Gut microbiome metatranscriptomics for identifying novel drug targets in inflammatory bowel disease

Inflammatory Bowel Disease (IBD), which includes Crohn’s disease and ulcerative colitis, is a lifelong remitting and relapsing inflammatory condition that affects the gastrointestinal (GI) tract. It is characterised by aberrant intestinal mucosal damage and repair in addition to increased epithelial permeability. The exact cause of IBD is unknown however strong associations have been identified, linking the disease to genetics, lifestyle factors and the microbiome. Changes in the microbiome are strongly correlated with IBD, and faecal microbiota transplant has demonstrated some efficacy in ulcerative colitis, however results are highly heterogenous because of the substantial inter-individual variability in microbiota taxonomy. Instead of studying taxonomy, we hypothesise that the best way of studying the involvement of the microbiome is by focussing on microbial gene activity. In our approach we use a discovery pipeline based on large RNAseq datasets to identify new proteins with biomarker properties or functional relevance in IBD. With this pipeline we endeavoured to produce recombinant proteins that are differentially expressed in disease and subsequently evaluate these for biomarker and functional properties. Production of proteins was performed in a E. coli vector expressing system. After purification, proteins were tested using a range of serological and human cell-based assays. Biomarker discovery included testing for seroreactivity of 20 upregulated microbial proteins in 90 serum samples derived from CD, UC and control groups. Although we were able to distinguish CD samples based on existing markers in our assay, no novel biomarkers were identified in our approach. A panel of 10 microbial proteins was tested to assess inducing and protecting effects on inflammation, intestine epithelial permeability, and wound repair effects in assays with humanised organoid models. From our unbiased screening assays, we identified one therapeutic candidate, that was downregulated in stool from patients with high disease activity, which exhibited protective effects on epithelial permeability. Further characterisation of this candidate revealed its specific inhibition of a neutrophilic protease that is pathologically linked to IBD and thereby demonstrated protective effects and therapeutic potential in IBD treatment. This thesis is one of the first to explore the role of the microbiome in IBD through synthesis of proteins identified through a differential gene expression analysis using a faecal MTX pipeline to study their potential as novel biomarkers and drug targets. Combined, these studies have outlined the potential of focusing on the activity of the microbial community by using MTX.