Identification of Campylobacter virulence and colonisation factors

Mu, X 2014, Identification of Campylobacter virulence and colonisation factors, Doctor of Philosophy (PhD), Applied Sciences, RMIT University.

Document type: Thesis
Collection: Theses

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Title Identification of Campylobacter virulence and colonisation factors
Author(s) Mu, X
Year 2014
Abstract Campylobacter jejuni is the most common Campylobacter species associated with campylobacteriosis. Contamination of poultry with C. jejuni during processing and subsequent poor food handling is the major cause of infection in humans. Effective strategies to control Campylobacter contamination and to prevent campylobacteriosis are still lacking. Since contaminated poultry is the major source of campylobacteriosis in industrialised countries, identification of virulence and colonisation factors will without doubt lead to new strategies for minimising colonisation of C. jejuni in poultry intestines. A starting point for identifying potential virulence factors is to identify proteins that are secreted from Campylobacter and therefore can interact with host molecules/cells.

In 2000, the complete genome sequence of C. jejuni subsp. jejuni NCTC 11168 was completed, which contains 1,654 coding DNA sequences (CDS). A range of bioinformatics techniques have been employed in this study to identify secreted and surface-exposed proteins, which may have the ability to interact with chicken immune cells. Results indicate that 6.96% of C. jejuni proteins have been predicted to contain a signal peptide, and 8.63% are predicted to be non-classically secreted using the web servers SignalP4.0 and Secretome2.0. Among them, a subset of 70 putative non-classically secreted proteins have been selected for further analysis, as they were predicted to be located on the cell surface or extracellularly using Gneg-PLoc and Gneg-mPLoc. They have been analysed further for the presence of transmembrane helices, transmembrane beta-barrels, putative proteases, enzymes, and virulence.

One of the proteins from the list, Cj0391c, was selected to be expressed as a recombinant protein in an E. coli BL21(DE3) pRSET-A expression system. This purified recombinant protein was shown to induce a reduction in viability of chicken macrophage HD11 cells, and induced apoptosis, suggestive of possible roles of this protein in immune system evasion or suppression of immune responses in poultry. Furthermore, this protein was computationally modelled and molecular dynamics simulations performed at different conditions. The constructed Cj0391c hexameric structural model was modelled, inserted and simulated in a phosphatidylethanolamine-phosphatidylglycerol bilayer. Under simulated conditions, the model maintained structural integrity at low temperature (300 K) without the presence of zinc, but not at higher temperatures, 310 K and 314 K. The helical bundle was maintained with the presence of zinc from low temperature 300 K to higher temperature 314 K. Simulations also suggest a wider pore in the presence of zinc, suggestive of higher channel conductance. Thus, similar to human dermcidin, zinc may play a role in enabling formation of a stable hexameric channel with a pore of sufficient size to cause pathological membrane permeability, leading to cell death.

Results from this study will help to improve our understanding of pathogenic mechanisms, more specifically, the virulence factors involved in C. jejuni pathogenicity. The molecular modelling and simulation revealed one possible mechanism of a protein toxic to chicken macrophage cells. These potential extracellular virulence factors can be used as virulence candidates for anti-Campylobacter strategies. By modelling identified proteins, antigenic determinants and/or functional regions/mechanisms may be found, which can then be used to design vaccines aimed at immunising poultry against Campylobacter colonisation.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Applied Sciences
Subjects Gene Expression (incl. Microarray and other genome-wide approaches)
Separation Science
Keyword(s) Virulence factors
Molecular Simulation
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Created: Fri, 24 Apr 2015, 12:43:32 EST by Keely Chapman
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