In vivo essential genome of Salmonella

沙门氏菌体内必需基因组

• 批准号: 10471446
• 负责人: YOUNG MIN KWON
• 金额: $ 7.18万
• 依托单位: DIVISION OF AGRICULTURE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-18 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10471446
• 关键词: Animal Model; Animals; Bacteria; CBA/J Mouse; Cecum; Cell Culture Techniques; Cells; Complex; Development; Disease; Enteral; Essential Genes; Gastroenteritis; Genes; Genetic; Genetic Determinism; Genome; Genotype; Goals; Human; In Vitro; Infection; Inflammation; Intraperitoneal Injections; Investigation; Lead; Libraries; Modeling; Mus; Mutagenesis; Pathogenesis; Performance; Population; Process; Protocols documentation; Research Personnel; Research Priority; Resistance; Resort; Route; Salmonella; Salmonella typhimurium; Site; System; Systemic infection; Therapeutic; Tissues; Virulence; Virulence Factors; animal model selection; fitness; functional genomics; genome-wide; genome-wide analysis; genomic tools; gut colonization; improved; in vivo; mutant; novel; novel strategies; oral infection; pathogen; pathogenic bacteria; screening; stressor; transposon sequencing

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.

总结 确定细菌病原体在宿主中适应性的全基因组遗传要求是一个基本的 病原体组学的目标。转座子测序（Tn-seq）是一种功能基因组学工具， 鉴定细菌中的必需基因和条件必需基因。当应用于细菌病原体时 通过适当的动物感染模型，Tn-seq可以加速发现感染的遗传决定因素。 一种细菌病原体，用于体内适应性。然而，这种强大的方法的全部潜力， 细菌毒力决定簇常常受到与动物感染模型相关的瓶颈的阻碍。 瓶颈导致突变体的随机损失，而没有它们的基因型的贡献，这可能导致 假阳性发现。在本申请中，我们将开发一种新的策略来克服这种限制， 使用转座子系统从细菌中产生基因组饱和的随机突变体文库， 细胞从诱导后在靶宿主组织中建立的小种子群体增殖， 解决瓶颈问题。从基因组饱和突变体获得的Tn-seq图谱 从宿主组织中回收的群体将允许通过以下方法在全基因组范围内鉴定体内必需基因： 以与体外必需基因鉴定相同的方式鉴定缺少插入的基因。这个新 一种方法将得到充分的开发和评估（Aim 1），并应用于全基因组的肠道识别 鼠伤寒沙门氏菌14028在小鼠中感染期间的定殖因子（目的2）。一旦建立， 经过验证，这种方法可以很容易地扩展到其他细菌病原体，以充分探索体内的基本 基因组