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The Central Hinge Link Truncation of the Antimicrobial Peptide

Fowlicidin-3 Enhances Its Cell Selectivity without Antibacterial
Activity Loss
Pei Qu,a Wei Gao,a Huixian Chen,a Dan Li,a Na Yang,a Jian Zhu,a Xingjun Feng,a Chunlong Liu,b,c Zhongqiu Lid
College of Animal Science and Technology, Northeast Agricultural University, Harbin, People’s Republic of Chinaa; Northeast Institute of Geography and Agricultural
Ecology, Chinese Academy of Sciences, Harbin, People’s Republic of Chinab; Collaborative Innovation Center for Development and Utilization of Forest Resources, Harbin,
People’s Republic of Chinac; Animal Husbandry Research Centre of Heilongjiang Academy of Agricultural Science, Harbin, People’s Republic of Chinad

Antimicrobial peptides (AMPs) have been paid considerable attention because of their broad-spectrum antimicrobial activity
and a reduced possibility of the development of bacterial drug resistance. Fowlicidin-3 (Fow-3) is an identified type of chicken
cathelicidin AMP that has exhibited considerable antimicrobial activity and cytotoxicity. To reduce cell toxicity and improve cell

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selectivity, several truncated peptides of fowlicidin-3, Fow-3(1-15), Fow-3(1-19), Fow-3(1-15-20-27), and Fow-3(20-27), were
synthesized. Our results indicated that neither the N- nor C-terminal segment alone [Fow-3(1-15), Fow-3(1-19), Fow-3(20-27)]
was sufficient to confer antibacterial activity. However, Fow-3(1-19) with the inclusion of the central hinge link (-AGIN-) re-
tained substantial cell toxicity, which other analogs lost. Fow-3(1-15-20-27) displayed potent antimicrobial activity for a wide
range of Gram-negative and Gram-positive bacteria and no obvious hemolytic activity or cytotoxicity. The central link region
was shown to be critically important in the function of cell toxicity but was not relevant to antibacterial activity. Fow-3(1-15-20-
27) maintained antibacterial activity in the presence of physiological concentrations of salts. The results from fluorescence
spectroscopy, scanning electron microcopy, and transmission electron microcopy showed that Fow-3(1-15-20-27) as well as fow-
licidin-3 killed bacterial cells by increasing membrane permeability and damaging the membrane envelope integrity. Fow-3(1-
15-20-27) could be a promising antimicrobial agent for clinical application.

B ecause the widespread use of antibiotics has brought a series of

side effects to public health, there is an urgent need to develop
new antimicrobials that are active against bacteria and less likely to
against Gram-positive and Gram-negative bacteria, including an-
tibiotic-resistant strains, with MICs in the 1 to 2 ␮M range. Addi-
tionally, the peptide was more toxic to eukaryotic cells. Fow-3
induce drug resistance (1). Antimicrobial peptides (AMPs) are an comprises 27 amino acid residues and adopts two predominantly
integral component of innate immunity and have been found in ␣-helical structures connected by a central hinge link between
virtually all living species (2). Acting as an important first line of residues 16 and 19 (-AGIN-) (15).
defense, these peptides are mostly produced by innate immune In the current study, a series of truncated peptides based on
cells such as phagocytes, mucosal epithelial cells, and skin kerati- Fow-3 were chemically synthesized, and the antimicrobial activity
nocytes in vertebrates and are capable of killing a broad range of and mechanism were examined. The secondary structures and
bacteria, fungi, and viruses, including resistant strains (3). They membrane-disrupting activities of these peptides in different buf-
are capable of killing a variety of pathogens and have similar effi- fer environments were measured. The antimicrobial properties of
ciencies against strains resistant and susceptible to antibiotics (4, these peptides were evaluated by determining the MIC against a
5). AMPs display multiple modes of action to kill bacteria that are broad selection of threatening microbes, including Gram-nega-
generally perceived as differing from those of conventional anti- tive and Gram-positive bacteria. The hemolytic activity, cytotox-
biotics (6). Because of the nonspecific membranolytic activities, icity, and toleration of salt were also measured. Finally, whole
AMPs have a low tendency to induce drug resistance, which is a bacteria were further employed to investigate potential membrane
desirable feature as a new class of antimicrobial agents. destruction mechanisms. Scanning electron microscopy (SEM)
However, systemic toxicity, in vivo stability, and production and transmission electron microscopy (TEM) were used to di-
costs are challenges for the further development of natural AMPs rectly observe changes in cell morphology as a result of the peptide
as therapeutic drugs (7, 8). To overcome these inherent adverse
effects, various strategies focusing on optimizing the peptide se-
quences have been employed to increase antimicrobial efficacy, Received 28 September 2015 Returned for modification 31 October 2015
specifically, the cell selectivity and in vivo stability. Many studies Accepted 15 February 2016
were carried out to do this by altering certain physicochemical and Accepted manuscript posted online 22 February 2016
structural properties, such as charge, amphipathicity, hydropho- Citation Qu P, Gao W, Chen H, Li D, Yang N, Zhu J, Feng X, Liu C, Li Z. 2016. The
central hinge link truncation of the antimicrobial peptide fowlicidin-3 enhances
bicity, degree of structuring (␣-helix or ␤-sheet), D-isomerization, its cell selectivity without antibacterial activity loss. Antimicrob Agents Chemother
and cyclization (9, 10, 11, 12). In addition, truncation of natural 60:2798 –2806. doi:10.1128/AAC.02351-15.
AMPs has been shown to be a simple and effective approach for Address correspondence to Xingjun Feng,, or
the design and/or optimization of AMPs (12, 13, 14). Chunlong Liu,
Fowlicidin-3 (Fow-3) is an ␣-helical peptide identified from Copyright © 2016, American Society for Microbiology. All Rights Reserved.
gallus (8). This peptide possesses broad-spectrum in vitro activity

2798 Antimicrobial Agents and Chemotherapy May 2016 Volume 60 Number 5
Hinge Truncation Improved Fow-3 Cell Selectivity

TABLE 1 Amino acid sequences, charges, lengths, molecular weights, and hydrophobicity values of the peptides used in this study
Length (no. of
Peptide Sequence Charge amino acids) Theoretical MW Measured MWa Hb uc
Fow-3 KRFWPLVPVAINTVAAGINLYKAIRRK-NH2 ⫹6 27 3,083.769 3,088.810 0.985 1.57
Fow-3(1-15) KRFWPLVPVAINTVA-NH2 ⫹2 15 1,699.083 1,704.124 ⫺0.707 0.63
Fow-3(1-19) KRFWPLVPVAINTVAAGIN-NH2 ⫹2 19 2,054.478 2,059.519 ⫺0.684 1.62
Fow-3(20-27) LYKAIRRK-NH2 ⫹4 8 1,047.306 1,052.347 ⫺1.277 0.64
Fow-3(1-15-20-27) KRFWPLVPVAINTVALYKAIRRK-NH2 ⫹6 23 2,728.374 2,733.415 0.729 0.55
Measured molecular weight (MW) values represent the molecular weight measured by mass spectroscopy (MS).
H values represent the hydrophobicity per residue of peptides calculated by the method of Kyte and Doolittle.
u values represent the mean hydrophobicity moment of peptides calculated by the method of Kyte and Doolittle.

treatment. The results indicated that the peptide Fow-3(1-15-20- 1 ⫻ 105 CFU/ml. Peptides were serially diluted (2-fold), dissolved in
27) derived from Fow-3 by removing the central hinge link re- 0.01% (vol/vol) acetic acid and 0.2% (wt/vol) bovine serum albumin
tained antimicrobial activities and increased cell selectivity. (Sigma), and added to each well of the 96-well plates in a volume of 50 ␮l,
followed by 50 ␮l of inoculum. Plates were incubated at 37°C for 24 h, and

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MATERIALS AND METHODS the MICs were determined as the lowest concentrations of peptide that
Peptide design and analysis. According to the three-dimensional struc- prevented visible turbidity. The broth with or without microbial cells was
ture, we designed a series of derivatives by truncation of Fow-3 (Table 1). used as the positive control or the negative control.
As previously described (15), glycine is located in the central hinge link of Measurement of hemolytic activity. The hemolytic activity of the
Fow-3. All the peptides were designed based on the central link (-AGIN-) peptides was measured as the amount of hemoglobin released by the
in the region from position 16 to position 19. Fow-3(1-15) and Fow-3(1- lysis of human erythrocytes/red blood cells (hRBCs) (17). The fresh
19) contained the amino acid sequence corresponding to the entire ␣-he- hRBCs were washed three times by centrifugation (1,000 ⫻ g, 5 min,
lical region of the N end of Fow-3 without and with the link, respectively. 4°C) in phosphate-buffered saline (PBS) solution (pH 7.2) and then
Fow-3(20-27) contained the amino acid sequence corresponding to the resuspended in PBS to obtain the erythrocyte with a dilution of ap-
entire ␣-helical region of the C end without the central link (-AGIN-). proximately 1% (vol/vol). Then, 50 ␮l of hRBC solution was incubated
Compared to the parent AMP Fow-3, Fow-3(1-15-20-27) comprises with 50 ␮l of different concentrations of the peptides dissolved in PBS
the sequence of two ␣-helixes, removing the link. Primary sequence anal- for 1 h at 37°C. After centrifugation (1,000 ⫻ g, 5 min, 4°C), the
ysis of the peptides was performed with the bioinformatics program Prot- supernatant was transferred to a new 96-well microtiter plate. Release
Param (ExPASy Proteomics Server: of hemoglobin was monitored by measurement of absorbance at 492
/protparam.html). The mean hydrophobicity moment and the hydro- nm. The control samples for 0% and 100% hemolysis consisted of
phobicity per residue of peptides were calculated by the method of Kyte hRBCs in PBS (Ablank) only and in 0.1% Triton X-100 (ATriton), respec-
and Doolittle. tively. The percentage of hemolysis was calculated according to the fol-
Peptide synthesis. The peptides were purchased from GL Biochem lowing equation:
Corporation (Shanghai, China) and were synthesized by solid-phase
methods using N-(9-fluorenyl) methoxycarbonyl (Fmoc) chemistry. The % hemolysis ⫽ 关共Asample ⫺ Ablank兲 ⁄ 共ATriton ⫺ Ablank兲兴 ⫻ 100
peptides were amidated at the C terminus. The purity of the peptides was Cytotoxicity assay. The colorimetric 3-(4,5 dimethylthiazol-2-yl)-
more than 95%, as analyzed by reverse-phase high-performance liquid 2,5-diphenyltetrazolium bromide (MTT) (Sigma) dye reduction assay
chromatography. Their identities were confirmed by electrospray ioniza- was used to determine the cytotoxicity of peptides on human skin epithe-
tion mass spectrometry (ESI-MS). lial cells (HaCaT) according to a previously described method (18). Yel-
CD analysis. The secondary structure of the peptides was deter-
low MTT is reduced by mitochondrial dehydrogenases of living cells to
mined on a Jasco-820 spectropolarimeter (Jasco, Tokyo, Japan) at
produce purple formazan, which is insoluble in aqueous solutions. The
25°C, using a 0.1-cm-path-length rectangular quartz cell. Peptide sam-
crystals can be dissolved in dimethyl sulfoxide (DMSO), and the resulting
ples were recorded at a final concentration of 150 mM in a mixture of
purple solution can be spectrophotometrically measured. An increase in
10 mM sodium phosphate buffer (pH 7.4, mimicking the aqueous
viable cell numbers results in an increase in the amount of MTT formazan
environment), 30 mM SDS micelles (mimicking the negatively
formed and an increase in absorbance. HaCaT were kindly provided by
charged prokaryotic membrane comparable environment; Sigma),
Ning Wang (College of Animal Science and Technology, Northeast Agri-
and 50% TFE (2,2,2-trifluoroethanol) (mimicking the hydrophobic
cultural University, China). Briefly, the cells were seeded on a 96-well
environment of the microbial membrane; Sigma). The spectra were
plate at a density of 2 ⫻ 105 cells/ml in Dulbecco modified Eagle medium
recorded at between 190 and 250 nm at a scanning speed of 10 nm/min.
(DMEM). After incubation for approximately 8 h, 10 ␮l of peptide solu-
At least three scans were conducted for each peptide sample. The ac-
quired circular dichroism (CD) signal spectra were then converted to tion at various concentrations (2, 4, 8, 16, 32, 64, 128, and 256 mM) was
mean residue ellipticity with the following equation: added. The cells were incubated under conditions of a fully humidified
atmosphere of 95% air and 5% CO2 at 37°C for approximately 22 to 24 h.
␪M ⫽ 共␪obs ⫻ 1, 000兲 ⁄ 共c ⫻ 1 ⫻ n兲 Cell cultures were incubated with MTT (50 ␮l, 5 mg/ml) for 4 h at 37°C.
where ␪M is the mean residue ellipticity (degrees ⫻ square centimeter per Then, the supernatants of cell cultures were discarded, 150 ␮l of DMSO
decimole), ␪obs is the observed ellipticity corrected for the buffer at a given was added to dissolve the formazan crystals that formed, and the optical
wavelength (in millidegrees), c is the peptide concentration (in milli- density (OD) was measured using a microplate reader (Bio-Tek Instru-
moles), l is the path length (in millimeters), and n is the number of amino ments Inc., USA) at 570 nm. Cells without peptides added served as con-
acids. trols. Cell viability was expressed as follows: (A570 of treated sample)/A570
Antimicrobial assay. The MICs of the peptides were measured ac- of control) ⫻ 100%.
cording to a modified version of the National Committee for Clinical Salt sensitivity. The peptides were analyzed for salt sensitivity using
Laboratory Standards (NCCLS) broth microdilution method as described Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213. Bac-
previously (16). Briefly, bacterial cells in mid-log phase were diluted to teria were incubated in the presence of different physiological salts (150

May 2016 Volume 60 Number 5 Antimicrobial Agents and Chemotherapy 2799
Qu et al.

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FIG 1 The CD spectra of the peptides. The peptides were dissolved in 10 mM PBS (pH 7.4; -}-), 50% TFE (-䊐-), or 30 mM SDS (-o-).

mM NaCl, 4.5 mM KCl, 6 ␮M NH4Cl, 8 ␮M ZnCl2, 1 mM MgCl2, 2.5 mM resuspended to an OD600 of 0.05 with buffer (5 mM HEPES and 20 mM
CaCl2, 4 ␮M FeCl3) as previously described (19). glucose, pH 7.4). The dye (diSC3-5) was added to give a final concentra-
Outer membrane permeability assay. The outer membrane permea- tion of 0.4 mM and incubated for 90 min. KCl was added to give a final
bility activity of the peptides was determined using the fluorescent dye concentration of 0.1 M for equilibrating the K⫹ concentration between
N-phenyl-1-napthylamine (NPN) assay, as previously described (20). the extracellular and intracellular environments, and then the bacteria
Briefly, E. coli UB1005 in mid-log phase was diluted to 1 ⫻ 105 CFU/ml in were incubated at room temperature for 10 min. The cell suspension (2
HEPES buffer (pH 7.2, containing 5 mM glucose). A final concentration ml) was placed in a 1-cm-path-length cuvette, and the peptides were
of 10 mM NPN was added, and the background fluorescence was recorded added. The fluorescence intensity changes were monitored using an
(excitation k ⫽ 350 nm, emission k ⫽ 420 nm). Changes in the fluores- F-4500 fluorescence spectrophotometer (Hitachi, Japan) with an excita-
cence were recorded with an F-4500 fluorescence spectrophotometer (Hi- tion wavelength of 622 nm and an emission wavelength of 670 nm. E. coli
tachi, Japan). The peptides were added, and the fluorescence was recorded UB1005 cells without treatment and E. coli UB1005 cells treated with 0.1%
as a function of time until no further increase in fluorescence was ob- Triton X-100 were employed as negative and positive controls, respec-
served. Values were converted to percent NPN uptake using the following tively.
equation: Scanning electron microscopy (SEM). As previously described (22),
% NPN uptake ⫽ 关共Fobs ⫺ F0兲 ⁄ 共F100 ⫺ F0兲兴 ⫻ 100 E. coli ATCC 25922 and S. aureus ATCC 29213 in exponential phase were
diluted to an OD600 of 0.2 with 10 mM PBS and incubated at 37°C for 1 h
where Fobs is the observed fluorescence at a given peptide concentration,
with 1⫻ MIC peptides. After incubation, the cells were centrifuged at
F0 is the initial fluorescence of NPN with E. coli cells in the absence of
5,000 ⫻ g for 5 min and washed 3 times with PBS. Bacterial pellets were
peptide, and F100 is the fluorescence of NPN with E. coli cells upon addi-
tion of 10 mg/ml polymyxin B, which was used as a positive control in this then fixed in 500 ml of 2.5% (vol/vol) glutaraldehyde–PBS at 4°C over-
assay. night. Thereafter, the bacteria were washed twice with PBS and dehy-
Inner membrane permeabilization assay. The cytoplasmic mem- drated through a graded ethanol series (50%, 70%, 90%, and 100%), for
brane permeabilization by Fow-3 and Fow-3(1-15-20-27) was deter- 15 min in each dilution. The samples were then transferred to a mixture
mined using E. coli UB1005, which constitutively expresses ␤-galactosi- (1:1) of ethanol and tertiary butanol and then to pure tertiary butanol for
dase in the cytosol, as previously described (18). The inner membrane 20 min each time. After lyophilization and gold coating, the specimens
disruption of E. coli UB1005 resulted in the release of ␤-galactosidase, were observed using a scanning electron microscope (Hitachi S-4800;
which in turn hydrolyzed ONPG (o-nitrophenyl-␤-D-galactopyrano- Hitachi, Japan).
side), a chromogenic substrate, to generate a color product, o-nitrophe- Transmission electron microscope (TEM). Preparation of the bac-
nol. Briefly, E. coli UB1005 cells cultured overnight were harvested and terial samples was conducted in the same manner as described for the
diluted to an OD at 600 nm (OD600) of 0.05 in 10 mM PBS (pH 7.4) SEM treatment. After fixing with 2.5% glutaraldehyde overnight was
containing 1.5 mM ONPG. Aliquots of the cells were incubated with 1/2⫻ performed, the bacterial pellets were washed three times with PBS and
and 1⫻ MICs of peptides at 37°C. OD measurements were made at 420 postfixed with 1% osmium tetroxide–PBS for 2 h. The fixed bacterial
nm every 2 min from 0 to 30 min, which reflected the ONPG influx into cells were washed three times with PBS, followed by dehydration for 15
the cells, and were taken as indicators of the permeability of the inner min in a graded ethanol series (50%, 70%, 90%, and 100%). After
membrane. E. coli UB1005 without peptide treatment was employed as a being placed in absolute acetone for 20 min, the samples were trans-
negative control. ferred to 1:1 and 1:3 mixtures of absolute acetone and epoxy resin for
Cytoplasmic membrane electrical potential measurement. The abil- 1 h in each mixture, followed by transferring to pure epoxy resin
ity of peptides to depolarize the bacterial cytoplasmic membrane was overnight. Ultrathin sections obtained using an ultramicrotome were
measured using E. coli UB1005 cells and 3,30-dipropylthiadicarbocyani- poststained with uranyl acetate and lead citrate. Specimens were ob-
neiodide (diSC3-5) (Sigma), a membrane potential-sensitive fluorescent served by transmission electron microscope (Hitachi H-7650; Hitachi,
dye, as previously described (21). E. coli UB1005 in logarithmic phase was Japan).

2800 Antimicrobial Agents and Chemotherapy May 2016 Volume 60 Number 5
Hinge Truncation Improved Fow-3 Cell Selectivity

TABLE 2 MICs of Fow-3 and its analogs against the tested bacteria
MIC (␮M)
Bacterial strain Fow-3 Fow-(1-15) Fow-(1-19) Fow-(20-27) Fow-(1-15-20-27)
Gram-negative bacteria
Escherichia coli ATCC 25922 2 ⬎128 ⬎128 ⬎128 2
Escherichia coli UB1005 4 64 ⬎128 ⬎128 4
Pseudomonas aeruginosa ATCC 27853 4 ⬎128 ⬎128 ⬎128 4
Salmonella enterica serovar Typhimurium 4 ⬎128 ⬎128 ⬎128 4
ATCC 14028
Salmonella enterica serovar Typhimurium 4 ⬎128 ⬎128 ⬎128 2
ATCC 7731

Gram-positive bacteria
Staphylococcus aureus ATCC 29213 4 ⬎128 ⬎128 ⬎128 4
Staphylococcus epidermidis ATCC 12228 2 128 ⬎128 ⬎128 4
Bacillus subtilis CMCC63501 2 ⬎128 ⬎128 ⬎128 4

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RESULTS concentration of 256 ␮M. Fow-3(1-19) also displayed higher he-
Design and characterization of the peptides. Fow-3 is composed molytic activity than the other three peptide derivatives, and
of 27 amino acid residues, with broad-spectrum antimicrobial 38.9% hemolysis was observed at the concentration of 256 ␮M.
activity but with significant hemolysis and cytotoxicity. To further Fow-3(1-15), Fow-3(19-27), and Fow-3(1-15-20-27) showed no
reduce the cell toxicity of Fow-3 and explore its antibacterial obvious hemolytic activity even at 256 ␮M (less than 5% hemoly-
mechanism, the derived peptides of Fow-3, Fow-3(1-15), Fow- sis), suggesting that the 4-amino-acid segment from position 16 to
3(1-19), Fow-3(20-27), and Fow-3(1-15-20-27) (Table 1) were position 19 in the central link region is of vital importance in
designed. The structure and molecular weight of the peptides were maintaining the hemolytic activity of Fow-3. Melittin, which is
verified by ESI-MS. The theoretically calculated and measured known to have strong antimicrobial activities against all bacteria,
molecular weights of each peptide are shown in Table 1. Each caused complete hemolysis at the concentrations of more than 32
peptide was observed to have a measured molecular weight value m⌴. A second control peptide, LL-37, which is known to possess
consistent with its theoretical value, suggesting that the peptides strong anti-inflammatory activity, also showed a certain level of
were successfully synthesized. hemolytic activity.
Structure variability of the peptides in different environ- Cytotoxicity. The cytotoxic activity of the peptides against
ments. The secondary structures of the peptides were investigated HaCAT was determined by the colorimetric MTT viability assay,
by CD spectroscopy in different environments (Fig. 1). The CD and the results are shown in Fig. 3. A dose-dependent decrease in
spectra showed that Fow-3 and Fow-3(1-15-20-27) formed ran- cell survival rates was observed for all peptides, and the peptides
dom coil structures in PBS. In 50% TFE and 30 mM SDS, the containing the central link region [Fow-3 and Fow-3(1-19)] dis-
spectrums of Fow-3 and Fow-3(1-15-20-27) showed two negative played significantly higher cytotoxic activities on HaCAT than the
dichroic bands at approximately 208 and 222 nm, which is the other Fow-3 analogs. Fow-3(1-19) displayed the highest cytotoxic
characteristic of an ␣-helical structure. The CD spectra of Fow- activity in the concentration range of 8 to 128 ␮M. At the highest
3(1-15) exhibited a ␤-sheet structure in TFE solution and SDS concentration of 256 ␮M, the cell survival rates seen with Fow-3
micelles and a random coil structure in PBS. Fow-3(1-19) dis- and Fow-3(1-19) were less than 30%. Fow-3(1-15), Fow-3(19-
played a ␤-sheet structure, and Fow-3(20-27) displayed a random 27), and Fow-3(1-15-20-27) displayed significantly lower cyto-
coil structure in various solutions as indicated by the CD spectrum toxic activity on cells than Fow-3 and Fow-3(1-19). At the con-
Antimicrobial activities. The antimicrobial activity of the de-
signed peptides was tested against three Gram-positive and five
Gram-negative bacterial strains. As shown in Table 2, Fow-3 was
potent against all strains, with MIC values in the range of 2 to 4
␮M. Fow-3(1-15), the analog containing only the N-terminal
segment of Fow-3, exhibited poor efficacy against all the tested
bacterial strains (MICs of ⱖ64 ␮M), whereas no antimicrobial
activity for tested microorganisms (⬎128 ␮M) was observed
for Fow-3(1-19) or Fow-3(19-27). The MIC values of Fow-3(1-
15-20-27) for the tested bacteria are approximated to those of
the parent peptide.
Hemolytic activity. The hemolytic activity of these peptides
against human erythrocytes was determined. As shown in Fig. 2,
Fow-3 and Fow-3(1-19) showed significant hemolytic activity in a
dose-dependent manner. Fow-3 displayed stronger hemolytic ac-
tivity than its derivatives, and its hemolysis reached 88.1% at the FIG 2 Hemolytic activities of the peptides.

May 2016 Volume 60 Number 5 Antimicrobial Agents and Chemotherapy 2801
Qu et al.

FIG 3 Cytotoxicity of the peptides against HaCat. FIG 4 The outer membrane permeability of the peptides.

centration of 256 ␮M, the cell survival rates seen with Fow-3(1-

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nol, which has marked absorbance at 420 nm. As shown in Fig. 5,
15), Fow-3(19-27), and Fow-3(1-15-20-27) exceeded 60%. Fow-3 and Fow-3(1-15-20-27) induced a rapid increase in the
Melittin, used as a control peptide, was very toxic against mam- permeability of inner membrane at their 1⫻ MICs within 30 min.
malian cells. The cytotoxicity of LL-37 was similar to that of Fow- However, the absorption value for Fow-3 and Fow-3(1-15-20-27)
3(1-19) at high concentrations. at 1/2⫻ MIC was significantly lower than that at 1⫻ MIC. As
Stability of peptides. The antimicrobial activities of two pep- control peptides, melittin showed the strongest inner membrane
tides, Fow-3 and Fow-3(1-15-20-27), treated with different salts permeability, whereas the permeability of LL-37 was very weak.
were investigated. As shown in Table 3, the MIC values of Fow-3 The results indicated that Fow-3 and Fow-3(1-15-20-27) dam-
and Fow-3(1-15-20-27) against E. coli ATCC 25922 were not af- aged the cell inner membrane in dose- and time-dependent man-
fected or increased 2-fold in the presence of different salts. How- ners.
ever, for Pseudomonas aeruginosa ATCC 27853, sodium had no Cytoplasmic membrane electrical potential. The ability of the
effect or even promoted the antibacterial activity of the two pep- peptides to depolarize the bacterial cytoplasmic membrane was
tides. The monovalent (Na⫹, K⫹, and NH4⫹), divalent (Mg2⫹, investigated using diSC3-5, a membrane potential-dependent
Ca2⫹, and Zn2⫹), and trivalent (Fe3⫹) cations exhibited little or no probe. Upon permeabilization and disruption of the cytoplasmic
effect on the MIC values of the two peptides at physiological con- membrane, the membrane potential is dissipated and diSC3-5 is
centrations. released into the medium, causing a consequent increase in fluo-
Outer membrane permeability. The outer membrane of rescence. As shown in Fig. 6, the membrane depolarization was
Gram-negative bacteria performs the crucial role of providing an monitored over a period of 500 s. The results revealed a rapid
extra protection layer without compromising the exchange func- increase in Fow-3- and Fow-3(1-15-20-27)-induced relative fluo-
tion of the material required for sustaining life. The ability of the rescence. However, Fow-3 caused depolarization of the cytoplas-
peptides to permeabilize the bacterial outer membrane was exam- mic membrane that was faster and stronger than that seen with
ined using the NPN uptake assay. As shown in Fig. 4, Fow-3 and Fow-3(1-15-20-27). These effects of Fow-3 and Fow-3(1-15-20-
Fow-3(1-15-20-27) were detected in a dose-dependent manner in 27) were weaker than those of melittin at their 1⫻ MICs.
permeabilizing the outer membrane of E. coli UB1005. Both of the SEM and TEM. A direct visualization of bacterial membrane
peptides could permeabilize the outer membrane at different con-
centrations from 0.5 to 16 ␮M. Fow-3 and Fow-3(1-15-20-27)
had a weaker capability than melittin and LL-37 of permeabilizing
the outer membrane at different concentrations.
Inner membrane permeability. When permeabilization of the
inner membrane of bacteria occurs, ONPG can enter the cyto-
plasm and be degraded by ␤-galactosidase, producing o-nitrophe-

TABLE 3 MIC values of the peptides in different salt ions

MIC (␮M)
Strain and peptide Control NaCl KCl NH4Cl MgCl2 ZnCl2 FeCl3 CaCl2
E. coli ATCC 25922
Fow-3 2 4 4 4 2 4 4 2
Fow-3(1-15-20-27) 2 8 8 4 4 4 2 4
FIG 5 Cytoplasmic ␤-galactosidase-releasing activity of E. coli cells treated
P. aeruginosa ATCC with the peptides. The hydrolysis of ONPG due to the release of cytoplasmic
27853 ␤-galactosidase of E. coli UB 1005 treated by 1⫻ MIC of melittin (2 ␮M), 1⫻
Fow-3 4 4 4 4 2 2 4 4 MIC of LL-37 (2 ␮M), 1⫻ MIC of Fow-3, 1⫻ MIC of Fow-3(1-15-20-27),
1/2⫻ MIC of Fow-3, and 1/2⫻ MIC of Fow-3(1-15-20-27). AU, absorbance
Fow-3(1-15-20-27) 4 4 4 4 4 4 2 4

2802 Antimicrobial Agents and Chemotherapy May 2016 Volume 60 Number 5
Hinge Truncation Improved Fow-3 Cell Selectivity

cytoplasmic membrane of E. coli cells showed a slight rupture and

leakage of intracellular contents. The significant rupture of cell
membranes, the change of cellular morphology, and the release of
intracellular contents can be observed clearly in Fig. 8.

AMPs are important components of innate immune defense
which are mainly employed against microbial infections (23).
They are generally considered to be a potential substitute for an-
tibiotics due to their broad-spectrum antimicrobial activity and
lower likelihood of inducing bacterial resistance (24). Although
natural AMPs have good biological activity, several barriers, for
example, cytotoxic and/or hemolytic effects (7) and the antimi-
FIG 6 The cytoplasmic membrane potential variation of E. coli UB1005 crobial activity loss of AMPs induced by salt, serum, enzymes, etc.,
treated by the peptides. a, 0.1% Triton X-100; b, melittin; c, Fow-3; d, Fow- limit their use as therapeutic agents (25, 26). To circumvent these

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3(1-15-20-27); e, negative control. limitations of AMPs, technological approaches for the optimiza-
tion of AMPs in antibacterial activity, stability, and cytotoxicity
have been undertaken in recent years. Truncating the natural
damage following treatment with peptides was obtained by SEM.
Figure 7 shows SEM images of E. coli and S. aureus treated with AMPs is considered an effective method for developing novel
Fow-3 and Fow-3(1-15-20-27) at their 1⫻ MIC for 1 h. The mem- AMPs, especially for exploring the crucial structure groups and
brane surface of the control (without peptides) was bright and amino acid residues of AMPs (15).
smooth (Fig. 7A and D). In contrast, the treatment with peptides In the present study, we truncated the native AMP Fow-3 and
induced significant membrane damage in E. coli and S. aureus. The studied several Fow-3 analogs with deletions of certain structural
membrane surface of the E. coli cells became completely shrunken components to determine the significance of the N- and C-termi-
and ruptured, and the intracellular contents had dispersed in the nal segments and the central link region of Fow-3 in antibacterial,
surface (Fig. 7B and C). Compared with the control, the S. aureus cytotoxic, and hemolytic activities. The antibacterial assays dem-
cells treated with peptides displayed marked structural changes onstrated that the antimicrobial activities of truncated peptides
(Fig. 7E and F). Distortion, blebbing, or breakage of the cell mem- Fow-3(1-15), Fow-3(1-19), and Fow-3(20-27) from Fow-3 were
brane of cells treated with Fow-3 or Fow-3(1-15-19-27) was evi- almost lost (Table 2). However, the truncated derivative Fow-3(1-
dent. 15-20-27) still exhibited antimicrobial activity that was similar to
TEM was employed to visualize the morphology and intracel- that of the parent peptide. These results demonstrated that neither
lular alteration of bacteria (Fig. 8). After 1 h of treatment, the the N terminus nor the C terminus alone has sufficient antibacte-

FIG 7 Scanning electron micrographs of E. coli and S. aureus treated with Fow-3 and Fow-3(1-15-20-27). (A to C) SEM micrographs of E. coli 25922: (A) control,
no peptides; (B) Fow-3(1-15-20-27) treated; (C) Fow-3 treated. (D to F) SEM micrographs of S. aureus 29213: (D) control, no peptides; (E) Fow-3(1-15-20-27)
treated; (F) Fow-3 treated. Bacteria in mid-logarithmic growth were treated with peptides at 1⫻ MIC for 1 h.

May 2016 Volume 60 Number 5 Antimicrobial Agents and Chemotherapy 2803
Qu et al.

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FIG 8 TEM micrographs of E. coli and S. aureus treated with Fow-3 and Fow-3(1-15-20-27) at their 1⫻ MICs. (A to C) TEM micrographs of E. coli 25922: (A)
control, no peptides; (B) Fow-3(1-15-20-27) treated; (C) Fow-3 treated. (D to F) TEM micrographs of S. aureus 29213: (D) control, no peptides; (E) Fow-3(1-
15-20-27) treated; (F) Fow-3 treated.

rial activity and that the hinge link (-AGIN-) had no effect on the Most reports show conformational changes of natural ␣-heli-
antibacterial activity of the peptides. cal AMPs in various solutions (30, 31). In the current study, the
The cytotoxicity of AMPs to erythrocytes and mammalian so- secondary structure of peptides in both water and membrane-like
matic cells is often considered to be the main side effect of AMPs environments was analyzed using CD spectra. The data showed
(27). Hemolysis and cytotoxicity are often thought to be among that Fow-3 and Fow-3(1-15-20-27) retained a random coil struc-
the main bottlenecks for the application of AMPs as future anti- ture in sodium phosphate buffer and changed to the typical ␣-he-
microbials. It is important to evaluate and attenuate the cytotoxic lical structure in SDS and TFE buffer. Structural changes of pep-
effect of AMPs. As shown in Fig. 2 and 3, all the peptides except for tides from an atypical structure in water (PBS) to an ␣-helix in
Fow-3(1-19) had significantly lower hemolytic and cytotoxicity membrane-like (SDS, TFE) environments play an important role
activity than the parent peptide. These results revealed that the in the function of AMPs at the bacterial cell membrane, including
presence of the hinge link (-AGIN-) can increase the hemolytic its antibacterial activity (31). Surprisingly, Fow-3(1-15) and Fow-
and cytotoxicity activity of the peptides. 3(1-19), whose sequences confirmed the N-end ␣-helical molec-
It is believed that the cationic charge facilitates peptide binding ular structure in Fow-3 (15), adopted ␤-sheet structures, and
to the negatively charged membrane and or bacterial cell walls, Fow-3(20-27), which confirmed the C-end ␣-helical molecular
such as lipopolysaccharide (LPS), via electrostatic interactions structure in Fow-3 (15), showed a random coil structure in mem-
(24). Some cations may affect the antibacterial activity of AMPs, brane-like environments. Fow-3 and Fow-3(1-15-20-27) are
not only by initiating membrane binding competition between more likely to form an ␣-helical structure than Fow-3(1-15), Fow-
the peptides and cations but also by interfering with the electro- 3(1-19), and Fow-3(20-27), which can also partly explain why the
static attraction (28, 29). Unlike many cationic AMPs that are antibacterial activity of Fow-3 and Fow-3(1-15-20-27) was better
inactivated in the presence of physiological concentrations of salt, than that of Fow-3(1-15), Fow-3(1-19), and Fow-3(20-27).
the antibacterial activity of Fow-3 and Fow-3(1-15-20-27) was not Most AMPs play an antibacterial role by destroying the cell
significantly affected in the presence of various salt ions (Table 3). membrane of bacteria (24, 32, 33). In this study, outer and inner
Although some cations increased the MIC of Fow-3(1-15-20-27) membrane permeability and cytoplasmic membrane electrical
against E. coli by 2-fold, Fow-3(1-15-20-27) still can be described potential assays were performed to investigate the interaction be-
as tolerant to cations. tween the peptides [Fow-3 and Fow-3(1-15-20-27)] and the

2804 Antimicrobial Agents and Chemotherapy May 2016 Volume 60 Number 5
Hinge Truncation Improved Fow-3 Cell Selectivity

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ACKNOWLEDGMENTS peptide with in vivo activity. Antimicrob Agents Chemother 41:1738 –
This work was supported by the “Academic Backbone” Project of North- 17. Stark M, Liu LP, Deber CM. 2002. Cationic hydrophobic peptides with
east Agricultural University (15XG15) and by the Open Project of the Key antimicrobial activity. Antimicrob Agents Chemother 46:3585–3590.
Laboratory of Feed Biotechnology, MOA (X. Feng, 2014).
18. Jin X, Mei H, Li X, Ma Y, Zeng AH, Wang Y, Lu X, Chu F, Wu Q, Zhu
FUNDING INFORMATION J. 2010. Apoptosis-inducing activity of the antimicrobial peptide cecropin
This work, including the efforts of Xingjun Feng, was funded by the of Musca domestica in human hepatocellular carcinoma cell line BEL-
7402 and the possible mechanism. Acta Biochim Biophys Sin 42:259 –265.
“Academic Backbone” Project of Northeast Agricultural University
(15XG15). This work, including the efforts of Xingjun Feng, was funded 19. Maisetta G, Di Luca M, Esin S, Florio W, Brancatisano FL, Bottai D.
by the Open Project of the Key Laboratory of Feed Biotechnology, MOA 2008. Evaluation of the inhibitory effects of human serum components on
(X. Feng, 2014). bactericidal activity of human beta defensin 3. Peptides 29:1– 6. http://dx
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2806 Antimicrobial Agents and Chemotherapy May 2016 Volume 60 Number 5