Virulence determinants of Fusobacterium nucleatum

具核梭杆菌的毒力决定因素

• 批准号: 9982064
• 负责人: Hung Ton-That
• 金额: $ 44.39万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9982064
• 关键词: Adherence; Affect; Anaerobic Bacteria; Anti-Infective Agents; Antibiotic Resistance; Attenuated; Bacteria; Biochemical; Biogenesis; Biological Process; Cell Communication; Cell Proliferation; Cell physiology; Cells; Code; Colorectal Cancer; Complex; Cryo-electron tomography; Defect; Dental Plaque; Development; Electron Microscopy; Endothelial Cells; Epithelial Cells; Functional disorder; Fusobacterium nucleatum; Future; Gene Deletion; Generations; Genes; Genetic; Genome; Gingiva; Goals; Growth; Halitosis; Hydrogen Peroxide; Hydrogen Sulfide; Immune; Immune response; Immunomodulators; Impairment; Individual; Infection; Inflammasome; Inflammation; Inflammatory; Inflammatory Response; Investigation; Knowledge; Laboratories; Libraries; Link; Mass Spectrum Analysis; Mediating; Membrane; Methods; Microbial Biofilms; Microbiology; Molecular; Mutagenesis; Names; Odontogenesis; Open Reading Frames; Oxidative Stress; Pathogenesis; Pathogenicity; Pathway interactions; Periodontitis; Porphyromonas gingivalis; Premature Birth; Production; Proteins; Reporting; Rodent Model; Role; Saliva; Science; Signal Transduction; Streptococcus mutans; System; Technology; Time; Tube; Virulence; Virulence Factors; adverse pregnancy outcome; antimicrobial; cell growth regulation; colon carcinogenesis; cytokine; disorder prevention; fitness; forward genetics; human disease; in vivo; methionine sulfoxide reductase; mouse model; mutant; novel; oral biofilm; oral infection; oral pathogen; oxidative damage; pathogen; public health relevance; repaired; response; reverse genetics; therapeutic development; tool

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.

项目摘要 革兰氏阴性厌氧菌具核梭杆菌是口腔生物膜形成中的关键定植者， 或牙菌斑，并且还已知其与人类疾病（包括口腔感染、不良反应和/或炎症）的关联。 妊娠结局和结直肠癌。F.有核物质具有与之相互作用或聚集的固有能力， 许多早期和晚期的口腔生物膜殖民者。在啮齿类动物中诱导炎症反应和早产 感染模型，以及促进体内结直肠癌发生。尽管有致病潜力，我们 对梭菌毒力机制和相关因子的了解有限;迄今为止， 已经报道了梭菌因子，即FomA、FadA、Fap 2、RadD、aid 1和FAD-1，尽管超过100%的梭菌因子是梭菌因子。 在许多F.具核菌株一个主要的障碍， 进展是缺乏强有力的遗传工具和系统的调查。我们已经开始着手解决这个问题， 成功地建立了F. nucleatum，并生成一个大型的随机库， 10倍基因组覆盖率的转座子突变体。通过这些以前无法使用的工具，我们的目标是确定 F.毒力决定因子使用包括正向和反向在内的多种互补方法的核质 遗传学、冷冻电子断层扫描、生物化学方法和啮齿动物感染模型。通过电子 显微镜下，我们发现F.核质产生外膜小管（OMT），并确定了一个关键 形成OMT所需的因素。通过基因缺失，我们发现了介导氧化应激防御的途径 以及宿主细胞粘附和侵入。通过转座子诱变，我们发现了几个生物膜相关的 这些因素是产生硫化氢所必需的，硫化氢在很大程度上导致口臭或口臭。 在本申请中，我们的目的是阐明OMT生物发生和OMT介导的发病机制， 揭示了宿主免疫防御和宿主细胞相互作用的机制，并建立了生物膜的作用， 口臭、多种微生物相互作用和细菌毒力的相关因素。我们的研究将大大 有助于我们了解这种病原体的分子毒力机制， 随着新开发的遗传工具和先进技术的应用，该领域向前发展。结果 从这些研究中产生的药物也可能为未来的治疗药物开发提供有希望的靶点。 战略布局