Comparative Genomics of Francisella

该项目的重点是图拉氏弗朗西斯氏菌，它是图拉热症的病原体，也是A类生物恐怖主义选择剂。迫切需要能够立即用于疫苗和治疗剂的发现以及图拉氏丝虫和其他A类生物的免疫学诊断分析方法的数据和试剂。我们的总体目标是使用基因组和蛋白质组的方法来识别与图拉氏菌致病相关的基因和对图拉热病免疫至关重要的抗原。在这份提案中，我们将把新的测序和寡聚合成/阵列技术(由我们的分包合作伙伴开发)与传统的Sanger测序策略相结合，以创建一种经济高效的方法来 确定具有不同毒力和生活方式表型的重要弗朗西塞氏菌菌株之间的遗传差异。作为实现这一目标的第一步，我们将测定两株A型和一株B型弗朗西塞氏菌的基因组序列。这些序列将与三个已经完成的弗朗西塞氏菌基因组序列进行比较，以确定可能导致不同弗朗西塞氏菌表型的插入、缺失和单核苷酸多态。 基因组测序将与蛋白质组表达相结合，使用一种新的鸟枪库/表达质粒来承载用于弗朗西斯菌测序的克隆，并表达来自每个弗朗西斯菌蛋白质组的多肽。来自这三个蛋白质组的多肽将用免疫反应血清进行筛选，以确定它们的抗原谱的差异。对强毒和无毒的弗朗西塞拉菌株进行比较遗传分析，并结合针对这些细菌蛋白质组中的多肽的抗原筛选，将鉴定出假定的毒力。 作为疫苗和免疫诊断试剂的未来研究的因素和抗原。

The focus of this project is Francisella tularensis, the causative agent of tularemia and a Category A Bioterrorism Select Agent. There is an urgent need for data and reagents that immediately can be directed toward vaccine and therapeutic agent discovery as well as immunological diagnostic assay development for F. tularensis, and other Category A organisms. Our overall goal is to use genomic and proteomic approaches to identify genes involved in F. tularensis pathogenesis and antigens important for tularemia immunity. In this proposal, we will merge novel sequencing and oligosynthesis/array technologies (developed by our subcontract partners) with traditional Sanger sequencing strategies to create a cost effective method to identify genetic differences between important Francisella strains with different virulence and lifestyle phenotypes. As a first step toward this goal, we will determine the genomic sequences of two type A and one type B Francisella strain. These sequences will be compared to three already completed Francisella genome sequences to identify the insertions, deletions, and single-nucleotide polymorphisms that may lead to differing Francisella phenotypes. Genome sequencing will be coupled to proteome expression using a novel shotgun library/expression plasmid to both host the clones for Francisella sequencing and to express peptides from each Francisella proteome. Peptides from the three proteomes will be screened with immunoreactive sera to identify differences in their antigen profiles. The comparative genetic analysis of virulent and avirulent Francisella strains in conjunction with an antigen screen against peptides from the proteomes of these bacteria will identify putative virulence factors and antigens for future study as vaccine and immunodiagnostic reagents.