Article pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Targeting the Thioredoxin Reductase−Thioredoxin System from Staphylococcus aureus by Silver Ions Xiangwen Liao,†,‡ Fang Yang,† Hongyan Li,§ Pui-Kin So,∥ Zhongping Yao,∥ Wei Xia,*,† and Hongzhe Sun*,†,§ †
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-sen University, Guangzhou, China, 510275 ‡ Hunan Provincial Key Laboratory for Ethnic Dong Medicine Research, Hunan University of Medicine, Huaihua, China, 418000 § Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China ∥ State Key Laboratory for Chirosciences and Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR, China S Supporting Information *
ABSTRACT: The thioredoxin system, which is composed of NADPH, thioredoxin reductase (TrxR), and thioredoxin (Trx), is one of the major disulfide reductase systems used by bacteria against oxidative stress. In particular, this reductase system is crucial for the survival of the pathogenic bacterium Staphylococcus aureus, which lacks a natural glutathione/ glutaredoxin (Grx) system. Although silver ions and silvercontaining materials have been used as antibacterial agents for centuries, the antibacterial mechanism of silver is not wellunderstood. Herein, we demonstrate that silver ions bind to the active sites of S. aureus TrxR and Trx with dissociation constants of 1.4 ± 0.1 μM and 15.0 ± 5.0 μM and stoichiometries of 1 and 2 Ag+ ions per protein, respectively. Importantly, silver ion binding leads to oligomerization and functional disruption of TrxR as well as Trx. Silver also depleted intracellular thiol levels in S. aureus, disrupting bacterial thiol-redox homeostasis. Our study provides new insights into the antibacterial mechanism of silver ions. Moreover, the Trx and TrxR system might serve as a feasible target for the design of antibacterial drugs.
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INTRODUCTION Reactive oxygen species (ROS) are formed by aerobic organisms as natural byproducts during normal cell metabolism. High levels of ROS within cells induce oxidative stress and have the potential to cause significant damage to cellular structures. To balance the oxidative state, aerobic organisms have evolved complex antioxidant systems to counterbalance ROS production. The glutathione−glutaredoxin (Grx) and thioredoxin (Trx) systems are the two main antioxidant enzyme systems in aerobic organisms.1−3 The Grx system is composed of glutathione reductase (GR), glutathione (GSH), glutaredoxin (Grx), and NADPH.4 Electrons are transferred from NADPH to GR, then to GSH, and finally to Grx, which can further reduce disulfides in proteins.5 By contrast, the Trx system only contains thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH. The active site of Trx consists of a conserved CXXC motif, which can cycle between active (reduced) and oxidized (disulfide) forms. Active Trx can scavenge ROS and be oxidized while maintaining other proteins in a reduced state. Oxidized Trx can then be reduced by the action of TrxR, using NADPH as an electron donor.6 The Trx system is ubiquitous in all living © XXXX American Chemical Society
organisms, whereas the Grx system is lacking in some pathogenic bacteria, such as Helicobacter pylori, Mycobacterium tuberculosis, and Staphylococcus aureus.7−9 Staphylococcus aureus (S. aureus), a worldwide human pathogen, can cause a variety of human diseases, ranging from soft tissue infections to life-threatening toxic shock syndrome.10,11 In particular, the emergence of drug-resistant S. aureus strains, such as methicillin-resistant Staphylococcus aureus (MRSA), has posed a great threat to public health worldwide.12 It is reported that MRSA infections cause more deaths than AIDS each year in the US. Therefore, there is an urgent need for new antibacterial strategies to combat this “superbug”. It is worth noting that S. aureus is a GSH-negative pathogen, in which the Grx system is naturally deficient. Therefore, the S. aureus Trx system is extremely important for bacterial survival under oxidative stress conditions.13 In a previous study, it was found that ebselen, a competitive inhibitor of bacterial TrxR, is highly bactericidal for GSH-negative bacteria, indicating that Received: July 27, 2017
A
DOI: 10.1021/acs.inorgchem.7b01904 Inorg. Chem. XXXX, XXX, XXX−XXX
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Inorganic Chemistry the Trx system could be a feasible antibacterial drug target.14 Consistently, a recent study showed that Auranofin, an FDAapproved antirheumatic organo-gold drug, exhibited high activity against MRSA by targeting the Trx system.15−17 Silver and silver-containing materials have been widely employed as antimicrobial agents due to their broad-spectrum bactericidal activity.18,19 Recent studies have shown that silver ions could enhance antibiotic activity against Gram-negative bacteria as well as restore the antibiotic susceptibility of resistant bacterial strains, implying the potential application of silver in the treatment of drug-resistant bacterial infection.20 Silver could also eliminate Gram-positive bacteria, such as S. aureus. However, the inhibitory mechanism of silver is only partially known.21 Herein, we report that silver ions directly bind to cysteines at the active site of S. aureus Trx and TrxR. Binding of silver ions led to oligomerization of TrxR and Trx and abolished their functions in vitro. Cellular thermal shift assay (CETSA) data confirmed that silver ions targeted both Trx and TrxR in cellulo. Treatment with silver ions caused significant depletion of free intracellular thiols in S. aureus. Our results suggest that the Trx system in GSH-deficient pathogens is a viable antibacterial drug target and also provide insights into the antibacterial mechanisms of silver ions.
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remove DTT and chloride ions. The eluted protein fractions were immediately used for different assays. Ellman’s Assay. Ellman’s assays were carried out in Tris-HNO3 buffer. Different molar equivalents of Ag+ were added into 25 μM reduced Trx (or TrxR) and incubated for 20 min at room temperature (RT). Excess amounts of 5,5-dithiobis(2-nitrobenzoic acid) (Ellman’s reagent, DTNB) were subsequently added to a final concentration of 160 μM. After further incubation for 20 min at RT, the absorbance of each sample at 412 nm was measured by UV−vis spectroscopy. The absorbance at 412 nm was plotted against the Ag+/protein molar ratio. Isothermal Titration Calorimetry. Isothermal titration calorimetry (ITC) experiments were performed on a Malvern MicroCal iTC200 at 25 °C. The active Trx and TrxR were prepared in TrisHNO3 buffer with concentration of 25 μM. The Ag+ titrant was prepared by dissolving AgNO3 in Tris-HNO3 buffer (0.25 mM for TrxR and 0.5 mM for Trx). Typically, 40 μL of AgNO3 titrant was titrated into a 200 μL protein sample with 150 s intervals between each injection. AgNO3 was titrated into Tris-HNO3 buffer without protein for ligand-to-buffer subtraction. The ITC data were analyzed using Origin software and fitted by a one-set-of-sites binding model. Inductively Coupled Plasma Mass Spectrometry. All inductively coupled plasma mass spectrometry (ICP-MS) experiments were conducted on a Thermo Scientific iCAP Q ICP-MS spectrometer. Each sample was quantified three times, and the average value was used. Approximately 3 molar equiv of Ag+ was added into 200 μM reduced Trx (or TrxR) in Tris-HNO3 buffer. After incubation for 30 min at RT, excess amounts of Ag+ were removed by HiTrap desalting columns. The eluted protein concentration was measured by bicinchoninic acid (BCA) assay, and the bound Ag+ was determined by ICP-MS. Protein Oligomerization State Analysis. The oligomerization states of Trx and TrxR were analyzed by size-exclusion chromatography. Reduced and oxidized Trx (or TrxR) was incubated with different equivalents of Ag+ at room temperature for 30 min. The elution volumes of samples were subsequently measured on a Tricorn Superdex 75 10/300 GL column (GE Healthcare) pre-equilibrated with Tris-HNO3 buffer. The column was calibrated with a GE LMW calibration kit under the same buffer conditions. Thioredoxin Reductase Activity Assay. Thioredoxin reductase activity assays were performed as previously described.17 Standard reaction mixtures contained 50 nM TrxR, 200 μM DTNB, and 100 μM NADPH (Sangon, China) in Tris-HNO3 buffer at ambient temperature. Absorbance at 412 nm was recorded on a Biotek Cytation3 plate reader as an indication of enzyme activity. The initial rate during the first 5 min of the assay was calculated based on the change of A412nm over time. For TrxR activity assays, 50 nM TrxR was preincubated with or without Ag+ (0, 0.2, 0.4, 0.6, 0.8, 1.2 molar equiv); reactions were initiated by the addition of 50 μM oxidized Trx. For Michaelis constant (Km) of enzyme determination, 50 nM TrxR was preincubated with 0, 0.6, and 1.2 molar equiv of Ag+, respectively. Assays were performed in triplicate with variable concentrations of oxidized Trx (0 μM to 200 μM). Km was calculated by fitting the curve to the Michaelis−Menten equation using GraphPad Prism. To investigate the inhibitory effects of Trx by silver, 50 μM Trx was preincubated with different molar equivalents of Ag+ as indicated; excess amounts of Ag+ were subsequently removed by desalting columns. The Ag+-bound Trx was then added into the reaction mixture, which contained 50 nM active TrxR, 100 μM NADPH, and 200 μM DTNB in Tris-HNO3 buffer, for 15 min. The final absorption at 412 nm was recorded. Cellular Thermal Shift Assay (CETSA). CETSA was performed as previously described.22 A single colony of S. aureus Newman strain grown on a tryptic soy agar (TSA) plate was picked and cultured in tryptic soy broth (TSB) medium until the OD600 value reached 1. The bacterial culture was diluted 1:100 into fresh TSB for further growth until the OD600 reached 0.6. The bacterial culture was subsequently separated into two groups, the control group and the experimental group, which was treated with 10 μM Ag+. The two cultures were harvested after 2 h incubation and resuspended in Tris-HNO3 buffer. Equal amounts of the bacterial suspensions from each group were
EXPERIMENTAL SECTION
Materials. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. The thiol detection assay kit was purchased from Cayman Chemicals (Ann Arbor, MI, USA). Custom polyclonal antisera of S. aureus Trx and TrxR were prepared by immunizing rabbits with purified Trx and TrxR proteins (Huabio, China). The rabbit polyclonal antibodies were purified from antisera by Protein A sefinose (Sangon, China). Oligonucleotide primers for PCR reactions were purchased from Sangon (Shanghai, China) and are listed in Table S1. Protein Expression and Purification. The thioredoxin (trxA) and thioredoxin reductase (trxB) genes were amplified by PCR using S. aureus Newman genomic DNA as a template. The amplified gene fragments contained NdeI and BamHI restriction sites at the 5′- and 3′-ends, respectively. The fragments and the expression plasmid pET47b were double-digested with the corresponding restriction enzymes and ligated by T4 ligase. The generated expression plasmids pET47b-trxA and pET47b-trxB were verified by DNA sequencing (Sangon, China) and transformed into Escherichia coli (E. coli) BL21 (DE3) strain for protein expression. BL21 (DE3) cells, which harbored the expression plasmid, were grown in 1 L of Luria−Bertani (LB) medium for 3 h aerobically at 37 °C until the OD600 reading reached 0.6. IPTG was added to a final concentration of 0.5 mM to induce protein expression. The bacteria were harvested after further incubation for 16 h at 25 °C by centrifugation (5000g for 20 min at 4 °C). The pellets were resuspended in resuspension buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.4). Cell pellets were lysed by sonication at 4 °C with 1 mM PMSF as a protease inhibitor. The lysate was centrifuged at 15000g for 10 min, and the supernatant was collected. The supernatant was then dialyzed against 50 mM Tris-HCl (pH = 8.0) buffer overnight and subsequently applied to a 5 mL HiTrap Q column (GE Healthcare). Elution was performed with a linear gradient from 0 to 500 mM NaCl in 20 mM Tri-HCl buffer, pH 8.0. The eluted protein was pooled and further purified with a Hiload 16/60 Superdex 75 column equilibrated with Tris-HCl buffer (20 mM Tris-HCl, 300 mM NaCl, pH 7.4). The Trx and TrxR were both purified in their oxidized forms since no free cysteines were detected by Ellman’s assay. To prepare the active Trx and TrxR (reduced forms) for biochemical assays, the protein was incubated with excess amounts of DTT to reduce disulfide bonds. All protein samples were buffer-exchanged into Tris-HNO3 buffer (50 mM Tris-HNO3, 150 mM NaNO3, pH 7.4) by HiTrap desalting column (GE healthcare) to B
DOI: 10.1021/acs.inorgchem.7b01904 Inorg. Chem. XXXX, XXX, XXX−XXX
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Inorganic Chemistry aliquoted into PCR tubes and heated individually with a temperature gradient for 3 min, followed by immediate cooling on ice. The cells were then lysed by three freeze−thaw cycles. The cell lysate was subsequently centrifuged at 15000g for 10 min at 4 °C to pellet the denatured, precipitated proteins. The soluble proteins in the supernatant were analyzed by gel electrophoresis followed by immunoblotting using the appropriate corresponding polyclonal antibodies. Thiol Depletion Assay. S. aureus was incubated in TSB medium until the OD600 reached 0.6. Subsequently, the bacterial culture was treated for 30 min with 1 μg/mL ampicillin or different concentrations of silver as indicated. After treatment, the bacteria were washed twice in phosphate-buffered saline (PBS), pH 7.4, and the pellet was finally suspended in 100 mM potassium phosphate buffer, pH 7.4, containing 1 mM EDTA. S. aureus cell lysis was prepared via glass bead beating as described previously.23 The total thiol concentration in the supernatant was quantified using the Thiol Detection Assay Kit following the manufacturer’s instructions. Cellular thiol concentrations were calculated based on a volume of S. aureus cells of 4.2 × 10−15 L (diameter of S. aureus spherical cell is up to 1 μm) and a cell density of OD600 = 1 (equivalent to 1.5 × 108 cells/mL).24
Figure 2. Ag+ binding to reduced Trx (A) and TrxR (B) monitored by Ellman’s assay. Reduced Trx or TrxR was preincubated with various molar equivalents of Ag+ as indicated, and the remaining free thiols in protein samples were measured by Ellman’s assay. The absorbance at 412 nm was plotted against Ag+/protein ratios.
data indicating that TrxR and Trx could bind 1 and 2 molar equiv of Ag+, respectively. Intriguingly, although both Trx and TrxR contained a similar active redox center with conserved CXXC motifs, the two proteins bound Ag+ with different stoichiometries, which might be partially attributable to the different chemical environments of the active sites. S. aureus Trx structure analysis revealed that the CXXC motif is located on the protein surface,26 while the active site of TrxR is relatively buried and close to the FAD binding pocket. To further investigate the Ag+-binding properties of reduced Trx and TrxR, isothermal titration calorimetry (ITC) was utilized to monitor the titration of Ag+ into reduced Trx and TrxR. As shown in Figure 3, Ag+ binding to reduced TrxR resulted in a typical S-shaped curve. However, Ag+ caused significant precipitation of Trx protein after saturation. Therefore, lower dose of Ag+ was added in each injection to prevent Ag+ saturation during titration. By fitting the data with a one-site-binding model, the dissociation constants were determined to be 1.4 ± 0.1 μM with a stoichiometry of 1.10 ± 0.01 for Ag+ binding to reduced TrxR, and 15.0 ± 5.0 μM with a stoichiometry of 2.42 ± 0.20 for Ag+ binding to reduced Trx. The binding stoichiometries are consistent with the results obtained from both ICP-MS and Ellman’s assay. The binding of Ag+ to oxidized Trx and TrxR was also investigated by ITC. In line with ICP-MS data, oxidized Trx and TrxR had no detectable Ag+-binding capability. Oligomerization State Change of Trx and TrxR. Given that metal binding usually induces protein aggregation and subsequently disrupts its function, we wondered whether Ag+ binding to Trx and TrxR also induced protein aggregation. Therefore, size-exclusion chromatography was used to investigate the oligomerization states of Trx and TrxR before and after Ag+ binding. As shown in Figure 4A, reduced Trx was eluted as a single peak at an elution volume of 12.7 mL with an apparent molecular weight (MW) of 19.8 kDa, corresponding to a dimeric form of Trx (11.4 kDa for monomeric Trx). Upon addition of 0.6 molar equiv of Ag+, a small elution peak at 11.2 mL was observed with an apparent MW of 33 kDa, implying the formation of trimeric Trx. Further addition of Ag+ (1.2 and 2.0 equiv) led to significant decreases in the fraction of monomeric Trx, while more protein was simultaneously eluted at lower elution volumes, indicative of Trx protein aggregation. In contrast, preincubation with 2 molar equiv of Ag+ had no effect on the elution volume of oxidized Trx (Figure 4B), confirming that oxidized Trx had no Ag+-binding capability. Similarly, reduced TrxR was eluted as a dimeric form with a MW of 61 kDa (33 kDa for monomeric TrxR). Addition of Ag+ caused decrease in the fraction of dimeric TrxR but a significant
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RESULTS AND DISCUSSION Characterization of Ag+ Binding to Thioredoxin and Thioredoxin Reductase. To investigate whether S. aureus Trx and TrxR can bind Ag+, inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the Ag+-binding capacities of Trx and TrxR after preincubation with excess amounts of Ag+ as described in the Experimental Section. As shown in Figure 1, reduced TrxR bound 1.25 ± 0.13 molar
Figure 1. Ag+-binding capacity of Trx and TrxR as determined by ICPMS. All samples were treated with 3 molar equiv of Ag+. Excess amounts of Ag+ were removed by a desalting column. The bound Ag+ levels were determined by ICP-MS, and protein concentrations were measured by BCA assay.
equiv of Ag+, whereas reduced Trx bound 2.34 ± 0.24 molar equiv of Ag+. In contrast, the oxidized Trx and TrxR exhibited no detectable Ag+-binding capabilities. Reduced Trx and TrxR each contain two free cysteines at the active sites, whereas in the oxidized state, the two cysteines form an intramolecular disulfide bond (Figure S1). Given the highly thiophilic property of Ag+, and based on the ICP-MS results above, the free cysteines in the active site of Trx and TrxR are likely involved in Ag+ binding. To test this hypothesis, we used Ellman’s assay to monitor the number of free sulfhydryl groups in Trx and TrxR after preincubation with different molar equivalents of Ag+. The Ellman’s reagent DTNB specifically reacts with the free sulfhydryl group to yield NTB, which exhibits a maximum absorption at 412 nm in the UV−vis spectra.25 As shown in Figure 2, for both Trx and TrxR, the absorbance at 412 nm decreased with the increasing Ag+/ protein ratios, indicative of the direct binding of Ag+ to the free sulfhydryl group. However, the absorbance at 412 nm leveled off after the addition of approximately 1 or 2 molar equiv of Ag+ for TrxR and Trx, respectively, consistent with the ICP-MS C
DOI: 10.1021/acs.inorgchem.7b01904 Inorg. Chem. XXXX, XXX, XXX−XXX
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Inorganic Chemistry
Figure 3. Calorimetric titration of Ag+ to TrxR (A, B) and Trx (C, D). All protein samples were prepared in 50 mM Tris-HNO3 buffer supplemented with 150 mM NaNO3, pH 7.4. AgNO3 at concentrations of 0.25 mM and 0.5 mM was used as the titrant for TrxR and Trx, respectively. The titration curves were fitted to a one-set-of-sites binding model using Origin software.
the presence of other higher oligomeric species after Ag+ treatment (Figure S2d). Inhibition of TrxR Activity by Ag+ in Vitro. In the Trx system, electrons are transferred from NADPH via TrxR to the oxidized Trx, which further reduces protein disulfides or other substrates. The entire electron transfer pathway is highly dependent on the active sites of Trx and TrxR.27 As Ag+ can bind to the active sites of the reduced TrxR and Trx, the effects of Ag+ on their functional activities were subsequently investigated. The reductase enzyme activity of TrxR was assayed in the presence of oxidized Trx and NADPH. The regenerated reduced Trx was quantified via the rapid intramolecular disulfide exchange reaction with DTNB. The reaction progress was monitored by measurement of the absorbance at 412 nm. As shown in Figure 5, a dose-dependent effect of Ag+ on TrxR activity was investigated by preincubation
increase in aggregates at much smaller elution volumes (Figure 4C), implying the oligomerization of TrxR upon Ag+ binding. However, there was no obvious change in the elution volume of oxidized TrxR after incubation with 2 molar equiv of Ag+, indicative of no Ag+ binding (Figure 4D). The Ag+ induced oligomerization of Trx and TrxR was further confirmed by native electrospray ionization mass spectrometry (ESI-MS). Trx exhibited as monomer in the native ESI-MS spectrum with a molecular weight (MW) of 11.3 kDa (Figure S2a). The native ESI-MS spectrum of TrxR showed signals of both monomer and dimer in solution (Figure S2b). Ag+ binding significantly induced aggregation of both Trx and TrxR. Ag+-bound Trx mainly existed as a tetramer with a MW of 46.2 kDa in native ESI-MS spectrum (Figure S2c), while the signals of Ag+-bound TrxR indicated the formation of higher abundance of dimer and D
DOI: 10.1021/acs.inorgchem.7b01904 Inorg. Chem. XXXX, XXX, XXX−XXX
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Inorganic Chemistry
Figure 4. Effects of Ag+ binding on the oligomeric states of Trx (A, B) and TrxR (C, D). Size-exclusion chromatography profiles of reduced Trx (A) and TrxR (C) incubated with 0, 0.6, 1.2, and 2.0 molar equiv of Ag+, and oxidized Trx (B) and TrxR (D) incubated with 0 and 2.0 molar equiv of Ag+.
Figure 5. Inhibition of S. aureus TrxR and Trx activities by Ag+. (A) Approximately 50 nM purified recombinant TrxR was preincubated with different molar equivalents of Ag+ as indicated. Approximately 50 μM oxidized Trx, 100 μM NADPH, and 200 μM DTNB were added to initiate the reaction. The reaction rates within 600 s (inset) were calculated. (B) TrxR was preincubated with different molar equivalents of Ag+ as indicated. The TrxR reductase reaction rates were plotted against different concentrations of oxidized Trx. The curves were fitted to the Michaelis−Menten equation using GraphPad Prism. (C) Approximately 50 μM Trx was preincubated with different molar equivalents of Ag+ as indicated. Approximately 50 nM active TrxR, 100 μM NADPH, and 200 μM DTNB were subsequently added and incubated for 15 min. The final absorbance at 412 nm was recorded.
of 50 nM reduced TrxR with different molar equivalents of Ag+ as indicated. Approximately 50 μM oxidized Trx was added to initiate the reaction. The rate of TrxR reductase activity was linear over 600 s (inset of Figure 5A). With increasing amounts of preincubated Ag+, the TrxR activity was significantly inhibited in a dose-dependent fashion. The reductase enzyme activity was completely abolished when 1.2 molar equiv of Ag+ was added (Figure 5A). Similarly, the Km of the TrxR-catalyzed reaction was calculated with variable concentrations of oxidized Trx, as described in the Experimental Section. The Km for reduced Trx was 28.2 ± 7.0 μM without Ag+, whereas the value was determined to be 194.2 ± 41.4 μM when TrxR was preincubated with 0.6 molar equiv of Ag+ (Figure 5B).
Similarly, increasing amounts of Ag+ preincubated with Trx led to a decreased absorbance at 412 nm, suggesting that less Trx was available to react with DNTB (Figure 5C). These results clearly indicate that Ag+-bound Trx lost the capacity to regenerate the reduced form, even in the presence of active TrxR and NADPH. Collectively, these data demonstrate that Ag+ could effectively inhibit the functionalities of both Trx and TrxR in vitro. Trx and TrxR are Ag+ Targets in Cellulo. We have clearly demonstrated that Ag+ could abolish the physiological function of Trx and TrxR in vitro via binding to the redox active sites. To further verify whether Ag+ binds to TrxR and Trx in cellulo, a cellular thermal shift assay (CETSA) was employed. CETSA is E
DOI: 10.1021/acs.inorgchem.7b01904 Inorg. Chem. XXXX, XXX, XXX−XXX
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Inorganic Chemistry
demonstrated that the major low molecular weight (LMW) thiols in S. aureus include bacillithiol (BSH), coenzyme A (CoA), and cysteine.31 All of these LMW thiols had submillimolar cellular concentrations, which is consistent with the cellular concentration determined here. The amounts of cellular free thiols in S. aureus decreased by 17% and 31% after treatment with 40 μM and 60 μM Ag+, respectively. In addition to the inhibition of TrxR, the thiol depletion effect was probably due to the direct binding of Ag+ to free intracellular thiols. The intracellular free thiols provide bacteria a reducing capacity for defense against ROS. Thiol depletion severely impaired the bacterial capacity to cope with extracellular oxidative stress.15 In contrast, no obvious decrease in free thiols was observed in ampicillin-treated S. aureus. These results suggest that Ag+ treatment disrupted the thiol-redox homeostasis of S. aureus in vivo.
a method based on the biophysical principle of ligand-induced thermal stabilization of target proteins, which has been approved for evaluating drug binding to target proteins in cells and tissue samples.28 Sublethal doses of Ag+ with concentration of 10 μM were added into cell culture (Figure S3). Ag+ treatment resulted in a shift in the melting point of intracellular Trx from 54.3 to 50.7 °C, indicating that binding of silver destabilized the protein (Figure 6A). Similar results were
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CONCLUSIONS Drug-resistant S. aureus, especially methicillin-resistant (MRSA) and vancomycin-resistant S. aureus (VRSA), pose a great threat to human health globally. However, new antibiotics are no longer being developed at a rate that can keep pace with the rapid microbial evolution. Therefore, new antibacterial strategies are urgently needed. Repurposing of existing drugs or combination of appropriate non-antibiotic drugs with antibiotics to resensitize antibiotics could be feasible approaches to combat drug-resistant microbes.32,33 Ag+ and related materials have a long history of being used as antimicrobial agents.34,35 Previous studies have revealed that Ag+ was able to restore antibiotic susceptibility to resistant bacterial strains and even expanded the antibacterial spectrum of antibiotics against Gram-negative bacteria, which is indicative of their potentials in tackling drug-resistant bacteria. 20 However, in spite of their long-term use, the underlying mechanism of antimicrobial activity of Ag+ is not fully understood. ROS are toxic to bacterial cell as they cause oxidative damage to cellular constituents, such as DNA, lipids, and proteins.36 Human immune system cells, such as neutrophils, defend against invading pathogens by producing ROS through specific metabolic pathways.37 S. aureus has developed efficient pathways to defend against the oxidative stress when attacked by neutrophils.38 The Trx system of S. aureus plays a critical role in defense against ROS. The transcription levels of the Trx (trxA) and TrxR (trxB) genes are significantly increased in S. aureus under oxidative stress.13 Trx scavenges ROS and helps to maintain other proteins in their reduced forms. Oxidized Trx is subsequently reduced by the flavoenzyme TrxR and NADPH.6 Recent studies have shown that targeting the Trx system is effective in inhibition growth of GSH-deficient bacteria, indicating that the Trx system is a viable antibacterial drug target.14,17 Herein, we investigated the effects of Ag+ on the two protein components, Trx and TrxR, of the S. aureus Trx system, which are indispensable for bacterial growth, especially under oxidative stress.36 We show that Ag+ directly binds to cysteines at the active sites of Trx and TrxR. TrxR can bind one molar equivalent of silver ions with a dissociation constant (KD) of 1.4 ± 0.1 μM, whereas Trx can bind two molar equivalents of Ag+ with a relatively lower affinity (dissociation constant KD = 15.0 ± 5.0 μM). Binding of Ag+ induced oligomerization and dysfunction of both Trx and TrxR in vitro. Importantly, Ag+ also binds to Trx and TrxR in cellulo, as evidenced by the
Figure 6. Cellular thermal shift assay of Trx (A) and TrxR (B). S. aureus cultures with or without Ag+ treatment were heated over a temperature gradient. The soluble fractions of intracellular Trx or TrxR were quantified by Western blot. The band intensities at different temperatures are normalized to those at 37 °C for Trx and 49 °C for TrxR.
obtained for TrxR upon incubation with Ag+; the melting point of TrxR shifted from 53.7 to 51.2 °C after Ag+ incubation (Figure 6B). In contrast, a negative control α-hemolysin had no obvious shifts on its melting point after Ag+ treatment (Figure S4). It is worth noting that the thermal stability of the target protein decreased upon Ag+ binding, which is a nonphysiologically relevant metal ion. This observation is different from that for small molecule drugs, which usually increase the stability of target proteins.29 Thiol Depletion Assay. The Trx system plays an important role in thiol-redox homeostasis, especially for S. aureus, which is GSH-deficient. It has been reported that antibiotic treatment induced bacterial intracellular reactive oxygen species (ROS) production and altered the cellular redox state.30 We therefore investigated the effect of Ag+ on the levels of intracellular free thiols in S. aureus using a thiol detection assay kit (1 μg/mL ampicillin was used as a positive control). As shown in Figure 7, in the absence of Ag+ treatment, the level of intracellular free thiols was calculated to be 1.25 mM. Previous studies have
Figure 7. Ag+ induces a decline in intracellular free thiol levels. S. aureus cultures were treated with the indicated concentrations of Ag+ or ampicillin for 30 min. The intracellular free thiol concentrations were determined using a thiol detection assay kit. F
DOI: 10.1021/acs.inorgchem.7b01904 Inorg. Chem. XXXX, XXX, XXX−XXX
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Inorganic Chemistry cellular thermal shift assay results. Moreover, binding of Ag+ to Trx and TrxR blocked the electron transfer pathway in the Trx system, leading to a remarkable depletion of intracellular free thiols and accumulation of harmful ROS (Figure 8). It is worth
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21501200, 21671203), Science and Technology Program of Guangzhou, China (201707010038), a starting fund from Sun Yat-sen University, the Fundamental Research Funds for the Central Universities, the Research Grants Council of Hong Kong (17305415, 17333616), and the University of Hong Kong (for an e-SRT on Integrative Biology).
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Figure 8. Proposed mechanism for Ag+-induced inhibition of the thioredoxin−thioredoxin reductase system in S. aureus.
noting that mammalian cells have a high molecular weight thioredoxin reductase, which contains a selenocysteine residue in its active site.39 It is recently reported that mammalian thioredoxin reductase was also sensitive to Ag+-containing complex. 40,41 Therefore, how to rationally design Ag + compounds that can selectively inhibit bacterial thioredoxin reductase may warrant further investigation. Possible strategies include usage of bacterial membrane targeted compound as Ag+ ligand or encapsulation of Ag+ compound in specific liposome, which can selectively recognize bacterial anionic membrane.42,43 Collectively, this work demonstrates that the Trx system in S. aureus is targeted by Ag+, which expands our understanding of the antibacterial mechanism of Ag+.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.7b01904. Bacterial strains, plasmids, and primers, crystal structure of Trx and TrxR from S. aureus, native ESI mass spectra of Trx and TrxR, IC50 value of Ag+ against S. aureus, and cellular thermal shift assay of α-hemolysin (PDF)
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AUTHOR INFORMATION
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Zhongping Yao: 0000-0003-3555-9632 Wei Xia: 0000-0001-6480-3265 G
DOI: 10.1021/acs.inorgchem.7b01904 Inorg. Chem. XXXX, XXX, XXX−XXX
Article
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DOI: 10.1021/acs.inorgchem.7b01904 Inorg. Chem. XXXX, XXX, XXX−XXX
