Diversity-Generation and Variable Protein Displays in Pathogens and Phage

描述（由申请人提供）：基于在靶基因中产生大量多样性的能力，在博德特拉噬菌体中发现了产生多样性的复古元件（DGRs）。它们通过模板依赖、逆转录介导的机制发挥作用，在蛋白质编码序列的特定位点引入核苷酸替代。可变残基显示在一个特殊支架的配体结合口袋中，该支架平衡了蛋白质多样性和结构稳定性，为潜在的配体-受体相互作用创造了大量的受体。使用博德特拉噬菌体DGR作为模板，我们已经在许多细菌，质粒和噬菌体基因组中鉴定了同源元件。大多数DGRs是细菌染色体元件，它们分布在整个细菌结构域。与健康和疾病特别相关的是，dgr作为染色体元件存在于人类共生体中，包括拟杆菌属、双歧杆菌属、真杆菌属和Ruminococcus属的成员，它们也被嗜肺军团菌、齿状密螺旋体和脆弱拟杆菌等病原体编码。尽管它们在自然界中分布广泛，但我们对DGR功能的机制和它们所赋予的选择优势的理解尚处于初级阶段。

DESCRIPTION (provided by applicant): Diversity-generating retro elements (DGRs) were discovered in Bordetella bacteriophage on the basis of their ability to generate vast amounts of diversity in target genes. They function through a template-dependent, reverse transcriptase-mediated mechanism that introduces nucleotide substitutions at specified sites in protein-coding sequences. Variable residues are displayed in the ligand-binding pocket of a specialized scaffold which balances protein diversity with structural stability, creating vast repertoires of receptors for potential ligand-receptor interactions. Using the Bordetella phage DGR as a template, we have identified homologous elements in numerous bacterial, plasmid, and bacteriophage genomes. Most DGRs are bacterial chromosomal elements and they are distributed throughout the bacterial domain. Of particular relevance to health and disease, DGRs are present as chromosomal elements in human commensals including members of the Bacteroides, Bifid bacterium, Eubacterium and Ruminococcus genera, and they are also encoded by pathogens such as Legionella pneumophila, Treponema denticola, and Bacteroides fragilis. Despite their widespread distribution in nature, our understanding of the mechanisms of DGR function and the selective advantages they confer is at a rudimentary stage. This application focuses on two DGR systems which provide complementary components of an experimental platform that will allow us to address some of the most compelling unanswered questions regarding these elements. The Bordetella BPP-1 phage DGR is by far the best characterized and it provides a paradigm for all members of this retro element family. L. pneumophila is a well studied pathogen amenable to genetic manipulation and it has become our prototype system for studying bacterial DGRs. Our specific aims are as follows: 1. Determine the mechanisms of adenine mutagenesis and retro homing by the Bordetella phage DGR. These are the hallmarks of DGR activity and they are essential for the creation of diversity. 2. Probe variable protein localization and structure, and the regulation of diversity by a L. pneumophila DGR. We will test the hypothesis that DGRs have been exploited for bacterial surface display of variable protein repertoires. Understanding DGRs will reveal new mechanisms for accelerated evolution by bacteria and phage and provide new paradigms for understanding adaptations of importance to human health and disease.