The long-term objective of these studies are to determine how an enteropathogenic bacterium is able to enter within host cells and to evaluate the role of mammalian cell receptors in promoting uptake. As a model system, Yersinia pseudotuberculosis is being studied in order to gain detailed information on the function of bacterial- and host-encoded internalization factors. Specifically, Y. pseudotuberculosis invasin- integrin interaction will be studied, and host-encoded factors that modulate receptor-mediated bacterial internalization will be identified. Uptake promoted by invasin depends upon high affinity binding to its receptors, regulated by the concentration of receptor available to participate in uptake. To further investigate the molecular mechanism of uptake, the following experiments will be performed: 1) A model for the binding of a single invasin molecule to multiple host receptors will be tested, by analyzing the subunit structure of invasin and determining the stoichiometry of invasin-receptor interaction; 2) the region of the integrin heterodimer involved in invasin binding will be determined by performing two novel mutant selection for altered receptor interaction; 3)the role in bacterial uptake of the beta-1 integrin will be investigated by analyzing the binding of mammalian cell cytoplasmic components to hybrid protein harboring this domain; 4) functional studies on the role of mammalian cytoplasmic components will be performed using a newly-developed perforated cell assay, allowing evaluation of the biological roles of factors identified in other Aims; and5) the role of invasin in intestinal infections will be analyzed, using invasin mutations resulting in partially functional proteins. Bacterial uptake by host cells is a common step in establishing disease by a number of bacterial pathogens. Investigation of this process will result in important information on how enteric diseases are initiated, and provide a potential source for 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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