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

Fred Hutchinson Cancer Center, Seattle WA

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Abstract

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.