Project Summary: Fusobacterium nucleatum is a significant oral pathobiont associated with periodontal diseases. When it spreads beyond the oral cavity, it is strongly linked to various conditions, including colorectal cancer, adverse pregnancy outcomes, inflammatory bowel disease, and rheumatoid arthritis. The current approaches to combat fusobacterial pathogenesis have primarily involved antibiotic treatments, such as metronidazole. However, while these broad- spectrum antibiotics are effective against F. nucleatum, they also disrupt the microbial balance, leading to dysbiosis and further health complications. This underscores the need for more targeted, narrow-spectrum antimicrobials that specifically inhibit or eradicate F. nucleatum. To develop such targeted antibiotics, knowledge of the essential genes encoding core proteins uniquely required for fusobacterial survival is needed. Our preliminary data show that fomA, encoding the well-studied fusobacterial major outer membrane porin protein, is essential for cell viability. This unexpected result challenges the previous report of its non-essential nature. Given the colocalization of fomA with lysine degradation pathway-associated operons and the substantial butyrate production by F. nucleatum via the lysine degradation pathway, we hypothesized that FomA is an integral porin responsible for transporting butyrate, a by-product of lysine degradation, from the periplasm to the cell's exterior. This proposal seeks to answer why fomA is essential for cell survival and to systematically identify the essential genes in F. nucleatum, with the long-term goal of developing targeted narrow-spectrum antibiotics against F. nucleatum. Aim 1 will investigate the molecular basis of FomA's essentiality in F. nucleatum with a comprehensive approach that includes a liposome swelling assay and a planar lipid biolayer assay. Aim 2 will systematically identify the essential genes in F. nucleatum by high-density Tn5- based transposon mutagenesis followed by high-throughput sequencing and create a CRISPRi mutant library for each identified essential gene or operon. This experiment will define the nature of fomA in F. nucleatum, along with a comprehensive understanding of its essentiome. Our findings will pave the way for new, targeted, narrow-spectrum antibiotics to treat F. nucleatum infections effectively.