Gonococcal Pilus Structure and Function

描述（由申请人提供）：淋病奈瑟氏革兰氏阴性菌是淋病的唯一病原体。淋球菌（Gc）菌毛是该人类病原体的重要毒力因子。由于Gc不具有II型或III型分泌器官，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的许多方面。首先，菌毛通过增强细菌粘附和与人类宿主细胞相互作用的能力，在定植的起始事件中起作用。其次，菌毛参与了DNA进入细菌细胞进行遗传转化的有效运输。第三，它参与抽搐运动，这是一种由所有IV型毛状细菌表达的特殊运动形式。在过去的几年里，人们对构成Gc菌毛组装装置的成分已经了解了很多，但是菌毛及其组装装置如何促进粘附、DNA转化和抽搐运动仍然不清楚。我们提出了一系列的实验，以进一步了解Gc毛是如何为发病机制提供这些功能的。在Aim中，我将利用饱和诱变分离分泌素基因PilQ的功能丧失和功能获得突变。功能丧失突变将定义表达、稳定性和多细胞化所需的PilQ结构域。功能获得突变将筛选影响DNA转化、抗生素敏感性和抽搐运动性的突变。突变体还将测试上皮细胞粘附性的变化。在Aim 2将进行生化，酵母双杂交和生物物理分析三个相关的预测NTPases参与协调毛的功能。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。

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.