Proteomic characterisation of surface proteins in Mycobacteria and Corynebacteria

2017-02-22T00:51:31Z (GMT) by Rees, Megan Anne
A proteomic approach was used to characterise proteins expressed by several species of the Corynebacteriacae family, including the significant human pathogen Mycobacterium tuberculosis. The surface of a bacterium is its first point of contact with the environment and importantly the host. Therefore identifying surface proteins should provide insights into the disease process. The membrane shaving approach has been developed to target the bacterial surface. This method was successfully optimised and applied to C. glutamicum, C. pseudotuberculosis and M. tuberculosis. The cell wall is not disrupted by this method, which allowed a series of experiments to characterise the topology of proteins within the cell wall. Strains of C. glutamicum with defined cell wall defects underwent membrane shaving, revealing a group of proteins identified only in these mutant strains. These proteins had features that suggested they resided in a deeper layer of the mycobacterial cell wall. The use of living cells in the membrane shaving technique allowed a cross-sectional characterisation of the surface proteome under different environmental conditions. The surface protein repertoire was examined in bacteria exposed to an acid environment and also bacteria directly harvested from the host. Additionally differences in the host protein milieu surrounding these bacteria were able to be characterised. M. tuberculosis is a successful human pathogen, and is likely to possess many features that may be absent from the non- pathogenic C. glutamicum. C. pseudotuberculosis was selected as a model organism for M. tuberculosis because of its genetic similarity to C. glutamicum. A comprehensive genome and surface proteome comparison was made between these species, demonstrating greater variation in expressed proteins than predicted differences in the genome, including the identification of known virulence factors. Thus, characterising features that may mediate pathogenicity. Differences were also demonstrated between strains of the same species. Isotopic labelling with dimethylation was used to perform a comprehensive quantitative analysis of the whole proteome of several field strains of C. pseudotuberculosis in contrast to a laboratory reference strain. This comprehensive cross-sectional analysis of expressed proteins identified several groups of proteins that were present in greater quantities in the field strains, these were often proteins with specific metabolic functions. Production of these proteins may be increased to deal with the hostile, nutrient-poor environment of the infected host. The surface proteome of the human pathogen M. tuberculosis was defined with the membrane shaving method. Differences between clinical and laboratory references stains were also observed. Several clinical isolates of two different genotypic lineages of M. tuberculosis were compared with a label-free quantitative analysis, identifying proteins with increased expression. The proteomic approach described in this thesis takes advantage of the dynamic nature of the bacterial proteome to identify significant differences that occur between related bacterial species, between different strains with in a species and under different environmental conditions. This has identified proteins involved in the disease process and has resulted in a list proteins of interest that would be worthy of further characterisation, and which ultimately may include novel therapeutic targets.