MINING THE ORAL MICROBIOME FOR NOVEL GLYCAN-BINDING MOLECULES

描述（申请人提供）：在口腔环境中，口腔细菌上的凝集素样粘附物与口腔宿主细胞和牙齿表面以及其他口腔细菌表面上的互补聚糖识别基序之间的相互作用对于初始细菌定植和随后形成多物种生物膜起重要作用，所述多物种生物膜有可能变成致病性栖息地，引起牙齿、口腔以及可能的全身性疾病。考虑到（a）口腔生物膜中鉴定的多种细菌物种，（B）唾液糖蛋白上发现的聚糖的丰富多样性，以及（c）其他细菌表面上表达的无数聚糖基序，除了目前已知的少数几种之外，口腔微生物组中可能存在更多未鉴定的凝集素样细菌粘附素。一旦被鉴定，这些细菌凝集素样粘附素，其中许多可能会表现出新的聚糖结合特异性，可以通过重组DNA技术伪造成高度特异性的工具，用于检测和定位相应的聚糖受体结构。本申请的总体目标是通过使用聚糖官能化的靶向探针来挖掘人类和其他哺乳动物的口腔微生物组中的新型聚糖结合细菌粘附。在这里，我们专注于一类特征明确的细菌粘连，即革兰氏阳性菌的富含丝氨酸重复序列（SRR）蛋白。通过确定一组新的聚糖结合蛋白具有独特的配体结合特性，我们的目标是证明作为一个原则的证明，它将成为可行的，以扩大目前可用的聚糖结合探针的范围极大。该项目的具体目标是1。从人类和其他哺乳动物物种的口腔中分离SRR粘附承载细菌，其特异性结合含有N-乙酰基和N-羟乙酰神经氨酸的4-或9-O-乙酰化形式的宿主唾液酸聚糖。2.从人口腔生物膜中分离携带SRR粘附素的细菌，其特异性结合含有不寻常凝集素识别基序（Rhaα1- 2 Rha和Galα1- 6 Glc）的细菌多糖受体。3.使用生物信息学来识别SRR蛋白中的新型聚糖结合区域，并使用细菌基因组学来生成重组聚糖结合探针，以检测各种聚糖。

DESCRIPTION (provided by applicant): In the oral environment, interactions between lectin-like adhesions on oral bacteria and complementary glycan recognition motifs on oral host cell and tooth surfaces and on the surfaces of other oral bacteria play important roles for initial bacterial colonization and the subsequent formation of multi-species biofilms that have the potential to turn into pathogenic habitats causing dental, oral, and perhaps systemic disease. Considering (a) the multitude of bacterial species identified in oral biofilms, (b) the rich diversty of glycans found on salivary glycoproteins, and (c) the innumerable glycan motifs expressed on the surface of other bacteria, it is likely that many more unidentified lectin-like bacterial adhesns exist in the oral microbiome, beyond the few ones currently known. Once identified, those bacterial lectin-like adhesins, many of which presumably will exhibit novel glycan-binding specificities, can be forged by recombinant DNA techniques into highly specific tools for the detecting and localization of corresponding glycan receptor structures. The overall objective of this application is to mine the oral microbiomes of humans and other mammals for novel glycan- binding bacterial adhesions by using glycan-functionalized targeted probes. Here, we focus on one well-characterized class of bacterial adhesions, the serine-rich repeat (SRR) proteins of Gram- positive bacteria. By identifying a novel set of glycan-binding proteins with unique ligand-binding properties, we aim to demonstrate as a proof of principle that it will become feasible to expand the range of currently available glycan-binding probes enormously. The specific aims of the project are to 1. Isolate SRR adhesion-bearing bacteria from the oral cavity of humans and other mammalian species that bind specifically to host sialoglycans containing 4- or 9-O-acetylated forms of N-acetyl and N-glycolylneuraminic acid. 2. Isolate SRR adhesin-bearing bacteria from human oral biofilms that bind specifically to bacterial polysaccharide receptors containing the unusual lectin-recognition motifs (Rhaα1-2Rha and Galα1- 6Glc). 3. Use bioinformatics to identify novel glycan-binding regions in SRR proteins, and use bacterial genomics to generate recombinant glycan-binding probes for detection of a diverse set of glycans.