Diversity-Generation and Variable Protein Displays in Pathogens and Phage

描述（由申请人提供）：基于其在靶基因中产生大量多样性的能力，在博德特氏菌噬菌体中发现了产生多样性的逆转录元件（DGR）。它们通过模板依赖性逆转录酶介导的机制发挥作用，该机制在蛋白质编码序列的特定位点引入核苷酸取代。可变残基显示在专门支架的配体结合口袋中，该支架平衡蛋白质多样性与结构稳定性，为潜在的配体-受体相互作用创造大量受体库。使用博德特氏菌噬菌体DGR作为模板，我们已经确定了许多细菌，质粒和噬菌体基因组中的同源元件。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生殖细胞中，包括拟杆菌属、双歧杆菌属、真杆菌属和瘤胃球菌属的成员，并且它们也由病原体编码，例如嗜肺军团菌、齿垢密螺旋体和脆弱拟杆菌。尽管它们在自然界中广泛分布，但我们对DGR功能机制及其所赋予的选择性优势的理解仍处于初级阶段。 该应用程序的重点是两个DGR系统，它们提供了一个实验平台的互补组件，使我们能够解决有关这些元素的一些最引人注目的未回答的问题。博德特氏菌BPP-1噬菌体DGR是迄今为止最好的表征，它提供了一个范例，为所有成员的这个复古元素的家庭。L.嗜肺菌是一种经过充分研究的适于遗传操作的病原体，它已成为我们研究细菌DGRs的原型系统。我们的具体目标如下： 1.确定波氏杆菌噬菌体DGR的腺嘌呤诱变和反向归巢的机制。这些都是DGR活动的标志，对于创造多样性至关重要。 2.探针可变蛋白的定位和结构，以及L.嗜肺菌我们将测试的假设，DGRs已被利用的细菌表面展示的可变蛋白库。 了解DGR将揭示细菌和噬菌体加速进化的新机制，并为理解对人类健康和疾病重要的适应提供新的范式。

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.