Rationale-based, de novo design of dehydrophenylalanine-containing antibiotic peptides and systematic modification in sequence for enhanced potency

Article abstract of DOI:10.1128/AAC.01493-10

Increased microbial drug resistance has generated a global requirement for new anti-infective agents. As part of an effort to develop new, low-molecular-mass peptide antibiotics, we used a rationale-based minimalist approach to design short, nonhemolytic, potent, and broad-spectrum antibiotic peptides with increased serum stability. These peptides were designed to attain an amphipathic structure in helical conformations. VS1 was used as the lead compound, and its properties were compared with three series of derivates obtained by (i) N-terminal amino acid addition, (ii) systematic Trp substitution, and (iii) peptide dendrimerization. The Trp substitution approach underlined the optimized sequence of VS2 in terms of potency, faster membrane permeation, and cost-effectiveness. VS2 (a variant of VS1 with two Trp substitutions) was found to exhibit good antimicrobial activity against both the Gram-negative Escherichia coli and the Gram-positive bacterium Staphylococcus aureus. It was also found to have noncytolytic activity and the ability to permeate and depolarize the bacterial membrane. Lysis of the bacterial cell wall and inner membrane by the peptide was confirmed by transmission electron microscopy. A combination of small size, the presence of unnatural amino acids, high antimicrobial activity, insignificant hemolysis, and proteolytic resistance provides fundamental information for the de novo design of an antimicrobial peptide useful for the management of infectious disease. Copyright

Full text of DOI:10.1128/AAC.01493-10

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 2011, p. 2178–2188
0066-4804/11/$12.00 doi:10.1128/AAC.01493-10
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 55, No. 5
Rationale-Based, De Novo Design of Dehydrophenylalanine-Containing
Antibiotic Peptides and Systematic Modification in Sequence
for Enhanced Potencyᰔ
Sarika Pathak and Virander Singh Chauhan*
International Centre for Genetic Engineering and Biotechnology, New Delhi, India 110067
Received 28 October 2010/Returned for modification 20 January 2011/Accepted 9 February 2011
Increased microbial drug resistance has generated a global requirement for new anti-infective agents. As
part of an effort to develop new, low-molecular-mass peptide antibiotics, we used a rationale-based minimalist
approach to design short, nonhemolytic, potent, and broad-spectrum antibiotic peptides with increased serum
stability. These peptides were designed to attain an amphipathic structure in helical conformations. VS1 was
used as the lead compound, and its properties were compared with three series of derivates obtained by (i)
N-terminal amino acid addition, (ii) systematic Trp substitution, and (iii) peptide dendrimerization. The Trp
substitution approach underlined the optimized sequence of VS2 in terms of potency, faster membrane
permeation, and cost-effectiveness. VS2 (a variant of VS1 with two Trp substitutions) was found to exhibit good
antimicrobial activity against both the Gram-negative Escherichia coli and the Gram-positive bacterium
Staphylococcus aureus. It was also found to have noncytolytic activity and the ability to permeate and depolarize
the bacterial membrane. Lysis of the bacterial cell wall and inner membrane by the peptide was confirmed by
transmission electron microscopy. A combination of small size, the presence of unnatural amino acids, high
antimicrobial activity, insignificant hemolysis, and proteolytic resistance provides fundamental information
for the de novo design of an antimicrobial peptide useful for the management of infectious disease.
The accelerated emergence of pathogenic bacteria resistant
to conventional antibiotics is a major threat today (1). There-
fore, the development of a new class of broad-spectrum anti-
biotics is an urgent need. Cationic antimicrobial peptides
(AMPs) could be one of the best possible alternatives (31).
AMPs are an important component of the natural defenses of
most living organisms against invading pathogens (12). Natu-
rally occurring AMPs are generally 12 to 50 amino acids in
length and are folded into several structural groups, including
helix, sheet, extended, and looped structures (43). Although
they show marked variability in length, amino acid composi-
tion, and structure, a majority of them share two common
features and functionally important characteristics, namely, a
net positive charge that facilitates interaction with negatively
charged microbial surfaces and the ability to form an amphi-
pathic secondary structure that permits incorporation into mi-
crobial membranes (38). Although the exact mechanism of
action of AMPs is still not completely understood, it has been
well established that AMPs interact with the cell membrane of
the susceptible microorganism, where either their accumula-
tion in the membrane causes increased permeability and loss of
barrier function or they enter the membrane to access cyto-
plasmic targets (4). Development of resistance against peptides
that act on the microbial membrane is unlikely, as it would
require substantial alterations in the lipid composition of the
cell membranes of the microorganisms (28).
and show toxicity to host cells (21). Moreover, due to their
peptidic nature, they suffer from poor bioavailability and poor
proteolytic stability (25). These features have significantly
hampered their pharmaceutical development as therapeutic
agents. Short designer AMPs with increased half-lives offer
excellent templates for future antibiotic drug design.
Different approaches are being followed in efforts to in-
crease the effectiveness of AMPs, including alteration of se-
quences, inclusion of unnatural or D-amino acids or beta-
amino acids, cyclization of peptides and peptoid mimics, and
synthesis of multivalent constructs of short peptides (9, 18, 27,
33, 37, 40). Short designer AMPs that are less likely to induce
resistance and that minimize damage to host cells or tissues
appear to be the most promising candidates.
In the present work, we focused on the use of a nonpro-
teinogenic amino acid, ␣,␤-dihydrophenylalanine (⌬Phe), in
designing relatively short antimicrobial peptides. The presence
of more than one ⌬Phe in peptides has been shown to con-
strain the peptide in a 310 or ␣-helical conformation, along with
providing enhanced resistance to enzymatic degradation com-
pared to their phenylalanine-containing counterparts (23, 29).
We used a de novo-designed prototype undecapeptide peptide
(VS1) incorporating ⌬Phe as a lead in an optimization strategy
to design three sets of peptides with the same basic motif,
where ⌬Phe is placed at two residue spaces. In order to identify
the most promising, cost-effective antibiotic peptide as a drug
candidate, the designed peptides were compared in terms of (i)
spectrum of activity, (ii) specificity, (iii) microbicidal proper-
ties, and (iv) membrane permeabilization.
However, most natural AMPs are large, have low potency,
* Corresponding author. Mailing address: International Centre for
Genetic Engineering and Biotechnology, New Delhi, India 110067.
Phone: 91-11-26741358. Fax: 91-11-26162316. E-mail: virander@icgeb
.res.in.
MATERIALS AND METHODS
Materials. Amino acid derivatives and resin for peptide synthesis were ob-
tained from Nova Biochem; diisopropylcarbodiimide (DIPCDI), piperidine, di-
ᰔ Published ahead of print on 14 February 2011.
2178

VOL. 55, 2011
DE NOVO DESIGN OF NOVEL ANTIMICROBIAL PEPTIDES
TABLE 1. Amino acid sequences of designed peptides
2179
Length
(residues)
MW
MW
Peptide
Amino acid sequence
Charge ⌬Phe Trp
(calculated) (observed)
VS1
Ac-K-A-⌬F-W-K-⌬F-V-K-⌬F-V-K-NH
11
ϩ4
3
1
1,466.8
1,468
Series I (analogs with variable
length)
VSL1
Ac-⌬F-K-A-⌬F-W-K-⌬F-V-K-⌬F-V-K-NH2
Ac-A-⌬F -K-A-⌬F-W-K-⌬F-V-K-⌬F-V-K-NH2
Ac-K-A-⌬F-K-A-⌬F-W-K-⌬F-V-K-⌬F-V-K-NH2
12
13
14
ϩ4
ϩ4
ϩ5
4
4
4
1
1
1
1,607.4
1,679.8
1,807.3
1,608
VSL2
1,679
VSL3
1,808.2
Series II (analogs with different
Trp contents)
VS2
Ac-K-W-⌬F-W-K-⌬F-V-K-⌬F-V-K-NH2
Ac-K-W-⌬F-W-K-⌬F-W-K-⌬F-V-K-NH2
Ac-K-W-⌬F-W-K-⌬F-W-K-⌬F-W-K-NH2
11
11
11
ϩ4
ϩ4
ϩ4
3
3
3
2
3
4
1,577.6
1,665.7
1,752.4
1,575.1
1,667
1,754.7
VS3
VS4
Series III (branched dimeric
analog)
VSD1
Ac-K-A-⌬F-W-K-⌬F-V-K-⌬F-V-K-NH{
Ac-K-A-⌬F-W-K-⌬F-V-K-⌬F-V-K-NH}
23
ϩ8
6
2
3,001
3,000.6
K
methyl formamide (DMF), dichloromethane (DCM), hydroxybenzotriazole
(HOBt), isobutylchloroformate (IBCF), trifluoroacetic acid (TFA), triisopropyl
silane (TIS), DL-threo-␤-phenylserine, sodium hydroxide, citric acid, 3-[4,5-
dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT), DiSC3-5 (3,3Ј-
dipropylthiadicarbocyanine) iodide, dimethyl sulfoxide (DMSO), bovine serum
albumin (BSA), and N-methyl morpholine (NMM) were from Sigma-Aldrich (St.
Louis, MO); sodium chloride, acetic anhydride, and tetrahydrofuran (THF) were
from Qualigens (Mumbai, India); ethyl acetate, diethyl ether, sodium acetate,
and sodium sulfate were from Merck (Mumbai, India); silica gel thin-layer
chromatography (TLC) plates (60F-254) were from Merck (Germany); acetic
acid was from SD Fine Chem Ltd. (Mumbai, India); acetonitrile was from
Burdick and Jackson (Muskegon, MI). and RPMI 1640 and fetal bovine serum
(FBS) were from Invitrogen (Carlsbad, CA).
Preparation of Fmoc-X-DL-threo-␤-phenylserine. Fmoc-X-DL-threo-␤-phenyl-
serine (where Fmoc is 9-fluorenylmethoxy carbonyl and X is Lys [Boc], Trp
[Boc], or Ala) was synthesized by a method of salt coupling using mixed anhy-
dride. Fmoc amino acid (15 mmol) dissolved in 15 ml of sodium-refluxed and
distilled THF was activated at Ϫ15°C for 10 min with IBCF and NMM (15 mmol
each). A solution of 15 mmol of DL-threo-␤-phenylserine made in 1 equivalent of
NaOH (15 ml) was added to the above-mentioned mixed anhydride, and the
reaction mixture was stirred at room temperature overnight. Following evapo-
ration of THF, citric acid was added to the aqueous solution to reach a solution
of pH 2.0. The precipitate obtained was dissolved in 100 ml ethyl acetate and
transferred to a separating funnel. Following the removal of the lower aqueous
layer, the ethyl acetate layer was washed extensively with water to remove the
citric acid. The complete removal of citric acid was confirmed by measuring the
pH. The ethyl acetate layer was further washed with brine and allowed to pass
through a bed of anhydrous sodium sulfate. Evaporation of ethyl acetate on a
rotary evaporator resulted in solid dipeptide acids.
Preparation of Fmoc-X-⌬Phe azalactone. Fmoc-X-DL-threo-␤-phenylserine
was mixed with recrystallized anhydrous sodium acetate (obtained by fusing the
salt and allowing it to cool in a desiccator) in freshly distilled acetic anhydride
and stirred overnight. The thick slurry obtained was mixed with ice and stirred at
8 to 10°C. Following trituration, the yellow dipeptide azalactone was filtered on
a sinter funnel and dried to constant weight. The authenticity and purity of the
azalactones were assessed by TLC, mass spectroscopy, and UV-visible spectros-
copy.
Peptide synthesis. Peptides were synthesized as C-terminal amides using stan-
dard Fmoc chemistry on rink amide MBHA (4-methylbenzhydrylamine hydro-
chloride salt) resin in the manual mode, with DIPCDI and HOBt as coupling
agents. Fmoc-Lys (Fmoc)-OH was used to make a branching core for the syn-
thesis of the lysine-branched dimer VSD1. Piperidine treatment of the lysine
derivative immobilized on the resin gave rise to two amino groups (␣ and ε),
allowing the synthesis of two identical peptide chains, as shown in Table 1. The
synthesis of the dendrimer VSD1 was accomplished on a K-K2 core generated by
coupling of Fmoc-Lys (Fmoc)-OH to the two amino groups of the lysine resin
synthesized as described above. The side chain protection used was Boc (Lys or
Trp). Couplings were carried out using DMF at a 4-fold molar excess at a final
concentration of ϳ500 mM. Removal of Fmoc was carried out using 20%
piperidine in DMF. Both the coupling of amino acids and the Fmoc deprotection
were monitored by the Kaiser test (16). ⌬Phe was introduced into peptides as an
Fmoc-X-⌬Phe azalactone (where X is Lys [Boc], Trp [Boc], or Ala) dipeptide
block (23), which was allowed to couple overnight in DMF. At the completion of
assembly of the peptides, following Fmoc removal, the amino termini were
acetylated using 20% acetic anhydride in DCM. After acetylation of the peptides,
the resin was washed extensively with DMF, DCM, and methanol and dried in a
desiccator under vacuum.
Synthesis of FITC-labeled peptides. To the free amino terminus of peptide
VS2 on resins, Fmoc-ε-aminohexanoic (Ahx) acid-OH was coupled using HOBt
and DIPCDI in DMF. Fmoc was removed by treatment with 20% piperidine-
DMF. The resin was washed with DMF and equilibrated in pyridine-DMF-DCM
(12:7:5 [vol/vol/vol]). A 1.1 equivalent of fluorescein isothiocyanate (FITC) in
pyridine-DMF-DCM (12:7:5 [vol/vol/vol]) was added to the resin and allowed to
couple overnight. An orange color on the resin and a negative Kaiser test
indicated coupling.
Cleavage of the peptides from resin. Peptides were cleaved by stirring the resin
in a cleavage mixture (95% TFA, 2.5% water, and 2.5% TIS) for 2 h at room
temperature. The suspension was filtered using a sinter funnel, the TFA was
rotary evaporated, and the peptide was precipitated by adding cold dry ether.
The ether was filtered through a sinter funnel, and the peptide on the funnel was
dissolved in 10% acetic acid and lyophilized.
Peptide purification and mass spectrometry. Crude peptides were purified by
reverse-phase high-performance liquid chromatography (RPHPLC) on a Delta-
pac C18 column (15-␮m inside diameter [i.d.], 300 by 19 mm) using an acetoni-
trile-water linear gradient of 5 to 65% acetonitrile (0.1% TFA)-water (0.1%
TFA) with a flow rate of 5 ml/min for 60 min, with detection at 214 and 280 nm.
The purified peptides were reinjected into an analytical reversed-phase C18
column (Phenomenex; C18; 5-␮m i.d; 250 by 4.6 mm) using an acetonitrile-water
linear gradient of 5 to 65% acetonitrile (0.1% TFA)-water (0.1% TFA) at a flow
rate of 1 ml/min over 60 min and were found to be 98% pure. The identity of the
purified (98%) peptides was confirmed by electrospray ionization mass spec-
trometry at the International Centre for Genetic Engineering and Biotechnology
(ICGEB), New Delhi, India.
Solubility measurements. Water was added to the purified peptide powders to
attain complete dissolution, and the concentration of the peptide in the spun
supernatant (13,000 rpm; 10 min) was determined by measurement of the ab-
sorbance at 280 nm. The extinction coefficient (ε) for ␣,␤-didehydrophenylala-
nine (⌬Phe) is 19,000 MϪ1 cmϪ1 (23) and for tryptophan is 5,050 MϪ1 cmϪ1
.
Antibiotic susceptibility testing. MICs against the Gram-negative bacterium
Escherichia coli ML35p and the Gram-positive bacterium Staphylococcus aureus
(ATCC 700699) were determined according to a modified MIC method for
cationic antimicrobial peptides (41). Bacterial cells grown overnight were diluted
in Mueller-Hinton (MH) broth to a density of 105 CFU/ml. One hundred mi-
croliters of this culture was aliquoted into the wells of a 96-well flat-bottom
microtiter plate (Costar), and 11 ␮l of stocks of each peptide (in 0.2% BSA and
0.01% acetic acid) was added. This mixture was incubated at 37°C in a rotary
shaker incubator (Kuhner, Switzerland) set at 200 rpm. After 18 h of incubation,
the optical density at 600 nm (OD600) was measured using a microtiter plate
reader (Versa Max tunable; Molecular Devices, Sunnyvale, CA). The MIC is
defined as the lowest concentration of a drug that inhibits the measurable growth

2180
PATHAK AND CHAUHAN
ANTIMICROB. AGENTS CHEMOTHER.
of an organism after overnight incubation. Peptide concentrations were deter-
mined spectrophotometrically at 280 nm (ε280, 19,000 MϪ1 cmϪ1 for ⌬Phe and
5,050 MϪ1 cmϪ1 for tryptophan). Each experiment was done in triplicate and was
repeated at least twice.
analysis; 25 ␮l of cells suspended in 470 ␮l of buffer with and without PI served
as controls.
Membrane depolarization assay (DiSC3-5) assay. The depolarization of the
cytoplasmic membrane of E. coli ML35p by the peptides was determined by a
modified version of the method of Wu et al. (42) using the membrane potential-
sensitive cyanine dye DiSC3-5. In this experiment, E. coli cells, in mid-exponential
phase (OD600, 0.35), were collected by centrifugation, washed once with buffer (5
mM HEPES, pH 7.2, 5 mM glucose), and resuspended to an OD600 of 0.05. The
cells were incubated with 1 ␮M DiSC3-5 for 2 h for maximal uptake of the dye,
after which 100 mM KCl was added to equilibrate the cytoplasmic and external
potassium ion concentrations. The cells were mixed with the desired concentra-
tion of peptide, and the fluorescence was monitored at an excitation wavelength
of 622 nm and an emission wavelength of 655 nm. Dye released with the addition
of 1% DMSO was monitored as a control.
Cellular uptake of peptide studied by confocal microscopy. E. coli ML35p cells
grown overnight were subcultured to an OD600 of 0.35 (1 ϫ 108 CFU/ml). The
cells were harvested by centrifugation (4,000 rpm for 10 min), washed, and
resuspended in 5 mM glucose-10 mM sodium phosphate buffer (pH 7.5) to yield
108 CFU/ml; 100 ␮l of E. coli suspension (108 CFU/ml) was mixed with 5 ␮l of
FITC-labeled peptide VS2 aqueous stock solution to yield a final peptide con-
centration of 5 ␮M (1ϫ MIC) and incubated (37°C; 200 rpm) for 30 min in a
Kuhner rotary shaker incubator. The cells were harvested by centrifugation
(4,000 rpm for 10 min) and washed twice with 5 mM glucose in 10 mM sodium
phosphate buffer (pH 7.5). A smear was made on a poly-L-lysine-coated slide.
For confocal microscopy, confocal laser scanning (Radiance 2100; Bio-Rad)
under a Nikon microscope (objective plane Apo 60ϫ/1.4 oil) was used to observe
the slide. The excitation wavelength for FITC was 494 nm (argon laser), and
fluorescence was detected through an HQ 515/30 emission filter (high-quality
band-pass). Image processing was conducted with Lazer Sharp (Bio-Rad), and
Photoshop 6.0 (Adobe Systems, San Jose, CA) was used for the final image
assembly.
Examination of bacterial-membrane damage by electron microscopy. (i) Scan-
ning electron microscopy (SEM). Overnight-grown cells were subcultured to an
OD600 of 0.35. The cells were harvested (4,000 rpm; 10 min; 4°C), washed, and
resuspended in HEPES buffer, pH 7.2, to obtain 106 CFU/ml. Fifteen microliters
of E. coli suspension (106 CFU/ml) was incubated for 30 min and 60 min in 135
␮l of 5 mM HEPES buffer, pH 7.2, containing the peptide at its MIC (5 ␮M).
The cells were spun down (4,000 rpm; 4°C; 10 min) and washed three times in 0.1
M phosphate buffer, pH 7.4. The cells were fixed in 2.5% glutaraldehyde buffered
with 0.1 M phosphate buffer, pH 7.4, at 4°C for 2 h. They were further washed
in 0.1 M phosphate buffer, postfixed with 1% OSO4, and dehydrated with graded
ethanol. The sample was dried with hexamethyldisilizane (HMDS) and coated
with gold (15 nm). Observations were made on a Zeiss EV040 scanning micro-
scope at the Jawaharlal Nehru University, New Delhi, India.
Hemolytic-activity testing. Human blood in 10% citrate phosphate dextrose
was obtained from the Rotary Blood Bank, New Delhi, India. Red blood cells
(RBCs) were harvested by spinning (1,000 ϫ g; 5 min; room temperature). They
were washed three to five times with phosphate-buffered saline (PBS). The
packed cell volume obtained was used to make a 0.8% (vol/vol) suspension in
PBS; 100 ␮l of this RBC suspension was transferred to each well of a 96-well
microtiter plate and mixed with 100 ␮l of peptide solution at twice the desired
concentration. The microtiter plate was incubated (37°C; 60 min) and centri-
fuged (1,000 ϫ g; 5 min; room temperature). The supernatant (100 ␮l) was
transferred to new wells, and the OD414 was measured with a microtiter plate
reader (Versa Max tunable; Molecular Devices, Sunnyvale, CA) to monitor RBC
lysis. Cells incubated with PBS alone served as the negative control, and RBCs
lysed using 0.1% Triton X-100 were used to measure 100% lysis (positive con-
trol).
Mammalian-cell cytotoxicity. The cytotoxicity of the antibiotic peptides was
determined using an MTT assay against HeLa cells. Briefly, cells (5 ϫ 104/well)
were cultured at 37°C overnight in RPMI 1640 containing 10% fetal bovine
serum in 96-well microtiter plates. The next day, peptides (prepared in RPMI
1640) at 1ϫ and 5ϫ MIC were added to the cells and incubated for 18 h at 37°C.
Ten percent DMSO was taken as the positive control, and untreated cells served
as the negative control; 20 ␮l of MTT solution (5 mg/ml) in PBS was added, and
the cells were incubated (37°C; 3 to 4 h). Supernatant (120 ␮l) was removed,
DMSO (100 ␮l) was added, and the resulting suspension was mixed to dissolve
the formazan crystals formed by MTT reduction. The ratio of the OD570 for
treated cells to the OD570 for untreated cells was used to calculate percent
viability.
Proteolytic stability of peptides to trypsin and chymotrypsin. The proteolytic
stability of the peptide VS1 was compared with that of its phenylalanine analog
(Ac-KAFWKFVKFVK-NH2) using reversed-phase HPLC. Peptide VS1 and
trypsin/chymotrypsin, taken at a ratio of 100:0.5 (mol/mol) in 0.1 M ammonium
bicarbonate, 0.1 mM CaCl2, pH 8.3, were incubated in a rotary shaker incubator
(37°C; 200 rpm; 30 min). An aliquot was injected into a reverse-phase analytical
C
18 column (Phenomenex; C18; 5-␮m i.d.; 250 by 4.6 mm), using an acetonitrile-
water linear gradient of 5 to 65% acetonitrile (0.1% TFA)-water (0.1% TFA) at
a flow rate of 1 ml/min over 65 min. An identically treated control peptide
(phenylalanine analog) sample was also injected into a reverse-phase analytical
C18 column using an acetonitrile-water linear gradient of 5 to 65% acetonitrile
(0.1% TFA)-water (0.1% TFA) at a flow rate of 1 ml/min over 65 min. For the
variable time-dependent assay, peptides were incubated with enzyme for 0, 1.5,
and 2.5 h, respectively.
Outer membrane permeabilization (NPN) assay. The membrane permeabili-
zation activity of the peptides was determined by the 1-N-phenylnaphthylamine
(NPN) assay of Loh et al. (20). In overview, an overnight culture of E. coli
ML35p was diluted in MH broth and grown to an OD600 of 0.5 to 0.6. The cells
were harvested, washed, and resuspended in the same volume of buffer (5 mM
HEPES [pH 7.2], 5 mM glucose). For the NPN assay, 2 ml of cells and 10 ␮M
NPN were mixed, and the fluorescence was measured with a fluorescence spec-
trophotometer (excitation wavelength, 350 nm; emission wavelength, 420 nm).
The increase in fluorescence due to partitioning of NPN into the outer mem-
brane (OM) was measured by the addition of various concentrations of peptide
(0.25 ␮M to 2 ␮M). Polymyxin B (PMB) was taken as a positive control. All
experiments were performed three times, and the trends that were observed were
reproducible.
Inner membrane permeabilization assay (PI and Syto9 uptake-based assay).
Overnight-grown E. coli ML 35p cells were subcultured to an OD600 of 0.35. The
cells were harvested (4,000 rpm; 10 min; 4°C), washed, and resuspended in buffer
(5 mM glucose in 10 mM sodium phosphate buffer, pH 7.5) to 108 CFU/ml.
Fifteen microliters of E. coli suspension (108 CFU/ml) was incubated (37°C; 200
rpm) in 135 ␮l of buffer (5 mM glucose, 5 mM HEPES, pH 7.2) containing
peptides at 1ϫ MIC for 90 min. The samples were then incubated with pro-
pidium iodide (PI) (2.7 ␮M) and Syto9 (6 ␮M) for 15 min. A smear was made,
heat fixed, and visualized under a Nikon fluorescence microscope. Cells without
peptide treatment served as a control for the experiment.
(ii) Transmission electron microscopy (TEM). A sample containing E. coli
(1 ϫ 106 CFU/ml) in Mueller-Hinton medium was incubated for 1 h with peptide
(VS2). The peptide was taken at its MIC value (5 ␮M). After being centrifuged
at 3,000 ϫ g for 10 min, the pellets were washed with 0.1 M phosphate buffer and
resuspended in the same buffer. E. coli cells were fixed with 2% glutaraldehyde
in 0.1 M phosphate buffer for 1 h at room temperature (20°C). The cells were
washed with 0.1 M phosphate buffer (pH 7.2) and postfixed with 1% OSO4 in 0.1
M phosphate buffer for 1 h at 4°C. For ultrastructure study, samples were
dehydrated with graded acetone, cleared with toluene, infiltrated with a toluene
and araldite mixture at room temperature and then finally in pure araldite at
50°C, and embedded in an Eppendorff tube (1.5 ml) with a pure araldite mixture
at 60o. Ultrathin-section cutting was done with an ultramicrotome (Ultrami-
cotome Lecia EM UC6). Sections were taken on a 3.05-mm-diameter 200-mesh
copper grid and stained with uranyl acetate and lead acetate. The grids were
examined under a transmission electron microscope (JEOL 2100F).
RESULTS
Rationale-based design of prototype peptide antibiotic. Ab
initio and de novo designing resulted in a short, cationic, am-
phipathic undecapeptide, VS1 (Ac-K-A-⌬F-W-K-⌬F-V-K-⌬F-
V-K-CONH2), where Ac is an acetyl group. In order to allow
initial electrostatic interaction with the negatively charged bac-
terial membrane, VS1 harbored four lysine residues on its
hydrophilic face. An excessive charge has been shown to have
a deleterious effect on activity (because it prevents structur-
FACS-based analysis for PI uptake. Overnight-grown cells were subcultured
to an OD600 of 0.35. The cells were harvested (4,000 rpm; 10 min; 4°C), washed,
and resuspended in buffer (5 mM glucose, 5 mM HEPES, pH 7.2) to 10 CFU/ml.
Twenty-five microliters of E. coli suspension (108 CFU/ml) was incubated in 480
␮l of buffer containing peptide and 5 ␮l of PI (1 mg/ml) for 15 min at room
temperature and then subjected to fluorescence-activated cell sorter (FACS)

VOL. 55, 2011
DE NOVO DESIGN OF NOVEL ANTIMICROBIAL PEPTIDES
2181
FIG. 1. (A) De novo design of antimicrobial peptides: prototypic, amphipathic, cationic undecapeptide sequence in 310 helical-wheel config-
uration. (B) Chemical structure of ⌬Phe.
ing), and the optimal charge for maximal antimicrobial activity
has been shown to be ϩ4 (10). Our idea to keep the ϩ4 charge
was derived from such studies. Since, valine is the amino acid
most commonly found in natural, as well as synthetic, antimi-
crobial peptides, two valine residues were incorporated on the
hydrophobic face to maintain amphipathicity (3). A single
alanine at the N-terminal hydrophobic face was incorporated
to reduce peptide hydrophobicity while maintaining its helicity.
Abiotic residues (⌬Phe) were placed 2 residues apart. In pep-
tides, such placements of ⌬Phe have been shown to induce a
helical conformation (22). Taking a cue from the membrane-
active properties of Trp, a single Trp was also incorporated in
the prototype peptide VS1. The helical-wheel representation
in Fig. 1A shows the segregation of polar and apolar faces of a
310 helix.
in a 5-fold increase in potency against E. coli (MIC, 5 ␮M for
VSL1, VSL2, and VSL3); however, there was no augmentation
in activity against the Gram-positive bacterium S. aureus.
Moreover, in VSL3, even the combined effect of additional
positive charge (ϩ5) and increased length of the peptide was
unable to broaden the spectrum of antibiotic activity. However,
in the second series of peptides, sequential inclusion of Trp
residues in VS1 resulted in substantial enhancement of the
antimicrobial activity of the peptides. Replacement of Ala by
Trp in VS1 led to VS2, which exhibited a 5-fold increase in
activity against E. coli (MIC, 5 ␮M). This replacement also
resulted in moderate activity against S. aureus (MIC, 50 ␮M).
VS3, where Ala and Val in the VS1 sequence were replaced
with two Trp residues, was equipotent with VS2 against E. coli
but showed enhanced activity against S. aureus (MIC, 25 ␮M).
Systematic modifications in the prototype peptide sequence.
Using the basic template of VS1 (11 residues; ϩ4), systematic
variations in the sequence resulted in three sets of peptides.
The first set included three peptides, VSL1 (12 residues; ϩ4),
VSL2 (13 residues; ϩ4), and VSL3 (14 residues; ϩ5). These
peptides were identical to VS1 up to 11 residues from the C
terminus and differed only in their N-terminal extensions. The
second set of peptides were Trp analogs of VS1: VS2 (two
Trp), VS3 (three Trp), and VS4 (four Trp). They were iden-
tical to VS1 in length, charge, and number of ⌬Phe residues
and differed only in their Trp contents. The third approach
resulted in a single peptide dimer of VS1; a dendrimer, VSD1,
consisted of two VS1 peptide sequences on a core lysine resi-
due. Thus, the branched dimer had a net charge of ϩ8 (ϩ4 per
chain). The physicochemical properties, such as length, charge,
and number of ⌬Phe and Trp residues, of the designed pep-
tides are provided in Table 1.
TABLE 2. MICs of peptide analogs against Gram-negative (E. coli
ML35p) and Gram-positive (S. aureus 700699) bacteria
MIC (␮M)b
Peptidea
S. aureus
E. coli ML35p
(ATCC 700699)
VS1
25
Ͼ50
Series I
VSL1
VSL2
VSL3
5
5
5
50
Ͼ50
Ͼ50
Series II
VS2
5
5
5
50
25
10
VS3
VS4
Effects of increasing length, Trp substitution, and dendrim-
erization of VS1 on antibiotic activity and cell selectivity. (i)
Antibacterial activity. The antibacterial activities of VS1 and
its analogs against E. coli ML35p and S. aureus are shown in
Table 2. The prototype peptide VS1 showed moderate potency
(MIC for E. coli, 25 ␮M), a narrow spectrum of activity (MIC
for S. aureus, Ͼ50 ␮M), and no hemolysis of RBCs or toxicity
to HeLa cells. An increase in the chain length of VS1 resulted
Series III
VSD1
1
5
4
5
1
Rifampin
Kanamycin
7.5
a Rifampin and kanamycin were taken as reference antibiotics.
b MICs are shown as the geometric means of three sets of determinations.

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FIG. 3. (A and B) Circular-dichroism-based conformational anal-
ysis of designed peptides (25 ␮M each) in 20 mM SDS-10 mM sodium
phosphate buffer, pH 7.5. (B) Histogram showing mean residue ellip-
ticity (MRE) values for the peptides (for the 267-nm band).
FIG. 2. (A) Percent hemolysis caused by peptides. Peptide antibi-
otics at MIC and 5ϫ MIC (E. coli) were incubated with 0.4% RBCs in
PBS. The results are expressed as percent hemolysis. RBCs incubated
with 0.1% Triton X-100 were considered to be 100% lysed. Standard
deviations from three observations are plotted. (B) Mammalian-cell
cytotoxicity (MTT assay) of peptide antibiotics at MIC and 5ϫ MIC
(E. coli) against HeLa cell lines. Test samples were incubated with cells
for 24 h in RPMI 1640. Untreated cells served as a negative control.
The ratio of the OD570 for peptide-treated cells to the OD570 for
untreated cells was used to calculate the percent viability of the cells.
Standard deviations from three observations are plotted.
trations, the peptides VSL2, VSL3, VS2, and VS3 showed
some degree of hemolysis and cytotoxicity. VSL2 was ϳ15%
hemolytic and 50% toxic to HeLa cells, whereas VSL3 was
ϳ13% hemolytic and 55% toxic at 5ϫ MIC values. VS2
showed 12% hemolysis and 30% cytotoxicity, and VS3 showed
19% hemolysis and 40% toxicity to HeLa cells at 5ϫ MIC
values. VSL1 was exceptional; it was nonhemolytic and cyto-
toxic at the MIC but showed 75% hemolysis and 60% cytotox-
icity at 5ϫ MIC values (Fig. 2A and B). These results indicated
that out of all the peptides, VS2 provided the right balance
between antimicrobial activity and hemolytic activity and was
chosen for further studies.
Circular-dichroism-based structures of peptides. Circular-
dichroism (CD) studies showed that all the peptides acquired
helical structures in a membrane-mimetic environment of 20
mM SDS (the critical micelle concentration for SDS is 8 mM)
(Fig. 3A). All the peptides exhibited an excitonic couplet at 267
nm (ϩ) and 298 nm (Ϫ), which is the signature for the forma-
tion of a right-handed 310 helix in ⌬Phe-containing peptides
(23). However, different degrees of helicity were observed for
the different peptides, and a particular trend was observed in
each series of peptides, as shown in Fig. 3B. In the first series,
increasing the lengths of the peptides resulted in increases in
VS4 (four Trp substitutions in VS1) was also equipotent with
VS2 and VS3 against the Gram-negative E. coli (MIC, 5 ␮M)
but showed further enhancement in activity against the Gram-
positive S. aureus (MIC, 10 ␮M). The dendrimer VSD1
showed excellent antimicrobial activity against both E. coli
(MIC, 1 ␮M) and S. aureus (MIC, 5 ␮M).
(ii) Cell selectivity (hemolysis and toxicity to HeLa cells).
None of the designed peptides were hemolytic or cytotoxic at
their MICs, except VS3 (negligible hemolysis at 5 ␮M, the
MIC for E. coli), VS4 (ϳ60% hemolysis at 5 ␮M, the MIC for
E. coli), and VSD1 (ϳ10% at 1 ␮M, the MIC for E. coli). At
these concentrations, none of the peptides showed any toxicity
to HeLa cells. However, we observed that at 5ϫ MIC values,
which are much higher than physiologically relevant concen-

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2183
FIG. 4. (A and B) HPLC profile of VS1 at 0 and 2.5 h of incubation with trypsin/chymotrypsin. (C and D) HPLC profile of Phe analog at 0
and 2.5 h of incubation with trypsin/chymotrypsin.
their helicities (measured in terms of the intensity of the
ϩ267-nm band), with the only exception being VSL2, which
showed no increase in helicity with increasing length. However,
an interesting and reverse trend was observed in the second
series of peptides, where the helical intensity (the intensity of
the ϩ267-nm band) decreased with increasing Trp content.
VSD1, from the third set, showed the highest molar ellipticity
value of the helix-associated excitonic couplet. The helicity in
VSD1 resulted from the combined helicities of two helical
peptides (VS1).
a hydrophobic environment, like the interior of a membrane,
and weakly in an aqueous environment (20). Normally, the
outer membrane of a bacterial cell is impermeable to NPN.
However, permeabilization of the outer membrane by cationic
peptides allows NPN to enter the hydrophobic environment of
the membrane, resulting in enhanced fluorescence of the NPN.
Figure 5 shows that the cationic peptides were able to perme-
Proteolytic stability of peptides. Peptide stability is one of
the most important parameters in the development of peptide
therapeutics (35). In order to determine the relative stability of
the ⌬Phe-containing prototype peptide VS1, its analog con-
taining phenylalanine (Phe) instead of ⌬Phe was synthesized,
and both were treated with trypsin/chymotrypsin under iden-
tical conditions. Analysis by RPHPLC showed that after 1 h,
the saturated (Phe) analog (peak at 38 min) disappeared, giv-
ing rise to two small peaks at 14 min and 25 min. On the other
hand, VS1 containing ⌬Phe was found to be stable to proteo-
lytic degradation; there were no observed changes in the re-
tention time (39 min) and peak intensity of VS1 after 2.5 h, as
shown in Fig. 4.
Increased bacterial-membrane permeabilization and mem-
brane depolarization caused by peptides. (i) Outer membrane
permeabilization assay (N-napthylphenyl amine assay). The
abilities of peptides to destabilize the outer membrane were
judged by the permeabilization of the outer membrane. NPN is
a hydrophobic fluorescent molecule that fluoresces strongly in
FIG. 5. Outer membrane permeabilization (NPN assay) by pep-
tide. Polymyxin B was taken as a positive control. A dose-dependent
increase in the fluorescence intensity was observed upon addition of
peptides at concentrations much lower (0.25 to 2 ␮M) than their MICs.

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PATHAK AND CHAUHAN
ANTIMICROB. AGENTS CHEMOTHER.
abilize the bacterial membrane at concentrations of 0.25 ␮M to
2 ␮M, which is well below their MICs. VS4 and VSD1 showed
maximum increase in NPN fluorescence at 2 ␮M. Indeed, for
VS4 and VSD1 at 2 ␮M concentration, the increase in fluo-
rescence was much higher than for polymyxin B at the same
concentration (2 ␮M), which was taken as the positive control.
PMB was taken as a control because it is a bactericidal anti-
biotic that kills almost all Gram-negative bacteria at rather low
concentrations. Moreover, it has been shown to permeate the
OM of E. coli, changing the packing order of lipopolysaccha-
rides (LPS) and increasing the permeability of the OM to a
variety of molecules, including its own uptake (self-promoted
uptake) (6). In addition, it is a decapeptide antibiotic with a net
positive charge (ϩ5) and a molecular mass of 1,200 Da (com-
parable to the sizes and charges of the designed peptides).
Outer membrane permeabilization by these peptides was re-
markable, as only very few peptides are able to permeabilize
membranes at such low concentrations (2).
(ii) Cytoplasmic membrane permeation (PI and Syto9 up-
take by cells). Florescence microscopic studies on cytoplasmic
membrane permeabilization by the designed peptides is shown
in Fig. 6A. PI is a membrane-impermeable red fluorophore
that enters only those cells that have disrupted cell membranes
and shows enhanced fluorescence upon binding to DNA. The
fluorescence emission maximum for DNA-bound PI is about
615 to 620 nm when excited by a 488-nm laser. Thus, cells with
disrupted membranes appear red when excited by the PI filter,
whereas the cells with intact membranes do not show any
fluorescence when excited using a PI filter. On the other hand,
Syto9 is a cell-permeant nucleic acid stain. Its absorption and
emission maxima are 485 to 486 nm and 498 to 501 nm, re-
spectively. It has permeability to virtually all cell membranes
(living or dead), including bacterial and mammalian cells. By
differential staining of cells with Syto9 and PI, we were able to
compare cells with disrupted membranes to the total number
of cells. Comparison of PI and Syto9 fluorescence shows that
peptides were able to permeabilize almost all bacterial cell
membranes at their respective MICs after 90 min. Figure 6A
shows that there was no significant difference between the
Syto9 (green) and PI (red) fluorescence levels in the peptide-
treated cells. However, cells without any peptide treatment
showed only Syto9 fluorescence and no PI florescence. Since
these cells have intact membranes, PI was unable to permeate
the cells.
dye is released, and the increase in fluorescence can be mon-
itored over time. Figure 7 shows disruption of the bacterial
membrane potential by VS2 and DMSO (positive control).
DMSO increases the membrane permeability and exerts a
marked inhibitory effect on a wide range of bacteria and fungi.
The graph clearly demonstrates that, similar to DMSO, VS2
also efficiently disturbed the potential gradient across the bac-
terial membrane in a dose-dependent manner.
(v) Confocal microscopic analysis of uptake of FITC-labeled
VS2 peptide. Z-sectioned confocal images of cells treated with
FITC-VS2 for 30 min showed the presence of the peptide in
the intracellular milieu of E. coli (Fig. 8). This was an inter-
esting observation, as it suggested that although the primary
target for the activity of VS2 was the bacterial membrane, it
might also have some intracellular targets, and the bacterial
killing by VS2 might be the result of the combined effects of
the two independent activities.
(vi) Examination of bacterial lysis by electron microscopy.
The effect of VS2 on the morphology of peptide-treated E. coli
was investigated by scanning electron microscopy. A change in
the cell morphology was observed. Increased roughness of the
cell wall was observed after 30 min of incubation, while cell
shrinkage was observed after 60 min of incubation with the
peptides (Fig, 9, left).
In TEM micrographs, VS2 appeared to have the most severe
effect on the bacterial cell wall and cell membrane. Cell wall
breakage and variability in wall thickness was observed after 30
min. Separation of the cytoplasmic membrane from the cell
wall was also observed. More severe effects were observed
after 60 min, when bacterial cell lysis due to membrane dam-
age was clearly visible. E. coli cells without any peptide served
as a control (Fig, 9, right).
DISCUSSION
The aim of our work was to identify candidates for devel-
oping novel potent and cost-effective antibiotics, starting with
a short cationic AMP as a template and then systematically
engineering its structure to enhance the degree and spectrum
of activity without imparting excessive hemolytic activity to the
peptide. Here, we describe our experimental results related to
the effectiveness of the three series of peptides that were de-
signed.
The key element in the design of the lead template 11-
residue peptide were three ⌬Phe residues, four lysine residues,
and one tryptophan, along with valine and alanine residues.
The three ⌬Phe residues were separated by two amino acid
residues, an arrangement that induces 310 helical structures in
peptides (22). Four lysine residues in such an arrangement
were expected to form a polar face, while ⌬Phe residues align
themselves to form a hydrophobic face, resulting in an amphi-
pathic helical structure. A tryptophan residue was included in
the design because of its known membrane-active properties
and association with antibacterial activity in peptides like in-
dolicidin and tritrypticin (24, 36). Peptides varying from 10 to
12 residues have shown significant antimicrobial activity (15).
Previous reports from our laboratory have demonstrated anti-
microbial activity of ⌬Phe-containing decapeptides (8).
Although increased length is often associated with enhanced
activity, there are no set rules that correlate length with the
(iii) A FACS-based PI uptake assay was used to study the
kinetics of inner membrane permeabilization by four selected
peptides (VS1, VS2, VSL1, and VSD1). As shown in Fig. 6B,
VS1 and VSL1 were very slow in inducing membrane permea-
bilization. At their respective MICs (25 ␮M and 5 ␮M), VS1
and VSL1 were able to permeabilize only ϳ10% of E. coli cells,
whereas VS2 showed very fast kinetics of inner membrane
permeabilization. At its MIC, it permeabilized ϳ96% of cells
in 10 min. However, VSD1 was even faster in action: they
permeabilized ϳ98% of cells in 5 min, but were hemolytic.
(iv) Membrane depolarization (DiSC3-5) assay. The dye
DiSC3-5 (3,3Ј-dipropylthiadicarbocyanine iodide) is known to
be distributed between bacterial cells and the surrounding me-
dium, depending on the membrane potential gradient. Once
inside the membrane, the dye aggregates and self-quenches.
With the addition of a membrane-permeabilizing agent, the

FIG. 6. (A) Fluorescence microscopy of peptide-induced permeability of E. coli ML35p cells visualized by Syto9 and PI staining at 90 min. After
90 min, all the peptide-treated cells had increased membrane permeability, as seen by PI (red) fluorescence, except control cells without peptide
treatment. On the other hand, Syto9 stained all the bacterial cells (live, dead, and membrane permeabilized) nonspecifically to give green
fluorescence. Peptides were taken at their respective MICs. (B) Kinetics of PI uptake as studied by FACS for four peptides, VS1, VSL1, VS2, and
VSD1, belonging to different series. The peptides were taken at their respective MICs. Cells without any peptide served as a control.
2185

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PATHAK AND CHAUHAN
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 8. (A) Confocal images of cells incubated with FITC-labeled
VS2 for 30 min. (B) Z section of an individual E. coli mL35p cell
showing intracellular presence of peptide.
FIG. 7. E. coli membrane depolarization caused by the VS2 pep-
tide. (‚) Disruption of the potential gradient across the membrane was
assessed by the increase in the fluorescence of the potential-sensitive
dye diSC3-5. DMSO (10%) (Ⅺ) served as a positive control.
hemolysis (ϳ7%) at its MIC, it was considerably hemolytic
(ϳ40%) at 5ϫ MIC values.
Structure-activity relationship studies on helical antibacte-
rial peptides have shown that most of the ␣-helical peptides are
unstructured in aqueous solution and acquire a helical struc-
ture in the presence of a lipid-like environment (5). Peptide
chain lengths have been associated with increased potency of
helical peptides, as long helices can better span the bacterial
lipid membrane (32). All ⌬Phe-containing peptides showed
some degree of helicity in SDS. For the first series of peptides,
where the length of the template was successively increased by
1 residue, it was observed that VSL1 (12 residues) showed an
increase in helicity and 5-fold enhancement in activity against
E. coli. VSL2 (13 residues), however, was equipotent to VSL1
and showed lower helicity than VSL1. In VSL3, with increased
length and higher helical content, there was no gain in antimi-
crobial activity. A gradual decrease in helicity was observed on
incorporation of 2, 3, and 4 Trp residues. However, helicity
taken as a parameter did not correlate with the activities of
peptides of both series. The results indicate that the helicity of
a similarly structured peptide alone may not correlate with
activity. There are studies in the literature that cite a similar
lack of clear structure-function correlation for AMPs (13). In
such studies, the activity of AMPs is shown to be derived from
a correct balance of charge, hydrophobicity, solubility, and
amphipathicity, collectively called the “interfacial activity”
(30). It has been shown that peptide composition is also an
important parameter for the antimicrobial activity of short
designer peptides (34).
The cell membrane is vital for any microorganism and is the
primary target for most antibacterial peptides (39). However,
the lethal action of a peptide could result from membrane
disruption alone or from translocation through the membrane
to a target receptor inside the cell, or it can be a concerted
effect of both activities (14). We determined the ability of these
peptides to permeabilize the cytoplasmic membrane, which is
the killing mechanism of a large number of AMPs (11). In a PI
uptake assay, we observed that all the peptides were able to
permeabilize the bacterial membrane in 90 min. However, the
results for the kinetics of membrane permeabilization by se-
lected peptides showed that they differed in these kinetics. VS1
and VSL3 were slow in their action on the inner membrane of
E. coli, as they were able to permeabilize only 10% of cells in
activity of antimicrobial peptides. We decided to synthesize
three more peptides, VSL1, VSL2, and VSL3, containing 12,
13, and 14 residues, respectively. We found that increase in
length did not result in any significant enhancement of anti-
bacterial activity. Next, we sequentially increased the Trp con-
tent of VS1, resulting in VS2, VS3, and VS4, with two, three,
and four Trp residues, respectively. Thus, VS4 was made up of
only ⌬Phe, Lys, and Trp residues. We observed remarkable
changes in the properties of these Trp-containing peptides.
The presence of two Trp residues in VS2 enhanced its activity
against E. coli (MIC, 5 ␮M) and S. aureus (MIC, 50 ␮M).
Addition of another Trp in VS3 did not alter its activity against
E. coli (MIC, 5 ␮M) but enhanced its potency against S. aureus
(MIC, 25 ␮M). VS4, with four Trp residues, was the most
potent antibacterial peptide. However, the results of hemolysis
experiments showed that VS4 was most hemolytic at the 5ϫ
MIC values. High Trp content in the antimicrobial peptides is
known to be associated with enhanced hemolysis, making them
unsuitable for use as antimicrobial agents (7). These results
indicate that the presence of two Trp residues in VS2 provided
the right balance of charge, hydrophobicity, and appropriate
membrane properties, making it a reasonably potent and
broad-spectrum antimicrobial peptide. Dendrimerization has
been used as a strategy to potentiate the activity of antimicro-
bial peptides, as it allows multivalent binding (due to doubling
of the charge) to the bacterial membrane and generates a
higher local concentration that results in membrane destabili-
zation (19). In support of this, our results showed that a
branched dimeric peptide, VSD1, exhibited excellent activity
against E. coli (MIC, 1 ␮M) and methicillin-resistant S. aureus
(MIC, 5 ␮M). Dewan et al., in their study on ⌬Phe-containing
peptides, have shown that, along with increased antimicrobial
properties, branched dimers show faster kill kinetics and in-
creased serum stability compared to the linear dimeric analogs
(8). Duplication of the vital antimicrobial structure in VSD1
resulted in very high antibacterial activity but simultaneously
compromised its cell selectivity. Although it showed very low

VOL. 55, 2011
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2187
FIG. 9. SEM and TEM micrographs of E. coli cells treated with VS2 at its MIC (5 ␮M) for 30 min and 60 min. (A) Untreated cells with smooth,
intact surfaces. (B) Peptide-induced breakage and roughness in the cell wall after 30 min. (C) Increased damage to the bacterial cell wall in the form of
cracks after 60 min. Cell shrinkage due to loss of turgor was also observed. Similarly, TEM micrographs of ultrathin sections of negatively stained,
untreated E. coli cells showed a normal cell shape with an undamaged structure of inner membrane and intact outer membrane (AЈ). (BЈ) Peptide-
induced breakage in the cell wall and cell membrane after 30 min. Leakage of the inner mass of the cell was also visible. (CЈ) Complete damage to the
cell wall and inner membrane and lysis of the bacterial cell after 60 min. Arrows indicate the sites of cell membrane damage on E. coli cells.
60 min. However, VS2 and VSD1 efficiently permeabilized the
outer membrane of E. coli at their MICs in 10 min of incuba-
tion. Also, in a DiSC3-5 assay, VS2 was able to initiate mem-
brane depolarization immediately at 0.5ϫ its MIC, which is
remarkable, as some AMPs do not depolarize the membrane
until they reach concentrations as high as 4 to 10 times the
MIC (42). However, the presence of a FITC-labeled VS2 pep-
tide inside the cytoplasm of bacteria shows that the peptide
translocates itself into the cell, where it might act on some
intracellular targets, as well. Our finding that these peptides
were able to permeabilize and depolarize the bacterial cell
membrane at concentrations much lower than the MIC sug-
gests that membrane permeabilization is not the sole cause of
bacterial death and that the designed peptides might have a
multimodal mechanism of action (17, 26). SEM images of
VS2-treated E. coli cells showed changes in the cell wall mor-
phology. Peptide-induced cell wall breakage was clearly visible,
and an increase in the coarseness of the cell surface compared
to the control was also observed. TEM images of the VS2-
treated cells also confirmed the cell wall and inner membrane
damage leading to cell lysis, thereby providing additional evi-
dence of a putative multimodal action of the peptides. These
observations suggest that the antibacterial activity of VS2
might be a multistep process involving initial permeabilization
and depolarization of the bacterial membrane, leading to its
destabilization, and then action on intracellular components of
cell, ultimately leading to cell death.
Our study with a set of designed peptides demonstrated
that synthetic peptides, such as VS2, containing a helico-
genic residue, ⌬Phe, that exhibited a broad spectrum of
activity showed increased protease resistance and faster kill-
ing kinetics and could be suitable candidates for further
development.
Conclusion. Short abiotic antimicrobial peptides are prom-
ising candidates for overcoming the critical and accelerating
problem of bacterial resistance to currently utilized antibiotics.
To provide a road map for the development of improved sec-
ond-generation therapeutics, we investigated a rationale-based
approach for optimizing the peptide sequence to significantly
enhance its antimicrobial activity. Increase in length did not
dramatically alter the antimicrobial activity of peptides, sug-
gesting that a length of 11 to 12 residues is optimum for
activity. The resultant study adds significantly to the field, pro-
viding a lead peptide, VS2, with high potency, a broad spec-

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trum of activity, enhanced proteolytic resistance, and faster
membrane permeabilization kinetics, based on which novel
potent antimicrobials could be produced.
ACKNOWLEDGMENTS
We thank the Council of Scientific and Industrial Research, India,
for financial assistance. We acknowledge core funding from ICGEB,
New Delhi, India.
We are grateful to Dinkar Sahal, ICGEB, New Delhi, India, for
providing strains of E. coli and S. aureus. We acknowledge Rashmi
Shrivastva from the Immunology Group, ICGEB, for help in mass
spectrometry and Ruchita Pal from the Advanced Instrumentation
Research Facility (AIRF), JNU, for providing instrumental support for
electron microscopy. We also thank Manjula Kalia for her help in
taking confocal images. We thank anonymous reviewers for their con-
structive criticism that has enriched our work.
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