ADHESION PROPERTIES OF PORPHYROMONAS GINGIVALIS FIMBRIAE

牙龈卟啉单胞菌菌毛的粘附特性

• 批准号: 6238421
• 负责人: ROBERT J GENCO
• 金额: $ 3.71万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6238421
• 关键词: Bacteroides gingivalis; SDS polyacrylamide gel electrophoresis; bacterial antigens; cell adhesion; chemical binding; chimeric proteins; disease /disorder model; epitope mapping; gene expression; genetic mapping; host neoplasm interaction; human subject; hydroxyapatites; laboratory mouse; laboratory rabbit; laboratory rat; microorganism culture; molecular cloning; mutant; nucleic acid sequence; periodontium disorder; pilus; polymerase chain reaction; protein purification; radiotracer; site directed mutagenesis; southern blotting; synthetic peptide; tritium

Our studies with purified fimbrial proteins have demonstrated an interaction with salivary-coated hydroxyapatite (sHAP) that involves the carboxyl third of the major 43 kDa fimbrillin subunit. In contrast, immunodominant regions appear to reside in the N-terminus of the fimbrillin subunit. The principal goal of this study is to further define the structure-function relationship of fimbriae and fimbrial-associated adhesins of P. gingivalis. Molecular genetic approaches to studying the fimbriae will be used to evaluate the genes specifying the P. gingivalis fimbrial-associated adhesins. DNA sequences adjacent to the 5' and 3' ends of the fimbrillin gene will be examined to determine whether the gene encoding for fimbrillin is part of a larger polycistonic unit that also contains other fimbrial-associated components. In order to further define the sHAP binding domains of the fimbrillin monomer, we propose to introduce site-directed mutations in the binding sites of fimbrillin domains identified in our previous studies. The mutant genes will be expressed as fusion proteins, purified, and tested in the in vitro system for adherence to sHAP or salivary components in solution. We will also pursue the direct approach of insertional inactivation of fimbrial gene and complementation studies. Mutant genes will be inserted into shuttle vectors which will be used to complement fimA gene deficient mutants generated in our laboratory. We will expand our studies to other strains of P. gingivalis by comparing the variable and constant epitopes of the 43 kDa fimbrillin subunit from several strains. Completion of these studies will provide, at the molecular level, an understanding of the antigenic heterogeneity observed among the strains. Synthetic peptides of the variable regions will be used to identify their role in attachment to salivary-coated HAP as well as in immune reactivity of fimbriae. Thus, the studies outlined in this subproject could provide important information needed for a rational approach for immunization to modulate colonization of the oral cavity.

我们对纯化的菌毛蛋白的研究表明， 与唾液包被的羟基磷灰石（sHAP）的相互作用， 43 kDa菌毛蛋白主要亚基的羧基三分之一。 与此相反， 免疫显性区域似乎位于菌毛蛋白的N-末端 亚单位 本研究的主要目的是进一步确定 菌毛及其相关菌毛结构与功能关系 牙龈卟啉单胞菌粘附素。 分子遗传学方法研究 菌毛将用于评估指定牙龈卟啉单胞菌的基因 菌毛相关粘附素 与5'和3'末端相邻的DNA序列 将检查菌毛蛋白基因的基因，以确定该基因是否 编码菌毛蛋白是更大的多顺反子单元的一部分， 含有其他菌毛相关成分。 为了进一步明确 的sHAP结合结构域的菌毛蛋白单体，我们建议引入 菌毛蛋白结构域结合位点的定点突变 在我们以前的研究中发现。 突变基因将表达为 融合蛋白，纯化并在体外系统中测试粘附性 sHAP或唾液成分。 我们还将继续直接 菌毛基因的插入失活及互补途径 问题研究 突变基因将被插入穿梭载体， 用于补充我们实验室中产生的fimA基因缺陷突变体。 我们将扩大我们的研究，以其他菌株的牙龈卟啉单胞菌， 43 kDa菌毛蛋白亚基的可变和恒定表位来自 几种菌株。 这些研究的完成将提供， 分子水平，了解观察到的抗原异质性 在各种菌株中。 将使用可变区的合成肽 以确定它们在附着唾液包被的HAP以及在 菌毛的免疫反应性。 因此，本报告中概述的研究 子项目可以提供合理性评估所需的重要信息， 免疫途径以调节口腔的定殖。