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.
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.