The long-term objectives of these studies are to determine how an enteropathogenic bacterium is able to enter within host cells and to determine the cellular route taken by such pathogens after oral inoculation. Yersinia pseudotuberculosis is being studied in order to gain detailed information on the function of bacterial- and host-encoded internalization factors. To this end, the binding of the bacterial invasin protein with its mammalian integrin receptors, and the consequences of this event within the host cell, will be studied. In addition, this proposal will analyze the nature of the events that occur shortly after the bacterium is ingested by the host, when invasin appears to exert its most important role in disease. Efficient uptake promoted by invasin is regulated by the host cell FAK and Rac1 proteins, and requires high affinity binding to integrin receptors as well as multimerization of the invasin cell adhesion domain. To probe the molecular mechanism of uptake promoted by the substrate-receptor pair, and analyze the events that occur during translocation of the organism across the intestinal epithelium, the following studies will be performed: 1) using the recently completed crystal structure, determinants within invasin that allow uptake and differentiate invasin from other integrin substrates will be identified; 2) the region of invasin that allows presentation of the cell binding motif will be identified and analyzed topologically using genetic strategies; 3) the role of FAK in promoting uptake will be determined by analyzing its role in regulating both the nature of the phagocytic cup and the mobility of integrins within the membrane; 4) the route that Y. pseudotuberculosis takes to cross the intestinal epithelium in the absence of invasin expression will be determined. It is now apparent that many bacterial pathogens initiate disease by entering within host cells, and after ingestion, enteropathogens are able to translocate across multiple sites in the intestine. Therefore, these studies will result in determining how an enteric disease by a bacterial pathogen is initiated, and provide insight into a step that could be blocked by chemotherapeutic agents prior to colonization by such microorganisms. 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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