Principle investigator: Haike Antelmann
Co-supervisor: M. Lalk

Bacillus and Staphylococcus species utilize as thiol redox buffer bacillithiol (BSH, Cys-GlcN-Mal) to maintain the redox balance. We recently discovered that BSH protects protein thiols against irreversible oxidation under ROS stress by S-bacillithiolation [1-4]. Mycobacterium smegmatis produces mycothiol (AcCys-GlcN-Ins, MSH) as redox buffer and is used as a model organism to study antituberculosis drug resistance mechanisms against isoniazid (INH). INH is a prodrug and activated by the catalase-peroxidase leading to production of ROS and isonicotinic-acyl radicals that bind NAD and inhibits the enoyl-ACP reductase required for mycolic acid biosynthesis. It has been shown that mshA mutants are resistant to INH, but the mechanisms are not known.

In this project, we will investigate the INH resistance mechanisms and the oxidative stress response in M. smegmatis. We aim to analyze the changes in the proteome, thiol-redox proteome and thiol metabolome of M. smegmatis wild type and msh mutants in response to ROS (H2O2, diamide, NaOCl, cumene hydroperoxide), RNS (sodium nitroprusside) and INH. ESI-LC-MS/MS analyses will be used to identify S-thiolated proteins together with the mass spectrometry group of Dörte Becher (Institute for Microbiology). The changes of protein functions and activities by S-thiolation of proteins shall be analyzed in detail. We hypothesize that S-thiolated redox-controlled proteins will be identified involved in drug resistance or in the defense of Mycobacteria against the host immune system. Recently, a novel mycoredoxin-1/MSH/Mtr electron pathway has been discovered in Mycobacteria for reduction of S-mycothiolated proteins [1,5]. S-mycothiolated substrates of mycoredoxin-1 (Mrx-1) will be identified by redox proteomics in collaboration with Joris Messens. The results contribute to a better understanding of antibiotic resistance mechanisms and the role of MSH for protection against ROS and RNS in Mycobacteria. 
We will use our redox proteomics expertise to collaborate with C.H. Lillig (A1) to identify glutaredoxin substrates, with Mayerle (A3) and Lenzen/Elsner (A4) to analyze thiol-oxidations during pancreatitis, with Stöhr (C3) to identify S-nitrosylated proteins in plants. Thiol-metabolomics in M. smegmatis will be performed with Lalk (B4) and the antioxidant effect of redox buffers investigated together with Kahlert (B4). The expertise in enzymology of Bornscheuer (A2) will be used for analysis of functions of S-thiolated proteins.

Literature

1 Antelmann, H. and Hamilton, C.J.: Mol. Microbiol. (2012) 86, 759. 

2 Antelmann, H., and Helmann, J.D.: Antioxid. & Redox Signal. (2011) 14, 1049.

3 Chi, B.K., Gronau, K., Mäder, U., Hessling. B., Becher, D. and Antelmann, H.: Mol. Cell. Proteomics (2011) 10, M111.009506. 

4 Chi, B.K., Roberts, A., Huyen, T.T.T., Gronau, K., Becher, D., Albrecht, D., Hamilton, C. and Antelmann, H.: Antioxid. & Redox Signal. (2013) 18, 1273.

5 Van Laer, K., Buts, L., Foloppe, N., Vertommen, D., Van Belle, K., Wahni, K., Roos, G., Nilsson, L., Mateos, L.M., Rawat, M., van Nuland, N.A. and Messens, J.: Mol Microbiol. (2012) 86, 787.