We have initiated studies to characterize the role of CD8+ T cells in immunity to the intracellular bacterial pathogen, Shigella flexneri. During infection, S. flexneri enter cells and escape into the host cell cytosol. We expected that proteins excreted from S. flexneri would be proteolytically degraded by the MHC-I processing machinery, and that the resulting peptides would be presented to CD8+ T cells in the context of surface MHC-I molecules. During infection with Listeria monocytogenes, another intracellular bacterial pathogen that escapes into the host cell cytosol, Listeria-derived peptides, in complex with host MHC-I, are recognized by CD8+ T cells and contribute to the generation of protective immunity. While CD8+ T cells have been shown to be required for protective immunity to L. monocytogenes, we have found no evidence to suggest that CD8+ T cells play a role in protective immunity to S. flexneri. Even when we engineered S. flexneri to constitutively secrete heterologous epitopes known to stimulate potent CD8+ T cell responses, those responses were not detected. Furthermore, we found that when cultured cells were infected with epitope-tagged S. flexneri, these cells were not recognized by established T cell clones specific for the epitope tag. Our results over the previous award period suggest that S. flexneri inhibit the presentation of MHC-I restricted antigens to CD8+ T cells, and it is this lack of antigen presentation that results in the failure of CD8+ T cells to respond to antigens secreted into the host cell cytosol by S. flexneri. The experiments in this proposal seek to identify and characterize the defect in MHC-I processing and/or presentation that occurs during S. flexneri infection. Specifically: 1) we will use genetic screens to identify S. flexneri gene product(s) responsible for the inhibition;and 2) we will use biochemical assays to determine the step or steps in the MHC-I processing pathway that are altered during infection with S. flexneri and that result in decreased antigen presentation on the host cell surface. Through these experiments, we expect to identify and describe the activity of a bacterial inhibitor of antigen processing and presentation. Such an inhibitor might represent a novel class of virulence determinants specifically able to interfere with development of protective T cell-mediated adaptive immunity. Understanding how this inhibition affects the balance between bacterial virulence and immune clearance will further our understanding of the complex interaction of this bacterial pathogen and its mammalian host.
Shigella flexneri is responsible for serious gastrointestinal disease throughout the world. We have found that this organism has the capacity to inhibit the immune response directed against it by the host. The work proposed in this application is directed at understanding the mechanisms through which the organism is able to avoid detection and identifying which genes in the organism are responsible for immune system inhibition. Our work may lead to therapeutic strategies that might undermine the ability of this organism to hide from the immune system, thereby allowing more effective clearance of the bacteria.
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