1. Published Ahead of Print 30 June 2014.
10.1128/AAC.03391-14.
2014, 58(9):5435. DOI:Antimicrob. Agents Chemother.
Pasha
Hemlata Gautam, Mohammed Shahar Yar and Santosh
Rikeshwer Prasad Dewangan, Seema Joshi, Shalini Kumari,
aureus
Methicillin-Resistant Staphylococcus
against Planktonic and Sessile
Spermine Backbone Dipeptidomimetics
N-Terminally Modified Linear and Branched
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3. 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 to
cause lipid clustering and segregation of domains, leading to bac-
terial cell death (10). It is difficult for bacteria to develop resistance
to HDCPs because most HDCPs kill bacterial cells quickly
through their actions on the entire cytoplasm, acting as pore
formers (11). HDCPs have been reported to efficiently eradicate
slow-growing cells from planktonic and biofilm cultures and thus
have been proposed as promising alternative agents for the cure of
biofilm-associated multidrug-resistant infections (12). However,
the challenges in the application of HDCPs have been their high
cost, protease instability, reduced activity in the presence of salts,
and poor bioavailability (13). Over the past decades, attempts
have 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 clinical
trials as antibacterial agents (14, 15).
The aim of the present study was to optimize our previously
designed N-terminally tagged dipeptide spermidine template
(16). Toward this goal, the roles of hydrophobicity and charge
distribution 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 to
eradicate clinically relevant MRSA biofilms have been deter-
mined.
MATERIALS AND METHODS
Chemicals. 9-Fluorenylmethoxy carbonyl (Fmoc)-protected amino acids
and 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 (catalog
no. 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) (catalog
no. 80826005001730) and 2-acetyldimedone (Dde-OH) (catalog no.
8.51015.0005) were purchased from Merck. All of the moieties used as
N-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 (catalog
no. 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 obtained
from 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 peptidomimetics
were synthesized on 2-chlorotrityl chloride resin as a solid support, as
described previously, with slight modifications (17). Briefly, on pre-
swelled resin, 5 eq of spermine dissolved in dichloromethane was added
under 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 primary
amino group of spermine was protected through overnight reaction with
2 eq of Dde-OH in DMF. After protection of the primary amino group,
the secondary amino groups were protected through reaction for 4 h with
6 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-protected
resin 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 a
procedure similar to that described above. Final deprotection of peptido-
mimetics from the resin in both series was performed using a cleavage
cocktail (DCM, TFA, ethanedithiol, triisopropylsilane, phenol, and water
in a ratio of 65:30:2:1:1:1). The solution was filtered, and cold ether was
added to the filtrate to precipitate the crude product, which was filtered
and washed with cold ether (2 ϫ 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 a
semipreparative column (7.8 by 300 mm, 125-Å pore size, 10-m particle
size) with a gradient of 10 to 90% buffer 2 over 45 min; buffer 1 was water
with 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 a
modified serial broth dilution method, as reported previously (19, 20).
The following bacterial strains were used in this study: S. aureus (ATCC
29213), methicillin-resistant S. aureus (ATCC 33591), Staphylococcus epi-
dermidis (ATCC 12228), methicillin-resistant S. epidermidis (ATCC
51625), Enterococcus faecalis (ATCC 7080), Escherichia coli (ATCC
11775), and Acinetobacter baumannii (ATCC 19606). The inocula were
prepared from mid-log-phase bacterial cultures. Each well of the first 11
columns of 96-well polypropylene microtiter plates was inoculated with
100 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 of
the microtiter plates. The microtiter plates were incubated overnight at
37°C, with agitation (200 rpm). After 18 h, absorbance was measured at
630 nm. Cultures without test peptidomimetics were used as positive
controls. Uninoculated MHB was used as a negative control. Tests were
carried out in duplicate on three different days. MIC was defined as the
lowest concentration of peptidomimetic that completely inhibited
growth. For comparison purposes, the standard peptide antibiotics VAN
and polymyxin B (PMB) were assayed under identical conditions. The
antibacterial activities of peptidomimetics and the standard antibiotic
VAN were evaluated against MRSA strain 33591 in the presence of 25%
(vol/vol) human serum and fetal bovine serum (FBS) in biofilm growth
medium (tryptone soy broth [TSB] supplemented with 0.5% NaCl and
0.25% glucose). A protocol similar to that described previously was used
(21). Briefly, MHB was adjusted to 25% (vol/vol) of a heat-inactivated
human 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 peptidomimetics
were evaluated with human red blood cells (hRBCs). Briefly, 100 l of a
fresh 4% (vol/vol) suspension of hRBCs in NaCl-Pi (35 mM phosphate
buffer [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
Dewangan et al.
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4. 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). Percent
hemolysis was calculated using the following formula: % hemolysis ϭ
100[(A Ϫ A0)/(At Ϫ A0)], where A represents the absorbance of sample
wells 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. Blood
from healthy human donors was collected in tubes containing the antico-
agulant sodium heparin, in accordance with institutional guidelines. The
blood was diluted 1:1 with NaCl-Pi (35 mM phosphate buffer, 150 mM
NaCl [pH 7.2]). Blood cells were separated over Histopaque separation
medium (Sigma-Aldrich) by centrifugation at 1,200 rpm for 30 min. The
peripheral blood mononuclear cells (PBMCs) were collected and washed
twice 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 by
trypan blue exclusion, with microscopic assessment. PBMCs (1 ϫ 106
cells/ml) in complete medium were seeded in a 24-well plate and left in the
incubator in 5% CO2 for 2 h at 37°C. The cells were then treated with
peptidomimetic 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 negative
control. 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 2,000
rpm 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 are
presented as mean Ϯ standard deviation (SD).
Tryptophan fluorescence. Small unilamellar vesicles (SUVs), which
were prepared following the standard method (20), were used for the
experiment. 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 membranes
or DPPC to mimic mammalian membranes were dissolved in a chloro-
form-methanol mixture in a 150-ml round-bottom flask. The solvent was
removed with a stream of nitrogen gas, to allow formation of a thin lipid
film on the walls of the glass vessel. The lipid film thus obtained was
lyophilized for 6 h to remove traces of solvent. Dried thin films were
resuspended in 10 mM Tris buffer (0.1 mM EDTA, 150 mM NaCl [pH
7.4]) preheated at 60°C, with vortex mixing. The lipid dispersions were
then 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 solutions
became opalescent. Titanium debris was removed by centrifugation. Each
peptidomimetic (final concentration, 5 g/ml) was added to 500 l of 10
mM Tris buffer (0.1 mM EDTA, 150 mM NaCl [pH 7.4]) or 0.5 g/ml
bacterial 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, and
the 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, and
DIPCDI in DCM-DMF (1:1), overnight; reaction 10, 30% TFA in DCM.
Membrane-Active Staphylocidal Peptidomimetics
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5. both excitation and emission. The experiment was repeated twice on the
same day, and representative data are presented here.
Membrane depolarization. The evaluation of membrane depolariza-
tion of MRSA was performed as described previously (16). Briefly, MRSA
that had been grown overnight was subcultured in MHB for 2 to 3 h at
37°C to obtain mid-log-phase cultures. The cells were centrifuged at 4,000
rpm for 10 min at 25°C, washed, and resuspended in respiration buffer (5
mM HEPES, 20 mM glucose [pH 7.4]) to obtain a diluted suspension of
optical density at 600 nm (OD600) of ϳ0.05. The membrane potential-
sensitive dye 3,3=-dipropylthiadicarbocyanine iodide (DiSC35) (0.18 M
in dimethyl sulfoxide [DMSO]) was added to 500-l aliquots of resus-
pended cells, and the mixtures were allowed to equilibrate for 1 h. Baseline
fluorescence was assessed using an Edinburg F900 spectrofluorometer,
with excitation at 622 nm and emission at 670 nm, in a cuvette with a 1-cm
path length. A bandwidth of 5 nm was employed for excitation and emis-
sion. Subsequently, increasing concentrations of test peptidomimetics
were added to the equilibrated cells, and the increase in fluorescence re-
sulting from dequenching of the DiSC35 dye was measured every 2 min, to
obtain the maximal depolarization. Increases in relative fluorescence
units (RFU) were plotted against increasing concentrations of different
peptidomimetics or PMB.
Bactericidal kinetics. The kinetics of bacterial killing of a MRSA strain
(ATCC 33591) by peptidomimetics at 2ϫ MIC and 4ϫ MIC were deter-
mined and compared with those of VAN as described previously (24).
Log-phase bacteria (1.2 ϫ 107
to 3.0 ϫ 107
CFU/ml) were incubated with
peptidomimetic 1c, peptidomimetic 1d, or VAN at 2ϫ MIC or 4ϫ MIC in
MHB. Aliquots were removed after 0.5, 1, 2, 3, and 6 h and diluted in
sterile normal saline solution before plating on Mueller-Hinton II agar;
CFU were counted after 24 h of incubation at 37°C. To decrease the limit
of detection, larger aliquots were removed and centrifuged to remove
antibacterial 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, samples
were prepared by following previously reported protocols (16, 25).
Briefly, freshly inoculated MRSA (ATCC 33591) was grown on MHB up
to an OD600 of ϳ0.5 (corresponding to 108
CFU/ml). Bacterial cells were
then centrifuged at 4,000 rpm for 15 min, washed three times with
NaCl-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 10ϫ MIC for 30 min. Controls were run in the
absence of antibacterial agents. After 30 min, the cells were centrifuged
and washed three times with NaCl-Pi. For cell fixation, the washed bacte-
rial pallet was resuspended in 0.5 ml of 2.5% paraformaldehyde in
NaCl-Pi and incubated overnight at 4°C. After fixation, cells were centri-
fuged, washed twice with 0.1 M sodium cacodylate buffer, and fixed with
1% osmium tetraoxide in 0.1 M sodium cacodylate buffer for 40 min at
room temperature (RT) in the dark. The samples were then dehydrated in
a series of graded ethanol solutions (30% to 100%) and finally dried in
desiccators under reduced pressure. Upon dehydration, the cells were air
dried for 15 min at RT in the dark after immersion in hexamethyldisi-
lazane. An automatic sputter coater (Quorum SC7640) was used to coat
the specimens with gold particles at a thickness of 30 Å. Then samples were
imaged 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) were
determined as described above. Bacterial suspensions (100 l) from du-
plicate wells at sub-MIC concentrations were then used to inoculate fresh
cultures. The cultures was grown to yield approximately 105
CFU/ml for
the next experiment. These bacterial suspensions were then incubated
with the desired concentrations of antibacterial agents for 18 h to deter-
mine new MICs. The same subculturing protocol was used for the next 16
passages, and MICs were determined using OD630 values as described
previously (23).
Biofilm susceptibility assay. For the biofilm inhibition assay, the
standard protocol was used as reported previously (26). Briefly, freshly
inoculated MRSA (ATCC 33591) was grown overnight on biofilm growth
medium (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 in
fresh biofilm growth medium. Two hundred microliters of diluted culture
was dispensed into wells of a 96-well polystyrene plate for biofilm forma-
tion. To evaluate the inhibition of biofilm formation, antibacterial agents
at the planktonic MIC in biofilm medium (MICb) and sub-MICb concen-
trations were added initially to diluted cultures following incubation at
37°C without shaking. Another set of experiments was performed with the
addition of fresh medium containing antibacterial agents at 10ϫ MICb
and 20ϫ MICb to 24-h-preformed biofilm, after gentle washing with ster-
ile NaCl-Pi (35 mM phosphate buffer, 150 mM NaCl [pH 7.4]). Biofilm
cultures 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 violet
assay), as follows.
For determination of metabolic activity, the plates were sonicated in a
ultrasonic bath (Elmasonic, Germany) for 5 min at 37°C, with sonication
at 30 kHz, to ensure detachment of bacteria from the biofilms before the
addition 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 the
percent reduction of alamarBlue (cell viability) was calculated by using a
formula provided in the manufacturer’s protocol. The experiment was
repeated three times on three different days, and results are given as
mean Ϯ SD.
For biomass quantification, the crystal violet (CV) staining protocol
was used as reported previously (27). Slime and adherent cells were fixed
for 20 min with 1 ml of 99% methanol and then stained for 20 min with
200 l of 0.1% crystal violet. Excess stain was removed by washing the
coverslips with NaCl-Pi, and then the coverslips were air dried. The
stained dye was redissolved with the addition of 33% acetic acid and
incubation for 1 h at room temperature without shaking. The optical
density at 570 nm (OD570) was measured spectrophotometrically, and
data are presented as percent biomass in comparison with the positive
control.
Confocal laser scanning microscopy of biofilms. For confocal mi-
croscopy, biofilm formation was induced on glass coverslips in a 6-well
plate, following a reported procedure (27). Briefly, overnight cultures
of 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 a
6-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 twice
with sterile NaCl-Pi and stained with reagent from the Molecular
Probes Live/Dead kit (Invitrogen, Eugene, OR), following the manu-
facturer’s instructions. This stain contains the DNA-binding dyes
SYTO 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 in
the SYTO 9 staining (green fluorescence). The biofilms were examined
with 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 measurement
of biofilm depths, z-stack images were acquired at approximately
0.4-m intervals, using a 100ϫ HCX PL APO oil immersion lens (nu-
merical aperture, 1.2); image analyses and export were performed with
FV10-ASW-1.7 software. For each sample, at least five different re-
gions on a single coverslip were scanned. The experiment was repeated
three times on three different days, and representative data are pre-
sented here.
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6. RESULTS
Rational design and synthesis of peptidomimetics. Antimicro-
bial peptidomimetics based on the defined pharmacophore with
at least ϩ2 charges at physiological pH and hydrophobicity have
been designed by various research groups (28, 29). Recently, we
reported a small series of potent peptidomimetics with broad-
spectrum antibacterial activity based on a template containing
N-terminally tagged dipeptidomimetics conjugated with spermi-
dine (16). In the present work, new N-terminal tags and cationic
spermine at the C terminus were conjugated to the same template
to 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 suitable
residue 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 the
relative hydrophobicity (Fig. 1). The peptidomimetics in series 2
(peptidomimetics 2a to 2f) were synthesized to investigate the
effects of Trp positioning on the spermine backbone on activity
and therapeutic index values. In peptidomimetics 2a to 2f, the
secondary N atoms of spermine were coupled with the carboxylic
acid end of Trp residues, leaving the alpha-amino group of Trp
residues ionizable at physiological pH (Fig. 1). All of the designed
peptidomimetics were Ͼ80% pure, and their masses were in the
range of 575 to 850 Da (Table 1).
Biological activities of designed peptidomimetics. The anti-
bacterial activities of the designed peptidomimetics against five
Gram-positive bacterial strains and two Gram-negative bacterial
strains were evaluated using the serial broth dilution method (Ta-
ble 2). The template peptidomimetic 1a showed moderate activity
against Gram-positive bacterial strains, while peptidomimetics 1b
to 1f displayed good activity against Gram-positive bacterial
strains, with MICs of Ͻ10 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, in
series 2, peptidomimetics 2a and 2b showed negligible growth
inhibition 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 0.8
to 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 inhibition
of 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 found
to cause minimal hemolysis up to the maximal concentration
TABLE 1 Purity, proportion of acetonitrile for RP-HPLC elution, and
molecular masses of designed peptidomimetics
Peptidomimetic Purity (%) Acetonitrile (%)a
Mass ([MϩH]ϩ
)
(Da)
Calculated Observed
1a 95 17.41 575.3816 575.3808
1b 99 46.42 737.4133 737.4139
1c 95 54.72 761.4109 761.4110
1d 95 61.57 729.5174 729.5178
1e 95 65.21 757.5487 757.5489
1f 98 70.36 837.6113 837.6097
2a 80 12.30 575.3816 575.3815
2b 80 44.34 737.4133 737.4140
2c 83 49.85 761.4109 761.4118
2d 99 57.92 729.5174 729.5181
2e 99 62.63 757.5487 757.5495
2f 99 69.78 837.6113 837.6113
a
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
(ATCC
29213)
MRSA
(ATCC
33591)
S. epidermidis
(ATCC
12228)
MRSEa
(ATCC
51625)
E. faecalis
(ATCC
7080)
E. coli
(ATCC
11775)
A. baumannii
(ATCC
19606)
1a 113.6 227.2 113.6 NDd
454.5 ND ND 4 ND
1b 3.5 7.1 3.5 7.1 113.6 14.2 ND 16 ND
1c 1.7 3.5 1.7 3.5 28.4 56.8 28.4 2 5.78
1d 1.7 1.7 1.7 1.7 3.5 14.2 113.6 9 17.5
1e 1.7 3.5 1.7 1.7 7.1 14.2 56.8 31 ND
1f 7.1 3.5 1.7 7.1 28.4 28.4 ND 30 ND
2a Ͼ454.4 Ͼ227.2 Ͼ454.4 227.2 ND Ͼ454.4 ND 0 ND
2b Ͼ454.4 454.4 ND ND ND Ͼ454.4 ND 5 ND
2c 14.2 28.4 7.1 14.2 ND 113.6 113.6 1 ND
2d 0.8 1.7 0.8 1.7 28.4 28.4 113.6 83 ND
2e 0.8 1.7 0.8 1.7 7.1 28.4 113.6 96 ND
2f 0.8 3.5 0.8 1.7 14.2 28.4 56.8 88 ND
PMB 14.2 28.4 7.1 28.4 113.6 0.4 ND ND ND
VAN 0.4 0.8 0.4 0.8 ND 113.6 56.8 ND ND
a
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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7. tested of 250 g/ml. Peptidomimetics 1e and 1f caused 31% and
30% hemolysis, respectively, at 250 g/ml. Peptidomimetics 2d,
2e, and 2f caused significant hemolysis, leading to 83%, 96%, and
88% damage to hRBCs, respectively, at 250 g/ml.
The antibacterial activities of nonhemolytic peptidomimetics
1c and 1d were also evaluated against MRSA in the presence of
25% (vol/vol) human serum or bovine serum. Fourfold and 8-fold
increases in MICs were observed for peptidomimetics 1c and 1d,
respectively, with human serum (Table 3).
The LDH release assay with PBMCs demonstrated 5.78% Ϯ
6.58% and 17.56% Ϯ 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% Ϯ 5.4% and 21.62% Ϯ 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 to
evaluate the effects of Trp positioning on the insertion depth of
designed peptidomimetics in bacterial and mammalian mimic
membranes. In buffer, all peptidomimetics showed fluorescence
emission maxima in the range of 356 to 362 nm (Table 4). In
bacterial mimic SUVs (DPPC-DPPG, 7:3 [wt/wt]), blue shifts in
emission maxima in the range of 5 to 12 nm, concomitant with
increases 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 nm
were 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 peptidomimetics
1a to 1f in both bacterial mimic and mammalian mimic mem-
branes. Noticeably, for peptidomimetics 2a to 2f in bacterial
mimic membranes, significant blue shifts (2 to 16 nm) were ob-
served subsequent to partitioning, and the concomitant increase
in emission intensity was not observed for peptidomimetics 2a, 2c,
and 2d, in comparison with buffer (see Fig. S3 in the supplemental
material).
Next, the ability of the designed peptidomimetics to compro-
mise the membrane potential in MRSA was evaluated by using the
membrane potential-sensitive dye DiSC35. Upon partitioning in
the membranes of live cells at sufficiently high concentrations,
DiSC35 self-quenches its fluorescence. Under the influence of a
membrane-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 in
relative 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 RFU
were observed up to the highest concentrations tested (Fig. 2).
Intermediate changes in fluorescence intensity were observed for
peptidomimetics 1d and 2d, whereas significant changes in RFU
were observed for peptidomimetics 1e, 1f, 2e, and 2f. The increases
in 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 was
repeated twice on two consecutive days, with similar results. Rep-
resentative results from one assay are presented here. Further-
more, interaction studies were performed with peptidomimetics
1c and 1d, which are active and cell-selective peptidomimetics
from 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 growing
S. aureus ATCC 33591 (Fig. 3). At 2ϫ MIC, both peptidomimetics
produced Ն3-log10 CFU/ml reductions within 3 h of incubation;
at 4ϫ MIC, bactericidal effects with Ͼ4-log10 CFU/ml reductions
within 30 min of incubation were observed. VAN did not show
TABLE 3 Effects of salt concentrations and serum on antibacterial
activities of compounds
Compound
MIC (g/ml) against MRSA 33591 in:
Biofilm
medium
(low salt)a
TSB with
high saltb
MHB with
human
serumc
MHB with
FBSd
Peptidomimetic 1c 7.1 28.4 14.1 7.1
Peptidomimetic 1d 3.5 7.1 14.1 7.1
VAN 0.8 1.7 0.8 0.8
a
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 designed
peptidomimetics in buffer, DPPC SUVs, or DPPC-DPPG SUVs
Peptidomimetic
Emission maximum (nm)a
Bufferb
DPPC
DPPC-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 with
the potential-sensitive dye DiSC35.
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8. any bactericidal activity even upon incubation at 4ϫ MIC under
the 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 as
a 3-log10 CFU/ml decrease, in comparison with the time zero
value.
Further, we visualized the effects on MRSA of 30-min incuba-
tions with peptidomimetic 1c, peptidomimetic 1d, and VAN, at
10ϫ 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, cell
bursting, 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 and
hole formation (Fig. 4C), indicating the membrane-active mode
of action for the designed peptidomimetics. Surprisingly, VAN-
treated cells mostly retained their smooth appearance, albeit with
slight 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-MIC
serial passages was evaluated (Fig. 5). Fourfold and 2-fold in-
creases in MIC values were observed for peptidomimetics 1c and
1d, respectively. The MIC of the standard antibiotic VAN was
increased 4-fold after 17 passages, whereas a radical 256-fold
change 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 further
evaluated the efficacy of peptidomimetics 1c and 1d to prevent the
formation of biofilms and to eradicate preformed MRSA biofilms
(24 h) by using alamarBlue as a redox indicator for assessment of
metabolic activity and crystal violet for biomass quantification. A
well-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, and
VAN in biofilm growth medium (TSB with 0.5% NaCl and 0.25%
glucose) were evaluated. The results showed pronounced effects of
a high salt concentration (supplemented with 3% NaCl) on MICs,
whereas 2-fold increases in the MICs of peptidomimetics 1c and
1d 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, initial
inocula were added with sub-MICb and MICb concentrations of
the tested agents (Fig. 6A and B). Peptidomimetics 1c and 1d were
able to inhibit biofilm formation at sub-MICb concentrations (ϳ4
g/ml and ϳ2 g/ml, respectively), causing reductions in meta-
bolic activity of up to 33.1% Ϯ 5.7% and 26.4% Ϯ 3.3%, respec-
tively. Under identical treatment conditions, biomass reductions
were found to be 19.8% Ϯ 5.6% and 28.2% Ϯ 11.1% for peptido-
mimetics 1c and 1d, respectively. At MICb, both peptidomimetics
were able to inhibit the adhesion of biofilm, causing Ͼ90% reduc-
tions in measured viability and biomass quantity. Metabolic ac-
tivity and biomass quantity were not reduced significantly with
VAN at sub-MICb concentrations (ϳ0.5 g/ml), compared with
control values, whereas VAN at MICb concentrations (ϳ1 g/ml)
inhibited biomass quantity to 27.4% Ϯ 1.3%.
The effects of peptidomimetics on the viability of 24-h-pre-
formed mature biofilms were also evaluated at concentrations
higher than MICb. At 20ϫ MICb, the designed peptidomimetics
1c (140 g/ml) and 1d (70 g/ml) showed better killing profiles
than did VAN (20 g/ml), showing 6.4% Ϯ 0.2% and 10.1% Ϯ
7.8% viable cells, respectively, versus 77.7% Ϯ 7.0% viable cells for
VAN at the indicated concentration (Fig. 6C).
In parallel with viability results, assessments of reductions in
the biomass quantities of 24-h mature MRSA biofilms showed a
reduction in biomass to 24.0% Ϯ 13.4% with peptidomimetic 1c
at 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 10ϫ MICb and 20ϫ MICb), i.e.,
66.7% Ϯ 8.2% and 21.4% Ϯ 9.2%, respectively. For VAN-treated
biofilms, the observed biomass quantities were 119.3% Ϯ 17.5%
at 10ϫ MICb (10 g/ml) and 83.7% Ϯ 24.1% at 20ϫ MICb (20
g/ml).
(ii) Visualization of effects of designed peptidomimetics on
biofilms using confocal laser scanning microscopy. We next vi-
sualized the effects of the designed peptidomimetics and VAN on
biofilm-embedded MRSA, making use of the membrane permea-
bility-sensitive, DNA-binding dyes SYTO 9 and propidium iodide
as 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 2ϫ MIC (A) or 4ϫ MIC (B) levels of peptidomimetic 1c, peptidomimetic 1d, or VAN
and 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 from
one of three independent experiments with similar results.
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9. The control biofilm (24 h) showed a lawn of viable (green) cells,
with an average thickness of 14.3 Ϯ 1.4 m (Fig. 7A; also see Table
S1 in the supplemental material). At MICb, peptidomimetics 1c
and 1d prevented the formation of biofilm; very few cells adhered
to the substratum, with observed average thicknesses of 3.9 Ϯ 1.1
m and 3.5 Ϯ 0.6 m, respectively. Furthermore, at sub-MICb
concentrations, the observed thicknesses were 5.2 Ϯ 0.3 m and
5.8 Ϯ 0.4 m, respectively (Fig. 7Ab and Ad). The measured bio-
film thickness was 11.4 Ϯ 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 lawn
of viable (green) cells with an average thickness of 23.6 Ϯ 2.5 m
(Fig. 7B; also see Table S2 in the supplemental material). Subse-
quent to treatment with peptidomimetics 1c and 1d at 10ϫ MICb,
there were visible decreases in the numbers of live cells and thick-
ness was reduced to 7.1 Ϯ 1.5 and 7.0 Ϯ 1.0 m, respectively (Fig.
7Bb and Bd). With peptidomimetics 1c and 1d, most of the cells
lost their integrity at 20ϫ 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, morphological
alterations produced. Insets, higher-magnification images (magnification, ϫ150,000).
FIG 5 Resistance development induced by antibacterial agents in S. aureus
(ATCC 33591) after 17 serial passages with sub-MIC levels of peptidomimetic
1c, peptidomimetic 1d, or antibiotic. The fold change in MIC is the ratio of the
MIC after 17 passages to the MIC before the first passage.
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10. ment, no significant differences in the numbers of live cells were
observed, inasmuch as mixed bacterial populations stained green
were visible at both tested concentrations. VAN had little effect on
24-h biofilm at 10ϫ MICb, with no distinction between control
biofilm and VAN-treated biofilms being visible. Only with VAN at
20ϫ MICb was a slight decrease in the height of mature biofilm
observed (Fig. 7Bf and Bg). The confocal imaging experiments
were repeated three times on three different days, and similar re-
sults were obtained (representative data from one set is shown
here).
DISCUSSION
Polyamines (putrescine, spermidine, and spermine) modulate
various processes in cells, including nucleic acid packaging, DNA
replication, transcription, and translation, and thus are required
for 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 polyamine
backbone 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 caffeoyl
polyamines, has been reported (15, 33, 37). Synergistic effects of
exogenous polyamines (added to growth medium) and various
antibiotics have also been investigated, and it was shown that 1
mM spermine caused up to 200-fold reductions in the MIC of
oxacillin 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 produces
no 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 MRSA
strain USA300 was ascribed to the arginine catabolic mobile ele-
ment (ACME), which harbors the spermidine acetyltransferase
gene (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 this
deadly strain.
Various valuable structure-activity relationships for antibacte-
rial peptidomimetics have been reported, and modifications in
charge 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 violet
staining assay (B). (C and D) Metabolic activity of 24-h mature biofilm-embedded MRSA using the alamarBlue assay (C) and biomass quantification using the
crystal 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 all
experiments, data are expressed as mean Ϯ SD. Statistical differences from control values were determined by one-way analysis of variance (ANOVA) with
Tukey’s multiple-comparison post hoc tests. All differences between the control and treated biofilms were considered statistically significant (P Ͻ 0.001).
Membrane-Active Staphylocidal Peptidomimetics
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11. molecules for therapeutic applications (41–44). Extending our
previous 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 the
effects on antibacterial activity and selectivity and the mode of
action. In our previous work, we established 50 to 70% hydropho-
bicity (based on reverse-phase [RP]-HPLC retention times) and at
least ϩ2 charges to be crucial for antibacterial activity and cell
selectivity (16). The comparative MIC data for series 1 and 2
showed that peptidomimetics 1b and 1c with linear arrangements
of 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 bacterial
strains tested. For lipidated N-tagged peptidomimetics, series 1
and 2 showed comparable inhibitory effects on all Gram-positive
bacterial strains; however, series 2 was more hemolytic than series
1, although the hydrophobicity ranges for the two series were the
same. As reported in the literature and observed in our previous
study, hydrophobicity above a threshold range plays a crucial role
in increased hemolytic activity (16). This finding also holds true in
the present study, since overall, the charges were the same and
differences in hydrophobicity among corresponding pairs in se-
ries 1 and 2 were not very significant (Ͻ0.6 to 6%, as measured by
RP-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, which
indicates 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 the
mode of action of these peptidomimetics with artificial mem-
branes and intact bacterial cells. Trp fluorescence measurements
have been used as a sensitive tool to probe the interactions of
peptides 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 was
observed 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 increases
in fluorescence intensity, compared with buffer (see Fig. S3 in the
supplemental material). Similar observations of blue shifts subse-
quent to peptide-lipid interactions without increases in emission
intensity were reported previously for the antimicrobial peptides
temporin L and nisin; the authors attributed the decreases in Trp
fluorescence intensity to quenching due to aggregation of peptide
in the vicinity of membranes or due to the quenching properties of
the 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 these
peptidomimetics led to better electrostatic interactions with the
negatively charged bacterial mimic membranes and vicinity-in-
duced aggregation, causing decreases in fluorescence intensity in
bacterial mimic membranes.
Further, the dependence of membrane depolarization ability
on 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 scanning
microscopy. (a) Control; (b) peptidomimetic 1c at sub-MICb level; (c) peptidomimetic 1c at MICb; (d) peptidomimetic 1d at sub-MICb level; (e) peptidomimetic
1d 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 using
confocal laser scanning microscopy. (a) Control; (b) peptidomimetic 1c at 10ϫ MICb; (c) peptidomimetic 1c at 20ϫ MICb; (d) peptidomimetic 1d at 10ϫ MICb;
(e) peptidomimetic 1d at 20ϫ MICb; (f) VAN at 10ϫ MICb; (g) VAN at 20ϫ MICb. After treatment at different concentrations, the biofilms were stained with
SYTO 9 (green; viable cells) and propidium iodide (red; dead cells), as described in the manufacturers’ protocol.
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12. results (Fig. 2). The untagged template peptidomimetics (ϩ4
charges and Ͻ20% hydrophobicity) were unable to alter mem-
brane potential up to the highest concentrations tested. Despite
good activity against MRSA, aromatic tagging in peptidomimetics
1c 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 of
hydrophobicity 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 membrane
potential than were less-hydrophobic peptidomimetics (peptido-
mimetics 1d and 2d). It was evident from Trp fluorescence and
membrane depolarization experiments that series 2 peptidomi-
metics with branched Trp residues on the spermine backbone
although more potent, were more prone to cause nondifferential
interactions, 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 killing
was studied by exposing MRSA to 2ϫ MIC and 4ϫ MIC levels of
peptidomimetics 1c and 1d (Fig. 3). At 4ϫ MIC, which is a ther-
apeutically relevant concentration, most of the bacteria were
killed within 30 min, as extremely rapid bactericidal effects are
often seen for antimicrobial peptides (20). The fast bacterial kill-
ing suggests that, at these concentrations, the antibacterial effects
are mediated through significant permeabilization or lysis of bac-
terial membranes, which was corroborated by scanning electron
microscopic images of MRSA showing distinct membrane dam-
age at 10ϫ MIC with greater numbers of bacteria (108
CFU/ml)
for both peptidomimetics (Fig. 4).
Several reports suggested that antimicrobial peptides and their
analogue peptidomimetics have novel membrane-active modes of
action 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 could
not 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 kill
MRSA 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 corroborated
the 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 with
RP-HPLC, and the data demonstrated that no degradation was
found for the peptidomimetics even with 72 h of incubation (see
Fig. S4 in the supplemental material). Peptidomimetics 1c and 1d
were further assessed for cytotoxicity against primary PBMCs and
demonstrated 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 particularly
virulent mechanism for Staphylococcus species that renders treat-
ment and cure difficult with invasion, with associated mortality
rates in severe cases of MRSA infections being about 20% (50).
Various strategies have been proposed to either kill microbes or
drive them out of biofilms. Among these strategies, targeting quo-
rum sensing and designing antiadhesion agents and antimicrobial
peptides 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 peptidomimetics
with membrane depolarization and disruption abilities against
biofilm-embedded MRSA (26, 53), we extended our studies
against 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 alamarBlue
assay (for measurement of viability) and crystal violet assay (for
quantification of biomass) (27). In the biofilm assay, no perfect
correlation 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 viability
and biomass than was VAN at 20ϫ MICb (Fig. 6C and D). Al-
though VAN showed better ability to inhibit growth in planktonic
cultures of MRSA under sessile conditions with 24-h mature bio-
films, the designed peptidomimetics 1c and 1d proved to be more
efficacious at the tested concentrations, exhibiting significant de-
creases in viability versus the positive control (P Ͻ 0.001).
Further, the effects of peptidomimetic treatment on biofilm
formation and killing were visualized using confocal microscopy,
which is a well-known method to assess such effects (26, 27). The
results revealed a marked difference in the viability of 24-h mature
biofilms with peptidomimetics 1c and 1d versus VAN (Fig. 7). For
VAN, a subpopulation of viable, predominantly green cells was
observed. 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 was
shown that VAN, even at a high concentration of 500 g/ml, was
unable to cause growth depletion of Staphylococcus haemolyticus
biofilms (27).
In summary, we designed new ultrashort N-terminally modi-
fied dipeptidomimetics with or without modifications on the
spermine backbone leading to linear or branched tryptophans,
which could effectively inhibit the growth of Gram-positive and
Gram-negative bacterial strains under planktonic conditions. Di-
rect effects of Trp positioning on the depth of insertion in artificial
membranes 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 for
series 2 peptidomimetics. We found the linear arrangement of Trp
residues without backbone spermine modification to be better for
therapeutically viable antibacterial peptidomimetics. Interest-
ingly, under identical experimental conditions, with the dual
modes 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 the
potential of membrane-active antibacterial peptidomimetics as
useful tools to eradicate clinically relevant biofilms. Overall, our
present work provides an impetus for the design of better mem-
brane-active, spermine-based, antibacterial peptidomimetics to
Membrane-Active Staphylocidal Peptidomimetics
September 2014 Volume 58 Number 9 aac.asm.org 5445
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13. treat recalcitrant biofilm communities of MRSA. At present, we
are exploring the ability of the most active peptidomimetics to
hamper biofilm formation on solid supports, which would
broaden the therapeutic applications of these peptidomimetics in
clinical 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 the
microbial facility in the Institute of Genomics and Integrative Biology. We
acknowledge Ashok Sahu (Advanced Instrumentation Research Facility,
Jawaharlal Nehru University, Delhi, India) for help in the acquisition of
confocal laser scanning microscopic images. V. Sabareesh and Richa Gul-
eria are acknowledged for high-resolution electrospray ionization–mass
spectrometry data acquisition. We thank Qadar Pasha (Institute of
Genomics and Integrative Biology) and Pradeep Kumar (Institute of
Genomics and Integrative Biology) for their contributions in improving
the manuscript. Finally, we are grateful to the reviewers for their frank and
insightful reviews, which significantly shaped the present article.
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