Emily A. Peluso, Matthew Scheible, Hung Ton-That, Chenggang Wu
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Abstract
Considered a commensal, the Gram-negative anaerobe Fusobacterium nucleatum is a key member of the oral microbiome due to its wide range of interactions with many oral microbes. While the periodontal pathogenic properties of this organism have widely been examined, its connotation with extra-oral infections, including preterm birth and colorectal cancer, has now become apparent. Nonetheless, little is known about the mechanisms of pathogenicity and the associated virulence factors of F. nucleatum , most likely due to limited genetic tools and facile methodology. Here, we describe molecular techniques for the genetic manipulation of F. nucleatum , including markerless, nonpolar gene deletion, complementation, and Tn5 transposon mutagenesis. Further, we provide methodology to assess virulence potential of F. nucleatum using a mouse model of preterm birth. © 2020 Wiley Periodicals LLC.
Basic Protocol 1 : Generation of a galK mutant strain
Basic Protocol 2 : Complementation of a mutant strain
Basic Protocol 3 : Tn5 transposon mutagenesis of F. nucleatum
Basic Protocol 4 : Mouse model of preterm birth
核梭杆菌的遗传操作及毒力评价
革兰氏阴性厌氧菌核梭杆菌被认为是一种共生菌，由于其与许多口腔微生物的广泛相互作用，它是口腔微生物组的关键成员。虽然这种微生物的牙周致病特性已被广泛研究，但它与口外感染(包括早产和结直肠癌)的关系现在已变得明显。然而，由于遗传工具有限和方法简单，对具核梭菌的致病性机制和相关毒力因素知之甚少。在这里，我们描述了分子技术的遗传操作的核仁，包括无标记，非极性基因缺失，互补和Tn5转座子突变。此外，我们提供的方法来评估毒力潜力具核梭菌使用小鼠早产模型。©2020 Wiley期刊有限公司基本方案1:galK突变株的产生基本方案2:突变株的补充基本方案3:F. nucleatum的Tn5转座突变基本方案4:早产小鼠模型
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