The high surface area per mass and small size of the nano-particles make silver nanoparticles (AgNPs) an effective antibacterial agent [1, 2, 3]. For this reason, AgNPs have been applied in diverse medical applications ranging from silver-based dressings to silver-coated medicinal devices and on numerous consumer products [4]. Although AgNPs have been widely used for antimicrobials, their mechanism is still not well understood. Possible antimicrobial mechanisms proposed include (1) interference with cell wall synthesis [5, 6, 7, 8], (2) inhibition of protein synthesis [6, 8], (3) interference with nucleic acid synthesis [6, 8], and (4) inhibition of a metabolic pathway [6, 8, 9]. To get a better insight on how AgNPs affect bacterial growth, the proteomic approach is concerned as an effective tool for protein analysis. Recently, only few studies have been reported on bacterial proteomes after AgNP exposure [10, 11, 12, 13, 14, 15, 16, 17], and most of the AgNPs previously described were stabilized with anionic citrate.
It is noteworthy that the stability of AgNPs synthesized can be increased by coating the nanoparticles with polymers, which can prevent particle agglomeration by steric hindrance. The differences in type of stabilizers could lead to different properties of AgNPs including the release or distribution of silver ions related to perhaps the protein activity [11, 12]. Thus, to study of how AgNPs affect the protein function, the capping agent of synthesized AgNPs should be mentioned. Poly (4-styrenesulfonic acid-co-maleic acid) or PSSMA is a polyelectrolyte copolymer of styrenesulfonic acid and maleic acid, which is widely used to develop multilayer thin films for bio-material or controlled drug release [18].
In this paper, AgNPs capped with anionic polystyrene-
Nanoscale silver particles (3.33 mM) stabilized by PSSMA (Aldrich, USA) were prepared using a chemical reduction and characterized as detailed in Tamiyakul et al. [19]. Briefly, 10 mM sodium borohydride (NaBH4; Fisher Scientific, USA) was rapidly added into a mixture of 10 mM silver nitrate (AgNO3; Carlo Erba Reagents, Italy) and 1 mM PSSMA in order to reduce silver ions to form nanoparticles. A dark brown solution of PSSMA-stabilized nanosilver (NS) showed a characteristic peak at a maximum wavelength (λmax) of 395 nm, as determined by Specord S 100 UV spectrophotometer (Analytikjena, Germany). The spherical shape of NS was seen from transmission electron microscope (TEM; JEM-2100, Jeol, Japan). The average paricle size of 5.21 ± 4.43 nm (n = 250) was obtained by further TEM analysis using equipped SemA-fore v.5.21 program. The value of surface charge of NS measured by Zetasizer NanoZS (Malvern Instruments, UK) was -35.5 ± 0.96 mV.
The strains of Gram-positive
The bacterial growth experiment was performed using
Bacteria collected at incubation times of 1, 15, 30, 45, 60, 120, and 180 min were centrifuged at 5000 rpm for 5 min, and the supernatant was discarded. The pellet was re-suspended with 0.5% w/v sodium dodecyl sulfate (SDS; Sigma-Aldrich, USA) solution, vortexed vigorously, and frozen in a freezer at –80°C overnight. After thawing, the suspension was centrifuged at 5000 rpm for 5 min at room temperature, and the supernatant was determined for the extracted protein by a method of Lowry using bovine serum albumin (BSA) as a standard [20].
The extracted proteins were mixed with a loading buffer (0.125 M Tris-HCl pH 6.8, 20% w/v glycerol, 5% w/v SDS, 0.2 M dithiothreitol [DTT], 0.02% w/v bromophenol blue) before loading onto polyacrylamide gels (12.5% separating gel, 5% stacking gel), which were prepared following the method of Laemmli [21]. An electrophoresis system was run at 50 V for stacking gel and 70 V for separating gel until the dye front reached approximately 1 cm from the edge of the gel. All gels were visualized by silver and Coomassie brilliant blue G staining and scanned with Image Scanner (Bio-Rad, USA).
In order to analyze the bacterial peptides by liquid chromatography-tandem mass spectrometry (LC–MS/MS), each lane of the gel was sectioned horizontally in order to acquire the entire population of proteins in the lane. Gel slices were excised to obtain gel plug with 1 mm3 in size. For in-gel digestion, the gel plugs were dehydrated with 100% acetonitrile (RCI Labscan, Thailand) and reduced with 10 mM DTT (USB Co. Ltd., USA) for 1 h at room temperature. Alkylation was further done in the dark using 100 mM iodoacetamide (GE Healthcare, UK) for 1 h at room temperature before dehydrated twice with 100% acetonitrile for 5 min. Finally, 10 ng of trypsin solution (Promega, USA) was added to the gels followed by overnight incubation at 37°C so as to digest proteins. Peptides were extracted, collected, and kept at -80°C prior to mass spectrometry analysis [22].
For the peptide analysis, the extracted peptides were re-suspended with 0.1% v/v formic acid (FA, [AppliChem, Germany]) and centrifuged at 10,000 rpm for 5 min. The supernatant was injected into Ultimate 3000 LC system (Dionex) coupled with ESI-ion Trap MS (HCT Ultra PTM Discovery System, BrukerDaltonics Ltd., UK). DeCyder MS Differential Analysis software (DeCyderMS, GE Healthcare) was used for protein quantitation [23, 24]. Mascot software (Matrix Science Ltd., London, UK) was used for the protein identification based on the National Center for Biotechnology Information (NCBI) database (
The proliferation of
In order to maintain the optimal level of bacterial protein prior to proteomic analysis, the cells cultured in the medium were collected at time intervals covering the early exponential phase, which were 15, 30, 45, 60, 120, and 180 min.
The extracted proteins of bacteria gathered from every incubation time were digested prior to analysis by LC/MS–MS. The proteins of NS-treated
The identified proteins by LC–MS/MS with known functions of
Protein number accession | Protein name | Protein symbol | Function | ||
---|---|---|---|---|---|
gi|170081960 | Long-chain fatty acid transport protein | fadL | Molecular transport | ||
gi|326798469 | 3-Deoxy-d-manno-octulosonic-acid transferase | waaA | Metabolic process | ||
gi|1786970 | 2,3-Bisphosphoglycerate-dependent phosphoglycerate mutase | gpmA | Metabolic process | ||
gi|1786964 | Quinolinate synthase A | nadA | Metabolic process | ||
gi|153844091 | Protein YhgF | yhgF | Metabolic process | ||
gi|146319043 | Threonine dehydrogenase and related Zn-dependent dehydrogenase | srlB | Metabolic process | ||
gi|308050024 | Flagellar hook-basal body protein | fliE | Metabolic process | ||
gi|156973219 | UDP- | murD | Metabolic process | ||
gi|1279404 | Peptide transport system ATP-binding protein SapF | sapF | Molecular transport | ||
gi|323524861 | Enoyl-CoA hydratase/isomerase | tdcF | Signal transduction | ||
gi|1787018 | Probable ATP-dependent helicase DinG | dinG | DNA replication | ||
gi|283778629 | DNA topoisomerase type IA Zn finger domain-containing protein | yrdD | Transcription | ||
gi|1789715 | 50S ribosomal protein L4 | rplD | Translation | ||
gi|227357988 | Siderophore biosynthesis IucA/IucC family protein | iucA | Metabolic process | ||
gi|226943115 | Isopropylmalate isomerase large subunit | leuC | Metabolic process | ||
gi|225010785 | PhoH family protein | phoH | Metabolic process | ||
gi|261854682 | Phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase | purH | Metabolic process | ||
gi|319762019 | Haloacid dehalogenase (HAD)-superfamily hydrolase | yqaB | Metabolic process | ||
gi|331007050 | Colicin V production protein | cvpA | Molecular transport | ||
gi|325067458 | Oligopeptide ATP-biding cassette (ABC) transporter periplasmic protein | oppA | Molecular transport | ||
gi|320353100 | OmpA/MotB domain-containing protein | motB | Molecular transport | ||
gi|257459944 | Putative TonB-dependent receptor | tonB | Signal transduction | ||
gi|333984533 | TonB-dependent siderophore receptor | yncD | Signal transduction | ||
gi|312196358 | IclR family transcriptional regulator | iclR | Transcription | ||
gi|384412003 | NusA antitermination factor | nusA | Transcription | ||
gi|85860692 | ATP-dependent RNA helicase | rhlB | Transcription | ||
gi|260893293 | Alanyl-tRNA synthetase | alaS | Translation | ||
gi|227495901 | Leucine—tRNA ligase | leuS | Translation | ||
gi|29653579 | 50S ribosomal protein L1 | rplA | Translation |
From proteomes analysis, AgNPs-PSSMA were able to downregulate long-chain fatty acid transport protein (fadL) of
The upregulated genes and downregulated genes of the bacteria were further studied for protein–protein interaction data via String database. The bioinformatics result was shown as a network of predicted functional associations for a group of proteins. The network nodes represent the proteins while the edges (lines) are the predicted associations of protein (
In this study, we investigate the role of AgNPs-PSSMA on the changes in protein patterns of Gram-positive
We used the proteomic approach as a tool to explore the bacterial proteins of the NS-treated cells. For
For NS-treated
In contrary to the downregulation, NS triggered the expression of approximately eight proteins in
Interestingly, we have found the upregulation of proteins dinG, rplD, and sapF, which are suggested to play a role in antimicrobial resistance. The ATP-dependent helicase dinG (dinG) is related to DNA repair and replication [43]. The process of DNA repair after exposure to NS might be consequences of cell resistance to antibacterials. Protein rplD is the 50S ribosomal protein functions as ribosome-mediated translation of mRNA into a polypeptide [44]. Protein sapF is an ATP-binding protein in plasma membrane involved in a peptide transport system [45]. The upregulation of proteins rplD and sapF of
The previous proteomic study in
The stable AgNPs-PSSMA could be used as antibacterial agents to Gram-positive and Gram-negative bacteria. NS caused both downregulation and upregulation of proteins in bacterial cells with the latter was more obviously found in
In this study, the NS without capping agent added was not performed due to the reasons dealing with the stability of AgNPs. The data obtained from the present study were the antibacterial effect AgNPs capped with PSSMA. The AgNPs could be further applied clinically as materials for antibacterial purposes. As the bacterial proteins change after AgNPs exposure seemed to be depend on the type of stabilizers [12], the further study should be on the comparison of bacterial protein change of AgNPs capped with different types of polymer. The information could be used to select the appropriate stabilizing polymer for the targeted bacterial proteins.