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Title: Towards accessing the proteolytic potential of the gut microbiota
Author: Morris, Laura
ISNI:       0000 0004 5352 537X
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2014
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Introduction: The human gut microbiota outnumbers our own human cells by 100-1 and is often considered an extension of our genome harboring fundamental functions of which we are genetically incapable. However, it can also be a significant liability and has been implicated in numerous diseases, particularly Inflammatory Bowel Disease (IBD). The efforts of this research entailed evaluating a specific molecular mechanism of the gut bacterial metagenome; proteolytic activity, collectively known as the Degradome due to their putative role as significant virulence factors in IBD. In order to access this degradome of the gut microbiota, firstly, novel functional metagenomic (FM) tools were developed with an aim of facilitating the isolation of proteases. Secondly, a comprehensive cohort study was conducted comparing faecal protease activity, 16S rRNA microbial community structure and the potential of the faecal degradome to act as virulence factors between a group of IBD patients and a group of healthy volunteers to begin to determine the role of microbial proteases in disease aetiology. Results:-The IBD cohort exhibited significantly higher protease activity than the healthy cohort. Inhibitor assays also showed that the IBD cohort contained different types of proteases to the healthy cohort with significantly higher levels of metalloprotease activity. 16S rRNA gene analysis of the microbial community also revealed a dysbiosis of the gut microbiota between the IBD cohort and the healthy cohort. Dysbiosis was also observed between the high protease producers and the low protease producers and protease activity in the IBD cohort was able to decrease trans epithelial resistance in an HT-29 cell line and increase cellular permeability. Functional metagenomics tools were also assessed for isolation of proteases from the gut microbiota. The ability of a protease deficient strain; Bacillus subtilis WB800N to express proteases was compared with E. coli to determine its usefulness as a host for FM screening for proteases. B. subtilis WB800N was able to express gelatinase E and neutral protease while E. coli was not suggesting B. subtilis WB800N was more suitable as a host. However, when the gut microbiota was screened for proteases using this host, none were isolated suggesting improvements still needed to be made. Conclusions: - Compositional alterations of the gut microbiota appear to be associated with high and low levels of protease activity. The IBD cohort had elevated activity and an expanded repertoire of faecal proteases which also appear to have the potential to act as a virulence factor by disrupting epithelial cell barrier integrity. Proteases remain elusive via FM screening; however this study has highlighted the areas that need improvement to optimize future screens for accessing the degradome of the gut microbiota.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QR Microbiology