Structural, genetic, and functional analyses of the glycoproteins of Bacteroides

描述(申请人提供)：哺乳动物的肠道是一个复杂而多样的生态系统，栖息着数百种不同的细菌。人类与肠道微生物群的共同进化导致了合作关系，这种合作关系塑造了这些共生伙伴的生物学和基因组。关于肠道微生物区系的主要成员用来建立人类肠道共生关系的机制，人们知之甚少。这项建议中概述的实验旨在表征一类新的和丰富的细菌糖蛋白，这些糖蛋白是由哺乳动物肠道的优势细菌--类杆菌--合成的。这些分子在许多方面都是独一无二的。首先，它们是唯一被证明含有L岩藻糖的细菌糖蛋白。其次，它们是据报道由细菌合成的最大数量的糖蛋白。最后，它们的合成涉及一种独特的细菌酶，只有类杆菌才能合成，以使用从宿主分子中清除的岩藻糖。本提案中概述的实验将涉及类杆菌岩藻糖化糖蛋白的三个方面。第一个目标是鉴定脆弱芽孢杆菌和稻瘟杆菌的岩藻糖化糖蛋白，并表征这些分子中的多糖部分。第二个目标是确定哪些岩藻糖基转移酶和其他基因产物参与了多糖的合成和与蛋白质的连接。第三个目标的目的是从功能上表征这些糖蛋白在体外和体内对细菌的功能以及它们对与宿主共生相互作用的贡献。最终，这些研究将使我们能够更好地了解这些微生物如何通过有益的(益生菌、免疫调节和耐受、上皮表面成熟)或有害的(转移抗生素耐药性和参与炎症性肠病)影响宿主。脆弱芽孢杆菌也是一种重要的条件致病菌。脆弱芽孢杆菌主要见于腹内脓肿和厌氧菌血症，已从骨和软组织感染患者中分离出来，已被确定为早产发生率增加的一个因素。因此，对脆弱芽孢杆菌岩藻糖化糖蛋白的分析也将有助于我们了解该微生物的致病潜力。

DESCRIPTION (provided by applicant): The mammalian gut is a complex and diverse ecosystem colonized with hundreds of different bacterial species. The coevolution of humans with their intestinal microbiota has resulted in cooperative relationships that have shaped the biology and the genomes of these symbiotic partners. Little is known about the mechanisms used by the predominant members of the gut microbiota to establish symbiotic relationships in the human intestine. The experiments outlined in this proposal are designed to characterize a novel and abundant class of bacterial glycoproteins that are synthesized by the predominant bacteria of the mammalian intestine, namely the Bacteroides. These molecules are unique in many regards. First, they are the only bacterial glycoproteins demonstrated to contain L-fucose. Second, they represent the largest number of glycoproteins reported to be synthesized by bacteria. Lastly, their synthesis of involves a unique bacterial enzyme, synthesized only by the Bacteroides, to use fucose scavenged from host molecules. The experiments outlined in this proposal will address three aspects of fucosylated glycoproteins of Bacteroides. The goal of the first aim is to identify the fucosylated glycoproteins of B. fragilis and B. distasonis, and to characterize the glycan portion of these molecules. The goal of the second aim is to determine which fucosyltransferases and other gene products are involved in the synthesis and linkage of the glycans to the proteins. The goal of the third aim is to functionally characterize,.both in vitro and in vivo, the functions of these glycoproteins to the bacteria and their contribution to the symbiotic interaction with the host. Ultimately, these studies will allow us to better understand how these microorganisms are affecting the host with either beneficial (probiotics, immune regulation and tolerance, maturation of the epithelial surface) or deleterious (transfer of antibiotic resistance and involvement in inflammatory bowel disease) effects. B. fragilis is also an important opportunistic pathogen. B. fragilis predominates in intraabdominal abscesses and cases of anaerobic bacteremia, and has been isolated from patients with bone and soft tissue infections, and has been identified as a contributing factor to increased incidence of preterm labor. Therefore, the analysis of the fucosylated glycoproteins of B. fragilis will also contribute to our understanding of the pathogenic potential of this organism.