Secretomes of Bacterial Foodborne Pathogens and Functional Assessment during Interaction with Intestinal Epithelium

This investigation is targeted towards improving the understanding of the fundamental basis of the differential pathogenicity of the main foodborne pathogens in livestock and human hosts, i.e. Campylobacter, Salmonella and E. coli O157:H7. We will take a novel experimental approach to test the effect of biological signals relevant to the intestinal environment on secreted bacterial determinants (secretome).<P>
To achieve this, intestinal epithelial cells of bovine and human origins will be cultured in vitro to produce preconditioned medium. This will necessitate culture of primary intestinal epithelial cells to produce relevant materials - this is a unique resource not previously utilised in studies of protein expression by these (or other) organisms. <P>
We have gained considerable experience in primary culture procedures over the past 4 years. Bacterial strains will be cultured in preconditioned and control media under standardised conditions and secretomes will be examined by SELDI-MS and 2d electrophoresis in combination with MALDI-MS to characterise secreted proteins. DIGE will be employed on selected samples for direct analysis of differential protein complement reciprocal studies of the function of selected bacterial secreted determinants identified through these procedures will be carried out using global (proteomic) and directed (signal transduction) procedures. Protein phosphorylation is a central process in the control of cellular function hence this will form the main focus for functional studies. Phosphoproteome will be characterised using a combination of SELDI-MS, 2d electrophoresis and MALDI-MS. Signalling cascades will be further defined by directed approaches including immunochemical assays, kinase assays and EMSA. Certain phenotypic aspects may be pursued subject to progress in other aspects of study.<P>
This investigation will therefore combine aspects of proteomics and cell biology to define aspects of both bacterial and host biology and assess functional consequence of selected determinants on intestinal epithelial cell function.