Gonococcal Pilus Structure and Function

描述(申请人提供)：革兰氏阴性菌淋病奈瑟氏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型分泌器，这种IV型菌毛在宿主与病原体的相互作用中具有重要作用。GC菌毛参与淋球菌致病的多个方面。首先，菌毛通过增强细菌附着和与人类宿主细胞相互作用的能力，在启动定植事件中发挥作用。其次，菌毛参与了DNA的有效运输，进入细菌细胞进行基因转化。第三，它与抽搐运动有关，这是一种特殊的运动形式，由所有IV型纤毛细菌表达。在过去的几年里，人们已经对GC菌毛组装装置的组成成分有了很多了解，但菌毛及其组装装置如何促进黏附、DNA转化和抽动运动在很大程度上仍不清楚。我们提出了一系列实验，以加深我们对GC菌毛如何为发病机制提供这些功能的理解。在目标1中，将利用饱和突变来分离分泌素基因PilQ的功能丧失和功能获得突变。功能缺失突变将定义表达、稳定和多聚体所需的PilQ结构域。将对功能获得突变进行筛选，以寻找影响DNA转化、抗生素敏感性和抽搐运动的突变。还将测试突变株上皮细胞粘附性的变化。在目标2中，将对参与编曲菌毛功能的三个相关预测的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。

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.