Published Ahead of Print 30 June 2014. 10.1128/AAC.03391-14.

2014, 58(9):5435. DOI:Antimicrob. Agents Chemother. PashaHemlata Gautam, Mohammed Shahar Yar and Santosh Rikeshwer Prasad Dewangan, Seema Joshi, Shalini Kumari, aureusMethicillin-Resistant Staphylococcus against Planktonic and SessileSpermine Backbone Dipeptidomimetics N-Terminally Modified Linear and Branched

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N-Terminally Modified Linear and Branched Spermine BackboneDipeptidomimetics against Planktonic and Sessile Methicillin-ResistantStaphylococcus aureus

Rikeshwer Prasad Dewangan,a,b Seema Joshi,a Shalini Kumari,a Hemlata Gautam,c Mohammed Shahar Yar,b Santosh Pashaa

Peptide Research Laboratory, CSIR-Institute of Genomics and Integrative Biology, Delhi, Indiaa; Department of Pharmaceutical Chemistry, Faculty of Pharmacy, JamiaHamdard, New Delhi, Indiab; Systems Biology Group, CSIR-Institute of Genomics and Integrative Biology, Delhi, Indiac

Toward the discovery of useful therapeutic molecules, we report the design and synthesis of a focused library of new ultrashortN-terminally modified dipeptidomimetics, with or without modifications in the spermine backbone leading to linear (series 1)or branched (series 2) tryptophans, as antimicrobial agents. Eight peptidomimetics in the library showed good antibacterial ac-tivity (MICs of 1.77 to 14.2 �g/ml) against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphy-lococcus epidermidis bacterial strains. Tryptophan fluorescence measurements on artificial bacterial or mammalian mimicmembranes and assessment of the MRSA potential depolarization ability of the designed compounds revealed membrane inter-actions dependent on tryptophan positioning and N-terminal tagging. Among active peptidomimetics, compounds 1c and 1dwere found to be nonhemolytic, displaying rapid bactericidal activity (at 4� MIC) against exponentially growing MRSA. Fur-ther, scanning electron microscopy of peptidomimetic 1c- and 1d-treated MRSA showed morphological changes with damage tocell walls, defining a membrane-active mode of action. Moreover, peptidomimetics 1c and 1d did not induce significant drugresistance in MRSA even after 17 passages. We also investigated the activity of these molecules against MRSA biofilms. At sub-MIC levels (�2 to 4 �g/ml), both peptidomimetics inhibited biofilm formation. At concentrations higher than the MIC (35 to140 �g/ml), peptidomimetics 1c and 1d significantly reduced the metabolic activity and biomass of mature (24-h) MRSA bio-films. These results were corroborated by confocal laser scanning microscopy (live/dead assay). The in vitro protease stabilityand lower cytotoxicity of peptidomimetics against peripheral blood mononuclear cells (PBMCs) support them being novelstaphylocidal peptidomimetics. In conclusion, this study provides two peptidomimetics as potential leads for treatment ofstaphylococcal infections under planktonic and sessile conditions.

Infectious diseases represent a major global health care concerndue to escalating multidrug resistance (MDR) against currently

available antibiotics (1). Multidrug-resistant strains such as me-thicillin-resistant Staphylococcus aureus (MRSA), vancomycin(VAN)-resistant enterococci (VRE), and carbapenem-resistantEnterobacteriaceae (CRE) in communities and nosocomial envi-ronments are rendering antibiotic therapy more difficult andcostly at an unprecedented rate (2, 3). The development of resis-tance is aggravated by the irrational use of antibiotics in livestockand medical practices, which has armed microbes with a multi-tude of novel drug resistance mechanisms. In the present scenario,no class of antibiotics with a fixed metabolic target in microbes isfree from the resistance development problem, as microbes areable to reinvent themselves, acquiring gene-encoded or plasmid-mediated drug resistance leading to better survival chances. A pas-sive known contributory lifestyle approach toward resistance de-velopment involves slow growth and a heterogeneous microbialpopulation, phenotypically as well as genetically, in the form ofbiofilms (4, 5).

Biofilms are microbial communities adhering to surfaces orfloating at air-water interphases in which the microbes are embed-ded in a self-produced exopolymeric substance (EPS), which iscomposed largely of proteins, DNA, and different extracellularpolysaccharides (6). Novel agents and strategies are needed toeradicate biofilms, as they play a major role in almost 80% ofinfections, including cystic fibrosis, dental plaques, chronicwounds, and infections involving implanted medical devices (7).Most antibiotics are active against log-phase bacterial cells, as they

target metabolic processes in bacteria to inhibit growth. However,biofilms act as recalcitrant infection reservoirs and contribute tovirulence, since the exopolymeric matrix and retarded metabolicactivity inside biofilm communities lead to increased persistenceof biofilms (8). Additionally, it is known that bacteria in biofilmsgenerally tolerate antibiotic treatment, and antibiotics can evenproduce a trigger for biofilm formation (9).

As an answer to MDR microbes, host defense cationic peptides(HDCPs) (12- to 60-mers) and their mimics, with a multitude ofnovel mechanisms, are commercial candidates that hold potentialto circumvent drug resistance (10, 11). HDCPs are produced byalmost all living organisms as a first line of defense against invad-ing microbes. Owing to global amphiphilicity, i.e., the balancebetween positive charge at physiological pH and hydrophobicity,HDCPs predominantly exhibit membrane-disruptive modes ofaction, although they have also been reported to be metabolicinhibitors in microbes (11). The positive charge on HDCPs helps

Received 22 May 2014 Returned for modification 9 June 2014Accepted 24 June 2014

Published ahead of print 30 June 2014

Address correspondence to Santosh Pasha, spasha@igib.res.in.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.03391-14.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AAC.03391-14

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them to become attracted to negatively charged surfaces of bacte-rial cells, facilitating primary interactions. After initial attach-ment, by virtue of their amphipathic nature, HDCPs are able tocause lipid clustering and segregation of domains, leading to bac-terial cell death (10). It is difficult for bacteria to develop resistanceto HDCPs because most HDCPs kill bacterial cells quicklythrough their actions on the entire cytoplasm, acting as poreformers (11). HDCPs have been reported to efficiently eradicateslow-growing cells from planktonic and biofilm cultures and thushave been proposed as promising alternative agents for the cure ofbiofilm-associated multidrug-resistant infections (12). However,the challenges in the application of HDCPs have been their highcost, protease instability, reduced activity in the presence of salts,and poor bioavailability (13). Over the past decades, attemptshave been made to mimic the structures and functions of HDCPs,leading to the design of potent synthetic mimics such as oligoacyl-lysines (OAKs), cationic steroid antibiotics (CSAs), and cyclic cat-ionic peptides, some of which are presently undergoing clinicaltrials as antibacterial agents (14, 15).

The aim of the present study was to optimize our previouslydesigned N-terminally tagged dipeptide spermidine template(16). Toward this goal, the roles of hydrophobicity and chargedistribution in activity have been assessed with different position-ing of tryptophan residues on the spermine backbone. Further-more, the mode of action and efficacy of the lead molecule toeradicate clinically relevant MRSA biofilms have been deter-mined.

MATERIALS AND METHODSChemicals. 9-Fluorenylmethoxy carbonyl (Fmoc)-protected amino acidsand resins were purchased from Novabiochem (Darmstadt, Germany),and N,N-diisopropylcarbodiimide (DIPCDI) (catalog no. D12,540-7),1-hydroxybenzotrizole (HOBt) (catalog no. 54804), diisopropylethyl-amine (DIPEA) (catalog no. D-3887), N-methylpyrrolidinone (NMP)(catalog no. 494496), piperidine (catalog no. 411027), spermine (catalogno. S3256), triisopropylsilane (catalog no. 23378-1), crystal violet (CV)(catalog no. C3886), glucose (catalog no. G7528), hydrazine (catalog no.225819), 3,3=-dipropylthiadicarbocyanine iodide (DiSC35) (catalog no.43608), and the Tox7 kit (lactate dehydrogenase [LDH] release assay kit)were obtained from Sigma-Aldrich. Trifluoroacetic acid (TFA) (catalogno. 80826005001730) and 2-acetyldimedone (Dde-OH) (catalog no.8.51015.0005) were purchased from Merck. All of the moieties used asN-terminal tags were purchased from Sigma-Aldrich. Tryptone soy broth(TSB) (catalog no. M011-500G) was purchased from HiMedia (India),and Mueller-Hinton broth (MHB) and Mueller-Hinton agar were pur-chased from Difco (Franklin Lakes, NJ). The alamarBlue reagent (catalogno. DAL 1025) and the Molecular Probes Live/Dead BacLight assay kit(L7012) were procured from Invitrogen (Eugene, OR). High-perfor-mance liquid chromatography (HPLC)-grade solvents were obtainedfrom Merck (Germany). Dimethylformamide (DMF) and dichlorometh-ane (DCM) were obtained from Merck (Mumbai, India). DMF was dou-ble distilled prior to its use.

Synthesis and purification of peptidomimetics. All peptidomimeticswere synthesized on 2-chlorotrityl chloride resin as a solid support, asdescribed previously, with slight modifications (17). Briefly, on pre-swelled resin, 5 eq of spermine dissolved in dichloromethane was addedunder an inert atmosphere for 4 h. Completion of the reaction was mon-itored by positive Kaiser test results (18). After coupling, capping of un-reacted resin with methanol was performed for 45 min. The primaryamino group of spermine was protected through overnight reaction with2 eq of Dde-OH in DMF. After protection of the primary amino group,the secondary amino groups were protected through reaction for 4 h with6 eq of t-butoxycarbonyl (Boc)-anhydride in the presence of DIPEA. Then

Dde-OH protection of primary amines was removed using 2% (wt/vol)hydrazine in DMF. Two additional couplings were performed with Fmoc-Trp(Boc)-OH in the presence of HOBt and DIPCDI in DCM-DMF (1:1).The N-terminal tagging was performed with 4 eq of unnatural tag, HOBt,and DIPCDI in DCM-DMF (1:1), leading to peptidomimetics 1a to 1f(Fig. 1). For synthesis of peptidomimetics 2a to 2f, Dde-OH-protectedresin was coupled with 4 eq of Boc-Trp(Boc)-OH, HOBt, and DIPCDI.Then, deprotection of the primary amino group was performed with 2%(wt/vol) hydrazine in DMF. The N-terminal tagging was performed by aprocedure similar to that described above. Final deprotection of peptido-mimetics from the resin in both series was performed using a cleavagecocktail (DCM, TFA, ethanedithiol, triisopropylsilane, phenol, and waterin a ratio of 65:30:2:1:1:1). The solution was filtered, and cold ether wasadded to the filtrate to precipitate the crude product, which was filteredand washed with cold ether (2 � 25 ml). The solid was dissolved in meth-anol and desalted using an LH-20 Sephadex column (Sigma). The pep-tidomimetics were further purified by reverse-phase (RP)-HPLC, using asemipreparative column (7.8 by 300 mm, 125-Å pore size, 10-�m particlesize) with a gradient of 10 to 90% buffer 2 over 45 min; buffer 1 was waterwith 0.1% TFA and buffer 2 was acetonitrile with 0.1% TFA. After puri-fication, the peptidomimetics were confirmed by either liquid chroma-tography-tandem mass spectrometry (LC-MS/MS) (Quattro Micro API;Waters) or ultra-high-performance liquid chromatography (UHPLC)(Dionex, Germany) with LTQ Orbitrap XL (Thermo Fisher Scientific)mass determination. Analytical HPLC traces and mass spectra of repre-sentative peptidomimetics are provided in Fig. S1 and S2 in the supple-mental material.

Antibacterial activity under planktonic conditions. The antibacte-rial activities of the designed peptidomimetics were evaluated by using amodified serial broth dilution method, as reported previously (19, 20).The following bacterial strains were used in this study: S. aureus (ATCC29213), methicillin-resistant S. aureus (ATCC 33591), Staphylococcus epi-dermidis (ATCC 12228), methicillin-resistant S. epidermidis (ATCC51625), Enterococcus faecalis (ATCC 7080), Escherichia coli (ATCC11775), and Acinetobacter baumannii (ATCC 19606). The inocula wereprepared from mid-log-phase bacterial cultures. Each well of the first 11columns of 96-well polypropylene microtiter plates was inoculated with100 �l of approximately 105 CFU/ml of bacterial suspension in Mueller-Hinton broth (MHB) (Difco). Then 10 �l of serially diluted peptidomi-metic in 0.01% (vol/vol) acetic acid and 0.2% bovine serum albumin(Sigma), over the desired concentration range, was added to the wells ofthe microtiter plates. The microtiter plates were incubated overnight at37°C, with agitation (200 rpm). After 18 h, absorbance was measured at630 nm. Cultures without test peptidomimetics were used as positivecontrols. Uninoculated MHB was used as a negative control. Tests werecarried out in duplicate on three different days. MIC was defined as thelowest concentration of peptidomimetic that completely inhibitedgrowth. For comparison purposes, the standard peptide antibiotics VANand polymyxin B (PMB) were assayed under identical conditions. Theantibacterial activities of peptidomimetics and the standard antibioticVAN were evaluated against MRSA strain 33591 in the presence of 25%(vol/vol) human serum and fetal bovine serum (FBS) in biofilm growthmedium (tryptone soy broth [TSB] supplemented with 0.5% NaCl and0.25% glucose). A protocol similar to that described previously was used(21). Briefly, MHB was adjusted to 25% (vol/vol) of a heat-inactivatedhuman serum pool obtained from two healthy volunteers. Growth con-trol experiments were conducted using MHB with and without 25% se-rum. MICs were determined as described above, according to CLSI stan-dard methods.

Hemolytic activity. The hemolytic activities of the peptidomimeticswere evaluated with human red blood cells (hRBCs). Briefly, 100 �l of afresh 4% (vol/vol) suspension of hRBCs in NaCl-Pi (35 mM phosphatebuffer [35 mM Na2HPO4 and 35 mM NaH2PO4·2H2O], 150 mM NaCl[pH 7.2]) was placed in a 96-well plate. After incubation of the peptido-mimetics (100 �l) in the hRBC suspension for 1 h at 37°C, the plates were

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centrifuged, and the supernatant (100 �l) was transferred to a fresh 96-well plate. Absorbance was read at 540 nm using an enzyme-linked im-munosorbent assay (ELISA) plate reader (Molecular Devices). Percenthemolysis was calculated using the following formula: % hemolysis �100[(A � A0)/(At � A0)], where A represents the absorbance of samplewells at 540 nm and A0 and At represent 0% and 100% hemolysis, respec-tively, determined in NaCl-Pi with 1% Triton X-100.

Cytotoxicity assay in peripheral blood mononuclear cells. Bloodfrom healthy human donors was collected in tubes containing the antico-agulant sodium heparin, in accordance with institutional guidelines. Theblood was diluted 1:1 with NaCl-Pi (35 mM phosphate buffer, 150 mMNaCl [pH 7.2]). Blood cells were separated over Histopaque separationmedium (Sigma-Aldrich) by centrifugation at 1,200 rpm for 30 min. Theperipheral blood mononuclear cells (PBMCs) were collected and washedtwice with NaCl-Pi (35 mM phosphate buffer, 150 mM NaCl [pH 7.2]).The cells were then resuspended in complete RPMI 1640 medium (Hi-Media) supplemented with 10% FBS (Sigma) and were quantified bytrypan blue exclusion, with microscopic assessment. PBMCs (1 � 106

cells/ml) in complete medium were seeded in a 24-well plate and left in theincubator in 5% CO2 for 2 h at 37°C. The cells were then treated withpeptidomimetic 1c, peptidomimetic 1d, or VAN at the desired concentra-tions (20 �g/ml and 50 �g/ml). Triton X-100 (2%) was used as a negativecontrol. After 24 h of incubation, the contents of each well were trans-ferred to sterile 1.5-ml Eppendorf tubes, and cells were pelleted at 2,000rpm for 10 min. The supernatants were assessed for the release of LDH by

using the Tox7 kit (Sigma), as described previously (22, 23). The experi-ments were carried out in duplicate on three different days, and data arepresented as mean � standard deviation (SD).

Tryptophan fluorescence. Small unilamellar vesicles (SUVs), whichwere prepared following the standard method (20), were used for theexperiment. Briefly, dry lipids 1,2-dipalmitoyl-sn-glycero-3-phospho-choline (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phospho(1=-rac-glyc-erol) (sodium salt) (DPPG) (7:3 [wt/wt]) to mimic bacterial membranesor DPPC to mimic mammalian membranes were dissolved in a chloro-form-methanol mixture in a 150-ml round-bottom flask. The solvent wasremoved with a stream of nitrogen gas, to allow formation of a thin lipidfilm on the walls of the glass vessel. The lipid film thus obtained waslyophilized for 6 h to remove traces of solvent. Dried thin films wereresuspended in 10 mM Tris buffer (0.1 mM EDTA, 150 mM NaCl [pH7.4]) preheated at 60°C, with vortex mixing. The lipid dispersions werethen sonicated on ice for 15 to 20 min using a titanium-tip ultrasonicator,with burst and rest times of 30 s and 10 s, respectively, until the solutionsbecame opalescent. Titanium debris was removed by centrifugation. Eachpeptidomimetic (final concentration, 5 �g/ml) was added to 500 �l of 10mM Tris buffer (0.1 mM EDTA, 150 mM NaCl [pH 7.4]) or 0.5 �g/mlbacterial or mammalian mimic SUVs, and the peptidomimetic-lipid mix-ture was allowed to interact at 25°C for 2 min in a cuvette. The fluores-cence measurements were performed with a Fluorolog spectrofluorom-eter (Jobin Yuvon, Horiba, Japan). Samples were excited at 280 nm, andthe emission was scanned from 300 to 400 nm, with a 5-nm slit width for

FIG 1 Reagents and conditions. Reaction 1, 5 eq spermine in DCM, 3 h; reaction 2, methanol, 30 min; reaction 3, 2 eq Dde-OH in DMF, overnight; reaction 4,6 eq (Boc)2O in DCM-DMF (1:1), 3 h; reaction 5, Boc-Trp(Boc)-OH, HOBt, and DIPCDI in DCM-DMF (1:1), overnight; reaction 6, 2% hydrazine in DMF;reaction 7, Fmoc-Trp(Boc)-COOH, HOBt, and DIPCDI in DCM-DMF (1:1), 1.5 h; reaction 8, 20% piperidine in DMF; reaction 9, 3 eq R-COOH, HOBt, andDIPCDI in DCM-DMF (1:1), overnight; reaction 10, 30% TFA in DCM.

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both excitation and emission. The experiment was repeated twice on thesame day, and representative data are presented here.

Membrane depolarization. The evaluation of membrane depolariza-tion of MRSA was performed as described previously (16). Briefly, MRSAthat had been grown overnight was subcultured in MHB for 2 to 3 h at37°C to obtain mid-log-phase cultures. The cells were centrifuged at 4,000rpm for 10 min at 25°C, washed, and resuspended in respiration buffer (5mM HEPES, 20 mM glucose [pH 7.4]) to obtain a diluted suspension ofoptical density at 600 nm (OD600) of �0.05. The membrane potential-sensitive dye 3,3=-dipropylthiadicarbocyanine iodide (DiSC35) (0.18 �Min dimethyl sulfoxide [DMSO]) was added to 500-�l aliquots of resus-pended cells, and the mixtures were allowed to equilibrate for 1 h. Baselinefluorescence was assessed using an Edinburg F900 spectrofluorometer,with excitation at 622 nm and emission at 670 nm, in a cuvette with a 1-cmpath length. A bandwidth of 5 nm was employed for excitation and emis-sion. Subsequently, increasing concentrations of test peptidomimeticswere added to the equilibrated cells, and the increase in fluorescence re-sulting from dequenching of the DiSC35 dye was measured every 2 min, toobtain the maximal depolarization. Increases in relative fluorescenceunits (RFU) were plotted against increasing concentrations of differentpeptidomimetics or PMB.

Bactericidal kinetics. The kinetics of bacterial killing of a MRSA strain(ATCC 33591) by peptidomimetics at 2� MIC and 4� MIC were deter-mined and compared with those of VAN as described previously (24).Log-phase bacteria (1.2 � 107 to 3.0 � 107 CFU/ml) were incubated withpeptidomimetic 1c, peptidomimetic 1d, or VAN at 2� MIC or 4� MIC inMHB. Aliquots were removed after 0.5, 1, 2, 3, and 6 h and diluted insterile normal saline solution before plating on Mueller-Hinton II agar;CFU were counted after 24 h of incubation at 37°C. To decrease the limitof detection, larger aliquots were removed and centrifuged to removeantibacterial agent carryover. The experiment was repeated on three dif-ferent days, and curves were plotted for log10 CFU/ml versus time.

Scanning electron microscopy. For electron microscopy, sampleswere prepared by following previously reported protocols (16, 25).Briefly, freshly inoculated MRSA (ATCC 33591) was grown on MHB upto an OD600 of �0.5 (corresponding to 108 CFU/ml). Bacterial cells werethen centrifuged at 4,000 rpm for 15 min, washed three times withNaCl-Pi (10 mM phosphate buffer, 150 mM NaCl [pH 7.4]), and resus-pended in an equal volume of NaCl-Pi. For scanning electron microscopy(SEM) experiments, larger bacterial inocula (108 CFU/ml) were used;therefore, the cells were incubated with test peptidomimetic 1c, peptido-mimetic 1d, or VAN at 10� MIC for 30 min. Controls were run in theabsence of antibacterial agents. After 30 min, the cells were centrifugedand washed three times with NaCl-Pi. For cell fixation, the washed bacte-rial pallet was resuspended in 0.5 ml of 2.5% paraformaldehyde inNaCl-Pi and incubated overnight at 4°C. After fixation, cells were centri-fuged, washed twice with 0.1 M sodium cacodylate buffer, and fixed with1% osmium tetraoxide in 0.1 M sodium cacodylate buffer for 40 min atroom temperature (RT) in the dark. The samples were then dehydrated ina series of graded ethanol solutions (30% to 100%) and finally dried indesiccators under reduced pressure. Upon dehydration, the cells were airdried for 15 min at RT in the dark after immersion in hexamethyldisi-lazane. An automatic sputter coater (Quorum SC7640) was used to coatthe specimens with gold particles at a thickness of 30 Å. Then samples wereimaged via scanning electron microscopy (Zeiss EVO LS15).

Drug resistance study. The initial MICs against MRSA of peptidomi-metics and the control antibiotics VAN and ciprofloxacin (CIP) weredetermined as described above. Bacterial suspensions (100 �l) from du-plicate wells at sub-MIC concentrations were then used to inoculate freshcultures. The cultures was grown to yield approximately 105 CFU/ml forthe next experiment. These bacterial suspensions were then incubatedwith the desired concentrations of antibacterial agents for 18 h to deter-mine new MICs. The same subculturing protocol was used for the next 16passages, and MICs were determined using OD630 values as describedpreviously (23).

Biofilm susceptibility assay. For the biofilm inhibition assay, thestandard protocol was used as reported previously (26). Briefly, freshlyinoculated MRSA (ATCC 33591) was grown overnight on biofilm growthmedium (TSB supplemented with 0.5% [wt/vol] NaCl and 0.25% [wt/vol] glucose). The next day, the cultures were diluted to 105 CFU/ml infresh biofilm growth medium. Two hundred microliters of diluted culturewas dispensed into wells of a 96-well polystyrene plate for biofilm forma-tion. To evaluate the inhibition of biofilm formation, antibacterial agentsat the planktonic MIC in biofilm medium (MICb) and sub-MICb concen-trations were added initially to diluted cultures following incubation at37°C without shaking. Another set of experiments was performed with theaddition of fresh medium containing antibacterial agents at 10� MICb

and 20� MICb to 24-h-preformed biofilm, after gentle washing with ster-ile NaCl-Pi (35 mM phosphate buffer, 150 mM NaCl [pH 7.4]). Biofilmcultures were reincubated at 37°C for 24 h. After removal of the medium,the biofilms were washed twice with sterile NaCl-Pi and assessed for met-abolic activity (alamarBlue assay) and biomass quantities (crystal violetassay), as follows.

For determination of metabolic activity, the plates were sonicated in aultrasonic bath (Elmasonic, Germany) for 5 min at 37°C, with sonicationat 30 kHz, to ensure detachment of bacteria from the biofilms before theaddition of 10% (vol/vol) alamarBlue reagent (according to the manufac-turer’s instructions). The plates were further incubated at 37°C for 2 h.After 2 h, absorbance was measured at 570 nm and 600 nm, and thepercent reduction of alamarBlue (cell viability) was calculated by using aformula provided in the manufacturer’s protocol. The experiment wasrepeated three times on three different days, and results are given asmean � SD.

For biomass quantification, the crystal violet (CV) staining protocolwas used as reported previously (27). Slime and adherent cells were fixedfor 20 min with 1 ml of 99% methanol and then stained for 20 min with200 �l of 0.1% crystal violet. Excess stain was removed by washing thecoverslips with NaCl-Pi, and then the coverslips were air dried. Thestained dye was redissolved with the addition of 33% acetic acid andincubation for 1 h at room temperature without shaking. The opticaldensity at 570 nm (OD570) was measured spectrophotometrically, anddata are presented as percent biomass in comparison with the positivecontrol.

Confocal laser scanning microscopy of biofilms. For confocal mi-croscopy, biofilm formation was induced on glass coverslips in a 6-wellplate, following a reported procedure (27). Briefly, overnight culturesof MRSA were diluted to 105 CFU/ml, and 3-ml volumes of this sus-pension were used to grow biofilms on glass coverslips in the wells of a6-well plate at 37°C. Biofilm growth conditions and treatment of bio-films with antibacterial agents were as described above for the alamar-Blue and crystal violet assays. Then the coverslips were washed twicewith sterile NaCl-Pi and stained with reagent from the MolecularProbes Live/Dead kit (Invitrogen, Eugene, OR), following the manu-facturer’s instructions. This stain contains the DNA-binding dyesSYTO 9 (green fluorescence) and propidium iodide (PI) (red fluores-cence). When used alone, SYTO 9 stains all bacteria in a population,i.e., those with intact or damaged membranes. In contrast, PI pene-trates only bacteria with damaged membranes, causing a reduction inthe SYTO 9 staining (green fluorescence). The biofilms were examinedwith an Olympus FluoView FV1000 confocal laser scanning micro-scope. For detection of SYTO 9 (green channel) and PI (red channel),488-nm and 561-nm lasers, respectively, were used. For measurementof biofilm depths, z-stack images were acquired at approximately0.4-�m intervals, using a 100� HCX PL APO oil immersion lens (nu-merical aperture, 1.2); image analyses and export were performed withFV10-ASW-1.7 software. For each sample, at least five different re-gions on a single coverslip were scanned. The experiment was repeatedthree times on three different days, and representative data are pre-sented here.

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RESULTSRational design and synthesis of peptidomimetics. Antimicro-bial peptidomimetics based on the defined pharmacophore withat least �2 charges at physiological pH and hydrophobicity havebeen designed by various research groups (28, 29). Recently, wereported a small series of potent peptidomimetics with broad-spectrum antibacterial activity based on a template containingN-terminally tagged dipeptidomimetics conjugated with spermi-dine (16). In the present work, new N-terminal tags and cationicspermine at the C terminus were conjugated to the same templateto expand and optimize the library. In the template peptidomi-metic 1a, two Trp residues were attached to the spermine moiety.The hydrophobic bulk, aromatic electron cloud, and lipid mem-brane anchorage ability of Trp residues have made Trp a suitableresidue for incorporation in novel antibacterial peptidomimetics(28, 30). In peptidomimetics 1b to 1f (series 1), different N-ter-

minal tags, i.e., caffeic acid, 4-(trifluoromethyl)phenylacetic acid,decanoic acid, lauric acid, and linoleic acid, were used to vary therelative hydrophobicity (Fig. 1). The peptidomimetics in series 2(peptidomimetics 2a to 2f) were synthesized to investigate theeffects of Trp positioning on the spermine backbone on activityand therapeutic index values. In peptidomimetics 2a to 2f, thesecondary N atoms of spermine were coupled with the carboxylicacid end of Trp residues, leaving the alpha-amino group of Trpresidues ionizable at physiological pH (Fig. 1). All of the designedpeptidomimetics were 80% pure, and their masses were in therange of 575 to 850 Da (Table 1).

Biological activities of designed peptidomimetics. The anti-bacterial activities of the designed peptidomimetics against fiveGram-positive bacterial strains and two Gram-negative bacterialstrains were evaluated using the serial broth dilution method (Ta-ble 2). The template peptidomimetic 1a showed moderate activityagainst Gram-positive bacterial strains, while peptidomimetics 1bto 1f displayed good activity against Gram-positive bacterialstrains, with MICs of �10 �g/ml against all tested strains except E.faecalis. Peptidomimetics in series 1 also showed activity against E.coli, with MICs in the range of 14.2 to 56.8 �g/ml. Similarly, inseries 2, peptidomimetics 2a and 2b showed negligible growthinhibition against all tested bacterial strains up to 454.4 �g/ml,while peptidomimetic 2c showed moderate activity and peptido-mimetics 2d to 2f exhibited good growth inhibition (MICs of 0.8to 28.4 �g/ml) of all of the bacterial stains except A. baumannii.PMB showed relatively poor activity against Staphylococcus spe-cies, although it showed excellent growth inhibition of Gram-negative bacterial strains. VAN showed potent growth inhibitionof Staphylococcus species but was ineffective against Gram-nega-tive strains under the experimental conditions.

The cell selectivity of the designed peptidomimetics on enucle-ated hRBCs was evaluated (Table 2). Most of the peptidomimet-ics, including peptidomimetics 1a to 1d and 2a to 2c, were foundto cause minimal hemolysis up to the maximal concentration

TABLE 1 Purity, proportion of acetonitrile for RP-HPLC elution, andmolecular masses of designed peptidomimetics

Peptidomimetic Purity (%) Acetonitrile (%)a

Mass ([M�H]�)(Da)

Calculated Observed

1a 95 17.41 575.3816 575.38081b 99 46.42 737.4133 737.41391c 95 54.72 761.4109 761.41101d 95 61.57 729.5174 729.51781e 95 65.21 757.5487 757.54891f 98 70.36 837.6113 837.60972a 80 12.30 575.3816 575.38152b 80 44.34 737.4133 737.41402c 83 49.85 761.4109 761.41182d 99 57.92 729.5174 729.51812e 99 62.63 757.5487 757.54952f 99 69.78 837.6113 837.6113a Percentage of acetonitrile for RP-HPLC elution.

TABLE 2 Antibacterial activities of peptidomimetics against Gram-positive and Gram-negative bacterial strains and cytotoxicity in blood cells

Peptidomimetic

MIC (�g/ml) of:

Hemolysis(%)b

LDH release(%)c

S. aureus(ATCC29213)

MRSA(ATCC33591)

S. epidermidis(ATCC12228)

MRSEa

(ATCC51625)

E. faecalis(ATCC7080)

E. coli(ATCC11775)

A. baumannii(ATCC19606)

1a 113.6 227.2 113.6 NDd 454.5 ND ND 4 ND1b 3.5 7.1 3.5 7.1 113.6 14.2 ND 16 ND1c 1.7 3.5 1.7 3.5 28.4 56.8 28.4 2 5.781d 1.7 1.7 1.7 1.7 3.5 14.2 113.6 9 17.51e 1.7 3.5 1.7 1.7 7.1 14.2 56.8 31 ND1f 7.1 3.5 1.7 7.1 28.4 28.4 ND 30 ND2a 454.4 227.2 454.4 227.2 ND 454.4 ND 0 ND2b 454.4 454.4 ND ND ND 454.4 ND 5 ND2c 14.2 28.4 7.1 14.2 ND 113.6 113.6 1 ND2d 0.8 1.7 0.8 1.7 28.4 28.4 113.6 83 ND2e 0.8 1.7 0.8 1.7 7.1 28.4 113.6 96 ND2f 0.8 3.5 0.8 1.7 14.2 28.4 56.8 88 NDPMB 14.2 28.4 7.1 28.4 113.6 0.4 ND ND NDVAN 0.4 0.8 0.4 0.8 ND 113.6 56.8 ND NDa MRSE, methicillin-resistant Staphylococcus epidermidis.b Hemolysis at 250 �g/ml.c LDH release at 20 �g/ml.d ND, not determined.

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tested of 250 �g/ml. Peptidomimetics 1e and 1f caused 31% and30% hemolysis, respectively, at 250 �g/ml. Peptidomimetics 2d,2e, and 2f caused significant hemolysis, leading to 83%, 96%, and88% damage to hRBCs, respectively, at 250 �g/ml.

The antibacterial activities of nonhemolytic peptidomimetics1c and 1d were also evaluated against MRSA in the presence of25% (vol/vol) human serum or bovine serum. Fourfold and 8-foldincreases in MICs were observed for peptidomimetics 1c and 1d,respectively, with human serum (Table 3).

The LDH release assay with PBMCs demonstrated 5.78% �6.58% and 17.56% � 10.15% LDH release caused by peptidomi-metics 1c and 1d, respectively, at 20 �g/ml. At 50 �g/ml, the re-lease was 20.81% � 5.4% and 21.62% � 5.04% with peptidomi-metics 1c and 1d, respectively.

Membrane insertion and depolarization potential of de-signed peptidomimetics. Trp fluorescence was used as a probe toevaluate the effects of Trp positioning on the insertion depth ofdesigned peptidomimetics in bacterial and mammalian mimicmembranes. In buffer, all peptidomimetics showed fluorescenceemission maxima in the range of 356 to 362 nm (Table 4). Inbacterial mimic SUVs (DPPC-DPPG, 7:3 [wt/wt]), blue shifts inemission maxima in the range of 5 to 12 nm, concomitant withincreases in fluorescence intensity, in comparison with buffer,were observed for all of the peptidomimetics in series 1. In mam-malian mimic DPPC SUVs, blue shifts in the range of 1 to 7 nmwere observed for peptidomimetics 1a to 1f. The emission max-

ima for series 2 peptidomimetics (peptidomimetics 2a to 2f)shifted more toward blue wavelengths than did peptidomimetics1a to 1f in both bacterial mimic and mammalian mimic mem-branes. Noticeably, for peptidomimetics 2a to 2f in bacterialmimic membranes, significant blue shifts (2 to 16 nm) were ob-served subsequent to partitioning, and the concomitant increasein emission intensity was not observed for peptidomimetics 2a, 2c,and 2d, in comparison with buffer (see Fig. S3 in the supplementalmaterial).

Next, the ability of the designed peptidomimetics to compro-mise the membrane potential in MRSA was evaluated by using themembrane potential-sensitive dye DiSC35. Upon partitioning inthe membranes of live cells at sufficiently high concentrations,DiSC35 self-quenches its fluorescence. Under the influence of amembrane-depolarizing agent, there is dye release with a signifi-cant increase in dye fluorescence, which is measured fluorometri-cally. For peptidomimetics 1a and 2a, no significant increases inrelative fluorescence units (RFU) were observed up to the maxi-mum concentrations tested, suggesting an inability of these pep-tidomimetics to alter membrane potential at concentrations be-low the MIC (data not shown). For peptidomimetics 1c and 2c,with aromatic N-terminal tags, only marginal changes in RFUwere observed up to the highest concentrations tested (Fig. 2).Intermediate changes in fluorescence intensity were observed forpeptidomimetics 1d and 2d, whereas significant changes in RFUwere observed for peptidomimetics 1e, 1f, 2e, and 2f. The increasesin fluorescence with lipid-tagged peptidomimetics were concen-tration dependent up to 9.9 �g/ml and then were saturated, re-sulting in plateau-like dose-response curves. The experiment wasrepeated twice on two consecutive days, with similar results. Rep-resentative results from one assay are presented here. Further-more, interaction studies were performed with peptidomimetics1c and 1d, which are active and cell-selective peptidomimeticsfrom series 1.

Bactericidal kinetics and membrane-disruptive mode of ac-tion. Bactericidal kinetic experiments with peptidomimetic 1c,peptidomimetic 1d, and VAN at 2 times and 4 times their respec-tive planktonic MICs were performed with exponentially growingS. aureus ATCC 33591 (Fig. 3). At 2� MIC, both peptidomimeticsproduced �3-log10 CFU/ml reductions within 3 h of incubation;at 4� MIC, bactericidal effects with 4-log10 CFU/ml reductionswithin 30 min of incubation were observed. VAN did not show

TABLE 3 Effects of salt concentrations and serum on antibacterialactivities of compounds

Compound

MIC (�g/ml) against MRSA 33591 in:

Biofilmmedium(low salt)a

TSB withhigh saltb

MHB withhumanserumc

MHB withFBSd

Peptidomimetic 1c 7.1 28.4 14.1 7.1Peptidomimetic 1d 3.5 7.1 14.1 7.1VAN 0.8 1.7 0.8 0.8a TSB supplemented with 0.5% (wt/vol) NaCl and 0.25% (wt/vol) glucose (i.e., MICb).b TSB supplemented with 3% (wt/vol) NaCl and 0.5% (wt/vol) glucose.c MHB with 25% human serum added.d MHB with 25% FBS added.

TABLE 4 Tryptophan fluorescence emission maxima of designedpeptidomimetics in buffer, DPPC SUVs, or DPPC-DPPG SUVs

Peptidomimetic

Emission maximum (nm)a

Bufferb DPPCDPPC-DPPG(7:3 [wt/wt])

1a 361 358 (3) 351 (10)1c 356 355 (1) 351 (5)1d 362 355 (7) 350 (12)1e 357 350 (7) 348 (9)1f 356 352 (4) 351 (5)2a 359 358 (1) 357 (2)2c 360 354 (6) 353 (7)2d 358 350 (8) 343 (15)2e 357 347 (10) 341 (16)2f 354 350 (4) 348 (6)a Blue shifts are indicated in parentheses.b The buffer contains 0.1 mM EDTA and 150 mM NaCl (pH 7.4).

FIG 2 Concentration-dependent cell membrane depolarization assessed withthe potential-sensitive dye DiSC35.

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any bactericidal activity even upon incubation at 4� MIC underthe same conditions and produced only 2-log10 CFU/ml reduc-tions over a period of 6 h. The lower limit of detection was deter-mined to be 100 CFU/ml, and bactericidal activity was defined asa 3-log10 CFU/ml decrease, in comparison with the time zerovalue.

Further, we visualized the effects on MRSA of 30-min incuba-tions with peptidomimetic 1c, peptidomimetic 1d, and VAN, at10� MIC, using SEM. Control MRSA cells exhibited a bright,smooth appearance, with intact cell membranes (Fig. 4A). Pep-tidomimetic 1c treatment caused rough damaged surfaces, cellbursting, leakage, and string-like substances, which are consid-ered to be cellular debris arising from cell lysis (Fig. 4B). Peptido-mimetic 1d-treated cells appeared distorted, with depressions andhole formation (Fig. 4C), indicating the membrane-active modeof action for the designed peptidomimetics. Surprisingly, VAN-treated cells mostly retained their smooth appearance, albeit withslight deformations in shape, compared with control cells(Fig. 4D).

Resistance development against peptidomimetics in MRSA.The ability of active peptidomimetics 1c and 1d to induce resis-tance development in MRSA strain ATCC 33591 in 17 sub-MICserial passages was evaluated (Fig. 5). Fourfold and 2-fold in-creases in MIC values were observed for peptidomimetics 1c and1d, respectively. The MIC of the standard antibiotic VAN wasincreased 4-fold after 17 passages, whereas a radical 256-foldchange in the MIC was observed for ciprofloxacin (CIP).

Activity against MRSA biofilms. (i) Quantification of viabil-ity and reduction in biomass. After establishing their antibacte-rial activity and mode of action on planktonic cells, we furtherevaluated the efficacy of peptidomimetics 1c and 1d to prevent theformation of biofilms and to eradicate preformed MRSA biofilms(24 h) by using alamarBlue as a redox indicator for assessment ofmetabolic activity and crystal violet for biomass quantification. Awell-characterized biofilm-producing reference strain of MRSA(ATCC 33591) was used for the experiments. Prior to this exper-iment, the MICs of peptidomimetic 1c, peptidomimetic 1d, andVAN in biofilm growth medium (TSB with 0.5% NaCl and 0.25%glucose) were evaluated. The results showed pronounced effects ofa high salt concentration (supplemented with 3% NaCl) on MICs,whereas 2-fold increases in the MICs of peptidomimetics 1c and1d in low-salt medium (supplemented with 0.5% NaCl) were ob-served. The MIC for VAN was increased only 2-fold even in the

high salt concentration. All biofilm-related experiments were per-formed with MICb measurements; MICb values were the plank-tonic MICs of peptidomimetics and VAN in biofilm growth me-dium (Table 3). For the biofilm formation inhibition assay, initialinocula were added with sub-MICb and MICb concentrations ofthe tested agents (Fig. 6A and B). Peptidomimetics 1c and 1d wereable to inhibit biofilm formation at sub-MICb concentrations (�4�g/ml and �2 �g/ml, respectively), causing reductions in meta-bolic activity of up to 33.1% � 5.7% and 26.4% � 3.3%, respec-tively. Under identical treatment conditions, biomass reductionswere found to be 19.8% � 5.6% and 28.2% � 11.1% for peptido-mimetics 1c and 1d, respectively. At MICb, both peptidomimeticswere able to inhibit the adhesion of biofilm, causing 90% reduc-tions in measured viability and biomass quantity. Metabolic ac-tivity and biomass quantity were not reduced significantly withVAN at sub-MICb concentrations (�0.5 �g/ml), compared withcontrol values, whereas VAN at MICb concentrations (�1 �g/ml)inhibited biomass quantity to 27.4% � 1.3%.

The effects of peptidomimetics on the viability of 24-h-pre-formed mature biofilms were also evaluated at concentrationshigher than MICb. At 20� MICb, the designed peptidomimetics1c (140 �g/ml) and 1d (70 �g/ml) showed better killing profilesthan did VAN (20 �g/ml), showing 6.4% � 0.2% and 10.1% �7.8% viable cells, respectively, versus 77.7% � 7.0% viable cells forVAN at the indicated concentration (Fig. 6C).

In parallel with viability results, assessments of reductions inthe biomass quantities of 24-h mature MRSA biofilms showed areduction in biomass to 24.0% � 13.4% with peptidomimetic 1cat 140 �g/ml (Fig. 6D). For peptidomimetic 1d, significant differ-ences in biomass quantities, in comparison with control values,were observed at both tested concentrations (35 �g/ml and 70�g/ml, corresponding to 10� MICb and 20� MICb), i.e.,66.7% � 8.2% and 21.4% � 9.2%, respectively. For VAN-treatedbiofilms, the observed biomass quantities were 119.3% � 17.5%at 10� MICb (10 �g/ml) and 83.7% � 24.1% at 20� MICb (20�g/ml).

(ii) Visualization of effects of designed peptidomimetics onbiofilms using confocal laser scanning microscopy. We next vi-sualized the effects of the designed peptidomimetics and VAN onbiofilm-embedded MRSA, making use of the membrane permea-bility-sensitive, DNA-binding dyes SYTO 9 and propidium iodideas markers. As a measure of biofilm formation/growth inhibition,the thickness of biofilm was measured using confocal microscopy.

FIG 3 Time-kill curves for S. aureus strain ATCC 33591 incubated with 2� MIC (A) or 4� MIC (B) levels of peptidomimetic 1c, peptidomimetic 1d, or VANand sampled at the indicated time points. The curves were plotted for log10 CFU/ml versus time as described in Materials and Methods. The data shown are fromone of three independent experiments with similar results.

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The control biofilm (24 h) showed a lawn of viable (green) cells,with an average thickness of 14.3 � 1.4 �m (Fig. 7A; also see TableS1 in the supplemental material). At MICb, peptidomimetics 1cand 1d prevented the formation of biofilm; very few cells adheredto the substratum, with observed average thicknesses of 3.9 � 1.1�m and 3.5 � 0.6 �m, respectively. Furthermore, at sub-MICb

concentrations, the observed thicknesses were 5.2 � 0.3 �m and5.8 � 0.4 �m, respectively (Fig. 7Ab and Ad). The measured bio-film thickness was 11.4 � 2.9 �m with VAN at MICb (Fig. 7Ag),whereas VAN was unable to reduce the biofilm thickness at sub-MICb levels.

Untreated 48-h mature biofilm (24 h plus 24 h) showed a lawnof viable (green) cells with an average thickness of 23.6 � 2.5 �m(Fig. 7B; also see Table S2 in the supplemental material). Subse-quent to treatment with peptidomimetics 1c and 1d at 10� MICb,there were visible decreases in the numbers of live cells and thick-ness was reduced to 7.1 � 1.5 and 7.0 � 1.0 �m, respectively (Fig.7Bb and Bd). With peptidomimetics 1c and 1d, most of the cellslost their integrity at 20� MICb, appearing red (Fig. 7Bc and Be),and a smear of permeabilized cells was observed. Upon VAN treat-

FIG 4 Scanning electron microscopic images of MRSA. (A) Untreated bacterial cells. (B) Cells treated with peptidomimetic 1c. (C) Cells treated with peptido-mimetic 1d. (D) Cells treated with VAN. The cells were exposed to various agents for 30 min at 10 times their respective planktonic MICs. Arrows, morphologicalalterations produced. Insets, higher-magnification images (magnification, �150,000).

FIG 5 Resistance development induced by antibacterial agents in S. aureus(ATCC 33591) after 17 serial passages with sub-MIC levels of peptidomimetic1c, peptidomimetic 1d, or antibiotic. The fold change in MIC is the ratio of theMIC after 17 passages to the MIC before the first passage.

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ment, no significant differences in the numbers of live cells wereobserved, inasmuch as mixed bacterial populations stained greenwere visible at both tested concentrations. VAN had little effect on24-h biofilm at 10� MICb, with no distinction between controlbiofilm and VAN-treated biofilms being visible. Only with VAN at20� MICb was a slight decrease in the height of mature biofilmobserved (Fig. 7Bf and Bg). The confocal imaging experimentswere repeated three times on three different days, and similar re-sults were obtained (representative data from one set is shownhere).

DISCUSSION

Polyamines (putrescine, spermidine, and spermine) modulatevarious processes in cells, including nucleic acid packaging, DNAreplication, transcription, and translation, and thus are requiredfor optimal growth in prokaryotes and eukaryotes (31). Poly-amines and their analogues exhibit versatile biological activities,including anticancer, antiparasitic, antiendotoxin, and antibacte-rial activities (32–34). Squalamine (from dogfish shark) and cino-dine (from Nocardia spp.) are natural antibiotics with a polyaminebackbone in their structures (35, 36). The role of polyamine con-

jugation in improving activity for a number of synthetic antibac-terial agents, such as ceragenins, acylpolyamines, and caffeoylpolyamines, has been reported (15, 33, 37). Synergistic effects ofexogenous polyamines (added to growth medium) and variousantibiotics have also been investigated, and it was shown that 1mM spermine caused up to 200-fold reductions in the MIC ofoxacillin against MRSA strain Mu50 under test conditions (38).Interestingly, it was recently reported that S. aureus lacks identifi-able genes for polyamine biosynthesis and consequently producesno spermine or spermidine or their precursors; therefore, poly-amines and their conjugates act as toxins to S. aureus (39). Sup-porting this, in a recent report, the exceptional virulence of MRSAstrain USA300 was ascribed to the arginine catabolic mobile ele-ment (ACME), which harbors the spermidine acetyltransferasegene (speG), imparting resistance to spermidine and other poly-amines (40). Therefore, for polyamine-sensitive MRSA, conjuga-tion of spermine might be a robust strategy to overcome thisdeadly strain.

Various valuable structure-activity relationships for antibacte-rial peptidomimetics have been reported, and modifications incharge distribution or hydrophobicity have led to optimization of

FIG 6 (A and B) Inhibition of MRSA biofilm formation by different agents using the alamarBlue assay (A) and biomass quantification using the crystal violetstaining assay (B). (C and D) Metabolic activity of 24-h mature biofilm-embedded MRSA using the alamarBlue assay (C) and biomass quantification using thecrystal violet assay (D). The MICb values for peptidomimetic 1c, peptidomimetic 1d, and VAN were 7.1 �g/ml, 3.5 �g/ml, and 0.8 �g/ml, respectively. For allexperiments, data are expressed as mean � SD. Statistical differences from control values were determined by one-way analysis of variance (ANOVA) withTukey’s multiple-comparison post hoc tests. All differences between the control and treated biofilms were considered statistically significant (P � 0.001).

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molecules for therapeutic applications (41–44). Extending ourprevious findings with the N-terminally tagged dipeptide spermi-dine template, in the present work we designed two series of pep-tidomimetics (series 1 and 2) with linear or branched arrange-ments of Trp residues on the spermine backbone to explore theeffects on antibacterial activity and selectivity and the mode ofaction. In our previous work, we established 50 to 70% hydropho-bicity (based on reverse-phase [RP]-HPLC retention times) and atleast �2 charges to be crucial for antibacterial activity and cellselectivity (16). The comparative MIC data for series 1 and 2showed that peptidomimetics 1b and 1c with linear arrangementsof tryptophan were more active than the corresponding peptido-mimetics with branched arrangements of tryptophan (i.e., pep-tidomimetics 2b and 2c) against all of the Gram-positive bacterialstrains tested. For lipidated N-tagged peptidomimetics, series 1and 2 showed comparable inhibitory effects on all Gram-positivebacterial strains; however, series 2 was more hemolytic than series1, although the hydrophobicity ranges for the two series were thesame. As reported in the literature and observed in our previousstudy, hydrophobicity above a threshold range plays a crucial rolein increased hemolytic activity (16). This finding also holds true inthe present study, since overall, the charges were the same anddifferences in hydrophobicity among corresponding pairs in se-ries 1 and 2 were not very significant (�0.6 to 6%, as measured byRP-HPLC). Therefore, the key determinant for activity and selec-tivity in the present study, besides hydrophobicity, was the place-ment of Trp residues at different positions in the template, whichindicates the role of the amino groups of spermine in activity.

To further elucidate the role of Trp moieties in the results de-

scribed above, we performed interaction studies and evaluated themode of action of these peptidomimetics with artificial mem-branes and intact bacterial cells. Trp fluorescence measurementshave been used as a sensitive tool to probe the interactions ofpeptides with artificial bacterial or mammalian mimic mem-branes. Partitioning of Trp residues into the hydrophobic mem-brane environment has been reported to result in blue shifts ac-companied by increases in emission intensity (45), as wasobserved for all peptidomimetics in series 1. In series 2, however,the emission intensity in mammalian mimic SUVs revealed more-pronounced increases for all of the peptidomimetics. Interest-ingly, for peptidomimetics 2a, 2c, and 2d in bacterial mimic mem-branes, blue shifts were observed without concomitant increasesin fluorescence intensity, compared with buffer (see Fig. S3 in thesupplemental material). Similar observations of blue shifts subse-quent to peptide-lipid interactions without increases in emissionintensity were reported previously for the antimicrobial peptidestemporin L and nisin; the authors attributed the decreases in Trpfluorescence intensity to quenching due to aggregation of peptidein the vicinity of membranes or due to the quenching properties ofthe negatively charged lipid head groups, which can interact di-rectly with orbitals of the indole ring in the Trp residue (46, 47).In the present study also, the positive charge distribution in thesepeptidomimetics led to better electrostatic interactions with thenegatively charged bacterial mimic membranes and vicinity-in-duced aggregation, causing decreases in fluorescence intensity inbacterial mimic membranes.

Further, the dependence of membrane depolarization abilityon Trp branching and N-terminal tagging was evident from the

FIG 7 Three-dimensional images of MRSA biofilms. (A) Effects of antibacterial agents on biofilm formation of MRSA, assessed using confocal laser scanningmicroscopy. (a) Control; (b) peptidomimetic 1c at sub-MICb level; (c) peptidomimetic 1c at MICb; (d) peptidomimetic 1d at sub-MICb level; (e) peptidomimetic1d at MICb; (f) VAN at sub-MICb level; (g) VAN at MICb. (B) Effects of antibacterial agents against 24-h-preformed mature MRSA biofilms, assessed usingconfocal laser scanning microscopy. (a) Control; (b) peptidomimetic 1c at 10� MICb; (c) peptidomimetic 1c at 20� MICb; (d) peptidomimetic 1d at 10� MICb;(e) peptidomimetic 1d at 20� MICb; (f) VAN at 10� MICb; (g) VAN at 20� MICb. After treatment at different concentrations, the biofilms were stained withSYTO 9 (green; viable cells) and propidium iodide (red; dead cells), as described in the manufacturers’ protocol.

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results (Fig. 2). The untagged template peptidomimetics (�4charges and �20% hydrophobicity) were unable to alter mem-brane potential up to the highest concentrations tested. Despitegood activity against MRSA, aromatic tagging in peptidomimetics1c and 2c did not allow significant changes in membrane poten-tial, which might have resulted from poor insertion of these pep-tidomimetics into hydrophobic membrane interiors. The effect ofhydrophobicity on membrane insertion was evident as more-hy-drophobic peptidomimetics (peptidomimetics 1e, 1f, 2e, and 2f)in both series 1 and series 2 were better able to alter membranepotential than were less-hydrophobic peptidomimetics (peptido-mimetics 1d and 2d). It was evident from Trp fluorescence andmembrane depolarization experiments that series 2 peptidomi-metics with branched Trp residues on the spermine backbonealthough more potent, were more prone to cause nondifferentialinteractions, whereas series 1 peptidomimetics were potent, cell-selective, membrane-depolarizing agents. In series 1, peptidomi-metics 1c and 1d were found to be more active and cell selective;therefore, further studies were carried out with these two mole-cules for optimization.

To establish whether bactericidal ability is inherent in the pres-ent designed peptidomimetics, the time course of bacterial killingwas studied by exposing MRSA to 2� MIC and 4� MIC levels ofpeptidomimetics 1c and 1d (Fig. 3). At 4� MIC, which is a ther-apeutically relevant concentration, most of the bacteria werekilled within 30 min, as extremely rapid bactericidal effects areoften seen for antimicrobial peptides (20). The fast bacterial kill-ing suggests that, at these concentrations, the antibacterial effectsare mediated through significant permeabilization or lysis of bac-terial membranes, which was corroborated by scanning electronmicroscopic images of MRSA showing distinct membrane dam-age at 10� MIC with greater numbers of bacteria (108 CFU/ml)for both peptidomimetics (Fig. 4).

Several reports suggested that antimicrobial peptides and theiranalogue peptidomimetics have novel membrane-active modes ofaction with multiple nonspecific targets, resulting in the develop-ment of resistance to bacteria (23, 48). The results for peptidomi-metics 1c and 1d after 17 serial passages at sub-MIC doses couldnot demonstrate resistance for MRSA. Poor serum protease sta-bility limits most of the developed antimicrobial peptides to top-ical application. Peptidomimetics 1c and 1d were found to killMRSA in the presence of human serum (25% [vol/vol]), with 4-and 8-fold increases in their MIC values, respectively (Table 3).The increases in MICs observed in the present study corroboratedthe previous report of short cationic antimicrobial peptides bind-ing with serum protein albumin (49). Further, to explain the in-creases in the MIC values of peptidomimetics, the stability of pep-tidomimetics 1c and 1d in human serum was evaluated withRP-HPLC, and the data demonstrated that no degradation wasfound for the peptidomimetics even with 72 h of incubation (seeFig. S4 in the supplemental material). Peptidomimetics 1c and 1dwere further assessed for cytotoxicity against primary PBMCs anddemonstrated mostly favorable nontoxic profiles at concentra-tions (20 �g/ml) higher than the MICs (Table 2).

MRSA is an extraordinary etiological agent due to its virulence,multidrug-resistant profile, and increasing prevalence in commu-nity and health care settings. Biofilm formation is a particularlyvirulent mechanism for Staphylococcus species that renders treat-ment and cure difficult with invasion, with associated mortalityrates in severe cases of MRSA infections being about 20% (50).

Various strategies have been proposed to either kill microbes ordrive them out of biofilms. Among these strategies, targeting quo-rum sensing and designing antiadhesion agents and antimicrobialpeptides are a few effective means that are currently being ex-plored (51, 52). Intrigued by the success of lipopeptide daptomy-cin, oritavancin, and other membrane-active peptidomimeticswith membrane depolarization and disruption abilities againstbiofilm-embedded MRSA (26, 53), we extended our studiesagainst MRSA biofilms and compared the activities of peptidomi-metics 1c and 1d with that of the standard drug VAN.

As a standard protocol for determination of biofilm forma-tion/killing abilities, we used a combination of the alamarBlueassay (for measurement of viability) and crystal violet assay (forquantification of biomass) (27). In the biofilm assay, no perfectcorrelation between cell viability and biomass quantity was ob-served, although similar patterns were seen in both experiments.At sub-MICb levels, the peptidomimetics 1c and 1d decreased bio-film formation, indicating the potential of these molecules to pre-vent MRSA adhesion to surfaces. For 24-h mature biofilm, pep-tidomimetics 1c and 1d were more effective in reducing viabilityand biomass than was VAN at 20� MICb (Fig. 6C and D). Al-though VAN showed better ability to inhibit growth in planktoniccultures of MRSA under sessile conditions with 24-h mature bio-films, the designed peptidomimetics 1c and 1d proved to be moreefficacious at the tested concentrations, exhibiting significant de-creases in viability versus the positive control (P � 0.001).

Further, the effects of peptidomimetic treatment on biofilmformation and killing were visualized using confocal microscopy,which is a well-known method to assess such effects (26, 27). Theresults revealed a marked difference in the viability of 24-h maturebiofilms with peptidomimetics 1c and 1d versus VAN (Fig. 7). ForVAN, a subpopulation of viable, predominantly green cells wasobserved. VAN has been reported to be less membrane depolariz-ing and less effective in reducing the viability of biofilm-embed-ded S. aureus, due to the slow growth of bacterial cells under bio-film conditions (26). Making use of live/dead cell staining, it wasshown that VAN, even at a high concentration of 500 �g/ml, wasunable to cause growth depletion of Staphylococcus haemolyticusbiofilms (27).

In summary, we designed new ultrashort N-terminally modi-fied dipeptidomimetics with or without modifications on thespermine backbone leading to linear or branched tryptophans,which could effectively inhibit the growth of Gram-positive andGram-negative bacterial strains under planktonic conditions. Di-rect effects of Trp positioning on the depth of insertion in artificialmembranes were observed. Furthermore, disruption of mem-brane potential in intact MRSA pointed to different charge-hy-drophobicity interactions leading to a lack of cell selectivity forseries 2 peptidomimetics. We found the linear arrangement of Trpresidues without backbone spermine modification to be better fortherapeutically viable antibacterial peptidomimetics. Interest-ingly, under identical experimental conditions, with the dualmodes of action of membrane depolarization and disruption,peptidomimetics 1c and 1d showed better efficacy than the con-ventional antibiotic VAN against biofilm formation and eradica-tion of 24-h mature MRSA biofilms. These findings highlight thepotential of membrane-active antibacterial peptidomimetics asuseful tools to eradicate clinically relevant biofilms. Overall, ourpresent work provides an impetus for the design of better mem-brane-active, spermine-based, antibacterial peptidomimetics to

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treat recalcitrant biofilm communities of MRSA. At present, weare exploring the ability of the most active peptidomimetics tohamper biofilm formation on solid supports, which wouldbroaden the therapeutic applications of these peptidomimetics inclinical settings.

ACKNOWLEDGMENTS

This work was financially supported by CSIR network project BSC-0120.R.P.D. and S.J. thank the CSIR for senior research fellowships.

We are grateful to Rita Kumar and Poornima Dhal for providing themicrobial facility in the Institute of Genomics and Integrative Biology. Weacknowledge Ashok Sahu (Advanced Instrumentation Research Facility,Jawaharlal Nehru University, Delhi, India) for help in the acquisition ofconfocal laser scanning microscopic images. V. Sabareesh and Richa Gul-eria are acknowledged for high-resolution electrospray ionization–massspectrometry data acquisition. We thank Qadar Pasha (Institute ofGenomics and Integrative Biology) and Pradeep Kumar (Institute ofGenomics and Integrative Biology) for their contributions in improvingthe manuscript. Finally, we are grateful to the reviewers for their frank andinsightful reviews, which significantly shaped the present article.

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