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Towards Understanding the Molecular Basis of Outer Membrane Vesicles Formation Open Access

Descriptions

Other title
Subject/Keyword
OMV cargo selection
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Elhenawy,Wael
Supervisor and department
Feldman, Mario (Biological Sciences, University of Alberta)
Examining committee member and department
Magor, Katharine (Biological Sciences, University of Alberta)
Valvano, Miguel (Centre for Infection and Immunity, Queen's University, Belfast)
Eitzen, Gary (Cell Biology, University of Alberta)
Raivio, Tracy (Biological Sciences, University of Alberta)
Department
Department of Biological Sciences
Specialization
Microbiology and Biotechnology
Date accepted
2015-09-25T09:00:04Z
Graduation date
2015-11
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
The release of outer membrane vesicles (OMV) is utilized by Gram-negative bacteria to perform a myriad of functions during pathogenesis and symbiosis. Despite their well-recognized roles, the mechanism responsible for OMV formation in bacteria remains elusive. Whether OMV are produced by an active mechanism or by the passive disintegration of the outer membrane (OM) is a still matter of controversy. If OMV are produced as a consequence to cell lysis, then their protein content should not differ from the parent OM. Nonetheless, many studies demonstrated the differences between the proteomes of OMV and the originating membranes, suggesting the presence of a directed mechanism for OMV formation and cargo selection. In this thesis, we compared the proteomes of OM and OMV purified from B. fragilis and B. thetaiotaomicron. Using mass spectrometry, we identified the proteins in the OM and OMV of both organisms. Our analysis revealed the presence of more than forty proteins exclusively-packed in B. fragilis OMV. In parallel, more than 30 proteins were excluded from OMV and were detected only in B. fragilis OM. Sugar hydrolases and proteases constituted a significant fraction of the OMV specific proteins. Using in vitro biochemical assays, we demonstrated the hydrolytic activity of B. fragilis OMV. Moreover, we were able to show that B. fragilis can induce OMV hydrolases in response to extracellular cues. Similar results were obtained with B. thetaiotaomicron OMV analysis. OMV cargo selection is not restricted to the protein content but was found to include the packed lipids. Previous studies demonstrated the uneven distribution of lipids between the OMV and the parent OM. In the dental pathogen, Porphyromonas gingivalis, deacylated lipid A species were preferentially enriched in the OMV. In this thesis, we investigated the role of the lipid A deacylase, PagL, in OMV production by the prominent enteric pathogen, Salmonella enterica serovar Typhimurium. The expression of PagL in vitro resulted in the exclusive accumulation of deacylated lipid A forms in S. Typhimurium OMV. Additionally, more OMV production was observed when PagL was recombinantly expressed in S. Typhimurium. On contrary, the expression of a catalytically inactive variant of the enzyme did not induce similar vesiculation levels. Moreover, we demonstrated that PagL is involved in OMV formation by intracellular S. Typhimurium. Our results suggest a role for PagL-mediated lipid A deacylation in OMV biogenesis. Unraveling the molecular mechanisms mediating OMV formation will greatly improve our understanding of bacterial pathogenesis.
Language
English
DOI
doi:10.7939/R3BR8MN0J
Rights
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
Citation for previous publication
Elhenawy, W., Debelyy, M. O., and Feldman, M. F. (2014). Preferential packing of acidic glycosidases and proteases into Bacteroides outer membrane vesicles. MBio 5 (2):e00909-14. doi:10.1128/mBio.00909-14

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