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

项目摘要/摘要 核梭杆菌(Fn)是人类口腔微生物区系中的重要成员，在口腔微生物区系中起着重要的作用。 作为早期牙面定殖者和与成熟菌斑相关的微生物之间的桥梁。然而， FN也被认为是几种口腔疾病(牙周炎和口腔鳞状细胞)的病原体 癌症、口腔鳞状细胞癌)和口腔外病理，包括结直肠癌(CRC)、阑尾炎、骨髓炎、 以及不良妊娠结局，如绒毛膜羊膜炎、胎盘感染和早产。然而，尽管 几十年来，FN在疾病状态和人类利基环境中的临床相关性几乎完全超出了 现代遗传学对其生理学、新陈代谢和发病机制进行基本研究的力量。 最近，我们的团队证明了由FN主导的肿瘤内微生物区系定植于特定的 人类口腔和结直肠肿瘤的微核，并有助于肿瘤空间和细胞的异质性。 然而，机械地理解FN是如何获得如此健康优势的，以至于它可以占据主导地位 在结直肠癌中，肿瘤中达到80%的相对丰度，但在健康的结肠中缺失，严重缺乏。 并代表着治疗进展的关键障碍。该提案的目的是利用泛表观基因组 来自158个不同的FN菌株的数据，包括临床分离的CRC，是为了使FN菌株系统地易于处理 并使用先进的基因系统在结直肠癌的背景下询问其发病机制。在目标1中，我们将 应用两种最先进的方法(SyngenicDNA和质粒人工修饰)来解决固有的 不同来源的FN菌株之间的Rm异质性问题。我们将设计、构建和验证FN- 基于转座子的随机突变的优化遗传工具(PFnTn)，CRISPR-Cas9靶向 突变(PFnCas9)和互补研究(PHS30MCSyn)。在目标2中，我们将构建一个基因组- 临床分离的结直肠癌菌株Fna SB010的100,000个条形码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，包括那些绝对需要生存的基因，以及那些 在特定环境中需要，包括在ApcMin/+小鼠模型中GI肿瘤定植期间 CRC。我们还将创建代表非必需基因组的单个突变体的有序文库 验证汇总结果和进一步筛选。我们的多学科团队汇集了遗传学方面的专业知识 工程学，泛基因组学，(Pi Johnston)，以及肿瘤内微生物区系，临床前癌症模型，宿主- 微生物原位成像(Co-I Bullman)，以及条形码方面的广泛知识和经验 转座子文库的构建、筛选、分析及阵列化文库的生成 黄)。这些目标的完成将为询问FN的生理、新陈代谢和 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，这些工具广泛适用，并将公开提供，以促进整个领域的研究 人类的生态位，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.