Shigella are gram negative enteric pathogens of man which are the causative agents of bacillary dysentery. The principal features of Shigella pathogenicity are: 1) the ability to invade epithelial cells and multiply within the cells and 2) coordinate regulation of virulence gene expression in response to temperature. This organism is invasive when grown at 37 degrees Celsius but phenotypically avirulent (non-invasive) when grown at 30 degrees Celsius. Shigella is an excellent model for the study of facultative intracellular parasitism as well as a model for analysis of virulence gene regulation. the long term objectives of this proposal are to address two broad questions of Shigella pathogenesis: how virulence genes are regulated in response to environmental stimuli and how virulence gene products function in pathogenesis.
Specific aims are: 1) examining the mechanism(s) by which virR regulates Shigella virulence genes in response to temperature; 2) testing the hypothesis that a subset of Shigella virulence genes is induced by the cytoplasmic environment of the host mammalian cell; and 3) studying the mechanism(s) by which the genes of the mxi (membrane expression of invasion plasmid antigens) operon export virulence factors into the external milieu. Operon fusions will be constructed to determine how virR is regulated. Interaction of the virR gene product with its proposed targets, virB and virF, will be studied by DNase I footprinting and deletion analysis of the promoter regions. Gene fusions will also be used to test the hypothesis of a regulon of intracellularly-induced Shigella virulence genes. Random operon fusions, using either beta-galactosidase or luciferase as reporter enzymes, will be generated to Shigella genes. Those operon fusions which are not expressed at 37 degrees Celsius will be screened for induced expression after penetration of the bacteria into tissue culture cells. The genes marked by the fusions will be cloned and their roles in intracellular survival, multiplication, and host cell killing will be examined. mxi mutants will be used to test models of protein export and the role of the exported products in pathogenesis. Genetic and biochemical approaches will be used to determine the cellular location of the mxi gene products and their interactions with the exported virulence proteins. These studies will enhance our understanding of how virulence genes are regulated and could lead to strategies for blocking expression of the genes. Characterization of new virulence genes will provide potential targets for intervention in the prevention and treatment of shigellosis.
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