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

口腔和口腔外病理学中具核梭杆菌的先进遗传系统

• 批准号: 10790572
• 负责人: Christopher D Johnston
• 金额: $ 48.14万
• 依托单位: FRED HUTCHINSON CANCER CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-19 至 2025-09-18
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10790572
• 关键词: ApcMin/+ mice; Appendicitis; Bar Codes; Biotechnology; CRISPR/Cas technology; Cancer Model; Classification; Clinical; Colon; Colorectal Cancer; Colorectal Neoplasms; Complement; DNA; DNA Restriction-Modification Enzymes; Data; Dental; Disease; Electroporation; Engineering; Environment; Epigenetic Process; Escherichia coli; Essential Genes; Fusobacterium nucleatum; Gastrointestinal Diseases; Gastrointestinal Neoplasms; Gene Deletion; Generations; Genes; Genetic; Genetic Engineering; Genome; Goals; Heterogeneity; Human; Human Pathology; In Vitro; Individual; Knowledge; Laboratories; Libraries; Malignant Neoplasms; Metabolism; Methods; Microbe; Modeling; Modernization; Modification; Molecular Mimicry; Mouth Diseases; Mouth Neoplasms; Mutagenesis; Oral; Osteomyelitis; Pathogenesis; Pathology; Periodontitis; Physiology; Plasmids; Premature Birth; Refractory; Reproducibility; Research; Role; Suicide; Surface; System; Techniques; Transposase; Urogenital Diseases; Validation; Variant; adverse pregnancy outcome; clinically relevant; cost effective; design; epigenome; experience; fitness; functional genomics; genetic manipulation; genome editing; genome-wide; gut colonization; in situ imaging; interest; intraamniotic infection; member; methylation pattern; methylome; microbial host; microbiota; mouse model; mouth squamous cell carcinoma; multidisciplinary; mutant; novel; oral microbial community; placental infection; plasmid DNA; pre-clinical; prevent; screening; tool; tumor; tumor microenvironment

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.

项目摘要/摘要 核细菌核（FN）是人口腔微生物群的关键成员，它在其中提供基本 作为早期牙齿表面殖民者和与成熟斑块相关的微生物之间的桥梁的角色。然而， FN还与多种口腔疾病（牙周炎和口腔鳞状细胞 癌; OSCC）和口外病理，包括大肠癌（CRC），阑尾炎，骨髓炎， 和不良怀孕结局，例如绒毛膜炎，胎盘感染和预期出生。但是，尽管如此 数十年来，疾病状态和人壁ches的临床相关性数十年，FN几乎完全超出了 现代遗传学对其生理学，代谢和发病机理的基本审查的力量。 最近，我们的团队表明，肿瘤内微生物群以FN为主导，将特定的菌群定居 人口腔和结直肠肿瘤的微观，有助于肿瘤空间和细胞异质性。 但是，对FN如何获得适应性优势的机械理解，以至于它可以占主导地位 在CRC中，肿瘤中相对丰度> 80％，但在健康结肠中不存在，严重缺乏 并代表了治疗进展的关键障碍。这个提案目标，利用泛斑模素组 来自158种不同FN菌株在内的数据，包括临床CRC分离株 并使用先进的遗传系统在CRC的背景下询问其发病机理。在目标1中，我们将 采用两种最先进的方法（SyngenicDNA和质粒人工修饰）来应对固有的 RM异质性跨FN菌株的各种起源的问题。我们将设计，构建和验证fn- 优化基于转座子的随机诱变（PFNTN）的遗传工具，CRISPR-CAS9针对 诱变（PFNCAS9）和互补研究（PHS30MCSYN）。在AIM 2中，我们将构建一个基因组 - 临床CRC分离物的FNA SB010的100,000个条形码转座子突变体的比例文库。使用此库， 我们将定义FNA的基本基因，既是生存绝对必需的基因，又 在特定环境中，包括在apcmin/+鼠模型中的胃肠道肿瘤定植期间 CRC。我们还将创建一个有序的单个突变体库，代表非必需基因组 验证合并结果和进一步筛选。我们的多学科团队汇集了遗传方面的专业知识 工程学，pangenomics，（Pi Johnston）和肿瘤内微生物群，临床前癌模型，宿主 - 微生物原位成像（Co-i Bullman），除了在条形码方面的广泛知识和经验外 Transposon库的构建，筛选和分析以及阵列的生成（sub-pi） 黄）。这些目标的完成将为询问FN生理学，代谢和 发病。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，将公开可用以推进整​​个研究 人壁细分市场，包括口腔，胃肠道和泌尿生殖器疾病。