖基化的fap样分子和基因在整个口腔链球菌中都非常保守，也存在于重要的病原体中，如肺炎链球菌和金黄色葡萄球菌。我们将以副鳗鲡为模型系统，研究fapl样蛋白在这些致病菌中的功能。Fapl是副鳗生物膜形成所必需的。我们已经确定了Fapl的n端多肽（rFapl）对于细菌在生物膜表面的初始附着是必不可少的。Fapl被糖基化，该蛋白的糖基化参与了生物膜的成熟。本应用的总体目标突出于两个特定目标：(1)确定初始细菌附着所需的rFapl粘附素的结构/功能关系；(2)确定rFapl侧翼基因在fapl糖基化和细菌生物膜形成中的功能。我们将结合遗传学、结构生物学（核磁共振波谱和x射线晶体学）、体外结合生化分析和体内动物模型研究来解决Fapl多肽的结构如何影响其功能。这些综合的方法将揭示Fapl与唾液受体结合的结构基础，以及预测其他医学上重要病原体的Fapl样分子。fapl侧翼区域的蛋白可能构成了fapl的耦合分泌和糖基化途径。为了开始理解所涉及的机制，我们将确定蛋白质在该位点的亚细胞本地化，并确定体内和体外可能的蛋白质-蛋白质相互作用。我们之前证明了Fapl糖基化参与了生物膜的成熟。结合分子和遗传分析，我们将剖析这些糖基化基因在生物膜形成中的功能。阐明Fapl糖基化的功能和Fapl多肽结构/功能关系的定义将有助于细菌在口腔环境中的定植领域和其他重要病原体的致病机制，因为它们的基因组表明它们也具有Fapl样分子和侧翼蛋白。

DESCRIPTION: Previous grant support has led to the discovery of a novel Streptococcus parasanguis fimbriae associated adhesion, Fapl. Fapl-like molecules and genes involved in Fapl glycosylation are strikingly conserved throughout oral streptococci and are also found in important pathogens, such as Streptococcus pneumoniae and Staphylococcus aureus. We will use S. parasanguis as a model system to study the function of Fapl-like proteins in these pathogenic bacteria. Fapl is required for the formation of a S. parasanguis biofilm. We have determined that the N-terminal polypeptide of Fapl (rFapl) is essential for initial attachment of bacteria to the biofilm surface. Fapl is glycosylated, and glycosylation of this protein is involved in maturation of biofilm. The overall goal of this application is highlighted by two Specific Aims: (1) to determine the structure/function relationship of the rFapl adhesin that is required for initial bacterial attachment and (2) to define the functions of the fapl flanking genes in Fapl glycosylation and bacterial biofilm formation. We will bring genetic, structural biology (NMR spectroscopy and X-ray crystallography), in vitro binding biochemical assays and in vivo animal model studies together to address how the structure of the Fapl polypeptide influences its function. These comprehensive approaches should shed light on the structural basis for binding of Fapl to salivary receptors, and for predicted Fapl-like molecules of other medically important pathogens. Proteins of the fapl flanking region may constitute a coupled secretion and glycosylation pathway for Fapl. To begin to understand the mechanisms involved, we will determine the subcellular Iocalization of proteins in this locus and identify putative protein-protein interactions in vivo and in vitro. We previously demonstrated that Fapl glycosylation is involved in maturation of the biofilm. Using a combination of molecular and genetic analyses, we will dissect the function of these glycosylation genes in biofilm formation. Elucidation of the function of Fapl glycosylation and definition of the Fapl polypeptide structure/function relationships will contribute to the field of bacterial colonization in the oral environment and to the pathogenic mechanism of other important pathogens as their genomes indicate that they also possess Fapl-like molecules, and the flanking proteins.