Protein S-Mycothiolation Functions as Redox-Switch and Thiol Protection Mechanism in Corynebacterium glutamicum Under Hypochlorite Stress

Abstract

Aims: Protein S-bacillithiolation was recently discovered as important thiol protection and redox-switch mechanism in response to hypochlorite stress in Firmicutes bacteria. Here we used transcriptomics to analyze the NaOCl stress response in the mycothiol (MSH)-producing Corynebacterium glutamicum. We further applied thiolredox proteomics and mass spectrometry (MS) to identify protein S-mycothiolation. Results: Transcriptomics revealed the strong upregulation of the disulfide stress r H regulon by NaOCl stress in C. glutamicum, including genes for the anti sigma factor (rshA), the thioredoxin and MSH pathways (trxB1, trxC, cg1375, trxB, mshC, mca, mtr) that maintain the redox balance. We identified 25 S-mycothiolated proteins in NaOCl-treated cells by liquid chromatography-tandem mass spectrometry (LC-MS/MS), including 16 proteins that are reversibly oxidized by NaOCl in the thiol-redox proteome. The S-mycothiolome includes the methionine synthase (MetE), the maltodextrin phosphorylase (MalP), the myoinositol-1-phosphate synthase (Ino1), enzymes for the biosynthesis of nucleotides (GuaB1, GuaB2, PurL, NadC), and thiamine (ThiD), translation proteins (TufA, PheT, RpsF, RplM, RpsM, RpsC), and antioxidant enzymes (Tpx, Gpx, MsrA). We further show that S-mycothiolation of the thiol peroxidase (Tpx) affects its peroxiredoxin activity in vitro that can be restored by mycoredoxin1. LC-MS/MS analysis further identified 8 proteins with S-cysteinylations in the mshC mutant suggesting that cysteine can be used for S-thiolations in the absence of MSH. Innovation and Conclusion: We identified widespread protein S-mycothiolations in the MSH-producing C. glutamicum and demonstrate that S-mycothiolation reversibly affects the peroxidase activity of Tpx. Interestingly, many targets are conserved S-thiolated across bacillithiol-and MSH-producing bacteria, which could become future drug targets in related pathogenic Gram-positives. Antioxid. Redox Signal. 20, 589-605.

Mycothiol/Mycoredoxin 1-dependent Reduction of the Peroxiredoxin AhpE from Mycobacterium tuberculosis

“…S-Mycothiolation affected CgTPx activity that was restored by incubation with CgMrx1 (17). MSH/Mrx1-dependent reduction has not been described in other Prxs from Mycobacteria (TPx and AhpC) studied so far, where NADPH/thioredoxin reductase is directly used to reduce different isoforms of thioredoxin, the reducing substrates for most of the bacterial Prxs (13,18).…”

“…Moreover, in Corynebacterium glutamicum, this protein participates in the arsenate resistance system by reducing the mycothiol arsenate adduct (16). More recently, twenty-five proteins of C. glutamicum including thiol peroxidase (TPx) were found mycothiolated under hypochloric stress conditions (17). The S-mycothiolation of Tpx inhibits its peroxidase activity, but could be restored after treatment with CgMrx1.…”

Corynebacterium diphtheriae Methionine Sulfoxide Reductase A Exploits a Unique Mycothiol Redox Relay Mechanism

“…We have previously shown in vivo that in the non-pathogenic actinomycete C. glutamicum, the Cys 86 of MsrA (the equivalent of Cys 87 in Cd-MsrA) was S-mycothiolated under oxidative stress (16). MSH is a low molecular weight thiol analog of GSH found in actinomycetes (38 -40).…”

“…Based on this observation, we investigated the role of S-mycothiolation as a possible catalytic mechanism of Cd-MsrA in vitro. We checked whether, aside from protecting vulnerable cysteines, as was shown for C. glutamicum thiol peroxidase (16), MSH might play a role as a catalytic electron transfer low molecular weight thiol during the reduction of L-Met-SO by Cd-MsrA. In the presence of the substrate L-Met-SO, we observed not only the nucleophilic Cys 52 of the WT Cd-MsrA to be S-mycothiolated (Table 3), as was shown for S-glutathionylation of poplar MsrA2 (14), but also Cys 206 and Cys 215 (Fig.…”

“…Actinomycetes, however, do not have the GSH/glutaredoxin system but rather the homologous mycothiol (MSH)/mycoredoxin-1 (Mrx1) system (15). Recently, we found MsrA to be S-mycothiolated in Corynebacterium glutamicum under hypochloric stress (16). The question remains whether and how this reduction system can act as part of the catalytic mechanism of MsrA in C. diphtheriae.…”