MECHANISMS OF BACTERIAL ADHERENCE & COLONIZATION

该项目的长期目标是了解 细菌在粘膜定植中的宿主大分子 口咽的表面。我们将主要关注这个角色 唾液分子在1型黏附的调节中发挥作用 流苏状革兰氏阴性杆菌医院内常见致病菌 肺炎)至粘膜上皮细胞。我们将提纯类型1 唾液菌毛结合糖蛋白(FBGs)的常规提取 层析程序。我们还将测试已知的样品 从其他研究人员那里获得的唾液糖蛋白 菲尔德。FBG将通过氨基酸分析来表征，两个- 十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法 作图和/或免疫交叉反应。低聚糖 将通过化学和凝集素结合分析来表征， 在有必要的情况下，通过质谱学和核磁学 共振光谱分析。产生的特异性抗体探针 将被用来测定各种物质的浓度 唾液中的分子。提纯的FBG将被放射性标记并使用 以确定结合位点的特异性、亲和力和数量 在1型菌毛状大肠杆菌和Fn.适当的控制措施将 包括缺乏或过度生产29 kDa的大肠杆菌突变体 甘露糖结合粘附素和其他表达1型的物种 粘附素。将注意pH、离子的影响 强度和二价阳离子。竞争性抑制研究 使用FN将用来进一步描述这些相互作用。 放射性标记的FBG也将用于结合研究，以确定 上皮细胞表面结合位点的特异性、亲和力和数量 细胞。将注意pH、离子的影响 强度、二价阳离子以及唾液和FN对 这种互动。由于FBG与类型1的相互作用 菌毛可能是通过低聚糖部分介导的。 糖蛋白，我们将研究糖苷酶对 大肠杆菌与纯化的FBG的相互作用及测定 唾液暴露于这些糖苷酶会暴露出额外的空腹血糖。 我们将使用一个动物模型来研究这一假设 创伤后革兰氏阴性菌定植增加是由于 至少部分与唾液或口腔细胞相关的变化有关 分子。上皮细胞和唾液的样本将被检测 原因：1)大肠杆菌(或其他革兰氏阴性菌)的变化 使用标准依从性分析的依从性，2)改变 在口腔细胞和唾液中使用FG、FN或FN碎片 免疫荧光、免疫印迹分析和定量 免疫学检测；3)空腹血糖或纤维蛋白原水平变化。 使用降级活动将大大增加信息 需要了解细菌致病的重要方面 在口咽腔里。

The long-term goal of this project is to understand the role of host macromolecules in bacterial colonization of the mucosal surfaces of the oropharynx. We will focus principally on the role salivary molecules play in the modulation of adherence of type 1 fimbriated gram negative bacteria frequent agents of nosocomial pneumonia) to mucosal epithelial cells. We will purify type 1 fimbrial-binding glycoproteins (FBGs) from saliva by routine chromatographic procedures. We will also test sample of known salivary glycoproteins obtained from other investigators in the field. The FBGs will be characterized by amino acid analysis, two- dimensional SDS polyacrylamide gel electrophoresis, peptide mapping, and/or immunological crossreactivity. Oligosaccharides will be characterized by chemical and lectin-binding assays and, where warranted, by mass spectrometry and nuclear magnetic resonance spectrometry. The specific antibody probes generated will be used to assay for the concentration of the various molecules in saliva. Purified FBGs will be radiolabeled and used to determine the specificity, affinity and number of binding sites on type 1 fimbriated E. coli and FN. Appropriate controls will include E. coli mutants which lack or overproduce the 29 kDa mannose-binding adhesin and other species which express type 1 adhesins. Attention will be given to the influence of pH, ionic strength and divalent cations. Competitive inhibition studies using FN will be used to further characterize these interactions. Radiolabeled FBGs will also be used in binding studies to determine the specificity, affinity and number of binding sites on epithelial cells. Attention will be given to the influence of pH, ionic strength, divalent cations and to the effects of saliva and FN on this interaction. Since the interaction of FBGs with type 1 fimbriae may be mediated through the oligosaccharide portion of the glycoprotein, we will study the effects of glycosidases on the interaction of E. coli with the purified FBGs and determine whether exposure of saliva to these glycosidases exposes additional FBGs. We will use an animal model to study the hypothesis that the increased gram negative colonization following trauma is due, at least in part, to changes in salivary or buccal cell-associated molecules. Samples of epithelial cells and saliva will be assayed for: 1) changes in E. coli (or other gram negative bacteria) adherence using standard adherence assays, 2) changes in levels of FBGs, Fn or Fn fragments on buccal cells and in saliva using immuno-fluorescence, Western blot analysis and quantitative immunological assays, and 3) changes in the levels of FBG- or Fn- degrading activity using will add significantly to the information needed to understand important aspects of bacterial pathogenesis in the oropharyngeal cavity.