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Role of protein splicing in immunity to infections with intracellular pathogens

Tuesday 28 July 2015

Role of protein splicing in immunity to infections with intracellular pathogens

Where Department of Infectious Diseases and Immunology/ division Immunology

Who Master student I&I

Duration 6-9 months

Techniques PCR, plasmid preps, bacterial transformation, DNA digestion, ligation, cell culture, generation retroviral vectors, transfection of cells with plasmid/cDNA constructs or transduction with retroviral vectors, mouse handling (under supervision), flow cytometry

Alice Sijts , e-mail: e.j.a.m.sijts@uu.nl

Detailed description
CD8 T cells play an important role in protection against intracellular pathogens. CD8 T cells recognize short protein fragments (peptide epitopes) that are presented by MHC class I molecules and are generated upon intracellular degradation of viral/bacterial proteins by a large protease complex, the proteasome. Recently, it was shown that proteasomes generate antigenic peptides not only through protein degradation, which leaves peptides with a length of 3-16 amino acids, but also by post translational protein splicing. Thus, in the latter case, two non-contignuous peptide products are ligated while still in the catalytic channel of the proteasome as illustrated below.

All epitopes described to be generated by protein splicing derive from self antigens (tumor associated antigens and a minor histocompatibility antigen). However, as the efficiency of epitope splicing increases with increasing antigen concentration, and as infected cells contain large quantities of pathogen-derived antigens, we assume that protein splicing plays an important role in the generation of MHC class I presented epitopes during bacterial infection as well. In balb/c mice the dominant CTL epitope of Listeria monocytogenes is known to be a linear epitope derived from an important virulence factor. In C57BL/6 mice, the mice have the same vigorous CTL responses but so far the epitope has not been identified. We hypothesize that spliced epitope generation plays a role in this infection. This project will test this hypothesis.
Cell lines expressing single bacterial antigens will be made and tested on recognition by CTLís from mice that have been infected with Listeria monocytogenes. In this way, we will determine which antigens contain still unidentified CD8 T cell epitopes that could well be generated by protein splicing. In order to specifically identify the epitopes, target cell lines expressing truncated and smaller fragments of these antigens will be generated and tested for recognition by Listeria-specific CD8 T cells. In this way, the range in which the epitope is located can be narrowed further to eventually have the identification of the dominant epitope.