Tuberculosis is a severe human disease and the appearance of a growing number of multiple-drug resistant Mycobacterium tuberculosis strains makes it a necessity to learn more about cell wall formation, drug uptake and drug mechanisms. A major hurdle for the development and improvement of newug candidates is the notorious difficulty in identification and characterization of the corresponding drug targets in M. tuberculosis. This limitation results mainly from the fact that we do not yet have the means to study protein function and protein-protein interactions within the mycobacterium itself. We therefore propose to develop a novel approach to study protein-protein interactions in living mycobacteria which addresses this important need. The approach is based on a recently developed fusion tag that can be labelled with a variety of chemically diverse compounds in living cells. The labelling of a protein of interest in living mycobacteria with a reactive oxygen species-generating photosensitizer and the subsequent proteome-wide detection of protein modifications by reactive oxygen species should allow the identification of proteins that are localized in the direct vicinity of the labelled protein of interest. The application of this technique to proteins associated with the outer membrane of mycobacteria should allows us to identify and characterize new mycobacterial outer membrane proteins and protein complexes, thereby yielding new insights into the biochemical features of the highly complex and unusual structure of the mycobacterial cell wall. Only the discovery of new mycobacteria-specific drug targets will lead to the development of new drugs for a better treatment of tuberculosis. In addition, this new technology should be applicable to the identification of protein-protein interactions in a variety of different host organisms and it has therefore the potential to become a general tool in functional proteomics.
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