UBC Theses and Dissertations
Proteomic method development for studying salmonella-host protein interactions Rattray, David Graeme
Salmonella species are a pathogen of humans and animals. Pathogenesis is mediated by effector proteins that are translocated into host cells. These effector proteins interact with host proteins and cellular processes to establish a replication permissive niche for Salmonella. Efforts to determine the roles of the effector proteins have revealed many insights, however the understanding of the interaction between host and pathogen is incomplete. To further investigate the impact of Salmonella effector proteins on host cells, we developed high throughput proteomic methods to assess changes that occur during infection. First, we describe a flow cytometry-based method to identify the Salmonella SPI-2 effector(s) that mediate suppression of MHC II surface complexes which interferes with Ag-dependent T-cell proliferation. Second, we develop an assay to identify protein-protein interactions that occur in a wild type Salmonella infection compared to the SPI-2 double effector mutant sifAsseJ strain which is deficient in the formation of membrane tubules that facilitate intracellular replication. This assay utilises Blue Native PAGE to fractionate proteins and protein complexes based on size through the gel matrix prior to mass spectrometry. A machine learning classifier then analyses the fractionation profile of every protein pair through the gel matrix to predict protein-protein interactions. Comparison of interactions between experimental conditions is achieved by the stable isotope labelling technique SILAC. Third, we adapt a protocol for analysing changes in protein subcellular distribution, LOPIT-DC, to compare the wild type Salmonella infection to the sifAsseJ strain. This protocol fractionates the cell lysate by differential centrifugation, and SILAC labelling enables comparison between the experimental conditions. This work was successful in further developing methodologies to investigate the host-pathogen interactions between Salmonella Typhimurium and host cells. The approaches described within were successful in identifying known host target proteins for Salmonella effectors, or otherwise recruited to the Salmonella containing vacuole and associated membranes. There was substantial overlap of identified proteins in the datasets generated with other published datasets, along with good coverage of other host proteins which may be involved in Salmonella pathogenesis.
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