Defined mutants of isogenic strains, carrying deletion derivatives of virulence plasmids, will be compared with parental strains for changes in cell wall components and virulence. Genetic approaches to be utilized include studies of spontaneous deletion mutants of a virulence plasmid by restriction mapping and insertion mutagenesis. Biochemical approach include 1D and 2D SDS-PAGE analysis of membrane proteins. These data will define which surface components are associated with adherence and invasiveness. Antibodies to these surface components (polyclonal and/or monoclonal) will be tested for ability to modify adherence and/or internalization. Receptor(s) on colonic epithelial cells or on HeLa cells for Shigella adhesion will be identified. SDS-PAGE gels of outer membranes of epithelial cells will be transferred to nitrocellulose and blotted with either intact radiolabeled bacteria or labeled bacterial cell wall components identified above as being involved in expression of virulence.
