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Biomaterials 33 (2012) 2327e2333

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Biomaterials

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The molecular mechanism of action of bactericidal gold nanoparticles onEscherichia coliq

Yan Cui a,b, Yuyun Zhao b, Yue Tian b, Wei Zhang b,2, Xiaoying Lü a,*, Xingyu Jiang b,1

a State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, 2 Si Pailou, Nanjing 210096, ChinabCAS Key Lab for Biological Effects of Nanomaterials and Nanosafety, National Center for NanoScience and Technology, 11 Beiyitiao, ZhongGuanCun, Beijing 100190, China

a r t i c l e i n f o

Article history:Received 31 October 2011Accepted 22 November 2011Available online 17 December 2011

Keywords:Gold nanoparticleMolecular mechanismTranscriptomic/proteomic analysisROS

q Yan Cui and Yuyun Zhao are equal contributors t* Corresponding author. Tel.: þ86 25 83793430; fa

E-mail addresses: zhangw@nanoctr.cn (W. Zhanxingyujiang@nanoctr.cn (X. Jiang).

1 Tel.: þ86 10 82545558; fax: þ86 10 82545631.2 Tel./fax: þ86 10 82545631.

0142-9612/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.biomaterials.2011.11.057

a b s t r a c t

This work examines the molecular mechanism of action of a class of bactericidal gold nanoparticles (NPs)which show potent antibacterial activities against multidrug-resistant Gram-negative bacteria by tran-scriptomic and proteomic approaches. Gold NPs exert their antibacterial activities mainly by two ways:one is to collapse membrane potential, inhibiting ATPase activities to decrease the ATP level; the other isto inhibit the subunit of ribosome from binding tRNA. Gold NPs enhance chemotaxis in the early-phasereaction. The action of gold NPs did not include reactive oxygen species (ROS)-related mechanism, thecause for cellular death induced by most bactericidal antibiotics and nanomaterials. Our investigationwould allow the development of antibacterial agents that target the energy-metabolism and tran-scription of bacteria without triggering the ROS reaction, which may be at the same time harmful for thehost when killing bacteria.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Multidrug-resistant (MDR) strains of pathogenic bacteriaemerge with increasing frequency in hospitals and the generalcommunity alike [1]. The need for effective antibacterial agents isundisputed. The rapid growth of nanotechnology has providedmany types of materials for biomedical applications, including inthe field of antimicrobes [2], cancer imaging and therapeutics [3,4],persistent biofouling [5], and so forth. Many metal- and carbon-based nanomaterials, such as Ag, TiO2, ZnO, fullerene derivatives,carbon nanotubes (CNT), and graphene show antimicrobial prop-erties [6e8]. The antibacterial mechanisms of Ag NPs includedamaging the membrane, binding to and inactivating proteins,inhibiting the replication of DNA and so forth [9]. TiO2 and ZnO NPsmainly kill bacteria via ROS-production under UV irradiation[10,11]. Carbon-based materials may exert oxidant or mechanicaldamage on bacteria [12,13]. Gold NPs may become useful in thedevelopment of antibacterial strategies because of their non-toxicity, versatility in surface modification, polyvalent effects and

o this paper.x: þ86 25 83792882.g), luxy@seu.edu.cn (X. Lü),

All rights reserved.

photothermal effects [14e19]. We have developed a strategy tofight against MDR bacteria via presenting inactive small organicmolecules, such as 4, 6-diamino-2-pyrimidinethiol on gold NPs(Au_DAPT NPs), which act on Escherichia coli and Pseudomonasaeruginosa via multiple means, such as disorganizing cellmembranes, binding to nucleic acids, and inhibiting proteinsynthesis [20,21]. To better understand the fundamental biologicalmechanisms of gold NPs to design NP-based antibacterial agents,we explore the changes of bacteria that take place on both gene andprotein levels.

Transcriptomic and proteomic analysis has been widely used instudying the molecular mechanisms of the action of antibacterialagents [22e24] and other fields, such asmechanisms of cytotoxicityof biomaterials [25,26]. Using these methods, researchers foundthat CNTs up-regulate stress-related sigma factor, genes involved insoxRS and oxyR systems, and genes related to glycolysis, fatty acidbeta-oxidation, and fatty acid biosynthesis pathways [12]. Ag NPsdissipate the proton motive force to accumulate envelope proteinprecursors in the cytoplasm of E. coli. This accumulation preventsthese proteins from properly functioning in the membrane. Ag NPscould also elevate formate acetyltransferase, implying an anaerobiccondition, and reduce recombinase A related to DNA repair inStaphylococcus aureus [27,28].

Here, we investigate themode of antibacterial action of gold NPson E. coli using gene expression microarrays and two-dimensionalpolyacrylamide gel electrophoresis (2D PAGE), supported with

Y. Cui et al. / Biomaterials 33 (2012) 2327e23332328

biochemical studies, focusing on oxidative phosphorylation, ribo-some, chemotaxis pathways, and ROS-related processes.

2. Materials and methods

2.1. Preparation of gold NPs

The synthesis of gold NPs followed published methods [20]. Briefly, we stirredthemixture of 4, 6-diamino-2-pyrimidinethiol (Merck China) dissolved inmethanol,acetic acid and Tween 80 and HAuCl4$3H2O (Jingke Chemical Co., Ltd., China) in ice-water bath, added NaBH4 dropwise with vigorous stirring and kept stirring for 1 h.We removed methanol in vacuum at 40 �C, added deionized water into the residue,dialyzed the solution in deionized water for 48 h, sterilized them through a 0.22 mmfilter (Millipore), and kept them at 4 �C for use.

2.2. Bacterial strain

Type strains of E. coli (ATCC 11775) were from China General MicrobiologicalCulture Collection Center (Beijing, China). We cultured E. coli in Luria-Bertani (LB)medium at 37 �C with a shaking speed of 200 rpm. Tryptone and yeast extract werefrom Oxoid (Biodee, Co., Beijing, China).

2.3. Bactericidal activity testing

We determined minimum bactericidal concentration (MBC) following theguidelines of the Clinical and Laboratory Standards Institute [29]. We plated theculture at or larger than the minimum inhibition concentration (MIC) onto the agarplate. The MBC is defined as the lowest concentration of gold NPs that resulted ina �99.9% reduction in growth compared with the starting test inoculums.

2.4. Microarray analysis

We added gold NPs at a final concentration of 10 mg/mL to E. coli in the early-logarithmic phase, cultured them for 4 h, and then collected bacteria by centrifu-gation at 8000 rpm for 5 min for the microarray experiment. The control wasuntreated E. coli. The extraction of total RNA, labeling with biotin, cDNA synthesis,terminal labeling, and hybridization were done by CapitalBio Corporation (Beijing,China). Samples were hybridized for 16 h on Affymetrix GeneChip E. coli Genome 2.0Array containing approximate 10,000 probe sets for all 20, 366 genes. The hybridizedgene chips were washed, fluorescently stained and scanned using an AffymetrixGeneChip fluidic station 450 with a Scanner 3000 7G.

2.5. 2D PAGE

We cultured gold NP-treated or untreated E. coli and collected them via theabove procedures. The extraction of proteins with lysis buffer (7 M urea, 2 M thio-urea, 4% tergitol-type NP-40) and the subsequent 2D PAGE were performed inBeijing Protein Innovation Co., Ltd. All gels were visualized by camasses staining andscanned with Power Look 2100xl (Amersham). Protein spots on the image files weredetected and quantified with ImageMaster 4.01 (Amersham). Experiments wereperformed in triplicates. Protein spots with consistent difference between theparallels were excised from the gels. Three-fold change was set as a cut-off limit.After digestion of the gel, the resulting peptides were identified by matrix assistedlaser desorption ionization-time of flight (MALDI-TOF). NCBI database search wasperformed manually using Mascot (http://matrixscience.com).

2.6. Data analysis

We analyzed the microarray data using Significance Analysis of Microarrays(SAM Version 2.23). Genes were considered as being regulated with greater than orequal to two-fold change, meanwhile the q-value must be below 0.05.

We performed two different programs for comparative analysis of expressiondata. For the generation of the GO enrichment graphs, we imported the lists ofregulated genes into the Gene Ontology Enrichment Analysis Software Toolkit(GOEAST, http://omicslab.genetics.cn/GOEAST/) and compared themwith all probe-sets on E. coli Genome 2.0 Array. To determine the concerned biological pathway ofthe expression data, we imported the probe-set ID and fold change of the differ-entially expressed genes into Molecule Annotation System (MAS, http://bioinfo.capitalbio.com/mas), and obtained biological pathways from Kyoto Encyclopediaof Genes and Genomes (KEGG).

We analyzed proteomic data by uploading GI numbers of proteins to the web-based software of annotation, visualization, and integrated discovery (DAVID,david.abcc.ncifcrf.gov/) and obtained biological pathway.

2.7. Determination of F-type ATPase activity, ATP levels, and membrane potential

We added gold NPs at a final concentration of 10 mg/mL to E. coli in the loga-rithmic phase, cultured them for 4 h, and collected bacteria by centrifugation at

8000 rpm for 5 min. Untreated E. coli was the control. We extracted membraneproteins and determined F-type ATPase activities of one part of collected bacteriaaccording to the protocol of the F-type ATPase activity assay kit (Genmed ScientificsInc., Shanghai, China). We extracted ATP of the other part of collected bacteria anddetermined ATP levels with the ATP assay kit (Beyotime Institute of Biotechnology,China). We used protein contents (BCA Protein Assay kit, Tiangen Biotech Co., Bei-jing, China) to correct the different samples for comparison. We performed theexperiment in triplicates.

We determinedmembrane potential with DiSC3(5) (Sigma), a lipophilic dye thatchanges its fluorescence intensity when the transmembrane potential changes. Thespheroplasts from E. coli in mid-logarithmic phase were prepared as previouslydescribed [30]. We collected E. coli, washed them with 150 mM Tris once, incubatedthem with 1 mM EDTA in 150 mM Tris buffer (PH 7.0) for 15 min at 25 �C, washedthem twice with 10 mM Tris (PH 7.0), finally diluted themwith 10 mM Tris buffer (PH7.0) to an optical density of 0.05 at 600 nm (OD600 nm 0.05). We incubated E. coli(OD600 nm 0.05) with DiSC3(5) at a final concentration of 0.4 mM in dimethyl sulf-oxide (DMSO) for an hour until uptake of the dye saturatedwith a stable reduction offluorescence.We used 100mMKCl to equilibrate the concentration of potassium ionsin the cytoplasmic and external environment. Membrane potential was monitoredby a change in fluorescence using Tecan Infinite 200 microplate reader (Excitationand emission wavelengths are 622 and 670 nm respectively) after 80 min of thetreatment with different concentrations of gold NPs. The untreated E. coli was thenegative control. DMSO itself did not induce any fluorescence change.

2.8. Determination of H2O2 scavenged by peroxidase

We cultured E. coli with or without 10 mg/ml gold NPs in logarithmic phase,collected them by centrifugation, washed and resuspended them with phosphate-buffer saline (PBS, 0.01 M, pH 7.4) at OD 600 nm 0.1. We determined H2O2-scav-enging according to reported protocols [31]. We added H2O2 (at a final concentrationof 1.5 mM) to the suspension of the above bacteria. After incubation for 25 min at37 �C, we immediately added 60 mL of amplex red reagent (10-acetyl-3, 7-dihydroxy-phenoxazine) (200 mM in 50 mM KPi, SinoBio Co., China), 60 mL of horseradishperoxidase (0.02 mg/mL in 50 mM KPi, SinoBio Co., China,) in 100 mL of H2O2-treatedbacteria, and determined fluorescence via a Tecan Infinite 200microplate reader.Wecalculated the concentration of H2O2 using a standard curve from standard samplesof H2O2 (Beyotime Institute of Biotechnology, China).

2.9. NADþ/NADH extraction, NADþ cycling assay, and reactive oxygen species (ROS)generation

We performed the dinucleotide extraction and cycling assay by the protocolsdescribed previously [23]. We collected 1 mL samples each for NADþ and NADHextraction by centrifugation at 13,000 rpm for 1 min from E. coli cultures every halfhour between 0 and 3 h after adding gold NPs (the final concentration is 10 mg/mL).We removed the supernatant, froze the pellets immediately in a dry ice-ethanolbath, and stored the pellets at �80 �C until finishing collecting all time-points.75 mL of 0.2 M NaOH (for NADH extraction) or 75 mL of 0.2 M HCl (for NADþ

extraction) were added to the ice-cold pellets. We heated the samples at 100 �C for10 min and subsequently centrifuged at 10,000 rpm for 5 min. The NADþ/NADH-containing supernatant were transferred to fresh tubes and stored in the dark on iceuntil use.

We performed the NADþ cycling assay via Spectra Fluor Plus in 96-well plateformat. The reaction mixture contained 16 mL of 1.0 M bicine (pH 8.0) (BiogenroBiotechnology Co., Ltd., China), 40 mL sample extract, 40 mL neutralizing buffer (0.1 MHCl for NADH, or 0.1 M NaOH for NADþ), 16 mL phenazine ethosulfate (PES, Sigma),16 mL 3-[4, 5-dimethylthiazol-2-yl]-2, 5- diphenyltetrazolium bromide (MTT,Beyotime Institute of Biotechnology, China), 16 mL 100% ethanoland, 30 mL of 40 mM

EDTA (pH 8.0). We finally added PES andMTT to the 96-well plate and incubated themixture for 3 min at 30 �C. We added 3.2 mL of alcohol dehydrogenase (500 U/mL,Sigma) in bicine buffer (pH 8.0) to the reaction mixture to begin the assay. Werecorded the increase absorbance at 570 within 10 min. The rate of MTT reduction isproportional to the concentration of NADþ or NADH in the sample. We used NADþ

and NADH standards (Sigma) between 0.0375 and 0.75 nM to calibrate the assay.We used an Reactive Oxygen Species Assay Kit (Beyotime Institute of Biotech-

nology, China) based on 20 , 70-dichlorodihydrofluorescein diacetate (DCFH-DA) todetermine the generation of ROS. We acquired E. coli samples in logarithmic phaseafter a treatment with 10 mg/mL of gold NPs for 4 h by centrifugation, and stainedthem with 10 mM DCFH-DA. We estimated the fluorescence intensity by the TecanInfinite 200 microplate reader with excitation at 488 nm and emission at 525 nm.

2.10. Chemotaxis

We performed the chemotaxis experiment on soft-agar swarming plate. Weadded different concentrations of gold NPs into the agar containing 1% tryptone,0.5% NaCl, and 0.3% bacto-agar (Becton Dickinson Medical Devices, China). Weinoculated E. coli in the logarithmic phase onto the agar plate, cultured them at37 �C, and observed the swarm size and density of the plaque. The control was theplate without NPs.

Table 2The most significantly up-regulated biological processes.

GO term Gene number

Chemotaxis 18Flagellum assembly 9Response to chemical stimulus 18Locomotion 32Cellular component movement 25

Y. Cui et al. / Biomaterials 33 (2012) 2327e2333 2329

3. Results

3.1. Determination of bactericidal activity

We confirmed the bactericidal property of gold NPs via testingthe MBC/MIC value according to reported methods [32,33]. Anti-biotics can be subdivided into two major categories, bactericidaland bacteriostatic. TheMBC of gold NPs ranges from 12 to 16 mg/mL,and theMIC is 4 mg/mL. TheMBC/MIC ratio is nomore than 4, whichsuggests that gold NPs present bactericidal activity against E. coli.

3.2. Microarray analysis

According to filtering criteria, 359 genes were differentiallyexpressed in E. coli on exposure to gold NPs, among which 119genes were up-regulated and 240 were down-regulated. Based onthe biological process, molecular function, and cellular componentclassification, these up- or down-regulated genes were linked todifferent GO terms (Table S1, S2, and S3). We obtained biologicalprocesses related to down-regulated and up-regulated genes by GOenrichment analysis [34] (Tables 1 and 2) and the concerned bio-logical pathway related to the differently expressed genes withminimal p value by analysis of KEGG pathway database (Table 3).

3.3. Proteomic analysis

We obtained proteomes of NP-treated or untreated E. coli by 2DPAGE with camasses staining, and identified 65 stimulated proteinsby peptide mass fingerprint (PMF). Down-regulated (Table S4) andup-regulated (Table S5) proteins were annotated by DAVID. Table 3indicates the pathways involving the differently expressedproteins.

3.4. ATP synthesis and membrane potential

Transcriptomic and proteomic data show the down-regulationof atpD and atpA, which are subunits of F-type ATP synthase. Thedown-regulation could lead to a decrease of the activity of F-typeATP synthase. To test this hypothesis, we extracted the membraneprotein of gold NP-treated or untreated E. coli and determined theactivity of F-type ATP synthase. Gold NPs can severely decrease theactivity of F-type ATP synthase (Fig. 1a). F-type ATP synthase playsa major role in the process of ATP synthesis; its decrease candirectly lead to the decrease of the ATP level. As expected, we foundthat the ATP level significantly decreased in NP-treated E. coli(Fig. 1b).

The biological function of ATP synthase is dependent on themembrane potential [35,36]. We further investigated the effects ofgold NPs on cytoplasmic membrane potential of E. coli via a fluo-rescent probe, DiSC3(5) dye, which can be quenched by the elec-trically polarized membrane. When the membrane potentialcollapses, the probe releases into the cytoplasm and leads to anincrease in fluorescence. The fluorescence of NP-treated E. coli wassignificantly increased compared with that of untreated E. coli

Table 1The most significantly down-regulated biological processes.

GO term Gene number

Energy-coupled proton transport, down electrochemical gradient 9Ion transport 26ATP biosynthetic process 9Purine ribonucleotide biosynthetic process 10ATP metabolic process 9Purine nucleoside triphosphate biosynthetic process 9

(Fig. 1c), which indicates a collapse of the membrane potential.Hence, the decrease of membrane potential also contributes to thedecrease of ATP level.

3.5. Ability of scavenging H2O2

From proteomic data, we found a down-regulated protein, alkylhydroperoxide reductase subunit C (AhpC), which is a peroxidaseand an efficient scavenger of low concentrations of H2O2 [31]. Wedetermined the concentration of H2O2 after 25 min treatment ofNP-treated or untreated E. coli with addition of original 1.5 mMH2O2. The H2O2-scavenging ability of NP-treated E. coli decreaseapproximately 20% compared with untreated E. coli (Fig. 2a),agreeing with the discovery of the down-regulation of AhpC inproteomic analysis.

3.6. Catabolic NADH and ROS generation

Since gold NPs decrease the low-level H2O2-scavenging abilityof E. coli, it seems to be possible for NPs to induce accumulation ofH2O2 and produce ROS damage. On the other hand, some reportsshow that bactericidal antibiotics induce cellular death via accu-mulation of excess hydroxyl radicals, belonging to an oxidativedamage-related pathway with the depletion of NADH and tricar-boxylic acid cycle [23]. Many nanomaterials generate ROS to inhibitbacterial growth [10,11,37,38]. To investigate if gold NPs act onbacteria with an oxidative damage-related pathway, we performedan NADþ cycling assay and determined the level of ROS. We foundthat bactericidal NP-treatment hardly induce an obvious change inNADþ/NADH ratio in E. coli (Fig. 2b).

We determined cellular ROS using a DCFH-DA probe (originallynonfluorescent), which can be hydrolyzed to the nonfluorescentdichlorofluorescein (DCFH) by esterase in cells and can further beoxidized to produce the fluorescent dichorofluorescein (DCF) bycellular ROS. Bactericidal gold NPs did not induce an obviousincrease of cellular ROS but instead led to a slight decrease within4 h treatment (Fig. 2c). In comparison, a distinguishable increase ofROS typically appear within 30 min for bactericidal antibiotics-treated bacteria [23]. Thus, it is a striking finding that bactericidalgold NPs induce cellular death without oxidative damage-relatedpathways from results of NADþ cycling, ROS generation, and tran-scriptomic/proteomic analysis, although they induce a slightdecrease of low-level H2O2-scavenging ability for E. coli.

3.7. Chemotaxis

Chemotaxis genes are up-regulated in NP-treated E. coli. Weperformed a swarm plate assay to test bacterial chemotaxis. In thepresence of gold NPs, E. coli swarmed faster through the agar, andformed a larger swarm size from the point of inoculation thanuntreated E. coli within 4 h (Fig. 3 a and b). With longer-termincubation (8 and 20 h), NP-treated E. coli swarmed ina decreasing rate and formed smaller swarm size than those ofuntreated ones (Fig. 3 c and d). We presume that bacteria tend torespond rapidly to the changes of chemical environment initiallyupon exposure to NPs until they lose spreading ability under the

Table 3

Pathways Proteomic data Microarray data

Oxidative phosphorylation SdhB;AtpD; AtpA fliH;atpH;atpB:atpD;atpG;atpF;atpC;atpA;atpERibosome RpsJ rpmD;rplC;rplD;rpsL;rpmE;rpsD;rplY;rpsB;rplR;rplN;rpsP;sra;rpsQ;rplO;rpmA;

rpsO;rplB;rpsI;rplS;ykg;rplA;rpsC;rplw;rplV;rpsR;rpll;rpsJ;rpsF;rplP;rpsA;rpmC;rpmH;rpsE;rplU;rplQ;ykgM;rplE;rplF;rpsG;rpsH;rplK;rpmG;rplX

Carbon fixation Rpe;Pgk fbaBGlycolysis / Gluconeogenesis PfkB;Pgk fbaB;adhEReductive carboxylate cycle (C02 fixation) SucC;SdhB fumB;fumC;ppsCitrate cycle (TCA cycle) SucC;SdhB fumB;fumCPentose phosphate pathway Rpe;PfkB fbaBOne-carbon pool by folate GlyA purNLysine degradation GlyA iucCSulfur metabolism CysK cysCLysine biosynthesis DapA iucCSelenoamino acid metabolism CysK cysCPropanoate metabolism SucC accC;accB;pflBButanoate metabolism SdhB pflB;adhE;gadBGlycine, serine and threonine metabolism GlyA thrA;thrCFructose and mannose metabolism PfkB fbaBTwo-component system - General ABR-0076261 narL;phoR;ompF;glnA;narIABC transporters ־ General ABR-0076815 pstS;kpsE;sitC;sitB;fepB;oppA;ybhS;oppF;fepG;oppB;sitA;fepC;oppDCyanoamino acid metabolism GlyAMethane metabolism GlyAC5־Branched dibasic acid metabolism SucCCysteine metabolism CysKMethionine metabolism LuxSBenzoatc degradation via CoA ligation SdhBPentose and glucuronale interconversions RpeGalactose metabolism PfkBBacterial chemotaxis chew;motA;fliG;cheB;fliN;aer;fliM;cheY;cheZ;tar;motB;cheR;tsr;cheA;tapFlagellar assembly fliC;fliD;flgG;fliH;fliI;fliJ;fliK;fliM;fliN;fliO;fliS;flit;flgB:flgD;flgE;flgF;flgH;

flgI;flgK;JlgL;flgM;flhB;motA;motBRNA polymerase rpoA;rpoB;rpoCType 111 sccrction system flhB;fliI;fliF;fliH;fliN;Pyrimidine metabolism rpoA;rpoB;rpoC;pyrB;pyrC;pyrD;cmk;carA;upp;pyrIPurine metabolism rpoA;rpoB;rpoC:purN;cysC;adk;allB

Y. Cui et al. / Biomaterials 33 (2012) 2327e23332330

antibacterial action of NPs. With the increase of concentration, thecolony became small (Fig. 3).

4. Discussion

We confirmed the bactericidal property of gold NPs againstE. coli. To investigate the effects of antibacterial gold NPs on E. coli ina global approach, we analyzed RNA expression by gene microarrayhybridization. Across the GO databases, we found differentlyexpressed genes in several classifications including physiologicalprocess, cellular process, catalytic activity, metabolism, binding,transporter activity, protein complex, localization, and so forth. Weassigned up- and down-regulated genes to specific processes using

Fig. 1. NPs inhibit ATP synthesis and dissipate membrane potential. (a) F-type ATP synthaseNP-treated E. coli indicated by the fluorescent dye DiSC3(5). The increase of fluorescence indimean � standard deviation of triplicate determinations.

GO enrichment analysis. The most significantly up-regulatedprocesses are associated with chemotaxis processes (Table 2).Many down-regulated processes are associated with energy-coupled proton transport, down electrochemical gradient, andATP biosynthetic process (Table 1). Pathway analysis can furtherclarify the molecular details. We list the concerned biologicalpathways (Table 3). We used 2D PAGE-MS proteomics to analyzeproteins from E. coli with or without treatment using gold NPs. Weidentified genes rpsJ, atpD, atpA, and their corresponding proteinswere down-regulated by NPs, which represent common pathways(Table 3).

Microarray analysis showed the down-regulation of 43 genesencoding two subunits of ribosome. Proteomic data showed the

activity and (b) ATP levels in membrane proteins. (c) Membrane potential changes ofcates the collapse of membrane potential. E. coli alone was the control. The data are the

Fig. 2. ROS-related biological processes. (a) H2O2-scavenging ability of NP-treated or untreated E. coli. We added H2O2 (a final concentration of 1.5 mM) to equal bacterialconcentrations of NP-treated or untreated E. coli, incubated them for 25 min, and determined the H2O2 concentration. The scavenging ability is indicated by the percent reduction ofthe H2O2. (b) The ratio of NADþ to NADH (NADþ/NADH) for NP-treated and untreated E. coli at the various treatment time. (c) Determination of ROS in NP-treated or untreated E. coliusing DCFH-DA dye. All data are the mean � standard deviation of triplicate determinations.

Y. Cui et al. / Biomaterials 33 (2012) 2327e2333 2331

down-regulation of ribosomal protein S10 (encoded by rpsJ) havinga tRNA-binding function. The modifier of gold NPs, 4, 6-diaminopyrimidine thiol as an analog of bacterial tRNA base, haspotential ability to inhibit tRNA function [39,40]. Here, our dataconfirmed that 4, 6-diaminopyrimidine thiol-modified gold NPs

Fig. 3. The chemotaxis of E. coli to gold NPs on a swarm plate a

can affect protein synthesis by inhibiting tRNA function, agreeingwith the results in our previous work [20].

Our transcriptomic and proteomic analysis showed the down-regulation of subunits of F-type ATP synthase, a and b subunit inF1 sector(encoded by atpA and atpD separately) (Table 3). The

ssay with varied concentrations of NPs and treatment time.

Fig. 4. Schematic diagram of mechanism of action of bactericidal gold nanoparticles onE. coli. Gold NPs induce the down-regulation of oxidative phosphorylation pathway (F-type ATP synthase and ATP level) and ribosome pathways, and the transient up-regulation of chemotaxis. Gold NPs do not induce the change of ROS-related processes.

Y. Cui et al. / Biomaterials 33 (2012) 2327e23332332

function of F-type ATP synthase includes catalyzing the synthesis ofATP in the terminal step of oxidative phosphorylations [41], oper-ating in reverse as an ATPase to generate the transmembraneproton electrochemical gradient required for molecular trans-location, and so forth [42]. F-type ATP synthase consists of multi-subunits including extramembrane and transmembrane domains,named F1 and F0 respectively. The F1 sector is composed of fivesubunits a3b3gdε, where ATP is synthesized and hydrolyzed [43].The F0 domain consists of three polypeptides ab2c10e12, by whichions move along with ATP synthesis/hydrolysis at F1 sector [43]. Wefound that proton-transporting ATPase activity was one of the mostsignificant processes effected by gold NPs (Table 1), which isaccomplished by F-type ATP synthase. Some antibiotics such asnorfloxacin and salicylate also induce the down-regulation of F-type ATP synthase genes [44]. The down-regulation of F-type ATPsynthase subunits led to a decrease of its activity, proven by the F-type ATP synthase activity assay (Fig. 1a). The decreased activity ofF-type ATP synthase led to the decrease of ATP levels in gold NP-treatedE. coli (Fig. 1b), resulting in a general decline in metabolism.ATP synthesis requires membrane potential, which generates tor-que in the F0 domain of F-type ATP synthase [36]We observed thecollapse of the membrane potential in gold NP-treated E. coli pro-bed by a fluorescent dye (Fig. 1c), which can further decrease ATPlevels

Ahp, a type of catalase, was down-regulated when treated withgold NPs. In bacterial aerobic metabolism, the accumulation ofdeleterious hydrogen peroxide debris tends to be converted toinnocuous products by enzymes [45]. Two catalases, alkyl hydro-peroxide reductase (Ahp) and hydroperoxidase I (HPI) have distinctroles in scavenging H2O2. Ahp scavenges low-level H2O2 and HPImainly scavenges higher concentrations of H2O2 when Ahp isinvalidated [31]. The mutation of Ahp decreases the ability ofscavenging low-level of H2O2 by less 18% compared with wild-type[31]. Ahp mutants are susceptive to growth inhibition by organichydroperoxides [46]. Proteomic analysis showed the down-regulation of Ahp while the level of HPI remains unchangedwhen bacteria was treated with gold NPs. By determining thecontents of H2O2, we confirmed that the ability of scavenging low-level H2O2 decreased, but it cannot change the NADþ cyclingreaction nor induce the accumulation of H2O2 by gold NPs (Fig. 2).We cannot find changes of biological pathway related to oxidativestress, SOS response, and DNA damage in transcriptomic and pro-teomic data, either. In comparison, reported bactericidal antibioticsinduce a transient depletion of NADH, stimulate the Fenton reac-tion, and produce excess hydroxyl radical formation to damage cell,which suggests a common mechanism of bacterial death [23].Many antibacterial nanomaterials, such as Ag NPs [37,38,47], TiO2

[47] and ZnO [10,11], also produce ROS to kill bacteria. Thesenanomaterials induced differentially expressed genes related todetoxification, SOS response, oxidative/redox stress, drug resis-tance/sensitivity and protein stress [47]. CNTs induced the up-regulation of bacterial oxidative stress response indicated bysoxRS and oxyR systems [12]. One kind of CNTs is also toxic toeukaryotes [48,49]. ROS-independent mechanism of action of goldNPs suggests their low toxicity to mammalian cells [20].

In this study, we found that the up-regulation of genes includethose related to chemotaxis and flagellar motility pathway in tran-scriptomic analysis. Chemotaxis is a mechanism by which bacteriaefficiently and rapidly respond to changes in the chemical compo-sition of their environment [50]. Chemotaxis is accomplished bytransmembrane signal transducers belonging to two-componentsignal transduction systems [51]. FliG, fliN, and fliM encode theproduction of components of flagella that propel the bacteriumforward. Tar, tsr, and tap encode the sensory receptor proteins in themembrane [52]. CheW, cheB, cheY, cheZ, cheR, cheA, andmotB encodethe genes in signaling bacterial movement [53]. The swarm plateassay confirmed bacterial chemotaxis and motility, whose pheno-types varied based on the treatment time and the concentration ofgold NPs (Fig. 3). In the early stage (within 4 h), E. coli formed a largecolony size in the presence of NPs (Fig. 3a and b). With treatmentexceeding 8 and 20 h, NP-treated E. coli swarmed slowly (Fig. 3c andd). With the increase of the concentration of gold NPs, the swarmsize became smaller and thinner but larger than the control in theearly stage (Fig. 3a and b). Bacteria probably initially respond rapidlyto NPs until they exhaust toomuch energy (motility requires ATP) tosustain motility due to the inhibition of ATPase activity and thecollapse of membrane potential by NPs. It has been reported thatantibacterial peptide octapeptin and silver NPs can induce bacterialchemotaxis at a low concentration and high concentrations lead tocomplete inhibition of bacterial motility [54,55]. Some compoundsthat inhibit bacterial motility can be used to enhance antibacterialefficacy [56]. To the best of our knowledge, our discovery representsan initial report where gold NPs can transiently increase bacterialmotility as they exert antibacterial actions. In addition, gold NPsinduce bacterial motility mainly dependent on the treatment timeinsteadof concentrations. Theup-regulation of chemotaxis pathwayin our analysis reflects the state of E. coli treatedwith gold NPs in theearly stage.

5. Conclusion

Through transcriptomic and proteomic approaches, we foundthat gold NPs exert their antibacterial action mainly by two ways:one is to change membrane potential and inhibit ATP synthaseactivities to decrease the ATP level, indicating a general decline inmetabolism; the other is to inhibit the subunit of ribosome for tRNAbinding, indicating a collapse of biological process (Fig. 4). Gold NPsalso enhance chemotaxis in the early-phase reaction. The multipletargets of action could help gold NPs to fight effectively againstMDRbacteria. In addition, a striking finding is that bactericidal gold NPsdid not induce any ROS-related process, while the generation of ROSis the cause of cellular death for most bactericidal antibiotics andantibacterial nanomaterials. ROS-independentmechanismof actionof gold NPs could partly explain the low toxicity of gold NPs tomammalian cells. Our investigationwould allowthedevelopmentofantibacterial agents that target the energy-metabolism and specifictranscription of bacteria without triggering the ROS reaction.

Acknowledgements

Thiswork is supported by theMinistryof Science andTechnology(2009CB930000, 2011CB933201), the Chinese Academy of Science

Y. Cui et al. / Biomaterials 33 (2012) 2327e2333 2333

(KJCX2-YW-M15), and the Nation Science Foundation of China(20890020, 90813032).

Appendix. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.biomaterials.2011.11.057

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