Project: The ejectosome: Deciphering its proteome
Cell-to-cell transmission of pathogenic organisms is equally important to maintain and spread an efficient infection as uptake and replication. However, little is known about the underlying molecular mechanisms, which allow pathogens to spread efficiently from cell to cell.
In the Dictyostelium- Mycobacterium marinum infection system the ejectosome, an actin-rich structure, represents the major mechanism of efficient cell-to-cell spreading. Ejection allows bacteria to exit their host cell directly from the cytosol through the plasma membrane in a non-lytic fashion. Little is known about the underlying molecular mechanism and about involved factors of the pathogen as well as host.
In the proposed project we plan to use two major approaches to decipher the protein composition of the ejectosome structure. First, we plan to express host and pathogen proteins, which we know are present in the ejectosome, as fusions to APEX2. Proteins, that come in close contact with these "bait-proteins" will be biotinylated and thus can be isolated and subsequently identified. In another approach we plan to make use of a Dictyostelium strain, that overexpresses an actin-binding domain which not only results in a higher number of ejectosomes but also more stable ejectosome structures. By optimizing the lysis conditions and subsequent enrichment steps we plan to purify ejectosomes, the protein composition of which then can be deciphered applying mass spectrometric analyses.
We expect that deciphering the proteome of the ejectosome will allow us to identify host and pathogen factors, which take part in the cell-to-cell transmission of bacteria and thereby elucidate the underlying molecular mechanism of this process. Furthermore, the acquired knowledge will highlight new strategies to contain infections on the cellular level, by interfering with egress. This will represent a further and complementary approach to counteract infections.