Role of the bacillithiol redox buffer for redox control in Firmicutes bacteria
Final Report Abstract
Bacillithiol is the major LMW thiol in Bacillus and Staphylococcus species. BSH maintains the reduced state of the cytoplasm and functions in detoxification of many reactive species. Staphylocococcus aureus BSH-deficient mutants are sensitive to the antibiotic fosfomycin and to the strong oxidant hypochlorite. Phenotype analyses of S. aureus natural bshC mutants of the NCTC8325 lineage uncovered an important role of BSH for stress resistance and under infection conditions. In phagocytosis assays using murine macrophages and human epithelial cell lines the survival of the SH1000 strain was decreased compared to the bshC complemented S. aureus strain. Thus, BSH is involved in the defense against the host-immune system and contributes to pathogen fitness in S. aureus clinical MRSA isolates under infectionrelated conditions. Bacillithiol is also involved in post-translational thiol-modification of proteins. Protein S- bacillithiolation was discovered as wide-spread thiol-protection and redox-regulatory mechanism. S-bacillithiolation functions as a redox-switch mechanism to control the activity of redox-sensing transcription factors and metabolic enzymes, including OhrR and MetE. In this project, 54 S-bacillithiolated proteins were identified in B. subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus megaterium and Staphylococcus carnosus. These include eight common and 29 unique S-bacillithiolated proteins across Bacillus and Staphylococcus species. The S-bacillithiolome contains mainly biosynthetic enzymes for amino acids (methionine, cysteine, branched chain and aromatic amino acids), cofactors (thiamine), nucleotides (GTP), as well as translation factors, chaperones, redox and antioxidant proteins. Among the most conserved protein-SSB were abundant and essential proteins like TufA, MetE, GuaB that are targets for S-thiolation also in MSH-producing bacteria. The methionine synthase MetE is the most abundant S-bacillithiolated protein in Bacillus species after NaOCl exposure. S-bacillithiolation of MetE occurs at its Zn-binding active site Cys730 and at the non-essential surface-exposed Cys719, leading to methionine starvation in NaOCl-treated cells. Similarly, methionine auxotrophy is caused by S-glutathionylation of MetE in E. coli after diamide stress. The active site Zn center of MetE is also S-mycothiolated in C. glutamicum as shown this project. Inactivation of MetE due to S-bacillithiolation could stop translation during the time of hypochlorite detoxification. S-bacillithiolations were observed under diamide and NaOCl stress, but not under control conditions. This confirms previous results about the mechanisms of S-glutathionylations which requires activation of protein thiols by ROS. Hypochlorite leads to chlorination of the thiol group to form sulfenylchloride that is unstable and rapidly reacts further to form mixed BSH protein disulfides. The increased BSSB level under NaOCl-stress might also contribute to S- bacillithiolation via thiol-disulfide exchange. Among the S-bacillithiolated proteins, the thioredoxin-like proteins YtxJ, YphP and YqiW were identified in B. subtilis and Staphylococcus. These Trx-like enzymes were suggested to function as bacilliredoxins (Brx) in the de-bacillithiolation process. We demonstrated in this project that BrxA and BrxB function in the reduction of the S-bacillithiolated substrates MetE and OhrR in vitro (14). The BrxBCxA resolving Cys mutant protein was able to reduce S-bacillithiolated OhrR to restore the DNA-binding activity of OhrR. However, the BrxBCxA mutant was unable to reduce S-cysteinylated OhrR. These results provide first evidence for the function of glutaredoxin-like enzymes in BSH-producing bacteria. However, phenotype analyses revealed that both, BrxA and BrxB are not essential and rather dispensable for oxidative stress resistance under conditions of S-bacillithiolations in B. subtilis. Thus, the bacilliredoxin pathway is redundant with other thiol-disulfide oxidoreductases or the thioredoxin pathway in vivo for reduction of BSH mixed disulfides. The redox regulation of enzymes and transcription regulators by S-bacillithiolation and bacilliredoxins has been studied in detail in the model bacterium B. subtilis. Future studies are directed to elucidate if S-bacillithiolation and bacilliredoxins control virulence functions and pathogen fitness in the major pathogen S. aureus.
Publications
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2013. Distribution and infectionrelated functions of bacillithiol in Staphylococcus aureus. Int. J. Med. Microbiol. 303: 114-23
Pöther, D.C., Gierok, P., Harms, M., Mostertz, J., Hochgräfe, F., Antelmann, H., Hamilton, C.J., Borovok, I., Lalk, M., Aharonowitz, Y. and Hecker, M.
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2013. Regulation of Bacillus subtilis bacillithiol biosynthesis operons by Spx. Microbiology 159: 2025-35
Gaballa, A., Antelmann, H., Hamilton, C.J., Helmann, J.D.
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2013. S-bacillithiolation protects conserved and essential proteins against hypochlorite stress in Firmicutes bacteria. Antioxid. Redox Signal. 18: 1273-1295
Chi, B.K., Roberts, A., Huyen, T.T.T., Gronau, K., Becher, D., Albrecht, D., Hamilton, C. and Antelmann, H.
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2014. Protein S-mycothiolation functions as redox-switch and thiol protection mechanism in Corynebacterium glutamicum under hypochlorite stress. Antioxid. Redox Signal. 20: 589-605
Chi, B. K., Busche. T., Van Laer, K., Bäsell, K., Becher, D., Clermont, L., Seibold, G., Persicke, M., Kalinowski, J., Messens, J. and Antelmann, H.
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2014. Redox regulation in Bacillus subtilis: The bacilliredoxins BrxA (YphP) and BrxB (YqiW) function in de-bacillithiolation of S-bacillithiolated OhrR and MetE. Antioxid. Redox Signal. 21: 357-367
Gaballa, A, Chi, B.K., Roberts, A.A., Becher, D., Hamilton, C.J., Antelmann, H., Helmann, J.D.
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2015. Redox regulation by reversible protein S-thiolation in bacteria. Front. Microbiol. 6: 187
Loi, V.V., Rossius, M. and Antelmann, H.
