Proteomic investigation of the genome-reduced pathogen, Mycoplasma hyopneumoniae

Publication Type:
Thesis
Issue Date:
2015
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Mycoplasma hyopneumoniae is a genome-reduced bacterium and an economically significant pathogen that chronically infects the respiratory tract of swine. This infection often leads to pneumonia and secondary infections, costing agricultural industries significantly in the use of antibiotics and vaccines, which are currently largely ineffective. An improved understanding of the molecular mechanisms behind the infection process is essential to our ability to rationally design better vaccine and therapeutic interventions. With fewer than 700 predicted protein coding sequences, M. hyopneumoniae possesses one of the smallest genomes of any free-living organism. As such, it lends itself well to thorough proteomic interrogation. In this thesis, a range of proteomic techniques have been used to investigate the M. hyopneumoniae global and surface proteome at the protein and peptide level, including surface shaving and labelling techniques, ligand and immuno-blotting and affinity chromatography, as well as N-terminal dimethyl labelling to determine true N-termini of mature proteins. This conceptually unbiased, function-oriented approach has revealed an unexpected level of complexity in the use of proteolytic processing, multifunctional proteins and moonlighting to compensate for reduced coding capacity at the genome level. While microarray and transcriptome studies suggest that under normal culture conditions, the majority of genes are transcribed; our analyses identified less than 400 detectable expressed protein products under similar conditions. A significant number of the expressed proteins were discovered to be multifunctional, post-translationally modified by proteolysis. Surface proteome analyses identified a range of proteins to be surface exposed, despite lacking known signal peptides. Even though many of these proteins had well-characterised functions in the cytoplasm, they were also identified to have secondary functions at the cell surface, a phenomenon known as moonlighting. Many of the proteins present at the cell surface were identified to be subjected to proteolytic cleavage events. These were predominantly cell surface adhesins, many of which have already been described in the literature, however a large number of cytoplasmic “housekeeping” proteins are also found to be post-translationally cleaved, multifunctional proteins or moonlighting proteins. These findings can be applied to improve the rational design and development of vaccines and therapeutics for the prevention and treatment of Mycoplasma hyopneumoniae, as well as having wider implications for the field of biology as a whole, if similar levels of post-translational regulation can be found in other bacterial pathogens.
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