Virulence determinants of Fusobacterium nucleatum

项目总结 革兰氏阴性厌氧菌核梭杆菌是口腔生物膜形成过程中的关键定植体， 或牙菌斑，也因其与人类疾病的关联而闻名，包括口腔感染、不良反应 妊娠结局和结直肠癌。F.核素具有与生俱来的相互作用或聚集的能力 许多早期和晚期的口腔生物膜殖民者。它能引起啮齿动物的炎症反应和早产 感染模型，以及促进体内结直肠癌的发生。尽管它具有致病潜力，但我们 对梭菌毒力的机制和相关因素的了解有限；到目前为止只有6个 已经报道了融合细菌因子，即FOMA、FADA、FAP2、RADD、AID1和FAD-I，尽管超过 在许多核盘菌的基因组中都注释了2000个开放阅读框。一个主要的障碍限制 进展是缺乏强大的遗传工具和系统的调查。我们已经开始解决这个问题， 成功地建立了一种简便的核盘藻基因缺失系统，并生成了一个大的随机文库 具有10倍基因组覆盖率的转座子突变体。通过这些以前不可用的工具，我们的目标是确定 利用包括正向和反向在内的多种互补方法确定核镰刀菌的毒力决定因素 遗传学、冷冻电子断层扫描、生化方法和啮齿动物感染模型。通过电子 显微镜下，我们发现核盘藻产生外膜小管(OMTs)，并确定了一个关键 OMT形成所需的系数。通过基因缺失，我们发现了介导氧化应激防御的途径 以及宿主细胞的黏附和入侵。通过转座子诱变，我们发现了几个与生物膜相关的 产生硫化氢所需的因素，这在很大程度上导致口臭或口臭。 在这一应用中，我们旨在阐明OMT的生物发生和OMT介导的致病机制。 揭示宿主免疫防御和宿主细胞相互作用的机制，并确立生物膜- 口臭、多菌相互作用和细菌毒力的相关因素。我们的学习将极大地 有助于我们理解这种病原菌的分子毒力机制，并显著地 随着新开发的基因工具和先进技术的应用，该领域向前发展。结果是 这些研究产生的结果也可能为治疗的未来发展提供有前途的目标 战略。

PROJECT SUMMARY The Gram-negative anaerobe Fusobacterium nucleatum is a key colonizer in the development of oral biofilms, or dental plaque, and also known for its association with human diseases including oral infections, adverse pregnancy outcomes, and colorectal cancer. F. nucleatum has an inherent ability to interact or aggregate with many early and late colonizers of the oral biofilms. It induces inflammatory responses and preterm birth in rodent models of infection, as well as promoting colorectal carcinogenesis in vivo. Despite its pathogenic potential, we have limited knowledge about the mechanisms of fusobacterial virulence and associated factors; to date only six fusobacterial factors have been reported, i.e. FomA, FadA, Fap2, RadD, aid1, and FAD-I, although more than 2,000 open reading frames are annotated in the genome of many F. nucleatum strains. A major obstacle limiting progress is the lack of robust genetic tools and systematic investigations. We have begun to tackle this problem, successfully developing a facile gene deletion system for F. nucleatum, and generating a large library of random transposon mutants with 10-fold genome coverage. With these previously unavailable tools, we aim to identify virulence determinants of F. nucleatum using multiple complementary approaches including forward and reverse genetics, cryo-electron tomography, biochemical methods, and rodent models of infection. By electron microscopy, we discovered that F. nucleatum produces outer membrane tubules (OMTs) and identified a key factor required for OMT formation. By gene deletion, we found pathways that mediate oxidative stress defense and host cell adherence and invasion. By transposon mutagenesis, we revealed several biofilm-associated factors that are required for production of hydrogen sulfide, which largely contributes to bad breath, or halitosis. In this application, we aim to elucidate the mechanism of OMT biogenesis and OMT-mediated pathogenesis, reveal the mechanisms of host immune defense and host cell interactions, and establish the role of biofilm- associated factors in halitosis, polymicrobial interactions, and bacterial virulence. Our studies will greatly contribute to our understanding of the molecular virulence mechanisms of this pathogen and significantly move the field forward with the application of newly developed genetic tools and advanced technology. The results generated from these studies may also provide promising targets for the future development of therapeutic strategies.