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Title: Investigating Fusobacterium pathogenesis using molecular and genomic methods to inform vaccine design
Author: Brewer, Matt L.
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2019
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Cellular adhesion is crucial in the pathogenesis of many bacterial species, where receptor-ligand interactions mediate host colonisation and/or invasion leading to host pathologies. Fusobacterium nucleatum (Fn) is a bacterial species gaining greater interest due to recent links with a variety of diseases, such as colorectal cancer. Fn is known to harbour a vast array of adhesive proteins (adhesins), however only a few have been studied in depth, such as FadA. This study aims to characterise a new previously unstudied subset of trimeric autotransporter adhesins (TAAs) found within Fn, responsible for binding the human receptor CEACAM1. These proteins were given the name CEACAM-binding proteins of Fusobacterium (CbpF). To examine the distribution of these receptors among Fusobacterium spp., we screened and sequenced a library of clinical isolates. From sequencing these strains, we identified two novel species of Fusobacterium (F. oralis sp. nov. and F. ovarium sp. nov.), both of which harboured CbpF. While performing the taxonomic analyses on the new strains we addressed the conflicting nomenclature and phylogenetic boundaries with respect to the genus. By utilising computational methods, we could confidently delineate species and show how the genus should be organised to better reflect the genomic differences and similarities between strains. Through screening two different types of CbpF from different species we confirmed the ability of both classes to bind CEACAM1 through proteomic- and cellular adhesion-based assays as well as showing that both classes of protein were capable of binding to CEA (CEACAM5), but not to other CEACAM variants examined. This highlighted the highly specific nature of these proteins, which was explored further by examining point mutants of CEACAM1, of which few showed any significant adhesion. As well as examining CbpF, we briefly looked at two other TAAs from Fn: FN0471 and FN0735; the former of which could bind indiscriminately to HeLa cells, thus indicating another important adhesin yet to be fully characterised. Structural analysis of CbpFs highlighted a gap in the literature with respect to TAA motifs and topologies, where no known structures showed significant homology to large portions of the proteins particularly in a region predicted to be occupied with a coiled-coil motif. X-ray crystallography, SAXS and CD were used to infer structural features of CbpF, however an atomic resolution structure could not be accurately produced from a protein crystal X-ray diffraction dataset. The work conducted here lays the foundation for additional studies into TAAs from Fusobacterium highlighting the requirement for increased detail on how these proteins contribute to pathogenesis and whether these proteins could be used as potential future vaccine candidates.
Supervisor: Hill, Darryl ; Brady, Leo Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Fusobacterium ; CEACAM1 ; TAA ; Hot-pathogen interaction ; CEA