https://orcid.org/0000-0001-7530-8593
Abstract
Silver nanoparticles (AgNPs) are the nanomaterials most widely used as antimicrobial agents in a range of consumer products, due to the environmental release of either the AgNPs themselves or silver ions. Although AgNPs appear to be more potent than silver ions, the mechanism behind the activity is not fully elucidated yet. The most common mechanism of toxicity of AgNPs proposed to date is the release of silver ions and/or the particle-specific functions. In this study, Pseudomonas aeruginosa (a model for Gram-negative bacteria) was treated with AgNPs, and its proteomic response was comprehensively characterized to elucidate the antimicrobial mechanism of AgNPs in the microorganism. In total, 59 silver-regulated proteins (27 up-regulated and 32 down-regulated proteins) and 5 silver-binding proteins were identified. Bioinformatic analysis revealed that interference with the cell-membrane function and generation of intracellular reactive oxygen species (ROS) were the main pathways for the antibacterial effect. The pattern of membrane proteins regulated by AgNPs was similar to that found for silver ions. In addition, the same silver-binding proteins were obtained with both AgNPs and silver ions, which indicated that AgNPs probably affect the cell membrane and react with proteins by releasing silver ions. The elevation of intracellular ROS relative to that with silver ions confirmed oxidative damage caused by AgNPs, which may be ascribed to the nano-characteristics and higher uptake efficiency of the particles. These results demonstrate that the antimicrobial activity of AgNPs is due to the synergistic action of release of dissolved silver ions and particle-specific effects. The proteomic analysis of silver-binding and silver-regulated proteins in the present study provides insight into the mechanism of antimicrobial activity of such nanomaterials.
Silver nanoparticles (AgNPs) are the nanomaterials most widely used as antimicrobial agents in a range of consumer products, due to the environmental release of either the AgNPs themselves or silver ions.
In this manuscript, the molecular mechanisms of antimicrobial activity of AgNPs in P. aeruginosa were investigated using a proteomics approach. Our results clearly suggested that interference with the cell-membrane function and generation of intracellular reactive oxygen species (ROS) were the main pathways for the antibacterial activity of AgNPs, in which both the nanoparticles themselves and the silver ions released from AgNPs play a crucial role. Overall, the proteomic and bioinformatic analysis of silver-binding and silver-regulated proteins in the present study provides new insight into the mechanism of antimicrobial activity of such nanomaterials.
Introduction
The antimicrobial activity of silver has been known since ancient times, and the metal has been commonly used for vessels or containers for liquid, coins, shavings, foils, sutures, and so on.1 Different forms of silver such as metals, salts, and nanoparticles have been applied to medical products to prevent wound infections, burns, and chronic ulcers caused by microorganisms. In recent years, silver nanoparticles (AgNPs) have attracted more attention in health related fields, due to their unique nano-characteristics and broad spectrum antimicrobial properties.2 Hence, AgNPs are used in biomedical applications, clothing, food industry, water purification, cosmetics and domestic appliances.3 AgNPs have already been reported to be effective in various microbes, including Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium), Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecium, and Bacillus subtilis), and fungi (Aspergillus niger, Candida albicans, and Saccharomyces cerevisiae).4
Previous studies had manifested that AgNPs act against bacteria in a similar way to silver ions. However, the effective concentrations of AgNPs and silver ions were at nanomolar and micromolar levels, respectively.5 The mechanism of toxicity of silver ions in bacteria has been investigated for a long time.6 Much of the research on silver ions has reported that the penetration of silver ions into bacterial cells turns DNA molecules into a condensed form and hinders the ability to replicate, which leads to cell-function disorder.7–9 In addition, it has been proven that silver ions react with proteins by binding with the sulfhydryl group (–SH) quickly, which leads to a decrease or loss of activity of multiple enzymes and then causes sterilization.10 AgNPs appear to be more toxic than silver ions due to their large surface area, providing better contact with the cells.11 With transmission electron microscopy (TEM), scanning electron microscopy (SEM), and proteomic analysis, it has been demonstrated that AgNPs interact with the bacterial membrane and penetrate inside the cell, which causes structural damage, drastic disturbance in proper cell function, and cell death.12–14 Specifically, AgNPs cause oxidative stress through the generation of reactive oxygen species (ROS), cause damage to proteins and nucleic acids, and finally inhibit cell proliferation.15–18 Furthermore, the nanoparticles release silver ions, which will enhance the bactericidal effect of AgNPs.7,19,20 Earlier studies have presented the results on the toxicity of AgNPs to biological systems; however, the mechanism of bactericidal action has not been fully elucidated from a proteomics perspective. Quantitative proteomic and bioinformatic analysis can provide comprehensive insight into the global responses of bacteria to AgNPs, which will help to elucidate the underlying antibacterial mechanism.
In this study, the antimicrobial activity of AgNPs was evaluated using P. aeruginosa, a model for Gram-negative bacteria, through integral analysis of silver-regulated and silver-binding proteins by a combination of proteomic and bioinformatic analysis. Compared with Gram-positive bacteria, Gram-negative bacteria have an outer membrane that makes them more resistant against antibodies. The isobaric tag for relative and absolute quantitation (iTRAQ) method, combined with two-dimensional liquid chromatography–tandem mass spectrometry (2D-LC-MS/MS), was used for the quantitative analysis of silver-regulated proteins. Gel electrophoresis (GE) coupled with inductively coupled plasma mass spectrometry (ICP-MS) was applied to separate and detect the silver-binding proteins. Additionally, we also compared the differential protein expression signatures in P. aeruginosa under exposure to AgNPs and silver ions (as a control), and hence a possible action mechanism for AgNPs was suggested.
Experimental
Bacterial culture and sample preparation
A freeze-dried culture of P. aeruginosa (ATCC 10145) was purchased from the China General Microbiological Culture Collection Center and reconstituted in Luria-Bertani medium at 37 °C for 24 h with shaking. The bacteria were cultured to the stationary phase (optical density at 600 nm (OD600) of 1.5) overnight and diluted to an absorption value of 0.1 at 600 nm before exposure experiments. AgNP solution was sonicated for 10 min and centrifuged at 10 000g for another 10 min to ensure its good dispersion and remove silver ions. AgNPs were characterized by TEM (2100F, Japan) and the image is shown in Fig. S1 in the ESI.† The stock solutions of AgNPs and AgNO3 (1.0 mg mL−1 as Ag) were freshly prepared in sterile deionized water and stored at 4 °C in the dark. The bacterial suspension (100 μL) was added to sterile 96-well plates containing solutions of AgNPs or AgNO3 (100 μL) at various concentrations (from 0.1 to 50 μg mL−1). The mixtures were then incubated at 37 °C in the dark for 24 h. The bacterial viability was evaluated by monitoring the OD600 value.
For sample preparation for iTRAQ and GE-ICP-MS, the growing bacterial culture (OD600 of 0.1) was added to sterile Erlenmeyer flasks containing solutions of AgNPs and AgNO3 to final concentrations of 1.2 μg mL−1 and 0.6 μg mL−1, respectively. The mixtures were then incubated at 37 °C in the dark for 24 h. After incubation, the bacteria were harvested by centrifugation at 5000g for 10 min and then washed with cold phosphate buffered saline three times. The cell pellets were resuspended with 10 mM tris(hydroxymethyl)aminomethane (Tris, pH 7.5) and a protease inhibitor cocktail (10 μg mL−1). The suspension was then lysed through sonication and further centrifuged at 10 000g for 30 min at 4 °C, and the supernatants were collected for further use. The protein concentration was determined by using the BCA Protein Assay Kit (Thermo). Protein samples were stored at −80 °C until use.
Silver adsorption in the bacterial cells
ICP-MS was used to measure the silver concentrations present in bacteria treated with AgNPs and AgNO3. The bacterial suspension was prepared and treated with AgNP and AgNO3 solutions (0.1, 0.2, 0.4, and 0.6 μg mL−1) as described above. The pellets from bacterial suspensions (1.0 mL) were digested with 1 mL of concentrated ultrapure nitric acid (65%) and 1 mL of ultrapure hydrogen peroxide (30%). The concentrations of adsorbed silver were evaluated against a calibration curve for AgNO3 ranging from 0.1 to 100 ng mL−1. The release of silver ions from AgNPs was measured by ICP-MS (Agilent 8800), as described previously (ESI†).21
iTRAQ labeling and 2D-LC-MS/MS detection
Protein digestion and labeling were performed by using the 8-plex iTRAQ reagent kit (AB SCIEX, Framingham, MA, USA) according to the manufacturer's protocol. Protein (100 μg) from each bacterial sample was reduced by using 4 μL of reducing reagent at 60 °C for 1 h. Cysteine residues were blocked with 2 μL of cysteine-blocking reagent at room temperature for 10 min. After that, the solutions were filtered to remove the denaturing detergent and small molecules by using a 10 kDa ultrafiltration system. The digestion procedure was conducted overnight at 37 °C with trypsin at a mass ratio of 1/50 (trypsin/protein), and the proteins were then labeled with iTRAQ tags. The labeling was stopped by water, and the samples were pooled and dried using a SpeedVac system.
The peptides were resuspended in 100 μL of buffer A (10 mM KH2PO4, 25% acetonitrile, pH 3.0) and fractionated by strong cation-exchange chromatography (polysulfoethyl column, 2.1 mm × 100 mm, 5 μm, 200 Å, Columbia, MD, USA) on a high-performance liquid chromatography system (Agilent, USA). The peptides were eluted with a linear gradient from 100% buffer A to 100% buffer B (10 mM KH2PO4, 500 mM KCl, and 25% acetonitrile, pH 3.0) for 60 min at a flow rate of 0.3 mL min−1. A total of 12 fractions were collected, dried, and stored at −20 °C until analysis. Each of the fractions was analyzed by using a nanoLC-Ultra™ 2D system (AB SCIEX) coupled with a TripleTOF 5600 system (AB SCIEX). The dried samples were redissolved in buffer C (2% (v/v) acetonitrile and 0.1% formic acid) and desalted with a C18 trapping column (C18, 100 μm × 3 cm, 3 μm, 150 Å; Eksigent, USA) with buffer C at a flow rate of 2 μL min−1 for 10 min. The peptides were then separated on a reversed-phase BEH130 C18 analytical capillary column (C18, 75 μm × 15 cm, 3 μm 120 Å; Eksigent, USA) by using a solvent system with mobile phase A containing 99.9% water and 0.1% formic acid, and mobile phase B containing 99.9% acetonitrile and 0.1% formic acid. A 70 min linear gradient (from 5% to 35% mobile phase B) was utilized to elute peptides at a constant flow rate of 400 μL min−1. The quadrupole time-of-flight (QTOF) instrument was operated in information-dependent analysis mode with a Nanospray III source (AB SCIEX). Data were acquired with the following parameters: ion-spray voltage of 2500 V, curtain gas at 30 psi, nebulizer gas at 15 psi, and interface heater temperature of 150 °C. Analysis survey scans were acquired from 350 to 1500 Da in 250 ms, and as many as 35 product-ion scans were collected when a threshold of 150 counts per s was exceeded.
Identification and data analysis for silver-regulated proteins
The original MS/MS data were analyzed by using ProteinPilot Software 5.0 (AB SCIEX). The searching parameters were as follows: iTRAQ as sample type, trypsin as enzyme, methyl methanethiosulfonate as cysteine alkylation, and biological modifications ID focus as analysis method. For protein identification, a strict criterion was applied with a cumulative confidence >95% based on ProtScore >1.3. For analysis of protein quantification, proteins were considered differentially abundant if the fold-change ratios were >1.50 or <0.67 and the p-value was <0.05.
The information on protein function, classification, and localization was statistically analyzed by using Gene Ontology (http://www.geneontology.org).
GE-ICP-MS detection and identification of silver-binding proteins
The separation and detection of silver-binding proteins in P. aeruginosa was conducted according to the previously reported protocol.22 Protein samples (10 μL, 2 μg μL−1) from bacterial cells exposed to silver were mixed with 10 μL of sample buffer (0.06 M Tris buffer, 25% glycerol, 2% sodium dodecylsulfate) and injected into the GE system by using a small syringe with a Teflon tube affixed. The separation was carried out with a three-step procedure: voltage of 100 V for 10 min, 200 V for 20 min, and then 800 V for 100 min. The chromatograms of silver-binding proteins were acquired by using the time-resolved analysis mode. More details are provided in the ESI.†
The silver-binding proteins were collected and applied to a gel slab for concentration and visualization. The molecular weights of silver-binding proteins were precalculated by the synthesized I-markers (ESI†). The protein bands were excised, washed, and digested with trypsin overnight at 37 °C. In-gel digests were analyzed by nanoscale LC-electrospray ionization-QTOF MS/MS. For the identification of proteins, the Mascot 2.0 server (http://www.matrixscience.com) was used to search for the ion spectra. The data were searched against P. aeruginosa entries in the National Center for Biotechnological Information and Swiss-Prot databases.
Intracellular ROS assay
P. aeruginosa was exposed to AgNP and AgNO3 solutions (0.1, 0.2, 0.4, and 0.8 μg mL−1) to evaluate the difference in intracellular ROS generation. After exposure, P. aeruginosa cells from each sample were collected, washed twice, and then resuspended in phosphate buffer at a density of two million cells per mL. 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA; 2 μL, 10 mM; Sigma-Aldrich, USA) was added to a 1 mL bacterial sample, and the mixture was incubated at 37 °C for 30 min. The fluorescence signal at λex/λem of 488 nm/525 nm was measured to evaluate intracellular ROS production in the exposed P. aeruginosa cells.
Statistical analysis
All data were acquired from three independent experiments. The statistical analysis was performed by using Student's t-test. P < 0.05 was considered to be a statistically significant difference.
Results
Bacterial viability and silver adsorption in the bacterial cells
The bacterial viability was analyzed after treatment with various concentrations of AgNPs and AgNO3 for 24 h. Both forms of silver induced concentration-dependent inhibition of bacterial viability (Fig. 1A). The half-maximal inhibitory concentrations (IC50) of AgNPs and AgNO3 were 0.88 μg mL−1 and 2.24 μg mL−1, respectively. The survival rate reached 80% under exposure to AgNPs and AgNO3 at concentrations of 0.60 μg mL−1 and 1.26 μg mL−1, respectively.
(A) Bacterial viability in P. aeruginosa induced by various concentrations of AgNPs and AgNO3. (B) Silver adsorption by P. aeruginosa after treatment with AgNPs and AgNO3 (0.1, 0.2, 0.4, and 0.6 μg mL−1) for 24 h. Significantly different from control: *p < 0.05.
Silver adsorption by P. aeruginosa was evaluated by measuring the total silver concentration in the bacterium with ICP-MS. Exposure to both forms of silver resulted in significant silver accumulation in P. aeruginosa. Fig. 1B illustrates that silver was taken up by the bacteria in a dose-dependent manner for both AgNPs and AgNO3 after 24 h exposure. The silver concentrations accumulated in P. aeruginosa were approximately the same for treatment with either AgNPs or AgNO3 at 0.1 μg mL−1. Notably, higher concentration levels of silver were detected in the AgNP-treated groups in comparison with the AgNO3-treated groups at 0.2, 0.4, and 0.6 μg mL−1, respectively.
The results of the measurement of released silver ions from AgNPs are shown in Fig. S2 (ESI†). The percent of silver ions released was generally from 5% to 15% in different concentration groups. The values of silver ions released from AgNPs were lower than the levels adsorbed by P. aeruginosa treated with the same doses of AgNPs, suggesting that both forms of silver (i.e., nanoparticle and silver ion) can penetrate the bacteria.
Classification of differentially expressed silver-regulated proteins
To profile the protein changes in P. aeruginosa caused by AgNPs, quantitative proteomic analysis based on iTRAQ labeling was executed. The silver-regulated proteins in the groups treated with AgNPs and AgNO3 are shown in Tables S1 and S2, respectively (ESI†). In total, 16 proteins were regulated by treatment with both forms of silver: 8 were up-regulated and 8 were down-regulated. However, the response to each treatment was clearly different; 43 proteins were regulated exclusively by AgNPs, whereas only 12 proteins were regulated exclusively by AgNO3. For the solely regulated proteins, 19 proteins (44%) were up-regulated and 24 proteins (56%) were down-regulated after exposure to AgNPs; 8 proteins (67%) were up-regulated and 4 proteins (33%) were down-regulated after exposure to AgNO3.
For visual analysis of the proteins regulated by AgNPs and AgNO3, heat maps were created to determine whether those proteins exhibited similar functions (Fig. 2A). It was observed that the silver-regulated proteins from the two treatment groups showed a similar pattern. Specifically, the proteins in the AgNP-treated group showed significant changes in the expression level. Fig. 2B and C are the heat maps of the membrane proteins and ROS-related proteins among the silver-regulated proteins in the AgNP- and AgNO3-treatment groups. Gene Ontology term enrichment analysis was carried out for the proteins that were up- and down-regulated. The silver-regulated proteins were classified according to their location in the cellular architecture (Fig. 3). The results of the analysis of molecular functions and biological processes by using BLAST2GO software are summarized in Fig. S3 (ESI†). Distribution into major process and functional categories showed approximately the same patterns between proteins regulated by exposure to AgNPs and AgNO3. The represented biological processes for both AgNP- and AgNO3-regulated proteins were the cellular process, single-organism process, metabolic process, localization, locomotion, biological regulation, response to stimulus, and signaling (Fig. S1A, ESI†). The most represented functional categories for the silver-regulated proteins were binding, catalytic activity, transporter activity, nucleic acid binding transcription factor activity, antioxidant activity, molecular transducer activity, and electron carrier activity (Fig. S1B, ESI†). The results of the bioinformatic analysis also showed little difference between the regulated proteins from the AgNP and AgNO3 treatment groups.
(A) Heat maps of the silver-regulated proteins in AgNPs (1.2 μg mL−1) and AgNO3 (0.6 μg mL−1) treatment groups. Enlarged figure of membrane proteins (B) and ROS related proteins (C) among the silver-regulated proteins. The red rectangles represent the proteins that are up-regulated relative to the control; the green rectangles show the proteins down-regulated by AgNPs. Note that the value is on a log scale.
Classification of differentially regulated proteins affected by AgNPs (A) and AgNO3 (B) according to their location in the cellular architecture.
Identification of silver-binding proteins
For systematic characterization of the silver-binding proteins of P. aeruginosa, on-line coupling of GE with ICP-MS was applied in this study, and the results are presented in Fig. 4. The control protein sample, without the addition of silver, was used to exclude intrinsic silver-binding proteins. In total, five silver-binding proteins (60 kDa chaperonin (GroL), elongation factor Tu (TufA), flagellin (FliC), electron-transfer flavoprotein subunit alpha (EtfA), and the uncharacterized protein PA3309) were identified from P. aeruginosa in both silver treatments (Table S2, ESI†). Some genuine silver-binding proteins may be lost due to the low content of silver or ICP-MS sensitivity limitations. GroL belongs to the chaperone family and plays a major role in the stress response, mitochondrial protein transport, and the transmission and replication of mitochondrial DNA. The same protein was found to bind with iron in Helicobacter pylori by using haem-affinity chromatography.23 TufA is an abundant prokaryotic elongation factor and participates in the protein synthesis by catalyzing the binding of aminoacyl-tRNA to the ribosome. Binding with TufA may affect the synthesis and translocation of proteins. FliC is a globular protein containing abundant negatively charged amino acids, such as glutamic acid and aspartic acid, which make it more attractive to silver ions. EtfA is located on the inner mitochondrial membrane and is involved in the process of electron transfer. Aside from the above proteins, another silver-binding protein was detected with a molecular weight of around 29 kDa. However, the amount of collected protein was too low for further identification.
Profiles of silver-binding proteins in P. aeruginosa measured with a GE-ICP-MS system after treatment with 1.2 μg mL−1 AgNO3 (A) and 0.6 μg mL−1 AgNPs (B).
Effects of silver-mediated ROS generation
To further examine the oxidative damage to the cells induced by AgNPs, we detected the expression levels of ROS generation in P. aeruginosa by using the DCFH-DA staining assay. Fig. 5 shows AgNP-induced intracellular ROS generation in a time- and dose-dependent manner. AgNP treatment induced more pronounced ROS generation in P. aeruginosa than AgNO3 treatment at the corresponding concentrations. Furthermore, a low, but detectable, increase in cellular DCFH oxidation in P. aeruginosa was observed with 0.6 μg mL−1 AgNO3 only after 24 h (Fig. 5B).
Intracellular ROS generation in P. aeruginosa treated with AgNO3 and AgNPs. (A) Dose–response for intracellular ROS generation in P. aeruginosa exposed to AgNO3 and AgNPs (0.1, 0.2, 0.4, and 0.6 μg mL−1) for 24 h; (B) time course for intracellular ROS generation in P. aeruginosa stimulated by AgNO3 and AgNPs (0.6 μg mL−1). *p < 0.05 and **p < 0.01 versus control.
Discussion
Based on previous studies, the mechanisms of AgNP toxicity can be attributed to the release of silver ions and/or nanoparticle deposition inside the cells, and detailed mechanisms mainly involve cellular membrane damage, ROS generation, disruption of energy metabolism, and disruption of gene transcription.12,15,16,24,25 Despite many studies regarding the toxicity of AgNPs, the reported mechanism of antibacterial activity remains controversial. Thus, a comprehensive study is crucial to distinguish the antimicrobial activity of AgNPs caused by the released silver ions, the silver nanostructures, or a combined action of both. The proteomics results from this study suggest that the effect on membrane proteins and the oxidative stress induced by AgNPs are the main mechanism responsible for the antimicrobial activity. The potential oxidative damage and membrane damage caused by the AgNPs were also confirmed in a previous study with two recombinant bioluminescent bacteria, an oxidative-stress damage sensitive strain, DS1 (sodA:luxCDABE), and a protein/membrane damage sensitive strain, DC1 (clpB:luxCDABE).26 However, that study verified the action solely with a stress-specific gene and a membrane-damage promoter, specifically, sodA and clpB; the actual mechanism of action in pathogens still needs to be explored.
The most remarkable finding in our proteomic analysis is the identification of many membrane proteins whose expression was apparently up- or down-regulated by the AgNPs. These proteins primarily function in ion binding, transport, flagellum assembly, pore formation, antibiotic resistance, and membrane stabilization. The top three up-regulated proteins (AtpE, PA2536, and PA4504) participate in adenosine triphosphate synthesis, phospholipid synthesis, and transmembrane transport. Several outer-membrane porins (OprH, OprD, and OprC) associated with the transport of cationic amino acids, peptides, antibiotics, and ions were significantly affected by AgNPs. Furthermore, a number of metal transporters, such as OprC (Cu), CcoO1 and CcoO2 (Fe), MgtE (Mg), and PA0372 (Zn), were all suppressed in P. aeruginosa upon exposure to AgNPs, which may assist with AgNP and/or Ag+ transport into the cell through the transmembrane pores. The regulated flagellin proteins (PilP, PilX, FlgE, and FliN) are rich in lipids and play an important role in many processes including motor activity, adhesion, biofilm formation, and phage infection.27 In previous studies, AgNPs were found to be present inside the bacteria and also attached to the outer membrane by using TEM combined with elemental analysis.28–31 Therefore, we can speculate that the AgNPs attached to the surface of the cell membranes and affected the expression of related proteins.
Oxidative stress that results from the increased ROS production has been indicated as a major mechanism of AgNP-induced toxicity in human cells.32–34 Superoxide dismutases and oxidoreductases are involved in intracellular oxidative damage caused by metal ions or other organic poisons. In our study, the expression levels of the related proteins (PA4133, Hmp, KatA, CcoP2, SodB, CcpA, RibC, EtfA, and PiuC) were specifically regulated in P. aeruginosa after exposure to AgNPs. Furthermore, the level of intracellular ROS in P. aeruginosa was found to be elevated after treatment with AgNPs in concentration- and time-related manners (Fig. 5). Notably, the cells exposed to AgNO3 did not show any perturbation in the intracellular ROS generation at low concentrations, which certifies that the oxidative stress was caused solely by the AgNPs at the corresponding concentrations.
Recent studies on AgNPs have debated the mechanisms that can be attributed for the toxicity to bacteria. Our proteomic analysis results have shown that AgNPs directly affected membrane proteins and ROS-related proteins, which will cause membrane damage, oxidative stress, and even cell death. The observed molecular functions and biological processes of silver-regulated proteins were basically similar in response to both AgNPs and AgNO3, which is ascribed to the release of silver ions from the AgNPs. Nevertheless, a comparison of protein expression levels in P. aeruginosa showed that AgNPs caused more intense alterations than silver ions. On the other hand, a number of differentially expressed proteins were found only in the AgNP-treated group, which reflects a particle-specific effect. Except for the 16 common regulated proteins, 12 proteins were specifically regulated in the AgNO3-treated groups, which is due to the different effective concentration of particles and ions. Furthermore, intracellular ROS generation in P. aeruginosa was significantly induced solely by AgNPs. This may partly be due to higher bioaccumulation of AgNPs than silver ions. The regulation of ROS-related proteins and the generation of oxidative stress by AgNPs can mainly be ascribed to the nano-characteristics of the particles. In this study, AgNPs were found to be more cytotoxic than AgNO3 at the equivalent concentration; the IC50 value for AgNPs was three times that for AgNO3 (Fig. 1A). We believe that this result is attributable to the test bacteria (Gram-negative) and the usage of small-sized AgNPs (5 nm). The bioavailability of silver ions is also affected by environmental conditions, such as the presence of chloride, sulfide, and phosphate in the medium. In addition, the total amount of silver in P. aeruginosa exposed to AgNPs was higher than that with AgNO3, which indicates the relatively high bio-uptake efficiency of AgNPs. The bacterial uptake of AgNPs to the cellular interior might provide the basis for their actual biological effects. The interaction of silver with proteins is also believed to be an important mechanism of toxicity of AgNPs.35–37 By using a GE-ICP-MS method, the same five silver-binding proteins were obtained from both the AgNP and AgNO3 groups. Based upon our findings, along with the specific chromatograms of AgNPs, we speculate that the AgNPs react with the proteins by releasing silver ions. Overall, AgNPs exert their antimicrobial activity by interference with the functions of the cell membrane and induction of oxidative stress, typically through the release of silver ions and the nano-characteristics of the particles.
Conclusion
In summary, we employed proteomic methods to analyze the silver-regulated and -binding proteins in P. aeruginosa exposed to AgNPs. Analysis of the protein profiles showed that the cell membrane was the main target of AgNPs and cellular oxidative stress was induced by ROS generation. The release of silver ions and particle-specific effects synergistically achieve the antibacterial action of AgNPs. We also found five silver-binding proteins in P. aeruginosa, which could be used as new direct targets and biomarkers. These results provide valuable knowledge for further investigations on the bactericidal activity of AgNPs on pathogenic bacteria. Furthermore, these proteomic techniques can be applied for exploring the molecular mechanisms of the action of metals and metal-based drugs in microorganisms.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (91743203, 21677153, 21577153, 21777179 and 21522706) and The Thousand Talents Plan for Young Professionals, China and the Frontier Research Key Project of the Chinese Academy of Sciences (QYZDB-SSW-SLH034).
References
Footnotes
Electronic supplementary information (ESI) available. See DOI: 10.1039/c7mt00328e
