| dc.description.abstract | Fusobacterium nucleatum is Gram negative anaerobe of the oral cavity, where it plays a key role as a bridging organism in the plaque biofilm between early colonisers of the tooth surface, such as streptococcal species, and late colonisers, such as Porphyromonas gingivalis. While F. nucleatum is generally considered to be a commensal organism, research has increasingly found it to be associated with a variety of diseases, from periodontitis in the oral cavity, to cancer at various extra-oral sites around the body, including colorectal cancer and breast cancer. This bacterium has also been found in increased abundance at sites of oral leukoplakia (OLK) relative to healthy mucosa. These are premalignant sites, which have the potential to transform to oral squamous cell carcinoma (OSCC). Therefore, with the association between F. nucleatum and cancer, this study aimed to investigate the isolates found on these sites, using both genotypic and phenotypic methods.
The first section of this work involved the recovery of isolates from OLK and healthy sites by direct culture on selective media. Using this method, the most commonly isolated subspecies was F. nucleatum subsp. polymorphum, with 73.9% of the isolates identified, belonging to this subspecies. To investigate the genomes of these isolates, whole genome sequencing, using Illumina short-read sequencing, was carried out on 60 isolates. Pangenome analysis using Panaroo was carried out on these isolates and 16 additional genomes from GenBank. This revealed a small core genome of 1,604 core genes were shared among the isolates, with a much larger accessory genome of 6,032 genes. Based on the alignment of the core genes, a phylogenetic tree divided the isolates into three clades, but there was no association with disease and phylogeny. Closer investigations into the adhesins, specifically the Type Va adhesins, showed great heterogeneity in the distribution of these proteins across the isolates, with some isolates possessing only one of these adhesins, and other possessing as many as eight. The genotypic diversity seen in the isolates was mirrored in phenotypic analysis carried out, with isolates displaying variation in serum resistance, haemagglutination and adherence to oral keratinocytes.
Following on from this work, further analysis of the TVa adhesins was carried out. Using a combination of short read Illumina sequences and long read MinION sequences, hybrid assemblies were generated for a selection of isolates. This allowed for visualisation of the position of the adhesins, relative to one another and allowed for better comparison between adhesins of other subspecies. Analysis of recombination events between subspecies polymorphum, animalis, nucleatum and vincentii at specific genetic loci was carried out using fastGEAR. This revealed that both ancestral and recent recombination events had occurred between the subspecies at the genes of several Type Va adhesins, including fap2 and radD, resulting in genetic mosaicism at these loci. Further work in characterising the Type Va adhesins was to be undertaken by generating deletion mutants, but due to the recalcitrance of F. nucleatum, this was not possible in this study.
Finally, the effect of F. nucleatum infection on oral keratinocytes was investigated, with a specific focus on the oncogenic micro-RNA, miR-21. Infection of H376 cells with F. nucleatum resulted in a 2-fold increase in the production of miR-21 after 8 hours of infection. To then assess the impact of miR-21 on cytokine production and RNA expression profiles of epithelial cells after F. nucleatum infection, cells were treated with a miR-21 inhibitor before infection. Infection with F. nucleatum resulted in increased expression of several cytokines and chemokines, in a dose-dependent manner, including granulocyte-macrophage colony stimulating factor (GM-CSF), IL-18 and the matrix metalloproteases, MMP-1 and MMP-9. Inhibition of miR-21 in cells resulted in significantly less MMP-9 secretion, suggesting that F. nucleatum infection can induce MMP-9 secretion via increased expression of miR-21. RNA-seq results also indicated pathways involved in cytokine signalling and cell motility were significantly upregulated in F. nucleatum infection, with inhibition of miR-21 specifically impacting the transforming growth factor-beta (TGF-beta) signalling pathway in F. nucleatum infection.
Overall, this work highlights the heterogeneity seen in isolates of F. nucleatum subsp. polymorphum in the oral cavity, both in terms of their genotype and phenotype. It also shows the variation seen between genes, as a result of recombination with other closely related subspecies, and that the adhesins of this bacteria are very varied. The potential pathogenicity of F. nucleatum is also demonstrated here, with its ability to induce miR-21 and modulate the tumour microenvironment. | en |
