Diversity-Generation and Variable Protein Displays in Pathogens and Phage

描述(由申请人提供)：在波氏噬菌体中发现了产生多样性的逆转元件(DGR)，这是基于它们在目标基因中产生大量多样性的能力。它们通过模板依赖、逆转录酶介导的机制发挥作用，该机制在蛋白质编码序列中的特定位置引入核苷酸替换。可变残基显示在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。以波氏杆菌噬菌体DGR为模板，我们已经在大量的细菌、质粒和噬菌体基因组中鉴定了同源元件。大多数DGR是细菌的染色体元件，它们分布在细菌领域。与健康和疾病特别相关的是，DGRs作为染色体元件存在于人类共生体中，包括类杆菌属、双歧杆菌属、真细菌属和瘤胃球菌属的成员，它们也由病原体编码，如嗜肺军团菌、齿密螺旋体和脆弱类杆菌。尽管它们在自然界中广泛分布，但我们对DGR功能的机制及其所提供的选择性优势的了解还处于初级阶段。 本申请侧重于两个DGR系统，它们提供了一个实验平台的补充组件，使我们能够解决与这些元素有关的一些最引人注目的悬而未决的问题。到目前为止，波氏杆菌BPP-1噬菌体DGR是最具特性的，它为这个复古元件家族的所有成员提供了一个范例。嗜肺性乳杆菌是一种可以进行基因操作的致病菌，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.确定了波氏杆菌噬菌体DGR对腺嘌呤的诱变和回巢机制。这些是DGR活动的特点，对创造多样性至关重要。 2.探索嗜肺乳杆菌DGR的蛋白质定位、结构变化及多样性调控。我们将测试这一假设，即DGRs已被用于细菌表面展示可变蛋白质谱系。 了解DGRs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具有重要意义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.