ADHESION PROPERTIES OF PORPHYROMONAS GINGIVALIS FIMBRIAE

牙龈卟啉单胞菌菌毛的粘附特性

• 批准号: 3732532
• 负责人: ROBERT J GENCO
• 金额: --
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3732532
• 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 与唾液包裹的羟基磷灰石(Shap)的相互作用涉及 羧基的三分之一，主要是43 kDa的Fimbrins亚基。相比之下， 免疫优势区似乎位于纤毛膜的N-末端 亚单位。这项研究的主要目标是进一步界定 菌毛及其伴生菌的结构与功能关系 牙龈假单胞菌的粘附素。分子遗传学方法在人类遗传研究中的应用 菌毛将被用来评估指定牙龈假单胞菌的基因 菌毛相关的粘附素。邻近5‘和3’端的DNA序列 将对芬布林基因进行检测，以确定该基因是否 Fimbrins的编码是一个更大的多克隆单位的一部分，该单位还 包含其他与菌毛相关的成分。为了进一步定义 Fimbrins单体的Shap结合结构域，我们建议介绍 苯丙西林结构域结合位点的定点突变 在我们之前的研究中确定了。突变基因将被表达为 融合蛋白，纯化，并在体外系统中进行粘附性测试 在溶液中塑造或形成唾液成分。我们还将追求直接的 菌毛基因插入失活及其互补的途径 学习。突变基因将被插入穿梭载体，这将是 用于补充本实验室产生的FIMA基因缺陷突变株。 我们将通过比较将我们的研究扩展到其他牙龈假单胞菌菌株。 青霉毒素43 kDa亚基的可变表位和恒定表位 有几个菌株。这些研究的完成将在 分子水平，对观察到的抗原异质性的理解 在这些菌株中。将使用可变区的合成肽 确定它们在黏附唾液包被的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Rational所需的重要信息 调节口腔定植的免疫方法。