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Arginine- and Lysine-rich Peptides: Synthesis, Characterization and

Antimicrobial Activity

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DOI: 10.2174/1570180814666170213161341

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Letters in Drug Design & Discovery, 2017, 14, 1-7 1

RESEARCH ARTICLE

Arginine- and Lysine-rich Peptides: Synthesis, Characterization and

Antimicrobial Activity

Adriano Mollica1,*, Giorgia Macedonio1, Azzurra Stefanucci2, Roberto Costante1,#,

Simone Carradori1, Valentina Cataldi1, Mara Di Giulio1, Luigina Cellini1, Romano Silvestri3,
Cesare Giordano4, Anita Scipioni2, Stefano Morosetti2, Pasqualina Punzi2,# and Sako Mirzaie5

1
Dipartimento di Farmacia, Università di Chieti-Pescara “G. d’Annunzio”, Via dei Vestini 31, 66100 Chieti, Italy;
2
Dipartimento di Chimica, Sapienza Università di Roma, P.le A. Moro, 5, 00187 Rome, Italy; 3Dipartimento di Chimica
e Tecnologia del Farmaco, Sapienza Università di Roma, P.le A. Moro, 5, 00187 Rome, Italy; 4Istituto di Biologia e
Patologia Molecolare del CNR, Università degli Studi La Sapienza, Piazzale Aldo Moro, 5, I-00185 Rome, Italy;
5
Department of Biochemistry, Sanandaj Branch, Islamic Azad University, Sanandaj, Iran; #Present address: IRBM

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Science Park S.p.A., Via Pontina Km 30.600, 00071 Pomezia, RM, Italy

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Abstract: In recent years, the multi-drug resistance of bacteria and fungi strains has become a
worldwide problem. The incidence of fungal and bacterial infections is experiencing a serious
is e
growth and the microorganisms have developed several new mechanisms of antibiotic resistance.
s
A R T I C L E H I S T O R Y   Antimicrobial peptides (AMPs) constitute one of the well-known barrier defense systems of plants.
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Received: December 23, 2015 They have been isolated from roots, seeds, flowers, stems and leaves of a wide variety of species
Revised: January 26, 2016
Accepted: January 31, 2016 and have activities towards phytopathogens, as well as against human pathogens. AMPs are often
Fo al

DOI:
10.2174/1570180814666170213161341
cationic peptides able to interact with the membrane through penetration or dissolving the biofilms.
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In this paper, we report the synthesis of several arginine and lysine-rich peptides and their evalua-
tion as antimicrobial agents.
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Keywords: AMPs, peptides, lysine- and arginine-rich peptides, Candida spp., multi-drug resistance, microorganisms.
Pe

1. INTRODUCTION capability of persisting in the host in respect to C. albicans

during mixed infection, also expressing higher resistant
Over the years the success of traditional antimicrobial
profile to antifungal drugs [3]. Among these species, C.
treatments against infections due to bacteria and yeasts, has
glabrata has been detected as a nosocomial pathogen in mu-
been affected by the increase of resistant strains. In particular cosal and systemic infections and its major incidence can be
among the yeasts, those belonging to the genus of Candida,
correlated to immunosuppressive and antimicrobial treat-
are the most common causes of human fungal infections,
ments [4].
involving major body organs [1].
As multi-drug resistance is becoming one of the major
Candida albicans represents the major pathogen, isolated
health cares of the 21st century, the development of novel
in 50-70% of the cases; however, more recently, other spe-
antimicrobial pharmaceuticals represents an urgent need.
cies such as non Candida albicans Candida (NCAC) spe- Recently, it has been demonstrated that the synthesis of new
cies, were notably detected and their emerging role in human
antimicrobial peptides (AMPs) could be a promising ap-
infection became of interest [2]. This increased number of
proach to the management of this problem [5].
NCAC species identified in human can reflect their major
To fight these pathogens, plants and mammalians pro-
duce a great quantity of toxic molecules, including AMPs,
*Address correspondence to these authors at the University “G. d'Annunzio” which have been recognized in a wide variety of species [6,
of Chieti-Pescara; Faculty of Pharmacy; Dipartimento di Scienze del
Farmaco; Via dei Vestini, 31; 66100 Chieti; Italy; Tel: +39-0871-3554476;
7]. Around 20% of plant’s AMPs are formed by cationic side
E-mail: a.mollica@unich.it chains of arginine and lysine residues, which have a crucial

1570-1808/17 $58.00+.00 ©2017 Bentham Science Publishers

2 Letters in Drug Design & Discovery, 2017, Vol. 14, No. 0
role in the bioactivity and chemical-physical properties [8,
9]. Being less harmful for host cells, AMPs can act as anti-
bacterial and antifungal agents, inducing minor resistance.
Generally, the presence of polar cationic residues helps their
solubilization in aqueous media, and the lipophilic portion
permits the positioning into lipid micelles, without toxicity
to mammalian cells [10]. The bioactivity of amphipathic α-
helical structures depends on their capacity to form mem-
brane channels through a well studied process of self-
assembling [11-13]. The great number of peptidic antibiotics
presents common structural features, such as 12-50 amino
acids in the skeleton, 50% of which are lipophilic residues
and cationic peptides. This distribution usually confers great
affinity and selectivity versus the membrane’s pathogen.
However, α-helical amphipatic model peptides of 8-12 resi-
dues composed of leucine and arginine amino acids can also
form ion channels [8,9], due to their ability to generate vari-
ous amphipathic structures, in a way that they can adhere to
the bacteria’s surface through electrostatic interactions, fol-
lowing membrane lysis [14].
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Among the strategies developed for obtaining antimicro-
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bial peptides, those characterized by regularly alternating
n
enantiomeric sequences are particularly interesting. Theo-
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retical aspects of such peptides were extensively studied by
De Santis et al. who first demonstrated that cyclic and linear
peptides with regularly alternating enantiomeric sequences
is e
generate rings or low-pitch helices with β-like conformations
s
stabilized by intrachain hydrogen bonds and van der Waals
interactions [15]. As a result, both cyclic and linear L,D al-
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ternating peptides can self-assemble in stacks directed and
stabilized by hydrogen bonds forming tubular assemblies
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where the side chains are located on the periphery. Under
conditions that favor hydrogen bonding, such β-sheet-like
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tubular structures can be absorbed onto lipid membranes [16-
18]. In fact, it was shown that properly designed six- and
eight-residues cyclic L,D-α-peptides act preferentially on
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Gram-positive and/or Gram-negative bacterial membranes
compared to mammalian cells, increase membrane perme-
Pe
ability, collapse transmembrane ion potentials, and cause
rapid cell death [19].
Stimulated by the above findings, we synthesized and
characterized 9 new cationic peptides, which present argin-
ine and lysine amino acids to introduce cationic charges and
phenylalanine and leucine residues to increase lipophilicity.
Four of them have regularly alternating enantiomeric
sequence to explore the effects of their different conforma-
tions with respect to omoconfigurational peptides. These
products have been tested for in vitro antimicrobial activity
against Gram-positive (e.g. Staphylococcus aureus and S.
epidermidis), Gram-negative (e.g. Escherichia coli and
Pseudomonas aeruginosa) strains, and against two panels of
Candida spp.
2. MATERIALS AND METHODS
2.1. Peptide Design
Peptides made of solely lysine and leucine possess a
strong tendency to form helical-structures in globular pro-
Froeba and Adolph
tein. In our design we limited the length of the sequence to
eight residues, dividing them into two main groups.
Products 1 and 3 have the same amphiphilic structure but
different cationic residues (lysine in product 1 and arginine
in product 3). Products 4 and 5 have also a strong am-
phiphilic structure, but differ for the position of the cationic
portion. Product 2 has an alternate sequence of arginines and
phenylalanines. In peptides 6-9, the hydrophilic residues are
L-lysines, while peptides 6 and 9 are characterized by regu-
larly alternating enantiomeric sequences where the hydro-
phobic residues are D-phenylalanines and D-leucines, re-
spectively. The omoconfigurational peptides 7 and 8 contain
L-phenylalanine residues. Furthermore, peptides 8 and 9 are
N-formylated.
2.2. Chemistry
Acetonitrile (MeCN), dichloromethane (DCM), dimeth-
ylformamide (DMF), trifluoroacetic acid (TFA), N,N’-
diisopropylcarbodiimide (DIC) 1-hydroxybenzotriazole
(HOBt), Fmoc-L-Lys(Boc)-Wang resin (200-400 mesh, 1%
DVB, loading: 0,4-0,6 mmol/g) and N-Fmoc-amino acids
(Fmoc-Xaa-OH) were purchased from Sigma-Aldrich as
peptide synthesis grade. Mass spectrometry (MS) was per-
formed on a Q-Tof Micro spectrometer (Micromass, now
Waters) with an electron spray ionization (ESI) source.
Peptides 1-5 were synthesized on 2-CT resin by solid
phase peptide synthesis employing Fmoc strategy. All cou-
plings were performed using amino acid building blocks of L
and D series, depending on the peptide structures: Fmoc-
Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-
Lys(Boc)-OH, For-Phe-OH (Scheme 1). Each coupling con-
sisted of Fmoc-deprotection with 20% piperidine in DMF,
washing with DMF and coupling of the next amino acid. The
completeness of each coupling was tested with “Kaiser” test.
Then, the terminal Fmoc group was removed from the pep-
tide, together with all the other protecting groups with a mix-
ture of TFA/TIPS/H2O 99.5/0.25/0.25, which simultaneously
cleaved the peptide from the resin. The mixture was then
collected in a round bottom flask, concentrated on rotavapor
and precipitated from cold Et2O. The crude products were
collected by centrifugation and resuspended five times in
cold ether. The resulting crude peptides were then purified
by RP-HPLC on C-18 semi-prep column. Analytical HPLC
was carried out on a Waters 600 HPLC (Waters Co., Mil-
ford, MA, USA) equipped with an X-Bridge BEH130 C-18,
5 µm, 4.6×250 mm column with Waters 2996 PDA detector,
using a solvent system of H2O/CH3 CN (0.1% TFA) in linear
gradient (from 5 to 80% of CH3 CN in 34 min and a flow rate
of 1 mL/min). The octapeptides 6-9 were manually synthe-
sized by conventional solid phase peptide synthesis on
Fmoc-L-Lys(Boc)-Wang resin (150 mmol scale, 250 mg)
[20]. Fmoc-Xaa-OH, DIC and HOBt (3 equivalents, respec-
tively) in DMF were used for couplings (3 h at room tem-
perature). Formylated L-phenylalanine (For-L-Phe) and D-
leucine (For-D-Leu) were prepared [21] and coupled accord-
ing to the usual procedure. “Kaiser” test was used to check
the completeness of each coupling; Fmoc deprotection was
performed with a piperidine (20%) solution in DMF. A solu-
tion of aqueous 95% TFA was prepared to deprotect and
Pharmacokinetics of Xenon
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Scheme 1. Synthetic procedure for the synthesis of products 1-9.
N rs
cleave the final peptides from resin, then peptides were pre-
cipitated with peroxide-free dry diethyl ether at 0 °C. The
Pe
precipitate was washed with ether, redissolved with water
and lyophilized. The crude peptides were purified by RP-
HPLC on a Waters 600E liquid chromatography system by
using a Waters µBondapak C-18 column (1:9 x 30 cm; 5
mm; 300 Å) for a semi-preparative scale with elution at 8
mL min-1 by a linear gradient of 10–60% MeCN in 0.1%
aqueous TFA in 30. The final peptide purity was >97% by
analytical HPLC (Waters µBondapak C-18 column, 0:39 x
30 cm; 5 mm; 300 Å). All products were obtained and tested
as TFA salts and the identity of new compounds was con-
firmed by LC-MS/MS measurements (Table 1).
2.3. Antimicrobial Activity
The antimicrobial activity of peptides 1-9 was deter-
mined against two reference Gram-positive strains (S. aureus
ATCC 29213 and S. epidermidis ATCC 35984), and two
Gram-negative (E. coli ATCC 8739 and P. aeruginosa
ATCC 9027) strains (Table 2).
In addition, the efficacy of peptides was also tested
against two reference strains of Candida spp. (C. albicans
ATCC 10213 and C. glabrata ATCC 2001) and against two
Letters in Drug Design & Discovery, 2017, Vol. 14, No. 0 3
clinical isolates for each species, characterized for their an-
timicrobial profile against the most common antifungal
drugs (resistant strains: C. albicans 2R, C. glabrata 21R;
susceptible strains: C. albicans 9S, C. glabrata 47S). Each
bacterial culture was grown at 37 °C in Tryptic Soy Broth
(Oxoid) overnight and then diluited 1:5 in Muller Hinton
Broth (Broth, Oxoid) and incubated aerobically for 2 h at 37
°C under shaking, to obtain the exponential bacterial growth.
Finally these cultures were adjusted to an optical density at
600 nm (OD600) of 0.1 corresponding to 108 cfu/mL and a
final dilution of 1:100 was performed for the experiments.
Candida spp. were growth at 37 °C in RPMI medium
(Oxoid) overnight and then were diluted 1:5 in RPMI with
2% glucose and incubated aerobically for 2 h at 37 °C under
shaking. The culture was adjusted to OD600 of 0.15 corre-
sponding to 5 x 107 cfu/mL and a final dilution of 1:10 was
performed. The minimum inhibitory concentration (MIC)
and the minimum bactericidal/fungicidal concentration
(MBC, MFC) of the peptides against all studies microorgan-
isms were performed using the broth microdilution method
according to CLSI guidelines [21]. The microdilution
method was carried out in 96-well microtitre plates and two-
fold dilutions of each peptides were performed to obtain the
final concentrations from 500 to 1.9 µg/mL. The MIC is de-
4 Letters in Drug Design & Discovery, 2017, Vol. 14, No. 0 Froeba and Adolph

Table 1. Analytical data for products 1-9.

AMPs Derivatives MW tR (min) pKa Yield

1 381.29 13.34 4.1 56%

2 1229.68 12.78 4.1 51%

3 1231.69 12.13 4.1 63%

4 1229.68 11.93 4.1 70%

5 1117.65 14.56 3.8 68%

6 1114.7 14.73 3.6 47%

7 1114.7 14.78 3.6 49%

8 1142.7 14.97 3.6 32%

9 1006.76 14.65 3.6 86%

Table 2. Antimicrobial activity of peptides 1-9 against Gram-positive and Gram-negative bacteria.

tio y
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Peptide S. aureus S. epidermidis P. aeruginosa E. coli

n
ATCC 29213 ATCC 35984 ATCC 9027 ATCC 8739

MIC
(µg/mL)
tri O MBC
(µg/mL)
MIC
(µg/mL)
MBC
(µg/mL)
MIC
(µg/mL)
MBC
(µg/mL)
MIC
(µg/mL)
MBC
(µg/mL)
is e s
1 >500 >500 >500 >500 >500 >500 >500 >500
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2 250 >500 >500 >500 >500 >500 500 >500

3 >500 >500 >500 >500 >500 >500 >500 >500

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4 500 >500 >500 >500 >500 >500 500 >500

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5 >500 >500 >500 >500 >500 >500 >500 >500

6 >500 >500 >500 >500 >500 >500 >500 >500

N rs

7 >500 >500 >500 >500 >500 >500 250 500

Pe

8 >500 >500 >500 >500 500 >500 500 500

9 >500 >500 >500 >500 >500 >500 >500 >500

-1 -1
Internal Quality Control: S. aureus ATCC 29213, Levofloxacin MIC: 0.24 µg mL ; S. epidermidis ATCC 35984, Levofloxacin MIC: 0.12 µg mL ; P. aeruginosa ATCC 9027,
Levofloxacin MIC: 1 µg mL-1; E. coli ATCC 8739, Amoxicillin MIC: 16 µg mL-1.

fined as the lowest concentration of product able to give a
full inhibition of visible growth if compared to the product-
free control and the MBC/MFC is defined as the lowest con-
centration at which no bacterial/fungal growth occurred on
Mueller–Hinton agar plates. For the internal quality control,
S. aureus ATCC 29213, S. epidermidis ATCC 35984, P.
aeruginosa ATCC 9027 were tested against levofloxacin, E.
coli ATCC 8739 was tested against amoxicillin and flucona-
zole was used against C. albicans ATCC 1023 and C.
glabrata ATCC 2001 by using the methodology previously
reported [22]. Data have been collected from two independ-
ent experiments performed in triplicate.
The antifungal activity was evaluated against six clinical
fungal isolates of Candida spp. (C. albicans ATCC 10213,
C. albicans X2 (R), C. albicans X9 (S), C. glabrata ATCC
2001, C. glabrata X21 (R), C. glabrata X47 (S)).
We observed that peptides 3 and 4 showed the best anti-
fungal activity (MICs values of 62.5 µg/mL) against C. albi-
cans ATCC 10213, X2 and C. glabrata ATCC 2001, X21
(Table 3). Peptides 7 and 9 possess antifungal activity but
are less potent than 3 and 4 and lack of specificity.
2.4. Monte Carlo Simulations
MCPep server (http://bental.tau.ac.il/MCPep/) [23] was
employed to perform Monte Carlo (MC) model of the inter-
actions between peptides and a simulated membrane bilayer,
tempting to distinguish between the transmembrane (TM)
and the surface mechanism of action. The analysis gave the
sequence input in FASTA, membrane width was fixed at 30
Å, anionic lipid content was fixed at 3 Å. The number of
independent MC runs was 3 and the number of MC cycles in
each independent was 500000. Molecular models were
Pharmacokinetics of Xenon Letters in Drug Design & Discovery, 2017, Vol. 14, No. 0 5

Table 3. Antimicrobial activity of peptides 1-9 against Candida spp.

Peptide C. albicans C. albicans C. albicans C. glabrata C. glabrata C. glabrata

ATCC 10213 2R 9S ATCC 2001 21R 47S

MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC
(µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL)

1 250 >500 500 >500 >500 >500 250 >500 >500 >500 500 >500

2 125 500 125 500 >500 >500 125 500 125 500 500 >500

3 62.5 250 62.5 250 >500 >500 62.5 250 62.5 250 >500 >500

4 62.5 250 62.5 250 >500 >500 500 >500 250 >500 >500 >500

5 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500

6 400 400 >800 >800 >800 >800 >800 >800 >800 >800 >800 >800

7 100 100 >100 >100 >100 >100 100 100 >100 >100 >100 >100

8 >800 >800 >800 >800 >800 >800 >800 >800 >800 >800 >800 >800

tio y
9 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50

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Internal Quality Control: C. albicans ATCC 10231, Fluconazole, MIC: 1 µg mL-1; C. glabrata ATCC 2001 Fluconazole, MIC: 16 µg mL-1.

n
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is e s
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Fo al
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N rs
Pe

Fig. (1). Monte Carlo simulations of peptide 3 as typical examples of helical obtained by MCPep [23].

 
6 Letters in Drug Design & Discovery, 2017, Vol. 14, No. 0
viewed and analyzed using Pymol [24]. Final images were
customized with GIMP (http://www.gimp.org).
3. CONCLUSION
We synthesized and tested AMPs derivatives 1-9 on dif-
ferent bacteria and fungi. Table 2 summarizes results regard-
ing the antimicrobial activity of peptides 1-9 of exemplifica-
tive Gram-(+) and Gram-(-) bacteria. The MIC values
against Gram-positive species were all >500 µg/mL, except
for peptides 2 and 4, that showed a detectable activity
against S. aureus ATCC 29213 (250 µg/mL and 500 µg/mL,
respectively). Moreover, the peptide 8 reported MIC value
against P. aeruginosa ATCC 9027 of 500 µg/mL, while
against E. coli ATCC 8739 the peptides 7, 8, 2 and 4 showed
the antimicrobial activity with 250 and 500 µg/mL MIC val-
ues. In general, the efficacy of all tested peptides against
Candida spp. was low in respect to bacteria (Table 3). In
particular, peptides 3 and 4 showed the best activity in the
MIC against C. albicans ATCC 10231, C. albicans (2R) and
tio y
C. glabrata ATCC 2001, C. glabrata 21 (R), with a value of
62.5 µg/mL. In all the other cases, the MIC values were
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higher and ranged from 50 to >800 µg/mL (Table 3). MC
n
simulations of peptide 3 with lipid membranes have been
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utilized to define the mode of action of our peptide in detail;
the so-obtained models differ in the lipid structure around
is e
the pores and their stability [25]. In the carpet model, which
was the most frequent in our simulations, peptides are con-
s
centrated on the membrane surface until transient pore’s
rD U
formation. When their concentration is high, membrane can
collapse due to the destabilization. In this condition, peptide
Fo al
adopts an oblique orientation within the membrane. Different
distribution of hydrophobicity determines interfacial or
ot on
transmembrane orientations. A number of results for the
most active peptide 3 are reported in Fig. 1.
As described for a transmembrane helix, the peptide’s
N rs
structure is perpendicular on the membrane. Indeed the
coiled structure in Fig. (3B) depicts a surface bound peptide
Pe
parallel to the plane of the membrane as expected for an am-
phiphilic peptide. Fig. (3C) depicts an oblique oriented pep-
tide. Although these simulations are indicative, they give
preliminary information on the nature of the lipid binding
[26].
In this paper, we have performed the solid phase peptide
synthesis (SPPS) [27], to develop a small-library of novel
antimicrobial peptides containing the arginine- and lysine-
rich peptides, with the exploration of D amino acids and
formylation at the N terminal; then we have studied their
effect against planktonic microbial populations. Among all
peptides tested, peptide 2 expressed an appreciable antimi-
crobial activity against Gram-positive bacteria.
Results obtained against the resistant C. albicans and C.
glabrata clinical strains with peptides 2, 3 and 4, which dis-
played inhibitory effect from 62.5 to 250 µg/mL, encourage
further studies to improve their profile as potential alterna-
tive/adjuvant antimicrobials, in case of multidrug yeasts re-
sistance [28-33].
Froeba and Adolph
Finally, these data confirmed the importance of the am-
phipatic distribution of amino acids and the preference of
peptides for some microorganisms, related to the specific
structural features of cell membrane.
CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flict of interest.
ACKNOWLEDGEMENTS
Declared none.
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