Antimicrobial Peptide from the Wild Bee Hylaeus signatus Venom and

Mar 21, 2016 - In the venom of the solitary bee Hylaeus signatus (Hymenoptera: ... The RP-HPLC profile of the bee venom extract at 220 nm (Figure 1) s...
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Antimicrobial Peptide from the Wild Bee Hylaeus signatus Venom and Its Analogues: Structure−Activity Study and Synergistic Effect with Antibiotics Ondřej Nešuta,†,‡ Rozálie Hexnerová,† Miloš Buděsí̌ nský,† Jiřina Slaninová,† Lucie Bednárová,† Romana Hadravová,† Jakub Straka,§ Václav Veverka,† and Václav Č eřovský*,† †

Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo náměstí 2, 166 10 Prague 6, Czech Republic ‡ Faculty of Food and Biochemical Technology, University of Chemistry and Technology Prague, Technická 5, 166 28 Prague 6, Czech Republic § Department of Zoology, Faculty of Science, Charles University in Prague, Viničná 7, 12843 Prague 2, Czech Republic S Supporting Information *

ABSTRACT: Venoms of hymenopteran insects have attracted considerable interest as a source of cationic antimicrobial peptides (AMPs). In the venom of the solitary bee Hylaeus signatus (Hymenoptera: Colletidae), we identified a new hexadecapeptide of sequence Gly-Ile-Met-Ser-Ser-LeuMet-Lys-Lys-Leu-Ala-Ala-His-Ile-Ala-Lys-NH2. Named HYL, it belongs to the category of α-helical amphipathic AMPs. HYL exhibited weak antimicrobial activity against several strains of pathogenic bacteria and moderate activity against Candida albicans, but its hemolytic activity against human red blood cells was low. We prepared a set of HYL analogues to evaluate the effects of structural modifications on its biological activity and to increase its potency against pathogenic bacteria. This produced several analogues exhibiting significantly greater activity compared to HYL against strains of both Staphylococcus aureus and Pseudomonas aeruginosa even as their hemolytic activity remained low. Studying synergism of HYL peptides and conventional antibiotics showed the peptides act synergistically and preferentially in combination with rifampicin. Fluorescent dye propidium iodide uptake showed the tested peptides were able to facilitate entrance of antibiotics into the cytoplasm by permeabilization of the outer and inner bacterial cell membrane of P. aeruginosa. Transmission electron microscopy revealed that treatment of P. aeruginosa with one of the HYL analogues caused total disintegration of bacterial cells. NMR spectroscopy was used to elucidate the structure−activity relationship for the effect of amino acid residue substitution in HYL.

T

components of the microbial cell envelope and cytoplasmic membrane; the hydrophobic part permits insertion of the peptide into the lipid bilayer of the membrane, thereby causing disruption of its structure in various ways, leading to leakage of cytoplasmic components and cell death. The intrinsic distinction in membrane compositions between bacterial and eukaryotic membranes provides the basis for the preference of AMP action toward a bacterial membrane.7−12 Some studies have revealed that the killing process in the case of certain AMPs may proceed with relatively little membrane disruption but occurs rather through interactions with putative key intracellular targets or by interfering with cell metabolism.1 Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen causing severe infections of airways, urinary tract, wounds, bones, and cystic fibrotic lungs.13 It is considered a

he negative impact of antibiotic resistance has prompted multidisciplinary research initiatives searching for new classes of antimicrobial agents. During the past three decades, studies have shown that antimicrobial peptides (AMPs), which are part of the innate defense system of all living organisms, represent a promising group of anti-infective agents as potential candidates to fight infections caused by multi-drug-resistant bacteria.1−6 AMPs exhibit activities comparable to those of conventional antibiotics even though the physical nature of their action is fundamentally different. The significant advantage of AMPs resides in their mechanism of action, which implies a faster killing process and is assumed not to develop bacterial resistance. In general, these prevalently positively charged peptides are able to fold into highly amphipathic conformations with hydrophobic and hydrophilic moieties segregated into distinct patches on the molecular surface. The hydrophilic cationic part electrostatically interacts with the anionic © XXXX American Chemical Society and American Society of Pharmacognosy

Received: December 18, 2015

A

DOI: 10.1021/acs.jnatprod.5b01129 J. Nat. Prod. XXXX, XXX, XXX−XXX

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in the monoisotopic mass of 1696.9 ± 0.1. The Edman degradation (see Supporting Information) gave the entire sequence in 18 cycles as follows: GIMSSLMKKLAAHIAK. The deconvoluted mass signifies that this peptide is C-terminally amidated. HYL, with a sequence rich in hydrophobic and basic amino acid residues, belongs to the category of cationic amphipathic α-helical peptides. When the HYL sequence is compared to the sequences of other α-helical AMPs obtained from natural sources, some obvious positional conservation in terms of residue types can be observed.36 This includes in particular Gly at position 1, Ser at position 4, Leu at position 6, Lys at position 8, the presence of a large hydrophobic residue near the N-terminus, and C-terminal amidation, which provides an extra hydrogen bond stabilizing helix. Such distribution of amino acid residues in the HYL sequence fits very well to the statistical analysis diagram of the residue distribution of naturally occurring α-helical AMPs as proposed in the literature.36 This also supports the fact that the sequence of HYL does not correspond to a truncated version of any other venom peptides. Sequences of AMPs that we previously identified in the venom of other wild bees, e.g., melectin (GFLSILKKVLPKVMAHMK-NH2),29 halictines (GMWSKILGHLIR-NH2, GKWMSLLKHILK-NH2),31 panurgin (LNWGAILKHIIK-NH2),32 and codesan (GMASLLAKVLPHVVKLIK-NH2),33 also match this diagram. Thus, HYL can be considered a new antimicrobial peptide whose sequence is not yet included in the antimicrobial peptide database at http://aps.unmc.edu/AP/main.php. Biological Activity of HYL and Its Analogues. Sequences, MS data, and physical properties of synthetic HYL and 26 analogues are shown in Table 1. HYL exhibited high antimicrobial activity against the sensitive Gram-positive bacteria M. luteus and Bacillus subtilis and moderate activity against the Gram-negative Escherichia coli and the yeast pathogen C. albicans (Table 2). However, its activity against all strains of pathogenic S. aureus and P. aeruginosa was weak. Toxicity to human red blood cells determined as LC50 > 400 μM in a hemolytic test was considered satisfactory. HYL is supposedly inclined to adopt an amphipathic αhelical secondary structure in the anisotropic environment, which is essential for its biological activity. When plotting its sequence onto an α-helical wheel projection (Figure 2), it shows a well-defined hydrophobic sector mostly with large aliphatic residues and a hydrophilic sector dominated by Lys residues. The ideal amphipathicity is disturbed by hydrophobic Ala12 and Ala15 residues present in the hydrophilic segment of the αhelix and by the presence of hydrophilic His13 in its hydrophobic segment. Taking this projection into consideration, we synthesized a set of 26 analogues (including three all-D analogues) in which selected amino acid residues in the sequence were replaced by other residues with the aim of increasing the cationicity,37 amphipathicity, and hydrophobicity38 of the peptide (Table 1). The hydrophobic moment (μH) as a quantitative measure of peptide amphipathicity and the mean hydrophobicity (H) as shown in Table 1 were calculated according to the formula given in the publication of Pathak et al.39 utilizing the Eisenberg consensus scale40 of hydrophobicity for each amino acid in the peptide chain. Increasing cationicity of the peptide by a single replacement of one of each Ala residue in the sequence by lysine (HYL-1, HYL-2, and HYL-3) had a negative or no effect on activity against G+ bacteria and slightly improved activity against G− P.

major pathogen of nosocomial infections in immunosuppressed patients.14−16 It also frequently infects foot ulcers of diabetic patients with serious health consequences.17 The ability of P. aeruginosa to form biofilms and its notorious resistance to antibiotics lead to the development of chronic conditions.18−20 The nonspecific disruption of bacterial cell membranes, including those of resistant strains, by AMPs signifies that these peptides should be regarded as an appealing alternative for combating infections associated with P. aeruginosa. Furthermore, several recent reports have shown a significant synergistic antimicrobial effect against resistant bacteria when some AMPs were used in combination with common antibiotics.21−28 Utilization of AMPs as a supplement to antibiotics seems to be a promising strategy for treatment of infections caused by multi-drug-resistant bacteria such as P. aeruginosa.23,24,26 In the course of our search for new AMPs, we have identified several cationic α-helical AMPs in the venom of Hymenoptera insects that show potent antimicrobial activity against both Gram-positive and Gram-negative bacteria,29−33 that are active against fungi, and that lyse some cancer cells.34,35 In the present work, we describe the isolation, synthesis, and structure− activity relationships of a new hexadecapeptide named HYL that we isolated from the venom of the solitary wild bee Hylaeus signatus (Panzer, 1798) (Hymenoptera: Colletidae). We tested HYL and 26 of its analogues against several strains of pathogenic bacteria as well as against Candida albicans and studied the mechanism of their action. Another focus of the work was on the synergism of HYL and its analogues with antibiotics such as tetracycline, rifampicin, and amoxicillin against multiresistant strains of P. aeruginosa and Staphylococcus aureus. In addition, we observed the effect of secondary structure modification on biological activity using NMR spectroscopy.



RESULTS AND DISCUSSION Peptide Isolation and Sequence Determination. The RP-HPLC profile of the bee venom extract at 220 nm (Figure 1) shows several peaks, but from all collected fractions only the

Figure 1. RP-HPLC profile of Hylaeus signatus venom extract at 220 nm. An elution gradient of solvents from 5% to 70% CH3CN/H2O/ 0.1% TFA was applied for 60 min at a flow rate of 1 mL/min.

component of the most intense one labeled as HYL exhibited antimicrobial activity against Micrococcus luteus in the drop diffusion test. The internally calibrated ESI-Q-TOF mass spectrum of the component showed the [M + 4H]4+ ion at m/z 425.2, the [M + 3H]3+ ion at m/z 566.6, and the [M + 2H]2+ at m/z 849.5 (see Supporting Information). The mass spectrum was deconvoluted using in-house software, resulting B

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Table 1. Amino Acid Sequences, MS Data, and Physical Properties of HYL and Its Analoguesa molecular mass (Da) peptide

sequence

calcd

foundb

tR (min)c

net charged

μH

H

HYL HYL-1 HYL-2 HYL-3 HYL-4 HYL-5 HYL-6 HYL-7 HYL-8 HYL-9 HYL-10 HYL-11 HYL-12 HYL-13 HYL-14 HYL-15 HYL-16 HYL-17 HYL-18 HYL-19 HYL-20 HYL-21 HYL-22 HYL-23 HYL-24 HYL-25 HYL-26

GIMSSLMKKLAAHIAK-NH2 GIMSSLMKKLKAHIAK-NH2 GIMSSLMKKLAKHIAK-NH2 GIMSSLMKKLAAHIKK-NH2 GIMSSLMKKLKKHIAK-NH2 GIMISLMKKLAAHIAK-NH2 GIMSSLMKKLAAIIAK-NH2 GIMSSLMKKLAKIIAK-NH2 GIMSSLMKKLKKIIAK-NH2 GIMSSLMKKLKKIIAK-NH2 GIMSSLMKKLAKIIKK-NH2 GIMSSLMKKLKKIIKK-NH2 GILSSLLKKLKKIIAK-NH2 GILSSLLKKLKKIIAK-NH2 GIXSSLXKKLKKIIAK-NH2 GILKSLLKKLKKIIAK-NH2 GILSKLLKKLKKIIAK-NH2 KILSSLLKKLKKIIAK-NH2 GIWSSLLKKLKKIIAK-NH2 GILSSWLKKLKKIIAK-NH2 GILSSLWKKLKKIIAK-NH2 GILSSLWKKLKKIIAK-NH2 GKLSSLWKKLKKIIAK-NH2 GILSSLLKKWKKIIAK-NH2 GILSSLLKKLKKWIAK-NH2 GILSSLWKKLKKWIAK-NH2 GILSSLWKKPKKIIAK-NH2

1696.98 1754.04 1754.04 1754.04 1811.10 1723.04 1673.01 1730.07 1787.13 1787.13 1787.13 1844.18 1751.21 1751.21 1751.21 1792.28 1792.28 1822.29 1824.21 1824.21 1824.21 1824.21 1839.22 1824.21 1824.21 1897.20 1808.18

1697.1 1754.0 1753.9 1754.0 1811.2 1724.1 1673.1 1730.0 1786.9 1787.1 1787.2 1843.9 1751.6 1751.2 1751.6 1792.3 1792.3 1822.2 1824.2 1824.2 1824.2 1824.2 1839.2 1824.2 1824.2 1897.2 1808.2

30.69 28.32 28.62 29.27 26.61 32.65 38.16 35.92 33.18 33.15 33.39 30.80 37.71 37.74 37.17 35.55 36.20 36.58 35.16 34.99 36.25 36.40 29.69 34.36 37.94 36.59 24.05

+4.1 +5.1 +5.1 +5.1 +6.1 +4.1 +4.0 +5.0 +6.0 +6.0 +6.0 +7.0 +6.0 +6.0 +6.0 +7.0 +7.0 +7.0 +6.0 +6.0 +6.0 +6.0 +7.0 +6.0 +6.0 +6.0 +6.0

0.239 0.223 0.323 0.278 0.307 0.249 0.248 0.332 0.334 0.334 0.386 0.401 0.359 0.359

−0.017 −0.101 −0.101 −0.101 −0.186 0.045 0.054 −0.031 −0.115 −0.115 −0.115 −0.199 −0.081 −0.081

0.385 0.403 0.438 0.350 0.352 0.354 0.354 0.359 0.349 0.350 0.344 0.316

−0.134 −0.134 −0.160 −0.091 −0.091 −0.091 −0.091 −0.206 −0.091 −0.104 −0.114 −0.129

a

Bold letters show amino acid residues replaced. D-Amino acids are shown in italics. X, norleucine. bMonoisotopic molecular masses of synthetic peptides measured by internally calibrated ESI-Q-TOF MS were manually calculated from the m/z values of multiple (2× , 3×, and 4×) charged molecular ions found in their mass spectra. cRetention times were determined on a Vydac C18 column using an elution gradient of solvents from 5% to 70% CH3CN/H2O/0.1%TFA over 60 min at a flow rate of 1 mL/min. dNet charges at pH 7 were calculated using Innovagen’s peptide property calculator (http://www.innovagen.se/custom-peptide-synthesis/peptide-property-calculator/peptide-property-calculator.asp).

aeruginosa. This was only in the cases of HYL-2 and HYL-3, however, where the substitution took place within the hydrophilic segment. Interestingly, a single substitution made in the vicinity of the C-terminus in HYL-3 resulted in a substantial increase of activity only against C. albicans. The further increment of peptide cationicity by substitution of both Ala11 and Ala12 with Lys (HYL-4) rendered a 4-fold increase in activity against P. aeruginosa but a significant decrease in activity against S. aureus (Table 2). The replacement of His13 by Ile in the hydrophobic face of the α-helix (Figure 2), enhancing both the overall hydrophobicity and amphipathic character of the peptide and resulting in the most hydrophobic analogue (HYL-6) of the series (H = 0.054), caused a remarkable increase in antimicrobial and antifungal activity against most of those microbes tested. That, however, came at the expense of a considerable increase in hemolytic activity (Table 2). This is in accordance with the literature data stating that increasing the hydrophobicity of the nonpolar face of amphipathic α-helical peptides increases not only their antimicrobial activity but also their hemolytic activity.38 On the other hand, the substitution of Ser4 by Ile made also within the hydrophobic face of HYL did not lead to improvement of the activities (HYL-5). A continuing trend in the improvement of activity against the strains of pathogenic S. aureus and P. aeruginosa was observed in the case of HYL-7 (Ala12 for Lys) and HYL-10 (Ala12 and

Ala15 for Lys), analogues with increased hydrophobicity and cationicity as well as high amphipathic character. While the former analogue was more active against P. aeruginosa, its hemolytic activity was still very high (Table 2). Other substitutions leading to an increment in peptide cationicity (Ala11 and Ala12 for Lys) resulted in an HYL-8 analogue with 8-fold higher activity against pathogenic S. aureus and P. aeruginosa compared to HYL as well as 3-fold greater activity against C. albicans. At the same time, it retained low toxicity against human red blood cells (equal to that of its parent peptide). The most cationic and ideally amphipathic analogue of the series, HYL-11, prepared by the replacement of all Ala residues in the sequence by Lys, exhibited improved antimicrobial activity against one strain of S. aureus and both strains of P. aeruginosa (Table 2) while still retaining low hemolytic activity. As described above for selected analogues, the increase of their antimicrobial activity while maintaining a low hemolytic activity was achieved by the incremental increase of cationicity up to a certain level 37 combined with simultaneous alterations of peptide hydrophobicity. A slight increase in the hydrophobic character of HYL-8 created by substitution of both Met residues with Leu (HYL-12) or Nle (HYL-14) led to a further enhancement of activity against S. aureus strains, albeit at the expense of increased hemolytic activity. C

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Table 2. Antimicrobial and Hemolytic Activity of HYL and Its Analoguesa antimicrobial activity MIC (μM) peptide

S.a. 6271

S.a.b

S.a.c

S.e.c

E.c.

P.a. 5482

P.a.b

C.a.d

hemolytic activitye LC50 (μM)

HYL HYL-1 HYL-2 HYL-3 HYL-4 HYL-5 HYL-6 HYL-7 HYL-8 HYL-9 HYL-10 HYL-11 HYL-12 HYL-13 HYL-14 HYL-15 HYL-16 HYL-17 HYL-18 HYL-19 HYL-20 HYL-21 HYL-22 HYL-23 HYL-24 HYL-25 HYL-26 tetracycline rifampicin amoxicillin

63 >100 63 85 >100 43 7.2 9 32 13 33 54 16 5.7 13 17 23 32 20 16 5.3 6.3 26 20 8 2.5 >100 0.9 100 35 77 >100 35 10 5.2 7.6 7 8.6 18 3.8 2.6 4 4.3 5.9 5.5 4.6 6.6 2 2.9 11 7.8 2.6 1.8 >100 1.1 100 63 90 >100 65 10 10 23 16 17 65 5 6.3 6.7 10 10 10 5.3 16 5 3.2 27 20 4 2 >100 2 100 >100 100 2.7 10 2.7

20 26 7.9 23 6.4 47 8 4.5 5 3.2 5.2 7.1 5 4.5 4 5 4.5 5 6.3 4 5.3 6.7 6.8 5 8 13 98 29 27 >100

68 47 20 46 17 >100 17 9.3 7.4 8.5 10 9.9 7.5 9.7 7.9 8.9 8.5 16 21 5.9 8.8 8.8 19 7.1 21 24 >100 70 19 >100

18 23 14 5 12 12 8.4 11 5.2 8 7.6 6.3 10 9 10 24 45 25 16 17 11 8 34 11 16 13 >100 n.t.f n.t. n.t.

>400 >400 >400 >400 >400 320 93 88 >400 >400 >400 >400 284 333 373 >400 >400 364 165 >400 197 191 >400 >400 115 57 >400 >200 n.t. n.t.

a

S.a., Staphylococcus aureus; S.e., Staphylococcus epidermidis; E.c., Escherichia coli; P.a., Pseudomonas aeruginosa; C.a., Candida albicans. bClinical isolates from Liberec Regional Hospital (Czech Republic). cClinical isolates from University Hospital in Motol (Prague, Czech Republic). dMIC of amphotericin B was 5.7 μM. eConcentration causing lysis of 50% of red blood cells. fn.t., not tested.

hemolytic activity while preserving antimicrobial activity, we replaced the centrally positioned leucine in its sequence by the helix-breaker proline. Against our expectations, and contrary to published data,41−44 this substitution resulted in significant loss of antimicrobial activity (HYL-26, Table 2). Note that HYL-26 eluted from the C-18 column with the shortest retention time (tR = 24.05, Table 1) of all analogues of the series. This clearly reflects its reduced hydrophobicity, amphipathicity, and the αhelical structure distortion, which consequently had a detrimental impact on its antimicrobial activity. The interactions of α-helical antimicrobial peptides with C-18 stationary phase groups during RP-HPLC are discussed in our former publication.31 Notably, the analogue HYL-19, which was prepared by a substitution of Leu6 for Trp in HYL-12, had low hemolytic activity, and its activities against all microbes tested were satisfactory. Incorporation of two Trp residues into the hydrophobic segment of the HYL-12 molecule led to the HYL25 analogue, which showed the highest hemolytic activity from the series. The all-D peptides (HYL-9, HYL-13, and HYL-21) exhibited biological activities equal to those of their L-isomers (Table 2). The MICs of HYL and all its analogues against M. luteus and B. subtilis did not exceed 3 and 7 μM, respectively (data not shown in Table 2). The only exception was HYL-26, which showed increased MIC values against these bacteria: 5.8 μM for M. luteus and 49 μM for B. subtilis.

Figure 2. Schiffer−Edmundson wheel projection of HYL. Hydrophobic amino acid residues are in red; hydrophilic residues in blue (lysines) and black.

The group of analogues with the highest net positive charge (+7) of the series (HYL-11, HYL-15, HYL-16, HYL-17, HYL22) exhibited activities the same on average as those having net positive charge +6. However, the high cationicity of these analogues had an unfavorable effect on their activity against C. albicans (Table 2). Substitution of Leu7 in HYL-12 for the bulky hydrophobic Trp residue resulted in the most powerful analogue (HYL-20) of the series, exhibiting activities against all bacteria tested with minimum inhibitory concentrations (MICs) < 10 μM. This was, however, associated with an increase in hemolytic activity. In an attempt to diminish its D

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Natural HYL and its two analogues HYL-8 and HYL-20 were selected for structural studies by NMR spectroscopy. Structural Characterization of HYL, HYL-8, and HYL20. In water, HYL provided electronic circular dichroism (ECD) spectra characteristic of an unstructured peptide with a broad minimum at 198 nm and low intensity. Detailed analysis showed that the peptide contained approximately 16% α-helical fraction. The ECD spectra (Figure 3) underwent a considerable

Figure 3. UV-ECD spectra of HYL in the presence of various concentrations of TFE (A) and SDS (B).

change with increasing concentration of 2,2,2-trifluoroethanol (TFE) or sodium dodecyl sulfate (SDS), demonstrating a predominantly α-helical structure as indicated by the appearance of typical bands at 208 and 222 nm of high intensity. The maximum α-helical content of 54% was reached in a solution containing 30% TFE and 61% in the presence of 4 mM SDS, as shown in Figure 3. Detailed information about the conformational behavior of HYL and its selected analogues HYL-8 and HYL-20 was obtained using NMR spectroscopy. We acquired NMR data for these peptides in the presence of TFE, as the studied molecules adopted a regular secondary structure only in this cosolvent, as suggested by ECD spectroscopy. All peptides provided good quality spectra, which allowed for complete sequence-specific resonance assignments using a combination of 2D homonuclear experiments followed by structural calculations using the NOEbased distance restraints. See the Supporting Information for the numbers of observed NOE peaks, distance constraints, and structural statistics for the obtained structures presented in Table S1 and detailed resonance assignments of peptides in 10% D2O/90% H2O and in 30% TFE-d2 shown in Tables S2− S7. In 30% TFE-d2, HYL adopted conformations having a secondary α-helical structure spanning from Met3 to Ala15 (Figure 4D). The preferred conformation for HYL is a curved helix (Figure 4A) with a convex surface covered mostly by hydrophilic amino acid residues, while its concave side is

Figure 4. NMR structures obtained for HYL, HYL-8, and HYL-20 in 30% TFE. (A, B, and C) Representative peptide structures; (D, E, and F) backbone topology for the sets of 30 convergent structures.

occupied prevalently by hydrophobic residues. The helical conformation is similar for all three peptides, but the absence of contacts between bulky amino acids at positions 11 and 12 within the HYL peptide enables the C-terminus to sample a larger conformational space (Figure 4D). The amphipathic character of HYL is further emphasized in its HYL-8 analogue by an extensive cationic patch formed by three lysines (Lys8, Lys11, and Lys12) and six hydrophobic side chains forming a large nonpolar area on the opposite side, as illustrated in Figure 4B. Such intervention involving the substitution of two Ala residues (positons 11 and 12) on both convex and concave surfaces for lysines led to the helix straightening by additional contacts of the hydrophobic parts of the introduced lysine side chains with the rest of the molecule and to the stabilization of the C-terminal tail. This structural rearrangement made in the C-terminal part of the peptide comprising the increment of its positive charge and amphipathicity, accompanied by a slight decrease in the hydrophobicity of the peptide (Table 1), apparently contributed to increased antimicrobial activity against all tested microbes. The substitution of Met3 and Met7 for Leu and Trp, respectively, in HYL-8 led to the HYLE

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20 analogue having an even better defined α-helix spanning from Ile2 to Ala15 (Figure 4 F) with side chains stabilized around the tryptophan aromatic ring. The presence of a large aromatic side chain resulted in a noticeably more curved helix than in HYL-8 due to the hydrophobic contacts between this newly introduced tryptophan ring and surrounding aliphatic side chains from Leu3, Lys8, Leu10, Lys11, and Ile14 (Figure 4C). These substitutions affecting also the hydrophobic character of the peptide led to substantial improvement in its activity against Gram-positive staphylococci, but that was at the expense of its toxicity against red blood cells. The helical conformation at the C-terminus of HYL as well as of HYL-8 and HYL-20 is supported by hydrogen bonds formed between the Lys16 amide group and a carbonyl group from residues His13 and Ile14 in HYL, Ile13 and Ile14 in HYL8, and a carbonyl group from only the Ile13 residue in HYL-20. The helical conformation of the C-terminal stabilizing hydrogen bond involving the C-terminal amide group has previously been observed in various α-helical AMPs.45 Synergistic Effect between HYL Peptides and Antibiotics. We tested the capability of various combinations of HYL and its analogues with three different types of antibiotics to inhibit the growth of P. aeruginosa and S. aureus. Amoxicillin is known to bind the penicillin-binding protein located on the cytoplasmic membrane, thus interfering with cell wall synthesis. Tetracycline targets bacterial protein synthesis on the ribosome, and rifampicin inhibits bacterial DNAdependent RNA polymerase,46 while the studied peptides are expected to disintegrate the bacterial cell membranes. The effects of the combinations were evaluated using fractional inhibitory concentration (FIC) indexes47 shown in Table 3. HYL and almost all of its analogues exhibited synergism (FIC ≤ 0.5) in combination with rifampicin against P. aeruginosa and only a few of them with tetracycline. We may speculate that peptides facilitated the entry of rifampicin into the P. aeruginosa cells to result in inhibition of the RNA polymerase. Some of the analogues showed synergistic activity also with amoxicillin against S. aureus. None of the combinations tested showed antagonism. Time Course of Bacteria Killing. We performed a time course killing experiment in order to compare the mode of action of the studied peptides with antibiotic on P. aeruginosa. We examined the effects of HYL-8, HYL-18, tetracycline, and rifampicin in concentrations of 8, 24, 80, and 20 μM, respectively (which means close to their MICs). Bacteria cultivated only in medium served as a growth control (Figure 5). The growth curve of P. aeruginosa in the presence of tetracycline demonstrated an unchanged quantity of viable bacteria within 60 min, followed by slight decrease over the following 3 h. This observation fully reflects the effect of the bacteriostatic antibiotic. In contrast, the bactericidal drug rifampicin, which is known to penetrate well into bacteria,46 showed a gradual decrease in cell count and achieved a reduction by 3 orders of magnitude over 4 h. The treatment with peptides resulted in a decrease by 2 orders of magnitude in surviving bacteria during the first 60 min, thus suggesting a fast killing effect of peptides. The structurally related HYL-8 and HYL-18 showed identical time courses, possibly reflecting their permeabilization effect on the bacteria as shown below. Propidium Iodide (PI) Uptake Study. To explain the synergistic effect, we hypothesized that HYL and its analogues are able to permeate the P. aeruginosa membranes (both outer

Table 3. FIC Indexes for the Combinations of Studied Peptides with Antibiotics against S. aureus and P. aeruginosaa FIC indexesb S. aureus

c

P. aeruginosac

peptide

AMX

TET

RIF

HYL HYL-1 HYL-2 HYL-4 HYL-6 HYL-7 HYL-8 HYL-9 HYL-10 HYL-11 HYL-12 HYL-13 HYL-14 HYL-15 HYL-16 HYL-17 HYL-18 HYL-19 HYL-20 HYL-21 HYL-22 HYL-23 HYL-24 HYL-25

n.t. n.t. 0.31 n.t. 0.56 0.50 0.33 0.54 0.38 0.17 0.69 0.63 0.55 0.54 0.33 0.29 0.40 0.37 0.56 0.73 0.25 0.44 0.56 0.58

0.56 0.31 0.54 0.39 0.76 0.79 0.65 0.67 0.54 0.63 0.54 0.75 0.58 0.52 0.65 0.71 0.35 0.65 0.65 0.58 0.54 0.58 0.63 0.42

0.26 0.21 0.25 0.23 0.51 0.43 0.43 0.32 0.42 0.36 0.39 0.30 0.93 0.33 0.34 0.26 0.27 0.40 0.44 0.29 0.29 0.42 0.27 0.29

a

Bold numbers indicate synergism. AMX, amoxicillin; TET, tetracycline; RIF, rifampicin; n.t., not tested. bThe FIC indexes were interpreted as follows: FIC ≤ 0.5 is synergistic, 0.5 < FIC ≤ 2 is additive, and FIC > 2 is antagonistic effect. cClinical isolates of S. aureus and P. aeruginosa were from Liberec Regional Hospital (Czech Republic).

Figure 5. Growth curves of P. aeruginosa in the presence of HYL-8, HYL-18, and antibiotics: (△) growth control, (red ▲) HYL-8 (8 μM), (blue ●) HYL-18 (24 μM), (green ●) tetracycline (80 μM), (purple ■) rifampicin (20 μM). All values are means from four independent measurements. Error bars represent standard error of the mean.

and inner), thus facilitating entrance of antibiotics into the cells. To confirm this, we used PI, which is known to increase its fluorescence upon interaction with nucleic acids inside the bacterial cell but cannot pass into the cell if the cell membrane is intact. The initial experiments indicated that the effect of F

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(108 vs 105 cfu/mL, respectively), PC50 values for all tested peptides were lower than their corresponding MICs. In the cases of HYL-18 and HYL-25, moreover, both peptides showed synergism with tetracycline and rifampicin, and their PC50 values were 30- and 19-fold lower, respectively, than were their MICs. This is in accordance with published data26,42 pointing to the fact that some lytic peptides can induce membrane perturbation at low peptide concentrations (permeabilization activity), whereas at high concentrations they cause serious damage to the bacterial membrane, leading to cell death (bactericidal activity). Conversely, those publications26,42 also described highly active peptides causing cell death at the same concentrations at which they permeabilized the bacterial membrane. In our work, almost all peptides showing high synergic effects with rifampicin (FIC < 0.3) against P. aeruginosa had rather moderate bactericidal activity, with MICs of >16 μM. As an exception, the peptide HYL-14, having both low MIC and low PC50, showed no synergistic effect in combination with any antibiotic. This exception is similar to that already described by others.26 Transmission Electron Microscopy (TEM). To examine the influence of HYLs on the morphology of P. aeruginosa cells, we treated the cells with the selected analogue HYL-8 and then visualized these cells by negative staining. Untreated control bacteria revealed native morphology represented by an electron-dense character and well-preserved envelope structure with a single polar flagellum (Figure 8A). The majority (about 60%) of bacteria treated with HYL-8 at a concentration of 30 μM still showed compact cells, but their envelopes were disrupted and detached from the cell body (Figure 8B, C, and D). Flagella were still attached to the cells. Some bacterial cells revealed leakage of inner bacterial content and breakdown in cell integrity (Figure 8C and D). Treatment of the bacteria with HYL-8 at a concentration of 150 μM revealed severe damage of their cells, detached flagella, and most cells completely destroyed (Figure 8E and F). Our results point to the potential for transforming a naturally occurring α-helical AMP with low antimicrobial activity into compounds with greater potency against pathogenic microorganisms, low toxicity against eukaryotic cells, and added benefit when used in combination with common antibiotics. Apparently acting on the bacterial cell surface, these peptides react quickly and cause disruption of both the outer and inner membrane of P. aeruginosa. This leads to augmented antibiotic uptake. The synergistic effect of some HYL peptides in combination with amoxicillin against S. aureus cannot be explained by similar actions inasmuch as penicillins do not need to cross the cytoplasmic membrane of Gram-positive bacteria to reach their target and exert a bactericidal effect.46

HYL-18 on membrane integrity was concentration dependent, as shown in Figure 6.

Figure 6. Time course for the uptake of propidium iodide by P. aeruginosa cells as a measure of its outer and inner membrane permeation at various concentrations of HYL-18: (blue □) 0.625 μM, (pink ○) 1.25 μM, (green ▲) 2.5 μM, (red ●) 5 μM, (blue −) 10 μM, (purple ▲) 20 μM, (◇) blank, (□) octenidine dihydrochloride (4 μM). All values are means from at least three independent measurements. For the sake of clarity, error bars are not shown.

HYL-18 was able to completely permeate the bacterial cell membranes at concentrations (5 and 10 μM) even lower than its MIC (21 μM) within the first 10 min, whereas in the case of octenidine dihydrochloride (ODDC, used as positive control) the fluorescence intensity grew slowly and reached its maximum after 90 min. Such a rapid permeabilization effect of HYL-18 is in accordance with the time−killing curve (Figure 5) showing a decrease in viable bacteria count by 2 orders of magnitude within the first 30 min of measurement. Taking these results into consideration, the fluorescence intensities of PI in the presence of all other peptides at different concentrations were acquired at the time point of 90 min. For the purpose of this work, we have introduced the term permeabilizing concentration (PC50), which means the concentration of a peptide causing permeation of 50% of bacterial cells. Permeabilization activity plotted against the logarithm of peptide concentrations resulted in sigmoid curves with good fitting. PC50 values were interpolated from such curves for each peptide tested (see Supporting Information). The low PC50 values for the majority of peptides as shown in Figure 7 indicate that even relatively low peptide concentrations enabled penetration of PI through the outer as well as inner membrane of bacterial cells. Considering the higher concentration of bacteria used in the PI uptake study than that used in the MIC determination assay

Figure 7. Concentration of each peptide analogue causing the permeation of 50% of P. aeruginosa cells expressed as PC50. G

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Peptide sequence by Edman degradation was determined on the Procise protein sequencing system (491 protein sequencer, PE Applied Biosystems) using the manufacturer’s pulse-liquid Edman degradation chemistry cycles. Fmoc-protected L-amino acids and Rink Amide MBHA resin were purchased from IRIS Biotech. Octenidine dihydrochloride was bought from TCI. Amoxicillin, amphotericin B, tetracycline, rifampicin, PI, brain-heart infusion, LB broth, and LB agar were purchased from Sigma−Aldrich. The sterile YPG medium for cultivation of C. albicans was prepared in our laboratory from 1% yeast extract (Difco Laboratories), 2% peptone (Oxoid), and 2% glucose. All other reagents, peptide synthesis solvents, and HPLC gradient grade CH3CN were of the highest purity available from commercial sources. As test organisms, we used the following: B. subtilis 168 was kindly provided by Prof. Yoshikawa (Princeton University); E. coli B and M. luteus No. CCM 144 were from the Czech Collection of Microorganisms, Brno, Czech Republic; P. aeruginosa (5482) ATCC 27853 and methicillin-resistant S. aureus (MRSA 6271) ATCC 43300 were purchased from the Czech National Collection of Type Cultures, the National Institute of Public Health, Prague, Czech Republic; S. aureus (MRSA (L)) and P. aeruginosa (L) were obtained as multiresistant clinical isolates from Liberec Regional Hospital, Czech Republic; methicillin-resistant S. aureus (MRSA (M)) and S. epidermidis (M) were obtained as clinical isolates from University Hospital in Motol, Prague, Czech Republic; and C. albicans (F7-39/IDE99) came from the mycological collection of the Faculty of Medicine, Palacky University, Olomouc, Czech Republic. Blood samples for determination of hemolytic activity were obtained from healthy volunteers. Stock solutions (1 mM) of tested peptides and antibiotics except for amphotericin B were prepared in sterile 0.9% NaCl. Due to the poor solubility of amphotericin B in H2O, its 1 mM stock solution was prepared by diluting its 10 mM solution in DMSO using sterile 0.9% NaCl. Collection of Bee Specimens and Isolation of HYL from Their Venom. Bee specimens of Hylaeus signatus were collected near Kladno, Czech Republic (GPS: 50.17715, 14.11379), during August 2010, database voucher Colletidae 2326 of Charles University in Prague collection. The specimens were kept frozen at −20 °C until dissected. The venom reservoirs of seven individuals were dissected, and their contents extracted using a 1:1 CH3CN/H2O mixture containing 0.5% TFA. The peptide was isolated from the extract by RP-HPLC as described in our preceding publications.29−33 The fraction active against M. luteus (Figure 1) was further analyzed by mass spectrometry and subjected to Edman degradation. Peptide Synthesis. HYL and its analogues were synthesized manually according to the standard Nα-Fmoc protocol on a Rink Amide MBHA resin (100 mg with 0.45 mmol/g substitution) in 5 mL polypropylene syringes with a Teflon filter on the bottom. Protected amino acids (4 equiv) were coupled using N,N′-diisopropylcarbodiimide (7 equiv) and 1-hydroxybenzotriazole (5 equiv) as coupling reagents in N,N-dimethylformamide (DMF) as a solvent. Deprotections of the α-amino groups were performed with 20% piperidine in DMF. The peptides were fully deprotected and cleaved from the resin with a 2 mL mixture of TFA/thioanisole/H2O/1,2-ethanedithiol/ triisopropylsilane (90:3:2.5:2.5:2) for 3.5 h. The peptides were then precipitated with tert-butyl methyl ether, yielding an average 100 mg of crude peptides. This material was further purified by preparative RPHPLC on a Thermo Separation Product instrument using a Vydac C18 column (10 × 250 mm; 5 μm) at a 3 mL/min flow rate using the solvent gradient as described above. The fractions were detected by UV absorption at 280 or 222 nm. The main dominant fraction containing the required peptide product was lyophilized. The purity by analytical HPLC ranged from 96% to 99%. The retention times and results of mass spectrometry analyses confirming the identity of peptides are shown in Table 1. Antimicrobial Activity Determination. The antimicrobial activity of RP-HPLC fractions collected in the course of HYL isolation (Figure 1) was detected using the drop diffusion test on a Petri dish with the sensitive bacterium M. luteus as previously described.29 MICs were established by observing bacterial growth in 100-well microtiter

Figure 8. Electron micrographs of negatively stained P. aeruginosa cells treated with different concentrations of HYL-8 for 60 min: (A) untreated cells, (B, C, and D) 30 μM peptide, and (E, F) 150 μM peptide. Scale bar: 500 nm. Bacterial cells are shown in different stages of disruption.



EXPERIMENTAL SECTION

General Experimental Procedures. Mass spectra of the peptides were acquired on a Micromass Q-Tof micro mass spectrometer (Waters) equipped with an electrospray ion source. A 1:1 CH3CN/ H2O mixture with 0.1% formic acid was continuously delivered to the ion source at a 100 μL/min flow rate. Samples dissolved in 20 μL of the mobile phase were introduced using a 2 μL loop. The measurement was performed in the positive mode, and the capillary voltage, cone voltage, desolvation temperature, and ion source temperature were 3.5 kV, 10 V, 150 °C, and 90 °C, respectively. RP-HPLC was carried out on an Agilent Technologies 1200 Series module with a Vydac C-18, 250 × 4.6 mm; 5 μm, column (Grace Vydac) at a 1 mL/min flow rate using a solvent gradient ranging from 5% to 70% CH3CN/H2O/0.1% trifluoroacetic acid (TFA) over 60 min. The elution was monitored by absorption at 220, 254, and 280 nm utilizing a diode-array detector. The instrument was controlled using ChemStation software. H

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plates.29,31 Different concentrations of tested compounds were prepared by 2-fold serial dilution in fresh LB medium from stock solutions and added to the wells (100 μL). The final concentrations in the wells after addition of bacterial suspension ranged from 0.1 to 100 μM for peptides and antibiotics. Test microorganisms were grown at 37 °C with continuous shaking for 3−5 h to mid log phase. Afterward, this bacterial suspension was diluted in fresh LB medium to a final concentration of approximately 5 × 105 cfu/mL, and 100 μL of that inoculum was added to the wells (final volume in the well was 200 μL). Fresh medium with bacteria only was used as growth control. For cultivation and dilution of C. albicans, fresh YPG medium was used. The plates were incubated at 37 °C for 20 h while being continuously shaken in a Bioscreen C instrument (Oy Growth Curves). The absorbance was measured at 540 nm every 15 min, and growth curves were constructed. Each peptide was tested at least three times in duplicates. Hemolysis Assays. Hemolytic activity is expressed as the concentration of a peptide required for lysis of 50% of human erythrocytes in the assay (LC50). The peptides in different concentrations ranging from 2.5 to 400 μM were incubated with a 4% suspension of human red blood cells in physiological solution at a final volume of 200 μL for 1 h at 37 °C. The samples were then centrifuged at 250g for 5 min, and the absorbance of the supernatant at 540 nm was measured on a Tecan Infinite M200 PRO reader. Red blood cells incubated with physiological solution were used as blank control for zero hemolysis, and 0.2% Triton X-100 served as control of 100% hemolysis. Each peptide was tested in duplicate in at least two independent experiments. Circular Dichroism. ECD spectra were measured on a Jasco 815 spectrometer in the spectral range 190−300 nm using a 0.1 cm quartz cell at room temperature. The experimental setup was as follows: 20 nm/min scanning speed, 8 s response time, and 1 nm spectral bandwidth. The final peptide concentration was held constant (0.2 mg/mL). The peptide spectra were measured in H2O, in H2O/TFE mixtures (from 10% to 50% v/v of TFE), and in the presence of SDS at concentrations of 0.016 to 8 mM. After baseline correction, the final spectra were expressed as a molar ellipticity θ (deg cm2 dmol−1) per residue. The α-helical fraction was calculated assuming a two-state model.48,49 NMR Spectroscopy and Structural Calculations (Molecular Modeling). NMR spectra were acquired from 0.66 mL samples of 5 mM HYL and its analogues HYL-8 and HYL-20 in an aqueous solution of 10% D2O/90% H2O (pH 4.0) and in the presence of 30% deuterated TFE (CF3CD2OH). All NMR data were collected at 25 °C on a 600 MHz Bruker Avance spectrometer equipped with a tripleresonance (15N/13C/1H) cryoprobe. A series of homonuclear spectra were recorded to determine sequence-specific resonance assignments for the studied peptides, in particular, 2D TOCSY spectra with a 90 ms mixing time and 2D DQF-COSY and 2D NOESY spectra, acquired with an NOE mixing time of 300 ms in 30% TFE. The combined automated NOE assignment and structure determination protocol implemented in Cyana 2.150 was used for automatic assignment of the NOE cross-peaks identified in 2D NOESY spectra. The families of converged structures for the studied peptides were obtained using a simulated annealing protocol with NOE-derived distance restraints. Subsequently, five cycles of simulated annealing combined with redundant dihedral angle constraints51 were used to produce a set of converged structures with no significant restraint violations (distance and van der Waals violations 2 antagonism.47 Killing Kinetics Assay. An inoculum of P. aeruginosa was prepared in fresh LB medium to a final concentration of approximately 5 × 105 cfu/mL, as described above. Then 1 mL of this inoculum was added to each sterile glass flask containing 1 mL of fresh LB medium (growth control), peptides, or antibiotics in LB medium. Their final concentrations were in their MIC (8 μM for HYL-8, 24 μM for HYL-18, 80 μM for tetracycline, and 20 μM for rifampicin). The mixtures were then incubated at 37 °C for 4 h with shaking. Aliquots (100 μL) were taken at 0, 10, 20, 30, 60, 90, 120, 150, 180, 210, and 240 min from the mixtures, serially diluted in sterile 0.9% NaCl, and 100 μL of each dilution was spread on an LB agar plate. Plates were incubated at 37 °C overnight, grown colonies were counted, and the number of viable cells for each time interval was expressed as cfu/mL. PI Uptake Study. An overnight inoculum of P. aeruginosa grown in 5 mL of LB medium at 37 °C was diluted 10 times in fresh LB medium and incubated with continuous shaking for another 3−5 h to mid log phase. Bacteria were transferred to sterile 15 mL Falcone tubes and centrifuged at 3500 rpm for 10 min. The supernatant was decanted, and a pellet was washed twice with 10 mL of sterile phosphate buffer saline (PBS; containing 61 mM Na2HPO4·2H2O, 39 mM NaH2PO4·2H2O, and 19.5 mM NaCl, pH 7). Finally, the pellet was resuspended in fresh PBS and diluted to contain the cells in the amount of 108 cfu/mL. PI from a stock solution (1 mg/mL of sterile MiliQ H2O) was added to the bacterial suspension so that its concentration was 10 μg/mL. This mixture (100 μL) was pipetted into the wells of a black 96-well microtiter plate containing 100 μL of tested concentrations of peptides prepared by 2-fold serial dilution in sterile PBS. Wells containing only PBS served as a blank control, and ODDC at a final concentration of 4 μM served as a positive control of the permeabilization of bacterial cells. Final concentrations of peptides ranged from 0.31 to 80 μM. The microtiter plate was incubated at 37 °C for 120 min in the dark, and fluorescence intensity was measured on a Tecan Infinite M200 PRO reader (Tecan Austria) every minute (for HYL-18 and ODDC) using excitation and emission wavelengths of 544 and 620 nm, respectively. In the case of all other peptides, the plates were incubated for 90 min at 37 °C in the dark, and then the fluorescence intensity was measured at the end of incubation. The permeabilization activity of each peptide concentration was expressed as a percentage of ODDC activity using the following equation:

Permeabilization activity [%] = (Ip − Ibl)/(IODDC − Ibl) × 100 where Ip is the fluorescent intensity of PI in the presence of peptide, Ibl is the fluorescent intensity of the blank, and IODDC is the fluorescent intensity of PI in the presence of ODDC considered to cause 100% permeability for PI. For each peptide, its percentage was plotted against the log of its concentration (see Supporting Information) and PC50 was interpolated as the concentration of the peptide causing cell permeability at 50% of bacterial cells. All compounds were tested at least three times in duplicates. Transmission Electron Microscopy. The effect of the HYL peptide on the structure of P. aeruginosa was studied by TEM after negative staining of the samples. Bacterial cells (108 cfu/mL) were treated with HYL-8 at two different concentrations (30 and 150 μM) for 60 min. Untreated bacteria were used as a control. Bacterial cells were centrifuged, washed twice with sterile 0.9% NaCl, and adsorbed on Parlodion-carbon-coated copper grids for 5−10 min. After a short washing, the samples were negatively stained using 0.25% phosphotungstic acid (pH 7.3) with 0.01% bovine serum albumin in dH2O for 30−40 s and then air-dried. The specimens were then examined using a JEOL JEM 1011 transmission electron microscope operating at 80 kV. I

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(19) Parameswaran, G. I.; Sethi, S. Future Microbiol. 2012, 7, 1129− 1132. (20) Garey, K. W.; Vo, Q. P.; Lewis, R. E.; Saengcharoen, W.; LaRocco, M. T.; Tam, V. H. Diagn. Microbiol. Infect. Dis. 2009, 63, 81− 86. (21) Park, Y.; Kim, H. J.; Hahm, K.-S. Biochem. Biophys. Res. Commun. 2004, 321, 109−115. (22) Giacometti, A.; Cirioni, O.; Kamysz, W.; Silvestri, C.; Del Prete, M. S.; Licci, A.; D’Amato, G.; Lukasiak, J.; Scalise, G. J. Antimicrob. Chemother. 2005, 56, 410−412. (23) Tré-Hardy, M.; Vanderbist, F.; Traore, H.; Devleeschouwer, M. J. Int. J. Antimicrob. Agents 2008, 31, 329−336. (24) Cirioni, O.; Silvestri, C.; Ghiselli, R.; Orlando, F.; Riva, A.; Mocchegiani, F.; Chiodi, L.; Castelletti, S.; Gabrielli, E.; Saba, V.; Scalise, G.; Giacometti, A. J. Antimicrob. Chemother. 2008, 62, 1332− 1338. (25) Anantharaman, A.; Rizvi, M. S.; Sahal, D. Antimicrob. Agents Chemother. 2010, 54, 1693−1699. (26) Sánchez-Gómez, S.; Japelj, B.; Jerala, R.; Moriyón, I.; Alonso, M. F.; Leiva, J.; Blondelle, S. E.; Andrä, J.; Brandenburg, K.; Lohner, K.; Martinez de Tejada, G. Antimicrob. Agents Chemother. 2011, 55, 218− 228. (27) Choi, H.; Lee, D. G. Res. Microbiol. 2012, 163, 479−486. (28) Hwang, I.; Hwang, J.-S.; Hwang, J. H.; Choi, H.; Lee, E.; Kim, Y.; Lee, D. G. Curr. Microbiol. 2013, 66, 56−60. (29) Č eřovský, V.; Hovorka, O.; Cvačka, J.; Voburka, Z.; Bednárová, L.; Borovičková, L.; Slaninová, J.; Fučík, V. ChemBioChem 2008, 9, 2815−2821. (30) Č eřovský, V.; Buděsí̌ nský, M.; Hovorka, O.; Cvačka, J.; Voburka, Z.; Slaninová, J.; Borovičková, L.; Fučík, V.; Bednárová, L.; Votruba, I.; Straka, J. ChemBioChem 2009, 10, 2089−2099. (31) Monincová, L.; Buděsí̌ nský, M.; Slaninová, J.; Hovorka, O.; Cvačka, J.; Voburka, Z.; Fučík, V.; Borovičková, L.; Bednárová, L.; Straka, J.; Č eřovský, V. Amino Acids 2010, 39, 763−775. (32) Č ujová, S.; Slaninová, J.; Monincová, L.; Fučík, V.; Bednárová, L.; Štokrová, J.; Hovorka, O.; Voburka, Z.; Straka, J.; Č eřovský. Amino Acids 2013, 45, 143−157. (33) Č ujová, S.; Bednárová, L.; Slaninová, J.; Straka, J.; Č eřovský, V. J. Pept. Sci. 2014, 20, 885−895. (34) Slaninová, J.; Putnová, H.; Borovičková, L.; Šácha, P.; Č eřovský, V.; Monincová, L.; Fučík, V. Cent. Eur. J. Biol. 2011, 6, 150−159. (35) Slaninová, J.; Mlsová, V.; Kroupová, H.; Alán, L.; Tůmová, T.; Monincová, L.; Borovičková, L.; Fučík, V.; Č eřovský, V. Peptides 2012, 33, 18−26. (36) Tossi, A.; Sandri, L.; Giangaspero, A. Biopolymers 2000, 55, 4− 30. (37) Jiang, Z.; Vasil, A. I.; Hale, J. D.; Hancock, R. E. W; Vasil, M. L.; Hodges, R. S. Biopolymers 2008, 90, 369−383. (38) Chen, Y.; Guarnieri, M. T.; Vasil, A. I.; Vasil, M. L.; Mant, C. T.; Hodges, R. S. Antimicrob. Agents Chemother. 2007, 51, 1398−1406. (39) Pathak, N.; Salas-Auvert, R.; Ruche, G.; Janna, M.-H.; McCarthy, D.; Harrison, R. G. Proteins: Struct., Funct., Genet. 1995, 22, 182−186. (40) Eisenberg, D.; Schwarz, E.; Komaromy, M.; Wall, R. J. Mol. Biol. 1984, 179, 125−142. (41) Suh, J.-Y.; Lee, Y.-T.; Park, C.-B.; Lee, K.-H.; Kim, S.-C.; Choi, B.-S. Eur. J. Biochem. 1999, 266, 665−674. (42) Zhang, L.; Benz, R.; Hancock, R. E. W. Biochemistry 1999, 38, 8102−8111. (43) Shin, S. Y.; Lee, S.-H.; Yang, S.-T.; Park, E. J.; Lee, D. G.; Lee, M. K.; Eom, S. H.; Song, W. K.; Kim, Y.; Hahm, K.-S.; Kim, J. I. J. Pept. Res. 2001, 58, 504−514. (44) Yang, S.-T.; Lee, J. Y.; Kim, H.-J.; Eu, Y.-J.; Shin, S. Y.; Hahm, K.-S.; Kim, J. I. FEBS J. 2006, 273, 4040−4054. (45) Sforca, M. L.; Oyama, S., Jr.; Canduri, F.; Lorenzi, C. B.; Pertinhez, T. A.; Konno, K.; Souza, B. M.; Palma, M. S.; Neto, J. R.; Azevedo, W. F., Jr.; Spisni, A. Biochemistry 2004, 43, 5608−5617. (46) Anderson, R. J.; Groundwater, P. W.; Todd, A.; Worsley, A. J. Antimicrobial Agents: Chemistry, Mode of Action, Mechanism of

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b01129. Cycles of Edman degradation; mass spectrum of natural HYL; NMR constraints and structural statistics for the studied peptides (Table S1); proton NMR parameters of peptides (Tables S2−S7); interpolation of PC50 values of HYL and its analogues from sigmoid curves of their permeabilization activity (PDF)



AUTHOR INFORMATION

Corresponding Author

*Tel: +420-220183378. Fax: +420-220183578. E-mail: [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was supported by Technology Agency of the Czech Republic, grant no. TA04010638, by the Ministry of Education of the Czech Republic, grant no. LO1304, and by research project RVO 61388963 of the Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic. We thank our technical assistant Mrs. L. Borovičková for her help with peptide synthesis and Mrs. L. Monincová, Ph.D., for mass spectrometry measurements. We also thank G. A. Kirking at English Editorial Services, s.r.o., for assistance with the English.



REFERENCES

(1) Giuliani, A.; Pirri, G.; Nicoletto, S. F. Centr. Eur. J. Biol. 2007, 2, 1−33. (2) Zaiou, M. J. Mol. Med. 2007, 85, 317−329. (3) Oyston, P. C. F; Fox, M. A.; Richards, S. J.; Clark, G. C. J. Med. Microbiol. 2009, 58, 977−987. (4) Baltzer, S. A.; Brown, M. H. J. Mol. Microbiol. Biotechnol. 2011, 20, 228−235. (5) Yeung, A. T. Y; Gellatly, S. L.; Hancock, R. E. W. Cell. Mol. Life Sci. 2011, 68, 2161−2176. (6) Brogden, N. K.; Brogden, K. A. Int. J. Antimicrob. Agents 2011, 38, 217−25. (7) Yeaman, M. R.; Yount, N. Y. Pharmacol. Rev. 2003, 55, 27−55. (8) Toke, O. Biopolymers 2005, 80, 717−735. (9) Huang, Y.; Huang, J.; Chen, Y. Protein Cell 2010, 1, 143−52. (10) Amiche, M.; Galanth, C. Curr. Pharm. Biotechnol. 2011, 12, 1184−1193. (11) Epand, R. M.; Epand, R. F. J. Pept. Sci. 2011, 17, 298−305. (12) Wimley, W. C.; Hristova, K. J. Membr. Biol. 2011, 239, 27−34. (13) Høiby, N.; Ciofu, O.; Bjarnsholt, T. Future Microbiol. 2010, 5, 1663−1674. (14) Bodey, G. P.; Bolivar, R.; Fainstein, V.; Jadeja, L. Clin. Infect. Dis. 1983, 5, 279−313. (15) Landman, D.; Bratu, S.; Alam, M.; Quale, J. J. Antimicrob. Chemother. 2005, 55, 954−957. (16) Mesaros, N.; Nordmann, P.; Plésiat, P.; Roussel-Delvallez, M.; Van Eldere, J.; Glupczynski, Y.; Van Laethem, Y.; Jacobs, F.; Lebecque, P.; Malfroot, A.; Tulkens, P. M.; Van Bambeke, F. Clin. Microbiol. Infect. 2007, 13, 560−578. (17) Sivanmaliappan, T. S., Sevanan, M. Int. J. Microbiol. 2011, 2011, 110.1155/2011/605195. (18) Høiby, N.; Johansen, H. K.; Moser, C.; Song, Z.; Ciofu, O.; Kharazmi, A. Microbes Infect. 2001, 3, 23−35. J

DOI: 10.1021/acs.jnatprod.5b01129 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Resistance and Clinical Applications; John Wiley & Sons: Chichester, 2012. (47) Meletiadis, J.; Pournaras, S.; Roilides, E.; Walsh, T. J. Antimicrob. Agents Chemother. 2010, 54, 602−609. (48) Rohl, C. A.; Baldwin, R. L. Methods Enzymol. 1998, 295, 1−26. (49) Whitmore, L.; Wallace, B. A. Biopolymers 2008, 89, 392−400. (50) Herrmann, T.; Güntert, P.; Wüthrich, K. J. Mol. Biol. 2002, 319, 209−227. (51) Güntert, P.; Wüthrich, K. J. Biomol. NMR 1991, 1, 447−456. (52) Harjes, E.; Harjes, S.; Wohlgemuth, S.; Müller, K.-H.; Krieger, E.; Herrmann, C.; Bayer, P. Structure 2006, 14, 881−888.

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