MYCOOMP · Outer membrane protein complexes of mycobacteria
FP6 — Marie Curie Actions (Human Resources and Mobility)
- Duration
- 2007-09-01 → 2009-08-31
- EU contribution
- €0
- Participants
- 1
- Scheme
- EIF
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Results in brief
Final Activity Report Summary - MycoOMP (Outer membrane protein complexes of mycobacteria)
The main goal of this project was to establish a novel method to explore protein interactions in live mycobacteria, which would be applicable for the study of not only soluble proteins but also membrane and hydrophobic cell wall proteins. Furthermore, the system should be easily adaptable for use in mycobacterium tuberculosis (Mtb). In order to identify interacting proteins, the protein of interest was planned to be fused to SNAP-tag, a small protein tag developed in the Johnsson group. Firstly, photosensitisers were coupled to SNAP-tag to specifically modify interacting proteins. This approach did not lead to promising results and the applied methodology was subsequently changed. By using two protein tags instead of one, namely SNAP-tag and CLIP-tag, it was possible to specifically cross-link interacting proteins in mycobacterium smegmatis, a model organism to study mycobacteria. To this end, mycobacterial expression vectors were designed and protein expression was optimised for fluorescence detection methods. Furthermore, it was possible to detect the interacting proteins via covalent cross-linking of the two tags in living cells. After having optimised the system we started looking at interactions of Pks13 with AccD4 and FadD32, i.e. with all enzymes involved in mycobacterial cell wall formation which were of interest as drug targets for tuberculosis treatment. These experiments were performed in cooperation with the group of Prof. Daffé at the Université Paul Sabatier in Toulouse. In a second cooperation with the group of Prof. Cole at the host institution Ecole Polytechnique Federale de Lausanne (EPFL) the applicability of our newly developed technology was tested in m. tuberculosis. We found that protein expression with the existing expression plasmids was possible and, most importantly, protein labelling could be performed in living Mtb cells.
Data: CORDIS, © European Union
Project objective
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.
Original text from CORDIS.
Participants
- ECOLE POLYTECHNIQUE FEDERALE DE LAUSANNE · LAUSANNECoordinatorSwitzerland
Links
Data: CORDIS, © European Union