MHC-ACCESSIBILITY OF SECRETED SALMONELLA PROTEINS

Intracellular bacterial pathogens adapt to their host cell environment by the selective secretion of proteins designed to alter the normal structural and metabolic machinery of the host cell, thus promoting bacterial survival and avoidance of host immune surveillance. These secreted bacterial proteins mediate their effects within the phago-lysosome or alternatively, directly within the host cell cytoplasm. Because access to the interior of the cell infers access to the proteolytic machinery of the host, we have collectively labeled these proteins MHC Class Accessible Proteins (CAPs). The identification of CAPs secreted in response to host cell invasion, would be an invaluable resource for the study of bacterial antigen processing and bacterial modification of the host-cell. In addition, since CAPs possess unique access to the host's antigen processing and presentation machinery, they represent potentially attractive vaccine targets and useful vehicles for the delivery of foreign epitopes by bacterial carrier vaccines. To identify CAPs in Salmonella typhimurium, we developed a novel approach, termed Disseminated Insertion of Class I Epitopes (DICE). DICE uses a resolvable Tn5-based transposon to randomly distribute the MHC-I H-2Kb-restricted ovalbumin epitope, SIINFEKL. When the resolved insertion is in-frame within a gene, CAPs released from the bacteria after infection are processed by the host cell, resulting in the presentation of the carried epitope in the context of H-2Kb. Salmonella strains containing epitope insertions within CAPs are then easily isolated by flow cytometry using H-2Kb/SIINFEKL specific antibodies. I will focus on bacterial proteins that are class I accessible and delineate precisely how they may reach the cytoplasm of infected cells. These studies have important implications for vaccine development and bacterial pathogenesis.