NrdH Redoxin enhances resistance to multiple oxidative stresses by acting as a peroxidase cofactor in Corynebacterium glutamicum

Abstract

NrdH redoxins are small protein disulfide oxidoreductases behaving like thioredoxins but sharing a high amino acid sequence similarity to glutaredoxins. Although NrdH redoxins are supposed to be another candidate in the antioxidant system, their physiological roles in oxidative stress remain unclear. In this study, we confirmed that the Corynebacterium glutamicum NrdH redoxin catalytically reduces the disulfides in the class Ib ribonucleotide reductases (RNR), insulin and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), by exclusively receiving electrons from thioredoxin reductase. Overexpression of NrdH increased the resistance of C. glutamicum to multiple oxidative stresses by reducing ROS accumulation. Accordingly, elevated expression of the nrdH gene was observed when the C. glutamicum wild-type strain was exposed to oxidative stress conditions. It was discovered that the NrdH-mediated resistance to oxidative stresses was largely dependent on the presence of the thiol peroxidase Prx, as the increased resistance to oxidative stresses mediated by overexpression of NrdH was largely abrogated in the prx mutant. Furthermore, we showed that NrdH facilitated the hydroperoxide reduction activity of Prx by directly targeting and serving as its electron donor. Thus, we present evidence that the NrdH redoxin can protect against the damaging effects of reactive oxygen species (ROS) induced by various exogenous oxidative stresses by acting as a peroxidase cofactor.

FIG 1

NrdH reduces disulfide bonds by the TrxR/NADPH pathway but not the MSH/Mtr/NADPH pathway. (A to C) Reduction of RNR (A), insulin (B), and DTNB (C) by NrdH (10 μM) coupled to the TrxR/NADPH regeneration system. Trx (10 μM) was used as a positive control. Negative controls are the omission of TrxR in the presence of NrdH (−Control 1), omission of TrxR in the presence of Trx (−Control 2), and omission of both Trx and NrdH in the presence of TrxR (−Control 3). (D to F) Reduction of RNR (D), insulin (E), and DTNB (F) by NrdH (10 μM) coupled to the MSH/Mtr/NADPH regeneration system. Mrx1 (10 μM) was used as a positive control. Negative controls are the omission of Mtr in the presence of NrdH (−Control 1), omission of Mtr in the presence of Mrx1 (−Control 2), omission of both Mrx1 and NrdH in the presence of Mtr (−Control 3), and omission of Mtr in the presence of Mrx1 and NrdH (−Control 4). The reduction of RNR (A and D) and insulin (B and E) were recorded by measuring the decrease of NADPH oxidation at 340 nm. The reduction of DTNB was recorded as an increase in absorption at 412 nm (C and F).

FIG 2

The active-site cysteines of NrdH are essential for reduction of disulfide bonds. RNR (A), insulin (B), and DTNB (C) reduction were measured in the presence of 10 μM of each NrdH mutant by using the TrxR/NADPH regeneration system as described in Fig. 1.

FIG 3

Effects of NrdH overexpression on multiple-stress resistance in C. glutamicum. (A) Survival for the C. glutamicum WT(pXMJ19), WT(pXMJ19-nrdH), and WT(pXMJ19-nrdH::SXXS) strains after challenging with H₂O₂ (55 mM), CHP (11 mM), IAM (40 mM), and CDNB (70 mM) for 30 min. (B) Survival for the C. glutamicum WT(pXMJ19), WT(pXMJ19-nrdH), and WT(pXMJ19-nrdH::SXXS) strains after challenging with ciprofloxacin (375 μg/ml), tetracycline (115 μg/ml), gentamicin (50 μg/ml), vancomycin (150 μg/ml), and neomycin (450 μg/ml) for 2 h. (C) The growth (OD₆₀₀) of the C. glutamicum WT(pXMJ19), WT(pXMJ19-nrdH), and WT(pXMJ19-nrdH::SXXS) strains after 24 h at 30°C in LB medium containing CdCl₂ (15 μM), CuSO₄ (2,400 μM), FeSO₄ (225 μM), and MnCl₂ (800 μM) was recorded. Mean values with standard deviations (error bars) from at least three repeats are shown. *, P ≤ 0.05.

FIG 4

Overexpressed NrdH reduces ROS production and protein oxidation under oxidative stress conditions. (A) The ROS levels in C. glutamicum strains expressing NrdH and NrdH::SXXS were measured by using the DCF fluorescence determination assay after exposure to the indicated oxidative reagents. The bars represent the fluorescence intensity in arbitrary units (A.U). *, P ≤ 0.05. (B) Protein carbonyl contents were analyzed by Western blotting with anti-DNP antibody after exposure to various oxidative reagents for 30 min at 30°C. A parallel run stained with Coomassie brilliant blue is shown in the bottom panel. Total proteins were extracted from vector-expressing (lanes 1, 3, 5, 7, and 9) and nrdH-expressing (lanes 2, 4, 6, 8, and 10) C. glutamicum cells.

FIG 5

Induction of the nrdH gene expression by multiple oxidative stresses. β-Galactosidase analyses of the nrdH gene expression in C. glutamicum strain containing the P_nrdH::lacZ chromosomal fusion reporter after challenging with H₂O₂ (A), CHP (B), IAM (C), CDNB (D), ciprofloxacin (E), neomycin (F), MnCl₂ (G), and CdCl₂ (H) at the indicated concentrations. Error bars represent the standard deviations (SD) from three different determinations.

FIG 6

NrdH acts as a Prx cofactor to facilitate its peroxidase activity. (A) NrdH-dependent peroxidase activity of Prx. The activity of Prx was monitored by recording NADPH oxidation at 340 nm in the presence of the TrxR/NADPH system and shown with the progress curves. Reaction mixtures contained 50 mM Tris-HCl buffer (pH 8.0), 2 mM EDTA, 200 μM NADPH, 3 μM TrxR, 2 μM Prx, and 5 μM NrdH in a final volume of 500 μl. Reactions were started by the addition of H₂O₂ (100 μM, left) or CHP (100 μM, right). Trx was used as a positive control, and reaction mixtures lacking NrdH, TrxR, or peroxidase in the assay system served as negative controls. (B) The active-site cysteines of NrdH are required for aiding the Prx peroxidase activity. The assay was carried out as described above. (C) Bacterial two-hybrid complementation assays were carried out to analyze interactions between Prx and NrdH. β-Galactosidase activities were assayed with ONPG as the substrate. Assays were performed in triplicate. (D) GST pulldown assay. His₆-NrdH::CXXS or His₆-NrdH::SXXS was incubated with GST-Prx or GST in PBS, and potential protein complexes captured with glutathione beads were detected with anti-His antibody. (E) NrdH-mediated protection against oxidative stress is dependent on Prx. C. glutamicum wild-type cells and prx mutant were transformed with pXMJ19 and pXMJ19-nrdH, respectively, and tested for sensitivity to H₂O₂ (55 mM), CHP (11 mM), CDNB (70 mM), and CdCl₂ (300 μM) challenge.