Easton, S.; (2009) Functional and metagenomic analysis of the human tongue dorsum using phage display. Doctoral thesis , UCL (University College London).

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Abstract

It is well established that mixed microbial communities contain organisms which have not been studied by conventional culture-based methods. In the human oral cavity this number is estimated at around 50%. Commensal bacteria develop and maintain an intimate relationship with human cells without triggering proinflammatory mechanisms and this study aims to explore this by searching for bacterial proteins which facilitate binding to the human tongue dorsum and wider oral cavity. Metagenomic DNA from the human tongue dorsum of 9 volunteers was extracted and a phage display library created, to our knowledge the first to incorporate metagenomic DNA. Phage display is an elegant molecular technique involving fusion of fragmented DNA to a phagemid coat protein, such that inserted DNA is encoded by the phage and displayed on the phage surface. The affinity selection technique panning, then exploited the natural affinity and specificity of the fusion proteins to identify bacterial binding proteins using, in this case, three ligands: IgA, Fibronectin and BSA. IgA is of special interest to this group as it interacts with bacterial proteins and is poised to respond to bacterial numbers in human secretions such as saliva. Proteins from panning were analysed in silico, however, the majority were discarded due to the presence of stop codons in the protein sequences. Remaining phagemid displaying fusion proteins of interest were assessed for function and binding assays carried out to confirm binding specificity. Due to the biased nature of phage display library production, a 16S rRNA gene analysis was also carried out in order to assess metagenomic DNA diversity prior to library construction. Because phage display was used successfully by colleagues with the genomes of single organisms, it was believed that including metagenomic DNA in a phage display library would cast a wide net over the tongue dorsum allowing capture of many more binding proteins occurring in this environment from a wide range of bacteria.

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