A fluorescent probe for specifically measuring the overall thioredoxin and glutaredoxin reducing activity in bacterial cells.

Thioredoxins (Trxs) and glutaredoxins (Grxs) are the two major thiol-dependent reductases, participating in many important cellular events such as defense against oxidative stress, DNA synthesis and repair. Both Trxs and Grxs have diverse disulfide-containing substrates in the cells to exert their activities, with overlapping functions. Specific methods for measuring the intracellular overall activities of Trxs and Grxs are still lacking. Here we find that TRFS-green, a disulfide containing fluorescent probe which was used to detect thioredoxin reductase (TrxR) in mammalian cells, is a substrate of bacterial Trxs and Grxs, but not a substrate of bacterial TrxR and GSH. This property made TRFS-green work as a probe to measure the overall activities of Trxs and Grxs in bacterial cells. Using various E. coli Trx or Grx null mutant strains, the contribution of different Trxs and Grxs to cellular redox regulation has been clarified, judged by the reducibility towards TRFS-green. E. coli Grx2 and Grx3 unexpectedly exhibited higher activity in reducing the disulfide probe than the other redoxins. In addition, the bacterial disulfide reductase activity was detected to be affected in the ofloxacin bactericidal process. These results show that TRFS-green may be a useful tool for investigating bacterial redox regulation and demonstrating the critical role of E. coli Grxs in maintaining the bacterial intracellular redox balance.

Disruption of Bacterial Thiol-Dependent Redox Homeostasis by Magnolol and Honokiol as an Antibacterial Strategy.

Some traditional Chinese medicines (TCMs) possess various redox-regulation properties, but whether the redox regulation contributes to antibacterial mechanisms is not known. Here, ginger juice processed Magnoliae officinalis cortex (GMOC) was found to show strong antibacterial activities against some Gram-positive bacteria, but not Gram-negative bacteria including E. coli, while the redox-related transcription factor oxyR deficient E. coli mutant was sensitive to GMOC. In addition, GMOC and its main ingredients, magnolol and honokiol, exhibited inhibitory effects on the bacterial thioredoxin (Trx) system, a major thiol-dependent disulfide reductase system in bacteria. The effects of magnolol and honokiol on cellular redox homeostasis were further verified by elevation of the intracellular ROS levels. The therapeutic efficacies of GMOC, magnolol and honokiol were further verified in S. aureus-caused mild and acute peritonitis mouse models. Treatments with GMOC, magnolol and honokiol significantly reduced the bacterial load, and effectively protected the mice from S. aureus-caused peritonitis infections. Meanwhile, magnolol and honokiol produced synergistic effects when used in combination with several classic antibiotics. These results strongly suggest that some TCMs may exert their therapeutic effects via targeting the bacterial thiol-dependent redox system.

The Role and Mechanism of Thiol-Dependent Antioxidant System in Bacterial Drug Susceptibility and Resistance.

Antibiotics play an irreplaceable role in the prevention and treatment of bacterial infection diseases. However, because of the improper use of antibiotics, bacterial resistance emerges as a major challenge of public health all over the world. The small thiol molecules such as glutathione can directly react and conjugate with some antibiotics, which thus contribute to drug susceptibility and resistance. Recently, accumulating evidence shows that there is a close link between the antibacterial activities of some antibiotics and Reactive Oxygen Species (ROS). Thioredoxin and glutathione systems are two main cellular disulfide reductase systems maintaining cellular ROS level. Therefore, these two thioldependent antioxidant systems may affect the antibiotic susceptibility and resistance. Microorganisms are equipped with different thiol-dependent antioxidant systems, which make the role of thioldependent antioxidant systems in antibiotic susceptibility and resistance is different in various bacteria. Here we will focus on the review on the advances of the effects of thiol-dependent antioxidant system in the bacterial antibiotic susceptibility and resistance.

Modulation of thiol-dependent redox system by metal ions via thioredoxin and glutaredoxin systems.

The thioredoxin and glutaredoxin systems possess a variety of biological activities in mammalian cells, including the defense against oxidative stress, regulation of DNA synthesis, the cell cycle and the mediation of apoptosis. The thioredoxin system, comprised of NADPH, thioredoxin reductase (TrxR) and thioredoxin (Trx), exerts its activities via a disulfide-dithiol exchange reaction. Mammalian TrxRs are selenoproteins; the thiols and selenols in the active site of these enzymes confer the thioredoxin system to work as soft bases, which have a high affinity with soft acids, including numerous metal ions. In this review we focus on recent advances in the modulation of thioredoxin and glutaredoxin systems by metal ion soft acids. Numerous clinical metal-containing drugs, such as platinum- and gold-containing compounds, show inhibitory effects on the thioredoxin system, providing strategies to develop novel anti-cancer drugs. Moreover, inhibition of the Trx system by soft acids, such as mercury-, chromium- and arsenic-containing compounds cause changes in the cellular redox state and contribute to their cell toxicity. In addition, metal ions are also involved in the regulation of the glutaredoxin system. Iron ions participate in regulating Grx2 activity via iron-sulfur cluster formation. Moreover, Grx5 in mitochondria contains a 2Fe-2S cluster stabilized by GSH, which can mediate cellular iron metabolism. Collectively, these results demonstrate that metal ions are major players in regulating the Trx and Grx systems-mediated cellular redox processes and thus, provide an opportunity to understand the functions of metal ions in thiol metabolism dysfunction-related diseases.