THe long term objective is to determine how an enteropathogenic bacterium is able to enter and survive within cells of the host. To this end, Yersinia pseudotuberculosis is being studied as a model system for cellular penetration, in order to gain detailed information on the mechanism of invasion into cultured mammalian cell lines. Specifically, the mechanism of action of the protein invasion will be studied, as will other factors encoded by this microorganism that are involved in cellular penetration and colonization of the mammalian cell surface. Invasion is a protein localized on the outer membrane of the bacterium that mediates attachment of the microorganism to the host mammalian cell as a first step in the entry process. To analyze cellular penetration by Y. pseudotuberculosis the following experiment will be performed: 1) The amino acid residues of invasin that contact its cellular receptor will be identified by isolating binding-deficient mutants and by analyzing proteolytic fragments that retain the ability to bind cultured cells; 2) the cell attachment domain of the protein will be dissected in order to determine if binding to the host cell is sufficient to promote entry of the bacterim; 3) the mammalian cell receptor for invasin will be identified by affinity chromatography and immunochemical techniques; 4) proteins that promote invasin-independent entry into cultured cells will be identified genetically; and 5) mutations that affect the invasin-independent pathways of cellular penetration will be analyzed for their role in pathogenesis, using a mouse infection model. Ingestion of bacteria by epithelial cells is the first step in the infection process of many enteropathogenic organisms. An understanding of how this occurs could allow the development of new chemotherapies that block this step in the infection process. In addition, identification of the components that allow a simple organism to enter an animal cell could result in new techniques to introduce therapeutic agents that would otherwise not be able to enter the host cell.
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