The study of leptospiral pathogenesis is hampered by the lack of efficient mutagenesis methodologies. Thus research has focused on alternative approaches including genome sequencing, comparative genomics, transcriptomics and proteomics. In this thesis a comparative proteomic approach was used to identify leptospiral proteins with a potential role in the leptospiral infection process. Identification of proteins was followed by characterization of target proteins with potential roles in the infection process and ultimately led to the identification of a novel leptospiral virulence factor.
Specifically, comparative proteomics using isobaric tags for relative and absolute quantitation complemented with two-dimensional gel electrophoresis were used for mass spectrometry-based protein identification and quantitation. These methodologies were utilised to identify and quantitate leptospiral proteins altered in expression in response to growth media limited in iron supply and/or supplemented with serum. These conditions were designed to mimic a subset of variables encountered by the bacteria within the host. These experiments led to the identification of five proteins with potentially novel roles in the leptospiral infection process.
One of these proteins was further characterized as a periplasmic catalase, KatE. Using insertion mutagenesis it was demonstrated that KatE enhances extracellular H2O2 resistance and is required for virulence in guinea pigs and hamsters.
Proteomic analyses also led to the identification of glutamic acid methylation of a protein that was further characterised to be surface exposed and expressed during leptospiral colonization of hamster liver and kidneys. This was the first description of glutamic acid methylation of a surface exposed protein in Leptospira. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/3732 |
| Date | 16 December 2011 |
| Creators | Eshghi, Azad |
| Contributors | Cameron, Caroline E. |
| Source Sets | University of Victoria |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Rights | Available to the World Wide Web |
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