Epitope mapping and functional analysis of Bacteroides fragilis ubiquitin

Abstract

Bacteroides fragilis is a Gram-negative, anaerobic bacterium localised to the lower regions of the human gastrointestinal (GI) tract where it acts as a key constituent of the intestinal microbiota. Some strains of this bacterium synthesise a protein with both structural and amino acid sequence homology to human ubiquitin (HUbb), a post-translational modification protein involved in virtually all aspects of cellular health, including proteasomal degradation. The structural homology that exists between B. fragilis ubiquitin (BfUbb) and its human counterpart, HUbb, is likely to account for the presence of circulating serum antibodies among patients suffering from various autoimmune diseases, including rheumatoid arthritis and systemic lupus erythematosus, which cross-react with both forms of ubiquitin (Ubb). Here, the current PhD project aimed to identify both shared and unique epitopes of BfUbb and HUbb through the isolation of 12mer peptide mimotopes using phage display technology. Two 12mer peptides of interest were isolated using phage display biopanning of anti-BfUbb and anti-HUbb antibodies. The unique binding activity of phage clones expressing these peptides to either anti-rBfUbb antisera or anti-HUbb antibodies was confirmed by phage binding ELISA. Peptides of interest were synthesised and subject to further in vitro analysis that involved ELISA assay optimisation and screening of autoimmune patient serum samples to determine the presence of reactive antibodies. To help elucidate the potential function of BfUbb, high content analysis and western blot were used to investigate its impact on cell health and tight junction protein expression, respectively. The prevalence of BfUbb among publicly available B. fragilis genomes was also determined using various bioinformatics approaches. During this work, division I and II B. fragilis were also confirmed as two distinct species, with these findings being subsequently published in the Journal of Medical Microbiology.

Date of Award	Jul 2024
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	Northern Ireland Department for the Economy
Supervisor	Linda Stewart (Supervisor), Lisa Connolly (Supervisor) & Sheila Patrick (Supervisor)