Identification of campylobacter proteins that interact with host cells

John, B 2012, Identification of campylobacter proteins that interact with host cells, Doctor of Philosophy (PhD), Applied Science, RMIT University.


Document type: Thesis
Collection: Theses

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Title Identification of campylobacter proteins that interact with host cells
Author(s) John, B
Year 2012
Abstract Elucidating the Campylobacter proteins (particularly orphan proteins) that interact with host cells and therefore the chicken humoral immune system (i.e. secreted proteins) may lead to new approaches for reducing or eliminating Campylobacter jejuni from chickens and hence reduce the incidence of human infection. Bioinformatics tools were used to identify the predicted secreted proteins that are non-classically secreted and where the function of the proteins is still unknown. It is conceivable that a protein of unknown function that is predicted to be secreted may be a virulence protein. Of the approximately 1623 proteins encoded by the genome, up to 407 are predicted to be secreted or inserted into the cell membrane, as identified by using Bioinformatics. The detailed Bioinformatics analyses were performed using SignalP 3.0, SecretomeP 2.0, Gneg-PLoc, Gneg-mPLoc, TMHMM and MemType-2L. These analyses reduced the number of C. jejuni proteins to 41 which are predicted to undergo extracellular secretion. Proteins selected for further analyses were non-classically secreted proteins with a Sec score above 0.5. Cj0391c, Cj0428, Cj1656c Cj0243c and Cj1450 were selected for knock-out analysis. No studies have been previously reported on these selected hypothetical.

This study identified such proteins, and developed gene knock-out strains that will no longer express the protein. The knock-outs were created in order to assess the role in cellular adhesion of the proteins. The gene inserts were first cloned into the pBluescript vector. Inverse PCR was used to delete part of the gene and to introduce a BamH1 site. The purified PCR product and the empty PMW2 vector were digested with BamH1. Then the products were ligated with the 1.4 Kb Kanamycin cassette of the PMW2 vector. The resulting suicide plasmid was transformed into Campylobacter. In vitro analysis of the resulting knock-outs was been performed to analyse the phenotype using INT 407 cells. Significant differences in phenotype were observed with one of the knock-out’s showing no adherence to epithelial cells.

Although the Bioinformatics analyses reduced the choice of possible antigens as vaccine candidates, another analysis was carried out to strengthen the choice of antigens for future work. This study concentrated on the differences in the proteomes of C. jejuni, which is a commensal organism in chickens, and C. concisus, which is a non-coloniser in chickens. Both however cause gastroenteritis in humans. If differences/similiarities in the secretomes is found, it may be possible to find out the actual proteins responsible for the high level of colonization by C. jejuni in chickens. Similarity between the two proteomes was investigated using BlastP. This comparative analysis has identified C. jejuni specific proteins with no homologue encoded by the C. concisus genome. Out of the 407 C. jejuni proteins analysed 109 were found to be with no C. concisus homologue. Bioinformatics tools were used to further explore the secretome of C.concisus. One hundred and seventy proteins were predicted to be at the top of the list of proteins that may be considered future vaccine candidates against this organism.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Applied Science
Keyword(s) Campylobacter
Knockout
Bioinfomatics
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