Genome biology of Francisella tularensis populations

描述（由申请人提供）：土拉菌弗朗西斯菌是一种高度传染性，风险a组选定的细菌制剂，具有武器化的潜力。它是土拉菌病的病因。申请人的长期目标是了解驱动土拉菌四个亚种独特毒力和传播特性的基因和基因产物的途径。本R21应用程序的目的是确定负责该细菌最毒亚种的毒力和传播性的基因。中心假设是，增加的毒力和传播性可归因于遗传差异。这一假设将通过追求三个具体目标来验证：1)设计和表征一个代表土拉菌亚种的参考菌株集合。Tularensis和supsp。具有时空表征的全北极；2)制作土拉菌亚种和全北极亚种具有代表性的参考微阵列。利用阵列技术系统地探测菌株收集的基因组多样性；3)利用对端序列作图来补充基于微阵列研究的数据。主要的方法和方法包括系统地编目亚种特异性基因组片段的替代策略和策略。这将使用DNA微阵列分析（从代表性亚种制备的霰弹枪文库衍生的阵列）来完成。土拉菌（tularensis）和全北极菌（holarctica）)，以及一种新的数据排序算法，随后进行DNA序列分析，并通过Southern印迹和PCR进行确认。其次，从一个具有代表性的全北极菌株中提取一个狭窄（10Kb）大小的文库，用于生成成对端序列读取，然后将该列表映射到子类群。土拉菌基因组序列。这将使亚种特有的长度差异得以识别。这些结果总体上具有重要意义，因为它们有望提供强有力的初步证据，证明毒力和传播力的菌株差异可归因于基因组差异。这些数据有望成为确定的r01水平研究的基础，包括候选基因的功能分析。这些研究将大大增加我们对这种生物的病理生物学的理解，为疫苗和治疗提供候选基因产物，并为开发改进的诊断和法医标记提供位点。

DESCRIPTION (provided by applicant): Francisella tularensis is a highly infectious, risk-group A select bacterial agent that has the potential to be weaponized. It is the cause of tularemia. The applicants' long-term goal is to understand the pathways of genes and gene products that drive unique virulence and transmissibility traits of the four subspecies of F. tularensis. The objective of this R21 application is to identify genes that are responsible for virulence and transmissibility of the most virulent subspecies of this bacterium. The central hypothesis is that increased virulence and transmissibility are attributable to genetic differences. This hypothesis will be tested by pursuing three specific aims: 1) Design and characterize a reference strain collection representing both F. tularensis subsp. tularensis and supsp. holarctica with temporal and spatial representation; 2) Fabricate representative reference microarrays of the tularensis and holarctica subsp. and systematically probe genome diversity of the strain collection using arrays; and 3) Use paired-end sequence mapping to complement data from the microarray-based studies. The principal approaches and methods to be used include alternative strategies and tactics to systematically catalogue subspecies-specific genome segments. This will be accomplished using DNA microarray analyses (arrays derived from shotgun libraries prepared from the representative subsp. tularensis and holarctica) and a novel algorithm for data sorting followed by DNA sequence analyses and confirmation by Southern blotting and PCR. Second, a narrow (10Kb)-size library from a representative holarctica strain will be used to generate paired-end sequence reads, after which the list will be mapped to the subsp. tularensis genome sequence. This will allow length differences that are subspecies-specific to be identified. The outcomes are collectively significant, because they are expected to provide strong preliminary evidence that strain differences in virulence and transmissibility are attributable to genomic differences. Such data are expected to become the foundation for definitive R01-level investigations involving the functional analyses of candidate genes. These studies will significantly increase our understanding of the pathobiology of this organism, provide candidate gene products for vaccine and therapeutics, and provide loci for development of improved diagnostic and forensic markers.