Monday, December 30, 2024
12/30/2024
PROJECT SUMMARY The Gram-negative oral anaerobe Fusobacterium nucleatum is associated with oral and extraoral diseases due to its remarkable ability to escape host immunity and spread to extraoral sites, including placenta and colon, promoting preterm birth and colorectal cancer. How this pathobiont adapts to various metabolically changing environments enabling its virulence potential is not well understood. To overcome the well-known genetic intractability of F. nucleatum, we employed multidisciplinary approaches, combining reverse and forward genetics, advanced electron microscopy, biochemical methods, and rodent models of infection to investigate the molecular assembly on the cell surface of F. nucleatum, since surface proteins and structures are important in many cellular processes, including host cell adherence/invasion, motility, and nutrient transport. Subsequently, we uncovered tubular structures produced by biofilm cells and planktonic cells under stress. Cryo-EM tomography revealed that these nanostructures are grown out from the outer membrane mainly at the cell pole, hence named outer membrane tubules or OMTs. Mass spectrometry analysis of isolated OMTs revealed a large number of proteins predicted to be involved in transport, metabolism, host cell interactions, and stress response, with many hypothetical outer membrane proteins/lipoproteins. Critically, genetic disruption of OMT-associated proteins identified two mutants defective in OMT formation, ∆omtA and ∆omtB. Informed by structural studies of OmtA and OmtB, we propose here to elucidate the mechanism of OmtA-mediated OMT biogenesis, examine the role of OmtB and its biochemically and genetically linked factors in OMT biogenesis and fusobacterial metabolism, and examine the role of OMT formation in the pathophysiology of F. nucleatum. The results generated from this study will not only provide insights into the pathophysiology of this oral pathobiont, as well as promising targets for therapeutic development, but also advance our knowledge of assembly mechanisms of membrane-derived appendages in other bacteria.
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