The role of outer membrane vesicles of Helicobacter pylori in pathogenesis

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AlSharaf, L.M.K.H.E., 2021. The role of outer membrane vesicles of Helicobacter pylori in pathogenesis. PhD, Nottingham Trent University.

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Abstract

Background: H. pylori continuously produces outer membrane vesicles (OMV) during active growth. These vesicles contain virulence factors and are cytotoxic, but the properties and contents of bacterial OMV can vary depending on the environmental conditions.

Aim: The study aimed to characterise H. pylori OMV isolated from different environmental conditions in terms of their quantity, size, cytotoxicity, and protein contents.

Methods: OMV were purified from H. pylori cultures in BHI broth with 0.2% β-cyclodextrin after 1 or 6 days or blood agar plates after 24 hours. OMV size and quantity were characterised by Zetaview. Their toxicity on human gastric epithelial (AGS) cells was determined by CellTiter, RealTime-Glo assays, IncuCyte and microscopy. Proteins were characterised and quantified by LC-MS/MS and SWATHMS, label-free, to determine changes in protein expression between growth conditions. A cagA mutagenesis plasmid was constructed in the 444A strain background.

Results: H. pylori produced more OMV in broth than on agar plates with differences also observed in average OMV diameter between conditions. OMV isolated from late broth and plate cultures were significantly more toxic to AGS cells than those from early broth (p <0.001). Vacuoles were seen in AGS cells treated with OMV of virulent strains isolated from early broth and plate cultures, indicating VacA activity. Hummingbirds (elongated gastric epithelial cells) were seen in AGS cells treated with OMV from cag+ strains, indicating CagA activity. There was ~2-fold more VacA protein in OMV from early broth and plate cultures than in OMV from late broth cultures. In contrast, there was more CagA toxin in OMV from early broth than in OMV from late broth and plate cultures. OMV isolated from plate cultures had completely different protein profiles compared with OMV from broth cultures. OMV from H. pylori 60190 WT strain were more toxic to AGS cells than those from the ΔcagA strain.

Conclusions: In summary, the characteristics of H. pylori OMV change dramatically, depending on the environment in which the bacterium is grown. This may influence bacterial virulence.

Item Type:	Thesis
Creators:	AlSharaf, L.M.K.H.E.
Date:	March 2021
Rights:	This work is the intellectual property of the author. You may copy up to 5% of this work for private study, or personal, non-commercial research. Any re-use of the information contained within this document should be fully referenced, quoting the author, title, university, degree level and pagination. Queries or requests for any other use, or if a more substantial copy is required, should be directed in the owner of the Intellectual Property Rights.
Divisions:	Schools > School of Science and Technology
Record created by:	Linda Sullivan
Date Added:	28 Jun 2022 11:21
Last Modified:	10 Feb 2023 03:00
URI:	https://irep.ntu.ac.uk/id/eprint/46495