Gonococcal Pilus Structure and Function

淋球菌菌毛的结构和功能

• 批准号: 6844919
• 负责人: Hank S. SEIFERT
• 金额: $ 29.7万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6844919
• 关键词: DNA binding protein; Neisseria gonorrhoeae; agglutination reaction; antibiotics; bacteria infection mechanism; bacterial proteins; cell component structure /function; cell line; cell motility; cell transformation; drug resistance; gene mutation; genetic screening; host organism interaction; membrane proteins; nucleoside triphosphate; pilus; protein protein interaction; protein structure function; secretin; site directed mutagenesis; virulence; yeast two hybrid system

DESCRIPTION (provided by applicant): The Gram-negative bacterium Neisseria gonorrhoeae is the only causative agent of the sexually transmitted disease gonorrhea. The gonococcal (Gc) pilus is an important virulence factor of this human pathogen. Since Gc do not possess a Type II or Type III secretion apparatus, this Type IV pilus has an important role in host-pathogen interactions. The Gc pilus is involved in many aspects of gonococcal pathogenesis. First, the pilus functions in the initiating events of colonization by enhancing the ability of the bacterium to adhere to and interact with cells of the human host. Second, the pilus is involved in allowing the efficient transport of DNA into the bacterial cell for genetic transformation. Third, it is involved in twitching motility, a specialized form of locomotion expressed by all Type IV piliated bacteria. Much has been learned about the components that make up the Gc pilus assembly apparatus in the past several years, but how the pilus and its assembly apparatus act to promote adherence, DNA transformation, and twitching motility remains largely undefined. We propose a series of experiments to further our understanding of how the Gc pilus provides these functions for pathogenesis. In Aim 1 will utilize saturating mutagenesis to isolate loss-of-function and gain-of-function mutations in the secretin gene, PilQ. Loss-of-function mutations will define domains of PilQ required for expression, stability, and multimerization. Gain-of-function mutations will be screened for mutations that effect DNA transformation, antibiotic sensitivity, and twitching motility. Mutants will also be tested for changes in epithelial cell adherence. In Aim 2 will undertake a biochemical, yeast two-hybrid and biophysical analysis of three related predicted NTPases involved in orchestrating pilus function. These studies will provide new findings and insights into the structure and functions of the gonococcal pilus.

描述（由申请人提供）：革兰氏阴性细菌淋病奈瑟菌是性传播疾病淋病的唯一病原体。淋球菌 (Gc) 菌毛是这种人类病原体的重要毒力因子。由于 Gc 不具有 II 型或 III 型分泌装置，因此这种 IV 型菌毛在宿主-病原体相互作用中具有重要作用。 Gc 菌毛参与淋球菌发病机制的许多方面。首先，菌毛通过增强细菌粘附人类宿主细胞并与其相互作用的能力，在定植的起始事件中发挥作用。其次，菌毛参与将 DNA 有效运输到细菌细胞中进行遗传转化。第三，它参与抽搐运动，这是所有 IV 型绒毛细菌表达的一种特殊运动形式。在过去的几年里，人们对 Gc 菌毛组装装置的组成部分有了很多了解，但菌毛及其组装装置如何促进粘附、DNA 转化和抽搐运动仍然很大程度上不清楚。我们提出了一系列实验来进一步了解 Gc 菌毛如何为发病机制提供这些功能。目标 1 将利用饱和诱变来分离促胰液素基因 PilQ 中的功能丧失和功能获得突变。功能丧失突变将定义表达、稳定性和多聚化所需的 PilQ 结构域。将筛选功能获得突变，以确定影响 DNA 转化、抗生素敏感性和抽搐运动的突变。还将测试突变体上皮细胞粘附的变化。在目标 2 中，将对参与协调菌毛功能的三个相关预测 NTPase 进行生化、酵母双杂交和生物物理分析。这些研究将为淋球菌菌毛的结构和功能提供新的发现和见解。