In vivo essential genome of Salmonella

摘要 确定细菌病原体在宿主中适合性的全基因组遗传要求是一个基本的 病理基因组学的目标。转座子测序(TN-seq)是一种功能强大的全基因组测序工具 细菌中必需基因和条件性必需基因的鉴定。当应用于细菌病原体时 通过合适的动物感染模型，TN-seq可以加速发现猪传染性支气管炎的遗传决定因素。 体内健康的细菌病原体。然而，这种强大的方法的全部潜力在于理解 细菌毒力决定因素常常受到动物感染模型瓶颈的阻碍。 瓶颈会导致突变体的随机丢失，而不是它们的基因类型的贡献，这可能导致 错误的积极发现。在本应用程序中，我们将开发一种新的策略来克服这一限制，方法是 利用转座子系统从细菌产生基因组饱和的随机突变文库 在诱导时从在目标宿主组织中建立的小种子种群中繁殖细胞， 绕过瓶颈问题。从基因组饱和突变体获得的TN-seq图谱 从宿主组织中恢复的群体将允许通过以下方式对体内必需基因进行全基因组鉴定 缺少插入的基因与在体外鉴定必需基因的方式相同。这是一项新的 将充分开发和评估方法(Aim1)，并将其应用于肠道的全基因组鉴定 鼠伤寒沙门氏菌14028在小鼠体内的定植因素(目标2)。一旦建立并 经过验证，这种方法可以很容易地扩展到其他细菌病原体，以充分探索体内必不可少的 基因组。

SUMMARY Defining genome-wide genetic requirements for fitness of a bacterial pathogen in the host is a fundamental goal of pathogenomics. Transposon sequencing (Tn-seq) is a power functional genomics tool for genome-wide identification of essential and conditionally essential genes in bacteria. When applied to a bacterial pathogen with an appropriate animal infection model, Tn-seq can accelerate the discovery of the genetic determinants of a bacterial pathogen for in vivo fitness. However, the full potential of this powerful approach to understand bacterial virulence determinants is often hampered by the bottlenecks associated with animal infection models. The bottlenecks cause stochastic loss of the mutants without contribution of their genotypes, which can lead to false positive discoveries. In this application, we will develop a novel strategy to overcome this limitation by employing a transposon system that generates a genome-saturating random mutant library from the bacterial cells multiplied from a small seeder population established in the target host tissue upon induction, circumventing the bottleneck problem. The Tn-seq profile obtained from the genome-saturating mutant population recovered from the host tissue would allow genome-wide identification of in vivo essential genes by the genes lacking insertions in the same manner by which in vitro essential genes are identified. This new approach will be fully developed and evaluated (Aim1) and applied for genome-wide identification of the gut colonization factors of Salmonella Typhimurium 14028 during infection in mice (Aim 2). Once established and validated, this approach can be easily extended to other bacterial pathogens to fully explore in vivo essential genomes.