Cloning, expression, purification and characterisation of the Helicobacter pylori outer membrane proteins BabC and HomB

Alissa, Mohammed Abdulaziz M (2022) Cloning, expression, purification and characterisation of the Helicobacter pylori outer membrane proteins BabC and HomB. PhD thesis, University of Nottingham.

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Abstract

Helicobacter pylori (H. pylori) is a pathogenic bacterium that colonises the gastric mucosa of more than 50% of all humans and a leading cause of peptic ulceration and gastric cancer worldwide. Despite the harsh conditions in the stomach, the bacterial adherence to the gastric mucosa and epithelium plays a significant role in initial colonisation and in long-term persistence by using multitude of adhesins that facilitate the adhesion to the host cells. Studies are greatly needed to understand the dynamic interplay between these adhesins, some of which have not been fully characterised.

This study is aimed at characterising the functional properties of the H. pylori outer membrane proteins BabC and HomB, which have not been studied well previously with unknown structure and function. Recombinant forms of BabC and HomB were expressed in the periplasm and cytoplasm of Escherichia coli, respectively. Carbohydrate microarray was used to identify host binding receptors for these putative adhesins, but no significant binding was detected. Structural analysis using Phyre2 suggested that HomB could be a metzincin, and indeed we were able to demonstrate proteolytic activity of recombinant HomB. However, we were unable to corroborate metzincin activity using divalent metal chelating agents such as EDTA and 1,10 phenanthroline, possibly due to the much higher binding affinity of the enzyme for zinc than the inhibitors. The recombinant HomB also induced IL 8 production from the gastric epithelial cells, which has not been previously reported. This indicates that HomB has a potential role in the pathogenesis of H. pylori and seems to be a marker associated with peptic ulcer and gastric cancer. The study presented in this thesis lays a strong foundation for future work and guides future design of specific attachment and/or inflammation inhibitors as a novel therapeutic approach, to limit the increasing resistance of this pathogen to conventional antibiotic treatment.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Falcone, Franco
Winkler, Sebastiaan
Keywords: Helicobacter pylori, H. pylori, Outer membrane proteins, BabC, HomB
Subjects: Q Science > QR Microbiology > QR 75 Bacteria. Cyanobacteria
Faculties/Schools: UK Campuses > Faculty of Science > School of Pharmacy
Item ID: 67244
Depositing User: Alissa, Mr Mohammed
Date Deposited: 31 Jul 2022 04:40
Last Modified: 31 Jul 2022 04:40
URI: http://eprints.nottingham.ac.uk/id/eprint/67244

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