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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
induce drug resistance (1). Antimicrobial peptides (AMPs) are an
integral component of innate immunity and have been found in
virtually all living species (2). Acting as an important first line of
defense, these peptides are mostly produced by innate immune
cells such as phagocytes, mucosal epithelial cells, and skin kerati-
nocytes in vertebrates and are capable of killing a broad range of
bacteria, fungi, and viruses, including resistant strains (3). They
are capable of killing a variety of pathogens and have similar effi-
ciencies against strains resistant and susceptible to antibiotics (4,
5). AMPs display multiple modes of action to kill bacteria that are
generally perceived as differing from those of conventional anti-
biotics (6). Because of the nonspecific membranolytic activities,
AMPs have a low tendency to induce drug resistance, which is a
desirable feature as a new class of antimicrobial agents.
However, systemic toxicity, in vivo stability, and production
costs are challenges for the further development of natural AMPs
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,
specifically, the cell selectivity and in vivo stability. Many studies
were carried out to do this by altering certain physicochemical and
structural properties, such as charge, amphipathicity, hydropho-
bicity, degree of structuring (␣-helix or ␤-sheet), D-isomerization,
and cyclization (9, 10, 11, 12). In addition, truncation of natural
AMPs has been shown to be a simple and effective approach for
the design and/or optimization of AMPs (12, 13, 14).
Fowlicidin-3 (Fow-3) is an ␣-helical peptide identified from
gallus (8). This peptide possesses broad-spectrum in vitro activity
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
comprises 27 amino acid residues and adopts two predominantly
␣-helical structures connected by a central hinge link between
residues 16 and 19 (-AGIN-) (15).
In the current study, a series of truncated peptides based on
Fow-3 were chemically synthesized, and the antimicrobial activity
and mechanism were examined. The secondary structures and
membrane-disrupting activities of these peptides in different buf-
fer environments were measured. The antimicrobial properties of
these peptides were evaluated by determining the MIC against a
broad selection of threatening microbes, including Gram-nega-
tive and Gram-positive bacteria. The hemolytic activity, cytotox-
icity, and toleration of salt were also measured. Finally, whole
bacteria were further employed to investigate potential membrane
destruction mechanisms. Scanning electron microscopy (SEM)
and transmission electron microscopy (TEM) were used to di-
rectly observe changes in cell morphology as a result of the peptide
Received 28 September 2015 Returned for modification 31 October 2015
Accepted 15 February 2016
Accepted manuscript posted online 22 February 2016
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
its cell selectivity without antibacterial activity loss. Antimicrob Agents Chemother
60:2798 –2806. doi:10.1128/AAC.02351-15.
Address correspondence to Xingjun Feng, fengxingjun@neau.edu.cn, or
Chunlong Liu, liuchunlong1976@163.com.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.

2798 aac.asm.org Antimicrobial Agents and Chemotherapy May 2016 Volume 60 Number 5

TABLE 1 Amino acid sequences, charges, lengths, molecular weights, and hydrophobicity values of the peptides used in this study
Peptide Sequence
Fow-3 KRFWPLVPVAINTVAAGINLYKAIRRK-NH2
Fow-3(1-15) KRFWPLVPVAINTVA-NH2
Fow-3(1-19) KRFWPLVPVAINTVAAGIN-NH2
Fow-3(20-27) LYKAIRRK-NH2
Fow-3(1-15-20-27) KRFWPLVPVAINTVALYKAIRRK-NH2
a
Measured molecular weight (MW) values represent the molecular weight measured by mass spectroscopy (MS).
b
H values represent the hydrophobicity per residue of peptides calculated by the method of Kyte and Doolittle.
c
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-
27) derived from Fow-3 by removing the central hinge link re-
tained antimicrobial activities and increased cell selectivity.
MATERIALS AND METHODS
Peptide design and analysis. According to the three-dimensional struc-
ture, we designed a series of derivatives by truncation of Fow-3 (Table 1).
As previously described (15), glycine is located in the central hinge link of
Fow-3. All the peptides were designed based on the central link (-AGIN-)
in the region from position 16 to position 19. Fow-3(1-15) and Fow-3(1-
19) contained the amino acid sequence corresponding to the entire ␣-he-
lical region of the N end of Fow-3 without and with the link, respectively.
Fow-3(20-27) contained the amino acid sequence corresponding to the
entire ␣-helical region of the C end without the central link (-AGIN-).
Compared to the parent AMP Fow-3, Fow-3(1-15-20-27) comprises
the sequence of two ␣-helixes, removing the link. Primary sequence anal-
ysis of the peptides was performed with the bioinformatics program Prot-
Param (ExPASy Proteomics Server: http://www.expasy.org/tools
/protparam.html). The mean hydrophobicity moment and the hydro-
phobicity per residue of peptides were calculated by the method of Kyte
and Doolittle.
Peptide synthesis. The peptides were purchased from GL Biochem
Corporation (Shanghai, China) and were synthesized by solid-phase
methods using N-(9-fluorenyl) methoxycarbonyl (Fmoc) chemistry. The
peptides were amidated at the C terminus. The purity of the peptides was
more than 95%, as analyzed by reverse-phase high-performance liquid
chromatography. Their identities were confirmed by electrospray ioniza-
tion mass spectrometry (ESI-MS).
CD analysis. The secondary structure of the peptides was deter-
mined on a Jasco-820 spectropolarimeter (Jasco, Tokyo, Japan) at
25°C, using a 0.1-cm-path-length rectangular quartz cell. Peptide sam-
ples were recorded at a final concentration of 150 mM in a mixture of
10 mM sodium phosphate buffer (pH 7.4, mimicking the aqueous
environment), 30 mM SDS micelles (mimicking the negatively
charged prokaryotic membrane comparable environment; Sigma),
and 50% TFE (2,2,2-trifluoroethanol) (mimicking the hydrophobic
environment of the microbial membrane; Sigma). The spectra were
recorded at between 190 and 250 nm at a scanning speed of 10 nm/min.
At least three scans were conducted for each peptide sample. The ac-
quired circular dichroism (CD) signal spectra were then converted to
mean residue ellipticity with the following equation:
␪M ⫽ 共␪obs ⫻ 1, 000兲 ⁄ 共c ⫻ 1 ⫻ n兲
where ␪M is the mean residue ellipticity (degrees ⫻ square centimeter per
decimole), ␪obs is the observed ellipticity corrected for the buffer at a given
wavelength (in millidegrees), c is the peptide concentration (in milli-
moles), l is the path length (in millimeters), and n is the number of amino
acids.
Antimicrobial assay. The MICs of the peptides were measured ac-
cording to a modified version of the National Committee for Clinical
Laboratory Standards (NCCLS) broth microdilution method as described
previously (16). Briefly, bacterial cells in mid-log phase were diluted to
May 2016 Volume 60 Number 5 Antimicrobial Agents and Chemotherapy
Hinge Truncation Improved Fow-3 Cell Selectivity
Length (no. of
Charge amino acids) Theoretical MW Measured MWa Hb uc
⫹6 27 3,083.769 3,088.810 0.985 1.57
⫹2 15 1,699.083 1,704.124 ⫺0.707 0.63
⫹2 19 2,054.478 2,059.519 ⫺0.684 1.62
⫹4 8 1,047.306 1,052.347 ⫺1.277 0.64
⫹6 23 2,728.374 2,733.415 0.729 0.55
1 ⫻ 105 CFU/ml. Peptides were serially diluted (2-fold), dissolved in
0.01% (vol/vol) acetic acid and 0.2% (wt/vol) bovine serum albumin
(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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the MICs were determined as the lowest concentrations of peptide that
prevented visible turbidity. The broth with or without microbial cells was
used as the positive control or the negative control.
Measurement of hemolytic activity. The hemolytic activity of the
peptides was measured as the amount of hemoglobin released by the
lysis of human erythrocytes/red blood cells (hRBCs) (17). The fresh
hRBCs were washed three times by centrifugation (1,000 ⫻ g, 5 min,
4°C) in phosphate-buffered saline (PBS) solution (pH 7.2) and then
resuspended in PBS to obtain the erythrocyte with a dilution of ap-
proximately 1% (vol/vol). Then, 50 ␮l of hRBC solution was incubated
with 50 ␮l of different concentrations of the peptides dissolved in PBS
for 1 h at 37°C. After centrifugation (1,000 ⫻ g, 5 min, 4°C), the
supernatant was transferred to a new 96-well microtiter plate. Release
of hemoglobin was monitored by measurement of absorbance at 492
nm. The control samples for 0% and 100% hemolysis consisted of
hRBCs in PBS (Ablank) only and in 0.1% Triton X-100 (ATriton), respec-
tively. The percentage of hemolysis was calculated according to the fol-
lowing equation:
% hemolysis ⫽ 关共Asample ⫺ Ablank兲 ⁄ 共ATriton ⫺ Ablank兲兴 ⫻ 100
Cytotoxicity assay. The colorimetric 3-(4,5 dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) (Sigma) dye reduction assay
was used to determine the cytotoxicity of peptides on human skin epithe-
lial cells (HaCaT) according to a previously described method (18). Yel-
low MTT is reduced by mitochondrial dehydrogenases of living cells to
produce purple formazan, which is insoluble in aqueous solutions. The
crystals can be dissolved in dimethyl sulfoxide (DMSO), and the resulting
purple solution can be spectrophotometrically measured. An increase in
viable cell numbers results in an increase in the amount of MTT formazan
formed and an increase in absorbance. HaCaT were kindly provided by
Ning Wang (College of Animal Science and Technology, Northeast Agri-
cultural University, China). Briefly, the cells were seeded on a 96-well
plate at a density of 2 ⫻ 105 cells/ml in Dulbecco modified Eagle medium
(DMEM). After incubation for approximately 8 h, 10 ␮l of peptide solu-
tion at various concentrations (2, 4, 8, 16, 32, 64, 128, and 256 mM) was
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.
Cell cultures were incubated with MTT (50 ␮l, 5 mg/ml) for 4 h at 37°C.
Then, the supernatants of cell cultures were discarded, 150 ␮l of DMSO
was added to dissolve the formazan crystals that formed, and the optical
density (OD) was measured using a microplate reader (Bio-Tek Instru-
ments Inc., USA) at 570 nm. Cells without peptides added served as con-
trols. Cell viability was expressed as follows: (A570 of treated sample)/A570
of control) ⫻ 100%.
Salt sensitivity. The peptides were analyzed for salt sensitivity using
Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213. Bac-
teria were incubated in the presence of different physiological salts (150
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Qu et al.
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
CaCl2, 4 ␮M FeCl3) as previously described (19).
Outer membrane permeability assay. The outer membrane permea-
bility activity of the peptides was determined using the fluorescent dye
N-phenyl-1-napthylamine (NPN) assay, as previously described (20).
Briefly, E. coli UB1005 in mid-log phase was diluted to 1 ⫻ 105 CFU/ml in
HEPES buffer (pH 7.2, containing 5 mM glucose). A final concentration
of 10 mM NPN was added, and the background fluorescence was recorded
(excitation k ⫽ 350 nm, emission k ⫽ 420 nm). Changes in the fluores-
cence were recorded with an F-4500 fluorescence spectrophotometer (Hi-
tachi, Japan). The peptides were added, and the fluorescence was recorded
as a function of time until no further increase in fluorescence was ob-
served. Values were converted to percent NPN uptake using the following
equation:
% NPN uptake ⫽ 关共Fobs ⫺ F0兲 ⁄ 共F100 ⫺ F0兲兴 ⫻ 100
where Fobs is the observed fluorescence at a given peptide concentration,
F0 is the initial fluorescence of NPN with E. coli cells in the absence of
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
assay.
Inner membrane permeabilization assay. The cytoplasmic mem-
brane permeabilization by Fow-3 and Fow-3(1-15-20-27) was deter-
mined using E. coli UB1005, which constitutively expresses ␤-galactosi-
dase in the cytosol, as previously described (18). The inner membrane
disruption of E. coli UB1005 resulted in the release of ␤-galactosidase,
which in turn hydrolyzed ONPG (o-nitrophenyl-␤-D-galactopyrano-
side), a chromogenic substrate, to generate a color product, o-nitrophe-
nol. Briefly, E. coli UB1005 cells cultured overnight were harvested and
diluted to an OD at 600 nm (OD600) of 0.05 in 10 mM PBS (pH 7.4)
containing 1.5 mM ONPG. Aliquots of the cells were incubated with 1/2⫻
and 1⫻ MICs of peptides at 37°C. OD measurements were made at 420
nm every 2 min from 0 to 30 min, which reflected the ONPG influx into
the cells, and were taken as indicators of the permeability of the inner
membrane. E. coli UB1005 without peptide treatment was employed as a
negative control.
Cytoplasmic membrane electrical potential measurement. The abil-
ity of peptides to depolarize the bacterial cytoplasmic membrane was
measured using E. coli UB1005 cells and 3,30-dipropylthiadicarbocyani-
neiodide (diSC3-5) (Sigma), a membrane potential-sensitive fluorescent
dye, as previously described (21). E. coli UB1005 in logarithmic phase was
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resuspended to an OD600 of 0.05 with buffer (5 mM HEPES and 20 mM
glucose, pH 7.4). The dye (diSC3-5) was added to give a final concentra-
tion of 0.4 mM and incubated for 90 min. KCl was added to give a final
concentration of 0.1 M for equilibrating the K⫹ concentration between
the extracellular and intracellular environments, and then the bacteria
were incubated at room temperature for 10 min. The cell suspension (2
ml) was placed in a 1-cm-path-length cuvette, and the peptides were
added. The fluorescence intensity changes were monitored using an
F-4500 fluorescence spectrophotometer (Hitachi, Japan) with an excita-
tion wavelength of 622 nm and an emission wavelength of 670 nm. E. coli
UB1005 cells without treatment and E. coli UB1005 cells treated with 0.1%
Triton X-100 were employed as negative and positive controls, respec-
tively.
Scanning electron microscopy (SEM). As previously described (22),
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
with 1⫻ MIC peptides. After incubation, the cells were centrifuged at
5,000 ⫻ g for 5 min and washed 3 times with PBS. Bacterial pellets were
then fixed in 500 ml of 2.5% (vol/vol) glutaraldehyde–PBS at 4°C over-
night. Thereafter, the bacteria were washed twice with PBS and dehy-
drated through a graded ethanol series (50%, 70%, 90%, and 100%), for
15 min in each dilution. The samples were then transferred to a mixture
(1:1) of ethanol and tertiary butanol and then to pure tertiary butanol for
20 min each time. After lyophilization and gold coating, the specimens
were observed using a scanning electron microscope (Hitachi S-4800;
Hitachi, Japan).
Transmission electron microscope (TEM). Preparation of the bac-
terial samples was conducted in the same manner as described for the
SEM treatment. After fixing with 2.5% glutaraldehyde overnight was
performed, the bacterial pellets were washed three times with PBS and
postfixed with 1% osmium tetroxide–PBS for 2 h. The fixed bacterial
cells were washed three times with PBS, followed by dehydration for 15
min in a graded ethanol series (50%, 70%, 90%, and 100%). After
being placed in absolute acetone for 20 min, the samples were trans-
ferred to 1:1 and 1:3 mixtures of absolute acetone and epoxy resin for
1 h in each mixture, followed by transferring to pure epoxy resin
overnight. Ultrathin sections obtained using an ultramicrotome were
poststained with uranyl acetate and lead citrate. Specimens were ob-
served by transmission electron microscope (Hitachi H-7650; Hitachi,
Japan).
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
Design and characterization of the peptides. Fow-3 is composed
of 27 amino acid residues, with broad-spectrum antimicrobial
activity but with significant hemolysis and cytotoxicity. To further
reduce the cell toxicity of Fow-3 and explore its antibacterial
mechanism, the derived peptides of Fow-3, Fow-3(1-15), Fow-
3(1-19), Fow-3(20-27), and Fow-3(1-15-20-27) (Table 1) were
designed. The structure and molecular weight of the peptides were
verified by ESI-MS. The theoretically calculated and measured
molecular weights of each peptide are shown in Table 1. Each
peptide was observed to have a measured molecular weight value
consistent with its theoretical value, suggesting that the peptides
were successfully synthesized.
Structure variability of the peptides in different environ-
ments. The secondary structures of the peptides were investigated
by CD spectroscopy in different environments (Fig. 1). The CD
spectra showed that Fow-3 and Fow-3(1-15-20-27) formed ran-
dom coil structures in PBS. In 50% TFE and 30 mM SDS, the
spectrums of Fow-3 and Fow-3(1-15-20-27) showed two negative
dichroic bands at approximately 208 and 222 nm, which is the
characteristic of an ␣-helical structure. The CD spectra of Fow-
3(1-15) exhibited a ␤-sheet structure in TFE solution and SDS
micelles and a random coil structure in PBS. Fow-3(1-19) dis-
played a ␤-sheet structure, and Fow-3(20-27) displayed a random
coil structure in various solutions as indicated by the CD spectrum
results.
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
concentration of 256 ␮M. Fow-3(1-19) also displayed higher he-
molytic activity than the other three peptide derivatives, and
38.9% hemolysis was observed at the concentration of 256 ␮M.
Fow-3(1-15), Fow-3(19-27), and Fow-3(1-15-20-27) showed no
obvious hemolytic activity even at 256 ␮M (less than 5% hemoly-
sis), suggesting that the 4-amino-acid segment from position 16 to
position 19 in the central link region is of vital importance in
maintaining the hemolytic activity of Fow-3. Melittin, which is
known to have strong antimicrobial activities against all bacteria,
caused complete hemolysis at the concentrations of more than 32
m⌴. A second control peptide, LL-37, which is known to possess
strong anti-inflammatory activity, also showed a certain level of
hemolytic activity.
Cytotoxicity. The cytotoxic activity of the peptides against
HaCAT was determined by the colorimetric MTT viability assay,
and the results are shown in Fig. 3. A dose-dependent decrease in
cell survival rates was observed for all peptides, and the peptides
containing the central link region [Fow-3 and Fow-3(1-19)] dis-
played significantly higher cytotoxic activities on HaCAT than the
other Fow-3 analogs. Fow-3(1-19) displayed the highest cytotoxic
activity in the concentration range of 8 to 128 ␮M. At the highest
concentration of 256 ␮M, the cell survival rates seen with Fow-3
and Fow-3(1-19) were less than 30%. Fow-3(1-15), Fow-3(19-
27), and Fow-3(1-15-20-27) displayed significantly lower cyto-
toxic activity on cells than Fow-3 and Fow-3(1-19). At the con-
FIG 2 Hemolytic activities of the peptides.

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Qu et al.
FIG 3 Cytotoxicity of the peptides against HaCat.
centration of 256 ␮M, the cell survival rates seen with Fow-3(1-
15), Fow-3(19-27), and Fow-3(1-15-20-27) exceeded 60%.
Melittin, used as a control peptide, was very toxic against mam-
malian cells. The cytotoxicity of LL-37 was similar to that of Fow-
3(1-19) at high concentrations.
Stability of peptides. The antimicrobial activities of two pep-
tides, Fow-3 and Fow-3(1-15-20-27), treated with different salts
were investigated. As shown in Table 3, the MIC values of Fow-3
and Fow-3(1-15-20-27) against E. coli ATCC 25922 were not af-
fected or increased 2-fold in the presence of different salts. How-
ever, for Pseudomonas aeruginosa ATCC 27853, sodium had no
effect or even promoted the antibacterial activity of the two pep-
tides. The monovalent (Na⫹, K⫹, and NH4⫹), divalent (Mg2⫹,
Ca2⫹, and Zn2⫹), and trivalent (Fe3⫹) cations exhibited little or no
effect on the MIC values of the two peptides at physiological con-
centrations.
Outer membrane permeability. The outer membrane of
Gram-negative bacteria performs the crucial role of providing an
extra protection layer without compromising the exchange func-
tion of the material required for sustaining life. The ability of the
peptides to permeabilize the bacterial outer membrane was exam-
ined using the NPN uptake assay. As shown in Fig. 4, Fow-3 and
Fow-3(1-15-20-27) were detected in a dose-dependent manner in
permeabilizing the outer membrane of E. coli UB1005. Both of the
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
P. aeruginosa ATCC
27853
Fow-3 4 4 4 4 2 2 4 4
Fow-3(1-15-20-27) 4 4 4 4 4 4 2
2802 aac.asm.org Antimicrobial Agents and Chemotherapy
FIG 4 The outer membrane permeability of the peptides.
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nol, which has marked absorbance at 420 nm. As shown in Fig. 5,
Fow-3 and Fow-3(1-15-20-27) induced a rapid increase in the
permeability of inner membrane at their 1⫻ MICs within 30 min.
However, the absorption value for Fow-3 and Fow-3(1-15-20-27)
at 1/2⫻ MIC was significantly lower than that at 1⫻ MIC. As
control peptides, melittin showed the strongest inner membrane
permeability, whereas the permeability of LL-37 was very weak.
The results indicated that Fow-3 and Fow-3(1-15-20-27) dam-
aged the cell inner membrane in dose- and time-dependent man-
ners.
Cytoplasmic membrane electrical potential. The ability of the
peptides to depolarize the bacterial cytoplasmic membrane was
investigated using diSC3-5, a membrane potential-dependent
probe. Upon permeabilization and disruption of the cytoplasmic
membrane, the membrane potential is dissipated and diSC3-5 is
released into the medium, causing a consequent increase in fluo-
rescence. As shown in Fig. 6, the membrane depolarization was
monitored over a period of 500 s. The results revealed a rapid
increase in Fow-3- and Fow-3(1-15-20-27)-induced relative fluo-
rescence. However, Fow-3 caused depolarization of the cytoplas-
mic membrane that was faster and stronger than that seen with
Fow-3(1-15-20-27). These effects of Fow-3 and Fow-3(1-15-20-
27) were weaker than those of melittin at their 1⫻ MICs.
SEM and TEM. A direct visualization of bacterial membrane
4
FIG 5 Cytoplasmic ␤-galactosidase-releasing activity of E. coli cells treated
with the peptides. The hydrolysis of ONPG due to the release of cytoplasmic
␤-galactosidase of E. coli UB 1005 treated by 1⫻ MIC of melittin (2 ␮M), 1⫻
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
4
units.
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.

FIG 6 The cytoplasmic membrane potential variation of E. coli UB1005
treated by the peptides. a, 0.1% Triton X-100; b, melittin; c, Fow-3; d, Fow-
DISCUSSION
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-
crobial activity loss of AMPs induced by salt, serum, enzymes, etc.,
limit their use as therapeutic agents (25, 26). To circumvent these

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3(1-15-20-27); e, negative control.
damage following treatment with peptides was obtained by SEM.
Figure 7 shows SEM images of E. coli and S. aureus treated with
Fow-3 and Fow-3(1-15-20-27) at their 1⫻ MIC for 1 h. The mem-
brane surface of the control (without peptides) was bright and
smooth (Fig. 7A and D). In contrast, the treatment with peptides
induced significant membrane damage in E. coli and S. aureus. The
membrane surface of the E. coli cells became completely shrunken
and ruptured, and the intracellular contents had dispersed in the
surface (Fig. 7B and C). Compared with the control, the S. aureus
cells treated with peptides displayed marked structural changes
(Fig. 7E and F). Distortion, blebbing, or breakage of the cell mem-
brane of cells treated with Fow-3 or Fow-3(1-15-19-27) was evi-
dent.
TEM was employed to visualize the morphology and intracel-
lular alteration of bacteria (Fig. 8). After 1 h of treatment, the
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
AMPs is considered an effective method for developing novel
AMPs, especially for exploring the crucial structure groups and
amino acid residues of AMPs (15).
In the present study, we truncated the native AMP Fow-3 and
studied several Fow-3 analogs with deletions of certain structural
components to determine the significance of the N- and C-termi-
nal segments and the central link region of Fow-3 in antibacterial,
cytotoxic, and hemolytic activities. The antibacterial assays dem-
onstrated that the antimicrobial activities of truncated peptides
Fow-3(1-15), Fow-3(1-19), and Fow-3(20-27) from Fow-3 were
almost lost (Table 2). However, the truncated derivative Fow-3(1-
15-20-27) still exhibited antimicrobial activity that was similar to
that of the parent peptide. These results demonstrated that neither
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 aac.asm.org 2803
Qu et al.
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
antibacterial activity of the peptides.
The cytotoxicity of AMPs to erythrocytes and mammalian so-
matic cells is often considered to be the main side effect of AMPs
(27). Hemolysis and cytotoxicity are often thought to be among
the main bottlenecks for the application of AMPs as future anti-
microbials. It is important to evaluate and attenuate the cytotoxic
effect of AMPs. As shown in Fig. 2 and 3, all the peptides except for
Fow-3(1-19) had significantly lower hemolytic and cytotoxicity
activity than the parent peptide. These results revealed that the
presence of the hinge link (-AGIN-) can increase the hemolytic
and cytotoxicity activity of the peptides.
It is believed that the cationic charge facilitates peptide binding
to the negatively charged membrane and or bacterial cell walls,
such as lipopolysaccharide (LPS), via electrostatic interactions
(24). Some cations may affect the antibacterial activity of AMPs,
not only by initiating membrane binding competition between
the peptides and cations but also by interfering with the electro-
static attraction (28, 29). Unlike many cationic AMPs that are
inactivated in the presence of physiological concentrations of salt,
the antibacterial activity of Fow-3 and Fow-3(1-15-20-27) was not
significantly affected in the presence of various salt ions (Table 3).
Although some cations increased the MIC of Fow-3(1-15-20-27)
against E. coli by 2-fold, Fow-3(1-15-20-27) still can be described
as tolerant to cations.
2804 aac.asm.org Antimicrobial Agents and Chemotherapy
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Most reports show conformational changes of natural ␣-heli-
cal AMPs in various solutions (30, 31). In the current study, the
secondary structure of peptides in both water and membrane-like
environments was analyzed using CD spectra. The data showed
that Fow-3 and Fow-3(1-15-20-27) retained a random coil struc-
ture in sodium phosphate buffer and changed to the typical ␣-he-
lical structure in SDS and TFE buffer. Structural changes of pep-
tides from an atypical structure in water (PBS) to an ␣-helix in
membrane-like (SDS, TFE) environments play an important role
in the function of AMPs at the bacterial cell membrane, including
its antibacterial activity (31). Surprisingly, Fow-3(1-15) and Fow-
3(1-19), whose sequences confirmed the N-end ␣-helical molec-
ular structure in Fow-3 (15), adopted ␤-sheet structures, and
Fow-3(20-27), which confirmed the C-end ␣-helical molecular
structure in Fow-3 (15), showed a random coil structure in mem-
brane-like environments. Fow-3 and Fow-3(1-15-20-27) are
more likely to form an ␣-helical structure than Fow-3(1-15), Fow-
3(1-19), and Fow-3(20-27), which can also partly explain why the
antibacterial activity of Fow-3 and Fow-3(1-15-20-27) was better
than that of Fow-3(1-15), Fow-3(1-19), and Fow-3(20-27).
Most AMPs play an antibacterial role by destroying the cell
membrane of bacteria (24, 32, 33). In this study, outer and inner
membrane permeability and cytoplasmic membrane electrical
potential assays were performed to investigate the interaction be-
tween the peptides [Fow-3 and Fow-3(1-15-20-27)] and the
May 2016 Volume 60 Number 5

membrane. The experimental results showed that peptides de-
stroyed the cell membrane in a concentration-dependent manner.
The inner membrane permeability assay indicated that peptides
induced a dose-dependent increase in fluorescence. Subsequently,
the cytoplasmic membrane depolarization assay indicated that the
peptides possessed the ability to destroy the bacterial cell mem-
brane.
The membrane permeability and cytoplasmic membrane elec-
trical potential experiments identified that the peptides were ca-
pable of destroying the bacterial cell membrane structure quickly,
thus increasing the permeability. To further investigate the inter-
action between the peptide and membrane, SEM and TEM studies
were performed. The results showed that peptides caused signifi-
cant vesicle and membrane blebbing or withering due to leakage
of fluid into the cytoplasm, suggesting that peptides kill bacteria
through membrane rupture.
In summary, a comparison between the functional properties
of Fow-3 and its analogs was performed to investigate the struc-
ture-activity relationship of the peptide and to identify short an-
alogs with better antimicrobial potential. The antibacterial activ-
ities of Fow-3(1-15), Fow-3(1-19), and Fow-3(20-27) were
attenuated or even nearly lost, and Fow-3(1-15-20-27) displayed
antibacterial activity similar to that of Fow-3. Fow-3(1-19)
showed obvious cytotoxicity compared with other analogs, which
demonstrated that the central hinge link (-AGIN-) was crucial to
the cytotoxicity of the peptide. Fow-3(1-15-20-27) showed good
tolerance to salt ions and a considerably higher level of cell selec-
tivity than Fow-3. The mechanism of action against bacteria was
determined through a combination of fluorescence and SEM and
TEM assays, and the results revealed that the peptide Fow-3(1-15-
20-27) kills bacterial cells, possibly by integrity disruption damag-
ing the cell membrane, leading to the cytosol leakage and eventual
cell lysis. Here, we showed that truncation is an effective approach
for the development of AMP analogs with improved bioactivity,
with the peptide Fow-3(1-15-20-27) representing a potential can-
didate in the therapeutic application.
ACKNOWLEDGMENTS
This work was supported by the “Academic Backbone” Project of North-
east Agricultural University (15XG15) and by the Open Project of the Key
Laboratory of Feed Biotechnology, MOA (X. Feng, 2014).
FUNDING INFORMATION
This work, including the efforts of Xingjun Feng, was funded by the
“Academic Backbone” Project of Northeast Agricultural University
(15XG15). This work, including the efforts of Xingjun Feng, was funded
by the Open Project of the Key Laboratory of Feed Biotechnology, MOA
(X. Feng, 2014).
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