MECHANISMS OF BACTERIAL ADHERENCE & COLONIZATION

本项目的长期目标是了解 粘膜细菌定植中的宿主大分子 口咽的表面。 我们将主要关注 唾液分子在调节1型糖尿病的粘附中起作用 革兰阴性菌菌毛医院常见病原体 肺炎）至粘膜上皮细胞。 我们将净化1型 唾液菌毛结合糖蛋白（FBG） 色谱程序。 我们还将测试已知的 唾液糖蛋白从其他研究者那里获得， 领域 FBG将通过氨基酸分析表征，两个- 双向SDS聚丙烯酰胺凝胶电泳，肽 作图和/或免疫交叉反应性。 寡糖 将通过化学和凝集素结合测定来表征， 在有保证的情况下，通过质谱和核磁共振 共振光谱法 产生的特异性抗体探针 将用于测定各种 唾液中的分子。 将对纯化的FBG进行放射性标记并使用 以确定结合位点的特异性、亲和力和数目 1型菌毛E. coli和FN。 适当的控制将 包括大肠缺乏或过度产生29 kDa的大肠杆菌突变体， 甘露糖结合粘附素和其它表达1型 粘附素。 将注意pH值、离子 强度和二价阳离子。 竞争性抑制研究 使用FN将用于进一步表征这些相互作用。 放射性标记的FBG也将用于结合研究，以确定 上皮细胞上结合位点的特异性、亲和力和数量 细胞 将注意pH值、离子 强度，二价阳离子和唾液和FN对 这种互动。 由于FBG与类型1的相互作用 菌毛可以通过微囊藻毒素的寡糖部分介导， 糖蛋白，我们将研究糖基化酶对糖蛋白的影响。 E.大肠杆菌与纯化的FBG，并确定是否 唾液暴露于这些糖苷酶会暴露额外的FBG。 我们将使用动物模型来研究这一假设， 创伤后革兰氏阴性菌定植增加， 至少部分地与唾液或口腔细胞相关的变化有关， 分子。 将对上皮细胞和唾液样本进行分析 （1）E.大肠杆菌（或其他革兰氏阴性菌） 使用标准粘附测定法的粘附，2）粘附水平的变化， FBG、Fn或Fn片段在颊细胞上和唾液中使用 免疫荧光、Western印迹分析和定量 免疫学测定，和3）FBG-或Fn-水平的变化， 使用降级活动将显著增加信息 需要了解细菌致病机理的重要方面 在口咽腔。

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.