Scientific Reports (Apr 2017)

Monitoring global protein thiol-oxidation and protein S-mycothiolation in Mycobacterium smegmatis under hypochlorite stress

  • Melanie Hillion,
  • Jörg Bernhardt,
  • Tobias Busche,
  • Martina Rossius,
  • Sandra Maaß,
  • Dörte Becher,
  • Mamta Rawat,
  • Markus Wirtz,
  • Rüdiger Hell,
  • Christian Rückert,
  • Jörn Kalinowski,
  • Haike Antelmann

DOI
https://doi.org/10.1038/s41598-017-01179-4
Journal volume & issue
Vol. 7, no. 1
pp. 1 – 20

Abstract

Read online

Abstract Mycothiol (MSH) is the major low molecular weight (LMW) thiol in Actinomycetes. Here, we used shotgun proteomics, OxICAT and RNA-seq transcriptomics to analyse protein S-mycothiolation, reversible thiol-oxidations and their impact on gene expression in Mycobacterium smegmatis under hypochlorite stress. In total, 58 S-mycothiolated proteins were identified under NaOCl stress that are involved in energy metabolism, fatty acid and mycolic acid biosynthesis, protein translation, redox regulation and detoxification. Protein S-mycothiolation was accompanied by MSH depletion in the thiol-metabolome. Quantification of the redox state of 1098 Cys residues using OxICAT revealed that 381 Cys residues (33.6%) showed >10% increased oxidations under NaOCl stress, which overlapped with 40 S-mycothiolated Cys-peptides. The absence of MSH resulted in a higher basal oxidation level of 338 Cys residues (41.1%). The RseA and RshA anti-sigma factors and the Zur and NrdR repressors were identified as NaOCl-sensitive proteins and their oxidation resulted in an up-regulation of the SigH, SigE, Zur and NrdR regulons in the RNA-seq transcriptome. In conclusion, we show here that NaOCl stress causes widespread thiol-oxidation including protein S-mycothiolation resulting in induction of antioxidant defense mechanisms in M. smegmatis. Our results further reveal that MSH is important to maintain the reduced state of protein thiols.