Advanced genetic systems for Fusobacterium nucleatum in oral and extra-oral pathologies

项目总结/摘要 具核梭杆菌（Fn）是人类口腔微生物群的关键成员，在那里它起到基本的作用。 作为早期牙齿表面定居者和与成熟菌斑相关的微生物之间的桥梁。然而，在这方面， Fn也被认为是几种口腔疾病（牙周炎和口腔鳞状细胞癌）的致病因子 癌; OSCC）和口腔外病理包括结肠直肠癌（CRC），阑尾炎，骨髓炎， 以及不良妊娠结局，如绒毛膜炎、胎盘感染和早产。但尽管 在疾病状态和人类小生境的数十年临床相关性中，Fn几乎完全超出了 现代遗传学的力量，其生理学，代谢和发病机制的基本讯问。 最近，我们的研究小组证明，以Fn为主的肿瘤内微生物群， 人类口腔和结肠直肠肿瘤的微生态系统，并有助于肿瘤的空间和细胞异质性。 然而，一个机械的理解Fn如何获得这样一个适应性优势，它可以占主导地位， 在CRC中达到>80%的相对丰度，但在健康结肠中不存在， 并且代表了治疗进展的关键障碍。该提案的目标是利用泛表观基因组 来自158个不同Fn菌株（包括临床CRC分离株）的数据，是为了使Fn菌株系统易处理 并利用先进的遗传学系统，以询问其发病机制的背景下，CRC。在目标1中， 应用两种最先进的方法（SyngenicDNA和质粒人工修饰）来解决固有问题 不同来源Fn菌株间RM异质性问题。我们将设计，建造和验证Fn- 用于基于转座子的随机诱变（pFnTn）的优化遗传工具，CRISPR-Cas9靶向 诱变（pFnCas 9）和互补研究（pHS 30 MCSyn）。在目标2中，我们将构建一个基因组- Fna SB 010（一种临床CRC分离株）的100，000个条形码化转座子突变体的规模文库。使用这个库， 我们将定义FNA的基本基因，包括生存绝对需要的基因和 在特定环境中需要，包括在ApcMin/+小鼠模型中GI肿瘤的定殖期间， 《儿童权利公约》。我们还将创建一个有序的个体突变体库，代表非必需基因组， 合并结果的验证和进一步筛选。我们的多学科团队汇集了遗传学方面的专业知识， 工程，泛基因组学，（PI约翰斯顿），和肿瘤内微生物群，临床前癌症模型，宿主- 微生物原位成像（Co-I Bullman），除了在条形码技术方面的广泛知识和经验外， 转座子库构建、筛选和分析以及阵列库（Sub-PI）的生成 Huang）。这些目标的完成将为研究Fn的生理、代谢和生物学特性提供重要的工具。 发病机制我们的长期目标是发现预防或治疗细菌相关癌症的新策略。 此外，这些工具具有广泛的适用性，并将公开提供，以促进跨学科的研究。 人类小生境，包括口腔、胃肠道和泌尿生殖系统疾病状态。

PROJECT SUMMARY/ABSTRACT Fusobacterium nucleatum (Fn) is a key member of the human oral microbiota, where it serves a fundamental role as a bridge between early dental surface colonizers and microbes associated with mature plaque. However, Fn has also been implicated as a causative agent in several oral diseases (periodontitis and oral squamous cell carcinoma; OSCC) and extra-oral pathologies including colorectal cancer (CRC), appendicitis, osteomyelitis, and adverse pregnancy outcomes such as chorioamnionitis, placental infections, and pre-term birth. Yet, despite decades of clinical relevance across disease states and human niches, Fn remains almost entirely beyond the power of modern genetics for fundamental interrogation of its physiology, metabolism, and pathogenesis. Recently, our team demonstrated that the intratumoral microbiota, dominated by Fn, colonizes specific microniches of human oral and colorectal tumors and contributes to tumor spatial and cellular heterogeneity. However, a mechanistic understanding of how Fn has gained such a fitness advantage that it can predominate in CRC to reach >80% relative abundance in tumors, but is absent within the healthy colon, is severely lacking and represents a critical barrier to progress for therapies. This proposal objective, leveraging pan-epigenome data from 158 distinct Fn strains including clinical CRC isolates, is to make Fn strains systematically tractable and to use advanced genetic systems to interrogate its pathogenesis in the context of CRC. In Aim 1 we will apply two state-of-the-art approaches (SyngenicDNA and Plasmid Artificial Modification) to tackle the inherent issue of RM heterogeneity across Fn strains of diverse origin. We will design, construct, and validate Fn- optimized genetic tools for transposon-based random mutagenesis (pFnTn), CRISPR-Cas9 targeted mutagenesis (pFnCas9), and complementation studies (pHS30MCSyn). In Aim 2 we will construct a genome- scale library of 100,000s of barcoded transposon mutants of Fna SB010, a clinical CRC isolate. Using this library, we will define the essential genes of Fna, both those that are absolutely required for survival, and those that are required in specific environments, including during colonization of GI tumors within the ApcMin/+ murine model of CRC. We will also create an ordered library of individual mutants representing the non-essential genome for validation of pooled results and further screening. Our multidisciplinary team brings together expertise in genetic engineering, pangenomics, (PI Johnston), and the intratumoral microbiota, preclinical cancer models, host- microbial in situ imaging (Co-I Bullman), in addition to extensive knowledge and experience in barcoded transposon library construction, screening, and analysis as well as generation of arrayed libraries (Sub-PI Huang). Completion of these aims will provide critical tools for interrogating Fn physiology, metabolism, and pathogenesis. Our long-term goal is to discover new strategies to prevent or treat bacterial-associated cancers. Moreover, these tools are broadly applicable and will be made openly available to advance research across human niches, including oral, gastrointestinal, and urogenital disease states.