Combating Drug-Resistant Fungi with Novel Imperfectly Amphipathic

Article pubs.acs.org/jmc

Cite This: J. Med. Chem. 2018, 61, 3889−3907

Combating Drug-Resistant Fungi with Novel Imperfectly Amphipathic Palindromic Peptides Jiajun Wang, Shuli Chou, Zhanyi Yang, Yang Yang, Zhihua Wang, Jing Song, Xiujing Dou, and Anshan Shan* Institute of Animal Nutrition, Northeast Agricultural University, Harbin 150030, P. R. China S Supporting Information *

ABSTRACT: Antimicrobial peptides are an important weapon against invading pathogens and are potential candidates as novel antibacterial agents, but their antifungal activities are not fully developed. In this study, a set of imperfectly amphipathic peptides was developed based on the imperfectly amphipathic palindromic structure Rn(XRXXXRX)Rn (n = 1, 2; X represents L, I, F, or W), and the engineered peptides exhibited high antimicrobial activities against all fungi and bacteria tested (including fluconazole-resistant Candida albicans), with geometric mean (GM) MICs ranging from 2.2 to 6.62 μM. Of such peptides, 13 (I6) (RRIRIIIRIRR-NH2) that was Ile rich in its hydrophobic face had the highest antifungal activity (GMfungi = 1.64 μM) while showing low toxicity and high salt and serum tolerance. It also had dramatic LPS-neutralizing propensity and a potent membrane-disruptive mechanism against microbial cells. In summary, these findings were useful for short AMPs design to combat the growing threat of drug-resistant fungal and bacterial infections.

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INTRODUCTION

different active mechanisms, and low resistance is urgently awaiting development. Antimicrobial peptides (AMPs) have received great attention as a promising solution to combat multiple-antimicrobial resistant microorganisms. As an important defensive line for the organism immune systems, AMPs show potent and broad spectrum antimicrobial activity, more rapid sterilization efficiency than conventional antimicrobial agents, target persistent bacteria persister cells, and have a lower likelihood of resistance development due to their unique physical membrane binding and disruption mechanism.7 In addition, many recent studies have demonstrated that peptide-based agents have attractive potential as antimicrobial implantation device coatings, since they have high biocompatible and have successfully been coated on a large variety of substrates.8−10 Specifically, the potent antifungal peptides, which have been subclassified as antifungal peptides (AFPs), have also been developed as surface-based strategies to prevent Candida albicans from forming a fungal biofilm on the surface of urinary catheters to reduce the incidence of fungal catheter-

With the global burden of antimicrobial resistant infections rising at an alarming pace, antimicrobial resistance cannot be ignored because difficulties with major surgery, transplant operations, and septicemia therapy would arise,1 specifically biomedical device-related infections. It is predicted that 10 million people will die from antimicrobial resistant infections a year worldwide by 2050, costing over 100 trillion USD in lost output.2Compared with bacterial resistance, antifungal resistance has not been widely recognized due to the low fungal infection rate until now. As medical development, HIV/AIDS and cancer treatment have led to a large number of immunosuppressive patients, who are highly susceptible to serious invasive fungal infections (IFI), for example, the major human fungal pathogen Candida albicans, that has had a rapid growth in morbidity and mortality.3,4 Amphotericin B, fluconazole, and other antifungal drugs have been widely used in clinical treatment of fungal infection; nevertheless, many antifungals have fungistatic rather than fungicidal activity while having high cytotoxicity.5 Because fungi in prolonged infections are not killed, serious antifungal resistant problems emerge.6 Therefore, a new antifungal agent with broad-spectrum activity, © 2018 American Chemical Society

Received: November 23, 2017 Published: April 12, 2018 3889

DOI: 10.1021/acs.jmedchem.7b01729 J. Med. Chem. 2018, 61, 3889−3907

Journal of Medicinal Chemistry

Article

Table 1. Peptide Design and Their Key Physicochemical Parameters compd

peptide

sequence

theoretical MW

measured MWa

net charge

Hb

μHrelc

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

L4 L4pfd F4 F4pf I4 I4pf W4 W4pf L6 L6pf F6 F6pf I6 I6pf W6 W6pf FITC-I6

RLRLLLRLR-NH2 LLRRLRRLL-NH2 RFRFFFRFR-NH2 FFRRFRRFF-NH2 RIRIIIRIR-NH2 IIRRIRRII-NH2 RWRWWWRWR-NH2 WWRRWRRWW-NH2 RRLRLLLRLRR-NH2 RLLRRLRRLLR-NH2 RRFRFFFRFRR-NH2 RFFRRFRRFFR-NH2 RRIRIIIRIRR-NH2 RIIRRIRRIIR-NH2 RRWRWWWRWRR-NH2 RWWRRWRRWWR-NH2 FITC-RRIRIIIRIRR-NH2

1207.56 1207.56 1377.64 1377.64 1207.56 1207.56 1572.82 1572.82 1519.94 1519.94 1690.01 1690.01 1519.94 1519.94 1885.19 1885.19 2022.51

1207.58 1207.58 1377.66 1377.66 1207.58 1207.58 1572.85 1572.85 1519.90 1519.90 1690.04 1690.04 1519.95 1519.95 1885.20 1885.20 2022.52

5 5 5 5 5 5 5 5 7 7 7 7 7 7 7 7 7

0.496 0.496 0.546 0.546 0.551 0.551 0.801 0.801 0.222 0.222 0.263 0.263 0.267 0.267 0.472 0.472

0.351 0.917 0.368 0.952 0.370 0.956 0.452 1.133 0.428 0.891 0.442 0.920 0.443 0.923 0.511 1.068

a

Molecular weight (MW) was measured by mass spectroscopy (MS). bHydrophobicity (H) values were calculated from http://heliquest.ipmc.cnrs. fr/cgi-bin/ComputParams.py. cRelative hydrophobic moment (μHrel) values were calculated from http://heliquest.ipmc.cnrs.fr/cgi-bin/ ComputParams.py. dSubscript pf represents the perfect amphipathicity peptides.

associated urinary tract infections (CAUTI).8,11 Thus, the AFPs have been considered to be effective candidates for the development of new generation of antifungal agents.12 However, AFPs have had limited success in clinical applications, primarily due to their low clinical antifungal activity, high systemic toxicity toward mammalian cells, and the high cost of isolation.13 Above all, a natural peptide and its derivatives would inevitably compromise a patient’s natural defenses, possibly causing a serious public health problem.14 Thus, de novo designed short AMPs/AFPs with a reasonable cost of production and low immunogenicity are exceptionally wellsuited as a new generation of antifungal drugs. Currently, α-helical peptides occupy an overwhelming majority in the recognized AMPs with a known structure. They share the common characteristics such as cationicity, hydrophobicity, and amphipathicity.15 Amphipathicity resulting from the dispersed or perfect segregation of hydrophobic and polar residues is a key structural characteristic of α-helical AMPs that favors peptide internalization and subsequent membrane perturbation, eventually affecting the membrane activity of the peptide.16 However, some controversy regarding the effect of perfect/imperfect amphipathicity on the biological activity of AMPs still exists.17−20 But most of these investigations studied the effect of perfect/imperfect amphipathicity via an amino acid substitution approach and did not sufficiently consider that such a modification of a peptide sequence generally alters greater than one structural characteristic that might modulate the antimicrobial activity, so it would be impossible to assign the observed effect exclusively to changes in perfect/imperfect amphipathicity. To overcome the difficulties presented by complex changes in structural characteristics due to peptide sequence modification, we employed an approach of minimal sequence modification, which allowed the modification of one structural characteristic while the others were kept largely constant. We recently constructed a centrosymmetric α-helical sequence template that conformed to the amphipathic palindromic structure distribution21 and found that centrosymmetric peptides with three units (containing seven net charges, due to the amidation) had

the greatest antimicrobial activity.22 However, the optimum peptide sequence was found to be long, and the primary unit had no activity due to a lack of sufficient cationicity, which was also important for membrane lytic function of AMPs/AFPs.23 Therefore, to develop a short but effective AMP as an antifungal and antibacterial agent, we redesigned an imperfectly amphipathic palindromic structure by adding one to two net charges at the C-terminus and N-terminus of the primary centrosymmetric α-helical template to disrupt the nonpolar face of the helix and decrease the amphipathicity. Arginine with highest cationicity was selected to provide the positive charges to enhance the antimicrobial activity of peptides.12 L, I, F, and W represented the different structural types of amino acids that were chosen to construct the hydrophobic core. The resulting simplified peptide structure was Rn(XRXXXRX)Rn (n = 1,2; X represents L, I, F, or W). To further explore the effect of perfect/imperfect amphipathicity on the biological activity of AMPs in this system, corresponding peptides with a perfectly amphipathic palindromic structure were also designed. All engineered peptides were treated with amidation modifications at the C-terminus to improve their stability. Circular dichroism (CD) was used to identify the secondary conformation of the engineered peptides in different solution environments. Then, the antimicrobial activity of the engineered peptides against various infectious fungi and bacteria (including clinically isolated fluconazole-resistant Candida albicans and MRSA), the salt and serum sensitivity, endotoxin neutralization, and cytotoxicity were also evaluated. Finally, liposome leakage, fluorescent spectrography, laser scanning confocal microscopy, Deltavision OMX system, scanning electron microscopy (SEM), atomic force microscopy (AFM), transmission electron microscopy (TEM), and reactive oxygen species (ROS) production were also employed to study the potential microbicide mechanisms of the peptides.

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RESULTS Characterization of Peptides. The peptide fidelity was first confirmed by MALDI-TOF MS and HPLC analysis as summarized in Table 1, Supplementary Figures S4−S6. As 3890



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Figure 1. Design of imperfect amphipathic antimicrobial peptides based on the model sequence Rn(XRXXXRX)Rn (n = 1, 2; X represents L, I, F, or W). (A) Sequence and schematic structure of the imperfect amphipathic peptides. (B) Three-dimensional structure projections of the imperfect and perfect amphipathic peptides. The hydrophobic residue and arginine are color coded as blue and red, respectively. (C) Helical wheel projection. The hydrophobic residue and arginine are color coded as yellow and blue, respectively.

shown in results, the measured molecular weight of each peptide almost had no difference from its theoretical molecular weight, showing that the peptides were successfully synthesized. All the imperfectly amphipathic peptides had the same mean hydrophobicity as their respective counterparts (Table 1). The wheel-diagram, 3D structure projection (Figure 1), and relative hydrophobic moments showed that the imperfectly amphipathic peptides had interrupted hydrophobic and cationic faces and had apparently lower relative hydrophobic moment values compared with the perfectly amphipathic peptides. Secondary Structures of Peptides. CD spectroscopy determined the structure conformations of the engineered peptides in different environments. The hydrophobic and helixstabilizing agent TFE was employed to assess the inherent helical propensity of the peptides, and an SDS micelle with a negatively charged surface was used to mimic anionic membrane environment.24 The spectra of the engineered peptides were not characteristic of α-helical conformation in 10 mM PBS. In membrane-mimetic environments, the CD spectra of the engineered peptides containing isoleucine and leucine showed two negative peaks at about 208 and 222 nm and indicated typical helical structure propensity, while phenylalanine-containing peptides displayed unusual α-helical secondary structure signal in membrane-mimetic environments, specifically in an SDS micelle (Figure 2). Because of close Trp-Trp interactions, the tryptophan-containing peptides

showed more tendency to form turn conformations in PBS and membrane-mimetic environments. Hemolytic Activity and Cytotoxicity. Table 4 and Figure 3A summarized the peptide hemolytic activities. It was desirable that all peptides cause less than 10% hemolysis at all concentrations. Compared with the typical α-peptide melittin, all engineered peptides had very significantly lower hemolytic activity (P < 0.01) (Figure 3A). The peptide cytotoxicity against RAW 264.7 cells and HEK 293T cells was evaluated using dose−response studies (Figure 4). In most cases, the cell viabilities induced by the engineered peptides were approximately 70−80% and no significant difference in the cell survival rate was seen within the imperfectly/perfectly amphipathic groups, except for 8 (W4pf) and 16 (W6pf) which eliminated the majority of the living cell at 64 μM. Furthermore, we also tested the toxicity of 17 (FITC-I6), the results showed that compared with 13 (I6), the hemolytic activity of 17 substantially increased to 9.26% at 128 μM but still less than 10%. Dramatically, the cell viabilities induced by 13 and 17 were both above 85%, indicating that fluorescein label had a little effect on the toxicity of the peptide (Supporting Information Figure S1). Antimicrobial Activity. The minimum inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs)/the minimum fungicidal concentrations (MFCs) were used to express the antimicrobial activity of the peptides, which are summarized in Table 2 and Table 3. Most peptides 3891



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Figure 2. CD spectra of all the peptides. All the peptides were dissolved in 10 mM PBS (pH 7.4), 50% TFE, or 30 mM SDS. The mean residual ellipticity was plotted against wavelength. The values from three scans were averaged per sample, and the peptide concentrations were fixed at 150 μM.

Figure 3. (A) Hemolytic activity of the engineered peptides against hRBCs. The graphs were derived from the average of three independent trials: (∗∗) P < 0.01, compared to values for Melittin. (B) Time−kill kinetic curves of 13 at 1 × MBC against E. coli ATCC25922 and C. albicans cgmcc 2.2086. (C) Resistance development in the presence of sub-MBC/MFC concentration of 13. 3892



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Figure 4. Cytotoxicity of the engineered peptides against RAW 264.7 cells and HEK 293T cells. The graphs were derived from average of three independent trials. Mean values in the same concentration with different superscript indicate a very significant difference (P < 0.01).

Table 2. MICsa (MBCsb) (μM) of the Engineered Peptides against Bacteria Gram-negative bacteria

Gram-positive bacteria

peptide

E. coli 25922

E. coli 1005

S. typhimurium 14028

S. pullorum 7913

S. aureus 29213

MRSAc 43300

S. epidermidis 12228

S. aureus 25923

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 polymyxin B melittin gentamicin ciprofloxacin

4(8) 16(32) 4(8) 8(8) 8(8) 32(32) 4(4) 8(32) 4(4) 4(16) 4(4) 8(8) 4(4) 16(32) 4(4) 16(16) 4(4) 2(4) 2(2) 1(2) 2(4)

4(8) 16(32) 4(8) 8(16) 4(8) 16(32) 2(4) 4(8) 4(4) 4(4) 4(4) 8(8) 4(4) 8(8) 4(4) 8(8) 8(16) 1(2) 2(2) 0.5(1) 2(4)

4(8) 16(32) 4(8) 4(8) 4(8) 64(>64) 4(4) 8(16) 4(4) 8(16) 4(8) 4(8) 4(4) 16(32) 4(8) 8(32) 4(8) 2(2) 1(2) 2(4) 8(16)

4(8) 32 (64) 2(8) 32(64) 4(16) 32(>64) 2(8) 8(16) 2(8) 8(32) 2(4) 4(8) 2(4) 16(64) 2(4) 4(8) 4(4) 1(2) 2(2) 1(2) 2(4)

8(8) 64(64) 4(8) 8(32) 4(8) 64(>64) 4(16) 4(16) 16(16) 4(16) 4(8) 8(16) 4(8) 64(>64) 2(4) 4(8) 4(4) 64(64) 8(8) 1(1) 4(4)

4(4) 8(16) 4(4) 4(4) 4(4) 64(>64) 2(2) 4(4) 8(16) 8(16) 4(8) 4(8) 4(4) 16(16) 4(8) 4(8) 4(4) 64(64) 1(1) 8(8) 1(1)

4(8) 16(16) 4(8) 4(8) 4(8) 64(>64) 4(8) 4(8) 4(4) 4(4) 4(4) 4(4) 4(4) 16(32) 4(8) 4(8) 2(4) 32(32) 0.5(1) 0.5(1) 2(4)

8(8) 16(32) 4(8) 4(8) 4(8) >64(>64) 4(8) 4(8) 4(8) 8(16) 8(16) 4(8) 4(8) 32(64) 4(8) 8(16) 8(8) 32(64) 4(4) 1(1) 8(8)

Minimum inhibitory concentration (MIC, μM) was determined as the lowest concentration of peptide that inhibited 95% of the bacterial growth. Data are representative of three independent experiments. bMinimum bactericidal concentrations (MBC, μM) were determined as the lowest peptide concentration that killed greater than 99.9% of the bacterial cells. The data were derived from representative value of three independent experimental trials. cMethicillin-resistant S. aureus.

a

antimicrobial level of a peptide to clinical microbial strains. Compared with the GM values of the corresponding perfectly amphipathic peptides, the imperfectly amphipathic peptides showed stronger antimicrobial activity in all strains. Further-

exhibited wide antimicrobial spectra against bacterial and fungal strains. Furthermore, the geometric means (GMs) of the MICs of the peptides against bacteria and fungi tested were calculated and presented in Table 4, which reflected the average 3893


3894

8(16) 32(32) 8(16) 16(32) 4(8) >64(>64) 8(16) 16(32) 8(16) 8(8) 4(8) 8(16) 2(4) 16(32) 4(8) 8(16) 4(4) 8(8) 1(64) 0.25(1) >64(>64)

8(16) 32(64) 8(16) 16(32) 4(8) 64(64) 8(16) 8(16) 8(16) 8(16) 4(16) 8(16) 2(2) 16(64) 4(8) 4(8) 2(2) 4(8) 1(2) 1(1) 0.25(1)

peptide

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 melittin fluconazole amphotericin B ketoconazole

8(8) 16(16) 4(4) 16(16) 2(8) >64(>64) 8(8) 4(4) 2(2) 4(4) 2(2) 4(4) 1(1) 4(4) 4(4) 2(2) 2(2) 4(4) 16(64) 0.125(1) >64(>64)

C. albicans SP3903 4(8) >64(>64) 4(4) 32(32) 2(2) >64(>64) 8(16) 8(8) 4(4) 4(8) 4(4) 8(8) 2(2) 16(16) 4(4) 4(4) 2(2) 2(2) 16(>64) 0.25(1) >64(>64)

C. albicans SP3902 4(4) 32(64) 8(8) 16(32) 1(1) >64(>64) 4(4) 4(4) 4(4) 4(8) 4(4) 8(8) 1(1) 8(16) 2(4) 2(2) 2(2) 4(4) >64(>64) 0.25(0.5) >64(>64)

C. albicans SP3876 8(16) 64(>64) 8(16) 16(32) 4(8) 64(>64) 8(16) 8(16) 4(8) 8(16) 4(8) 8(16) 2(4) 16(32) 4(8) 4(8) 4(4) 4(4) 2(8) 1(2) 0.5(2)

C. albicans isolated from alveolar fluid 8(8) 32(32) 4(4) 16(16) 4(4) 64(64) 4(4) 8(8) 4(8) 8(8) 4(4) 8(8) 1(1) 8(8) 4(4) 8(8) 2(2) 8(8) >64(>64) 1(2) 64(>64)

C. albicans 56452 8(16) 32(32) 8(16) 16(32) 2(4) 64(64) 4(8) 4(8) 4(8) 8(16) 4(8) 8(16) 1(2) 8(16) 4(8) 4(8) 2(4) 4(4) >64(>64) 1(2) 64(>64)

C. albicans 56214 8(8) 64(64) 8(8) 16(32) 2(2) >64(>64) 4(4) 8(8) 8(8) 4(4) 2(2) 4(4) 2(2) 8(16) 2(4) 2(4) 4(4) 4(4) 8(>64) 0.5(1) 1(>64)

C. albicans 58288 4(4) 32(>64) 8(8) 16(32) 2(2) >64(>64) 8(8) 8(8) 8(8) 8(8) 4(4) 8(16) 2(2) 8(8) 8(8) 4(4) 2(2) 4(8) 32(>64) 0.25(0.5) >64(>64)

C. albicans 14936 16(16) 64(>64) 4(4) 4(8) 2(2) >64(>64) 1(2) 2(4) 8(16) 8(16) 2(2) 4(8) 2(2) 16(16) 4(4) 4(4) 2(2) 8(8) >64(>64) 0.25(1) 32(64)

C. albicans 17546 2(4) 4(8) 2(4) 4(8) 2(4) 4(8) 2(2) 4(8) 2(4) 2(4) 1(2) 2(4) 1(2) 1(2) 2(4) 2(4) 4(8) 4(8) 4(32) 1(2) 0.25(1)

C. tropicalis cgmcc 2.1975 8(8) 8(8) 4(4) 4(4) 2(4) 16(16) 4(8) 4(4) 4(8) 4(8) 2(2) 2(4) 2(2) 4(8) 2(2) 2(4) 2(2) 2(4) 64(64) 1(1) 1(1)

C. krusei cgmcc 2.1857

>64(>64) >64(>64) 16(32) >64(>64) 8(16) >64(>64) 16(32) 32(64) 64(64) 8(16) 16(32) >64(>64) 4(8) >64(>64) 8(16) 16(32) 4(8) 2(2) 4(8) 2(2) 0.25(1)

C. parapsilosis cgmcc 2.3989

b

Minimum inhibitory concentration (MIC) was determined as the lowest concentration of peptide that inhibited 95% of the fungal growth. Data are representative of three independent experiments. Minimum fungicidal concentrations (MFC) were determined as the lowest peptide concentration that killed at least 99.9% of the fungal cells. The data were derived from representative value of three independent experimental trials.

a

C. albicans SP3931

C. albicans cgmcc 2.2086

Table 3. MICsa (MFCsb) (μM) of the Engineered Peptides against Fungi

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Table 4. MHC, GM, and TI Values of the Engineered Peptides GMb a

TIc

peptide

MHC

bacteria

fungi

all

bacteria

fungi

all

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 melittin

>128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 0.25

4.76 19.03 3.67 6.73 4.36 49.35 3.08 5.19 4.76 5.66 4.00 5.19 3.67 19.03 3.36 6.17 4.36 1.83

8.00 33.62 5.94 14.49 2.56 70.66 5.12 6.56 5.66 5.66 3.28 6.90 1.64 9.75 3.62 3.81 2.56 4.00

6.62 27.34 4.99 10.96 3.11 62.02 4.26 6.02 5.31 5.66 3.53 6.22 2.20 12.44 3.53 4.54 3.11 3.01

53.82 13.45 69.79 38.05 58.69 5.19 83.00 49.35 53.82 45.25 64.00 49.35 69.79 13.45 76.11 41.50 58.72 0.14

32.00 7.61 43.07 17.67 99.93 3.62 49.97 39.01 45.25 45.25 78.02 37.12 156.10 26.25 70.66 67.25 100 0.06

38.66 9.36 51.33 23.35 82.35 4.13 60.09 42.49 48.20 45.25 72.60 41.17 116.36 20.59 72.60 56.42 82.32 0.08

a

MHC is the minimum hemolytic concentration that caused 10% hemolysis of human red blood cells. Data are representative of three independent experiments. When no detectable hemolytic activity was observed at 128 μM, a value of 256 μM was used to calculate the therapeutic index. bThe geometric mean (GM) of the peptide MICs against bacteria and fungi was calculated. When no detectable antimicrobial activity was observed at 64 μM, a value of 128 μM was used to calculate the therapeutic index. cTI is calculated as MHC/GM. Larger values indicate greater cell selectivity.

Table 5. MIC Values of the Engineered Peptides against E. coli ATCC 25922 in the Presence of Physiological Salts peptide

controla

NaCla

KCla

NH4Cla

MgCl2a

CaCl2a

ZnCl2a

FeCl3a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 melittin polymyxin B gentamicin ciprofloxacin

4 16 4 8 8 32 4 8 4 4 4 8 4 16 4 16 2 2 1 2

4 16 4 8 8 32 4 8 4 4 4 8 4 16 4 16 4 8 2 2

4 16 4 8 8 32 4 8 4 4 4 8 4 16 4 16 2 2 1 2

4 16 4 8 4 32 4 8 4 4 4 4 4 16 4 16 2 2 2 2

4 16 2 8 8 32 2 4 2 2 2 2 2 16 2 8 4 4 1 8

8 64 4 32 16 64 4 32 4 8 4 8 8 32 8 32 8 2 1 8

8 16 4 8 8 32 2 16 4 8 2 8 4 16 4 16 2 2 2 2

4 16 4 8 8 64 4 4 8 16 4 8 4 16 4 16 2 2 1 4

a The final concentrations of NaCl, KCl, NH4Cl, MgCl2, CaCl2, ZnCl2, and FeCl3 were 150 mM, 4.5 mM, 6 μM, 1 mM, 2 mM, 8 μM, and 4 μM, respectively, and the control MIC values were determined in the absence of these physiological salts. The data were derived from representative value of three independent experimental trials.

(GMfungi = 1.64 μM, GMall = 2.2 μM). The MIC values for fungal strains were even better than or close to the melittin and traditional antifungal drugs, such as fluconazole and amphotericin B, which have a high hepatotoxicity in the treatment of fungal infections. The clinical isolates C. albicans SP3876, C. albicans 17546, C. albicans 56452, and C. albicans 56214 are both naturally resistant to fluconazole and other azoles but were highly sensitive to 13. In addition, the MBC/MFC values for a

more, the engineered peptides with 7 net charges had dramatically greater antimicrobial activity against fungal strains, with 1.41- to 7.25-fold lower GM values compared to the engineered peptides with 5 net charges (Supporting Information Table S1), yet against the bacterial strains, an increase in the net charge number did not always cause a positive effect on the antimicrobial potential of this structure. Overall, among all the engineered peptides, 13 had the greatest antimicrobial activity against all strains, specifically against fungal strains 3895



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Table 6. MIC Values of the Engineered Peptides against C. albicans cgmcc 2.2086 in the Presence of Physiological Salts peptide

controla

NaCla

KCla

NH4Cla

MgCl2a

CaCl2a

ZnCl2a

FeCl3a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 melittin fluconazole amphotericin B ketoconazole

8 32 8 16 4 64 8 8 8 8 4 8 2 16 4 4 4 1 1 0.25

32 64 32 16 16 64 16 16 64 16 16 32 4 64 8 16 8 1 1 0.25

16 64 32 16 4 64 16 16 32 16 16 16 2 64 8 8 4 1 1 0.25

8 32 8 8 4 64 8 8 8 4 4 4 2 16 4 4 4 1 1 0.25

8 64 16 16 4 64 8 8 8 32 4 8 2 32 4 8 4 1 1 0.25

64 64 64 32 32 64 32 16 64 8 16 64 4 64 8 8 8 1 2 0.5

8 64 8 32 4 64 8 8 8 8 4 8 2 64 4 4 4 1 1 0.25

8 64 8 32 4 64 8 8 8 8 4 8 2 16 4 4 4 1 1 0.25

The final concentrations of NaCl, KCl, NH4Cl, MgCl2, CaCl2, ZnCl2, and FeCl3 were 150 mM, 4.5 mM, 6 μM, 1 mM, 2 mM, 8 μM, and 4 μM, respectively, and the control MIC values were determined in the absence of these physiological salts. The data were derived from representative value of three independent experimental trials. a

obvious compromised antifungal activity in the presence of monovalent (Na+ and K+) and the divalent cation (Ca2+) against C. albicans 2.2086, with an increase of 2- to 8-fold compared to their MICs. Overall, 13 had great tolerance to the presence of physiological salts. We also examined the effect of serum on the MIC values of 13 against microbial cells (Table 7). It was noteworthy that the

peptide were equal to or 2−4 times higher than their MIC values. The ratio of the MHC to the GM of the MICs is usually referred to as the therapeutic index (TI), which indicates the cell selectivity of a peptide. Owing to its high MHC value and strong antifungal activities, 13 had the highest TI value against fungi of 156.1, which was 2600 times more than melittin (TIfungi = 0.06). Thus, 13 showed stronger cell selectivity toward fungal cells than erythrocytes, implying that it would have a larger therapeutic window. Furthermore, the kinetics of 13 in killing E. coli and C. albicans was further investigated at 1× MBC/ MFC concentrations at various exposure times. Figure 3B showed that 13 eliminated greater than 99.99% of bacterial and fungal cells within 5 min at 1× MBC/MFC, which was a high sterilization efficiency. The long-term exposures of microorganisms toward a drug at sub lethal concentrations can accelerate the emergence of drug resistance.25 And resistance to AMPs has been evolved in some resistant microorganisms.26 Thus, we also evaluated the potential of fungi to develop resistance to 13 through induction of drug resistance study. Following multiple exposures of fungal cells toward 13 at sub-MBC/MFC concentration, fungal resistance did not readily develop with 13-treatment as shown by the almost consistent MBC/MFC obtained over 30 passages (30 days) (Figure 3C). This result suggested that fungal resistance to 13 was not generated easily. Salt and Serum Sensitivity Assays. AMPs must retain activity in a physiological environment for clinical application. Thus, the antimicrobial activity of the engineered peptides was tested in physiological concentrations of various salts in a sensitivity assay. Tables 5 and 6 showed that cations at physiological concentrations had little or even a partial stimulating effect on the antibacterial activity of the peptides, except for divalent cations (Ca2+), which weakened the antibacterial activity of the most engineered peptides against E. coli 25922. However, the most engineered peptides showed

Table 7. MIC Values of the Peptide 13 against E. coli and C. albicans in the Presence of Serum peptide 13 melittin polymyxin B gentamicin ciprofloxacin 13 melittin fluconazole amphotericin B ketoconazole

control

12.5%

E. coli ATCC 25922 4 4 2 32 2 2 1 1 2 2 C. albicans cgmcc 2.2086 2 2 4 32 1 1 1 1 0.25 0.25

25%

50%

4 64 2 1 2

8 64 4 2 4

4 32 1 1 0.25

8 64 2 1 0.5

addition of serum had a slightly dose-dependent inhibitory effect on the antimicrobial activity of 13. In contrast, melittin almost lost its antimicrobial activity in the presence of serum. Overall, these results indicated that 13 could still keep excellent activity in a physiological environment. Cell Wall Permeabilization. On the basis of the TI value, 13 had the best antimicrobial potential and the greatest cell selectivity against all tested strains; hence, 13 was further studied for their active mechanism to kill the model microorganisms C. albicans 2.2086 and E. coli 25922. The conventional antibacterial and antifungal agents fluconazole and 3896



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Figure 5. (A) Cell wall permeability induced by 13. The uptake of NPN of E. coli and C. albicans in the presence of different concentrations of 13 was determined using the fluorescent dye (NPN) assay. The NPN uptake was monitored at an excitation wavelength of 350 nm and an emission wavelength of 420 nm. (B) Cytoplasmic membrane potential variation of E. coli and C. albicans treated by 1 × MBC/MFC 13, as assessed by the release of the membrane potential-sensitive dye DiSC3-5. The fluorescent intensity was monitored at an excitation wavelength of 622 nm and an emission wavelength of 670 nm as a function of time. (C) Concentration-dependent 13-induced calcein release from liposomes, Liposome composition: PC/PE/PI/ergosterol (5:4:1:2, w/w/w/w); PG/CL/PE (2:1:7, w/w/w); PC/cholesterol (10:1, w/w). (D) ROS levels in the presence of 13 in E. coli cells and C. albicans cells. Data shown are the mean ± SEM of three independent experiments: (∗∗) P < 0.01, compared to the untreated cells.

integrity. Similarly, melittin also showed strong cell wall permeabilization in E. coli, but the cell wall permeabilization of melittin was relatively weaker in C. albicans. However, the conventional antibacterial and antifungal agents ciprofloxacin and fluconazole showed little cell wall permeabilization. Liposome Leakage. A liposome leakage assay is usually used to monitor the interaction between phospholipid layer and peptides.24 To evaluate whether a peptide preferentially penetrates the bacterial or fungal membrane to exert antimicrobial activity, we measured the induction of calcein leakage from liposomes of different composition. Figure 5C showed that 13 induced good leakage activity from a negatively charged liposome in a concentration-dependent manner, inducing 63% and 62% leakage from PG/CL/PE and PC/ PE/PI/ergosterol at 32 μM, respectively, confirming the membrane-disrupting properties of 13. However, the vesicles composed of PC with cholesterol that mimic the mammalian cell membrane showed a negligible amount of leakage when they were exposed to 13. Moreover, ciprofloxacin and fluconazole showed almost no leakage activity; conversely, melittin had the strongest leakage activity against all LUVs. Cytoplasmic Membrane Electrical Potential Measurement. Antimicrobial peptide can perturb the electric potential

ciprofloxacin were used as the respective negative controls because their key intracellular targets inhibited specific biosynthetic pathways in microorganisms (i.e., fluconazole prevents fungal lanosterol from converting to ergosterol, thereby inhibiting membrane sterol synthesis27 and hinders fungal cell proliferation; ciprofloxacin inhibits DNA gyrase and topoisomerase IV28). The typical peptide melittin was chosen as the positive control. The cell wall provides a protective barrier against adverse environmental conditions in bacteria and fungi. Thus, the NPN uptake assay was conducted to assess the cell wall penetration capability of 13. The hydrophobic fluorescent probe NPN is usually blocked by the cell wall and quenched under aqueous conditions, but it is taken up if the cell wall is permeabilized and shows intense fluorescence intensity in a hydrophobic environment. 13 could permeabilize the fungal and bacterial cell wall in a concentration-dependent manner (Figure 5A). It induced the cell wall permeabilization of E. coli greater than 70% at 2 μM concentration. In contrast, the cell wall permeabilization induced by 13 against C. albicans was relatively weak, but dye uptake of 20% was still measured upon addition of 2 μM of 13, demonstrating that the dye penetrated into the hydrophobic environment because of the perturbation of the cell wall 3897



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Figure 6. Confocal fluorescence microscopic images and a Deltavision OMX system analysis of E. coli ATCC 25922 (A) and C. albicans cgmcc 2.2086 (B) treated with 17 and PI. From left to right the images in columns show the bright field images, green signal from FITC-peptides, red signal from PI, merged images, and Optical Deltavision OMX system 3D images.

showed that the fluorescence intensity due to 17 encompassed the entire E. coli and C. albicans cell and highly overlapped that due to nucleic acid stain PI, indicating that 17 killed the E. coli and C. albicans cells with compromised membrane integrity. However, the target site of 17 was not accurately determined. Thus, the localization of 17 was further studied by 3D-SIM super-resolution microscopy. The results distinctly showed that 17 presented green fluorescent signal distribution around the E. coli and C. albicans cell surface, indicating that 17 targeted the cell membrane surface (Supporting Information videos 1 and 2). Microscopic Observations. The cell morphology and membrane integrity were directly observed by SEM and AFM after peptide treatment (Figure 7A−H). Treatment with 13 at 1× MBC for 1 h induced obvious E. coli membrane damage compared to the control, which showed an unbroken surface (Figure 6A,E). The membrane surfaces of E. coli treated with 13 became completely roughened and broken (Figure 6B,F), showing obvious pore formation and membrane destruction. 13-treatment at 1× MFC played a similar role for the C. albicans membrane with evident membrane creping and deformation and intracellular content leakage (Figure 7D,H). The membrane morphology and intracellular ultrastructural alterations in E. coli and C. albicans after peptide treatment were further studied by TEM (Figure 7I−L). Compared to the E. coli control, 13-treatment caused obvious alteration of membrane

of cytoplasmic membrane to disrupt the functional membrane integrity.29 Thus, the membrane potential-sensitive dye DiSC35 was used to determine whether 13 affected fungal and bacterial membrane potential. DiSC3-5 normally accumulates in the cell to form nonfluorescent aggregations, once the cytoplasmic membrane potential changes due to the ion flux upon cytoplasmic membrane damage and disturbance, DiSC3-5 is released into the medium to form a monomer with a concomitant fluorescence increase. Figure 5B and Supplementary Figure S2 showed that the membrane potential changes of E. coli and C. albicans induced by various concentrations of 13 and melittin matched in a dose- and time-dependent manner within 800 s. The increase in the relative fluorescence induced by 13 and melittin at 1× MBC/MFC was both rapid and strong. Furthermore, an increasing trend in fluorescence in the presence of ciprofloxacin and fluconazole was not observed. Localization of FITC-Labeled Peptides. FITC-labeled peptides and propidium iodide (PI) dye were used to monitor peptide localization by confocal laser-scanning microscopy. PI is a membrane impermeable dye but can penetrate the damaged cell membrane and stain the nuclei. There is a good chance that the label will be detrimental to activity, so we first evaluated the antimicrobial activity of 17. The results indicated that fluorescein label had a slightly negative effect on the antimicrobial activity of the peptide, but 17 still kept great activity against most microbes tested (Tables 2−4). Figure 6 3898



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Figure 7. AFM (A−D), SEM (E−H), and TEM (I−L) micrographs of E. coli ATCC 25922 and C. albicans cgmcc 2.2086. From left to right the images in columns show the E. coli-control, E. coli treated with 13, C. albicans-control, C. albicans treated with 13. Microorganisms were treated with 13 at 1 × MBC/MFC for 1 h. The control was processed in the absence of 13. TEM: scale bar = 500 nm (E. coil), 1000 nm (C. albicans).

morphology and the loss of the intracellular contents resulting in sparse cytoplasmic distribution and clear zones. Inner and outer membrane separation was also observed (Figure 7J). Similarly, compared to the C. albicans control that kept intact membrane surface and dense cytoplasmic content, 13 distinctly broke the membrane structure of C. albicans and promoted content leakage and the empty regions formation of the cytoplasm (Figure 7L). ROS Production. ROS production is important for inducing apoptosis in fungi,30 which also has been reported to be a characteristic of apoptosis-like death in bacteria.31 In the case of fungi, the ROS levels in the fungal cells treated with 13 exhibited a dose dependent effect (Figure 5D) and increased 14-fold compared to the control cells at 32 μM. However, in the case of bacteria, no fluorescence increase in the bacteria cells treated with 13 was detected at low concentration (0.5−4 μM). Even at high concentration 32 μM, 13 only induced 2fold increase in ROS levels compared to the untreated cells. Overall, 13 could induce higher ROS levels in fungal cells than in bacteria cells. LPS-Neutralizing Activities. Lipopolysaccharide (LPS) is the major cell wall component of Gram-negative bacteria and can induce severe endotoxemia when it is abundant in the blood. We determined the LPS-binding ability of 13 with a fluorescence-based displacement assay with BODIPY-TR-

cadaverine and polymyxin B, which has well-known strong LPS-binding capability, as a reference compound. Figure 8A indicated that 13 showed a concentration-dependent increase in fluorescent intensity with 68% LPS-binding activity at a low peptide concentration (4 μM) and almost completely neutralized the LPS at 8 μM (95%). We also used SPR spectroscopy to real-time-investigate the interaction between 13 and LPS. Figure 8B showed that the RU of LPS binding to immobilized 13 was rapidly increased in a dose-dependent manner at concentrations of 3.125−25 μg/mL, indicating 13 had strong affinity for LPS. The endotoxin-neutralizing capability of 13 was further studied by stimulating RAW264.7 cells with 50 ng/mL LPS in the presence/absence of 13 at various concentrations. The levels of nitric oxide (NO) and tumor necrosis factor (TNF) α, which are typical inflammatory mediators, were chosen to assess the LPS neutralization activity of 13. Figure 8C showed that 13 inhibited NO production in a concentration dependent manner and had almost 100% LPS-neutralizing activity at 16 μM. The similar inhibitory effect for TNF-α production was determined with 13 (Figure 8D), which showed very significant LPS-neutralizing activity indicated by the noticeable attenuation TNF-α secretion at the low concentration of 2 μM. Furthermore, the ability of 13 to induce inflammation response was also assessed by stimulating RAW 264.7 cells with various 3899



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Figure 8. (A) Peptide binding affinity to LPS from E. coli 0111:B4. The fluorescence intensity was monitored at an excitation wavelength of 580 nm and an emission wavelength of 620 nm. The graphs were derived from the averages of three independent trials. (B) SPR spectroscopy of the interaction kinetics of 13 and LPS. The values were recorded in response unit (RU) for the indicated concentrations of LPS. (C, D) Effects of 13 on LPS-stimulated NO and TNF-α production in RAW 264.7 cells. Amounts of TNF-α and NO in cell culture supernatants were determined by ELISA and the Griess reagent, respectively. Data shown are the mean ± SEM of three independent experiments: (∗∗) P < 0.01, compared to the LPS-alone treated.

turn structures were driven not only by the formation of intramolecular H bonds but also through interactions of the bulky indole rings of the tryptophan residues.35 A major limitation for peptide-based biomaterial development is the possible deleterious activities of peptides in mammalian cells. Many studies indicated that perfect amphipathicity often results in increased cytotoxicity,36 yet in our study, only the cytotoxicity of 8 and 16 at 64 μM followed this result. The other perfectly amphipathic engineered peptides had similar hemolytic activity and cytotoxicity as their imperfect counterparts against mammalian cells at all concentrations. These results demonstrated that AMP toxicity was weakly associated with the perfect/imperfect amphipathicity in our system. As reported previously, hydrophobicity is the most critical structural factor that determines the toxicity of AMPs37 and is always positively correlated with it. Thus, it was easy to speculate that the perfectly amphipathic peptides had similar cytotoxic activity as their imperfect counterparts because they had the same amino acid composition and hydrophobicity, and the engineered peptides containing tryptophan with high hydrophobicity values showed relatively stronger cytotoxicity at the highest concentration. We also evaluated the possible cytotoxicity of the peptides by using a membrane model to mimic different organisms. The results showed that 13 preferred anionic membranes with PG and PI over zwitterionic membranes (Figure 5C), suggesting that 13 had highly selectivity for bacteria and fungi membranes over mammalian membranes. The distribution of perfect or imperfect amphipathic residues is always the bone of contention in structure−function

peptide concentrations. The results showed that 13 had little effect on NO and TNF-α production (data not shown), which were similar to the controls, indicating that 13 was not immunogenic.

■

DISCUSSION The conformational alteration of an amphipathic AMP from random coils in aqueous solution to a stabilized secondary structure in a membrane environment is important for its biological activity.32 Our results indicated that most engineered peptides adopted a nonordered structure in PBS and transformed to stabilized conformation in TFE and SDS, signifying that the peptide interacted with the cell membrane to exert antimicrobial activity.33 Figure 2 showed that the helicity of the peptide was affected by the hydrophobic amino acids composition of the peptide. The peptides containing phenylalanine showed weak α-helical secondary structure signal in membrane-mimetic environment. In comparison, the peptides containing leucine and isoleucine showed strong tendency to form α-helical structures. These results were consistent with the Michael Blaber amino acid α-helix propensity theory,34 implying that the side chain bulk and hydrophobicity of amino acids could modulate the helical propensity. Leucine and isoleucine residues have a stronger spiral tendency due to appropriate side chain hydrophobicity and bulk, while phenylalanine residues limit the formation of an α-helical conformation because of that steric hindrance induced by the bulky benzene ring present in phenylalanine residues, thus hindering the formation of stable α-helices.33 Furthermore, tryptophan-rich peptides presented turn conformations; these 3900



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7(W4) and 15(W6) that had the highest hydrophobicity in their respective imperfectly amphipathic series also had the best antibacterial activity. However, compared with the substantial increase of hydrophobicity, the increase in antibacterial activity of the peptides was not obvious. For example, compared with 13, the hydrophobicity of 15 increased by 77%; meanwhile its antibacterial activity only increased by 8.4%. Instead, the antifungal activity of 15 decreased dramatically by 54.6%. So the antifungal activity of the imperfectly amphipathic peptides could be fit to a quadratic function relationship with hydrophobicity in this system (Supporting Information Figure S3). According to the quadratic function, an increase of peptide hydrophobicity beyond 0.4 could compromise the antifungal activity of the imperfectly amphipathic peptides with 7 net charges. Some studies indicated that the peptide preforming a spiral structure due to high hydrophobicity has a negative effect on the penetration into the cell wall of microorganisms,43 and the fungal cell wall is more difficult to be penetrated by AMPs due to extensive cross-linking between the cell wall components in fungi.44 Thus, according to our CD results, 15 had a propensity for a turn structure in aqueous media, leading to a decrease in antifungal activity. Overall, we had come to the conclusion that 13 showed great specific selectivity for fungi. Electrostatic adsorption with the negatively charged surface of a microorganism is the basis for the selectivity of cationic peptides. Therefore, free ions, such as monovalent cations (Na+ and K+) and divalent cations (Ca2+) in the surrounding medium can hinder electrostatic interaction and decrease binding efficacy of a peptide by the charge screening effect, finally compromising the killing efficiency of a peptide,45 and multivalent cations (Fe3+) can compete with AMPs for binding to the anionic phosphate groups, such as those in LPS or glucan, while increasing membrane rigidity, and this effect slowly hinders pore formation, resulting in a decrease in antimicrobial activities of AMPs,46,47 but this is not always the case. Some literature has proposed that additional divalent cations will enhance the adsorption capacity of AMPs to the bacterial surface and increase the antibacterial activity of AMPs,12,13 suggesting that the effects of cations rely on the peptides and their concentrations.48 In our study, we found that only Ca2+ compromised the antibacterial activity of the engineered peptides; however, except for Ca2+, the monovalent cations Na+ and K+ also suppressed peptide antifungal activity, suggesting that the antifungal activity of the engineered peptides was much more salt sensitive than the antibacterial activity. A possible reason was that the fungal cell wall was more inherently difficult for the peptides to penetrate than the bacterial cell envelope, and the charge screening effect of monovalent cations further reduced the cell wall penetrability of the peptides.44 The recognition that AMPs activity is usually compromised in serum has prevented their further clinical applications. Fortunately, in our study, the inhibitory effect of serum on antimicrobial peptide of 13 was not obvious. This phenomenon could be explained by the existence of an equilibrium between free and protein-bound peptide molecules. As more free molecules became associated with their microbial targets, the equilibrium would shift toward the progressive release of more peptide molecules and contribute to maintaining their antimicrobial activities.49 Additionally, the antimicrobial activity of 13 decreased along increased serum concentrations. This was possibly attributed to that high concentration albumins compete with the bacterial surface for peptide binding and

relationship research on AMPs. Many studies have shown that a perfectly amphipathic residue arrangement effectively enhances the antimicrobial potency and selectivity, which has become a preferred strategy for the design of synthetic peptides.17 In contrast, other researchers have recently thought that perfect amphipathicity often induces an increase in cytotoxicity while promoting bacterial activity, so an appropriate imperfectly amphipathic residue distribution is more advantageous for optimizing the antimicrobial activity and selectivity.18,19,38,39 Due to the interactions of various structural characteristics, it is difficult to describe the complete relationship between the amphipathic residue arrangement and bioactivity. Nevertheless, there is still a clear indication that the interaction between the differential positive charge distribution and the hydrophobicity is a key factor in determining the bioactivity of AMPs, and the effect of perfect/imperfect amphipathicity on the activity is closely related to the amino acid composition of the peptide sequence.36 In our study, a comparison of perfectly and imperfectly amphipathic engineered peptides, which had the same amino acid composition but a different amino acid distribution in the primary sequence, indicated that most of the imperfectly amphipathic peptides that had an interrupted hydrophobic face on the helical wheel showed better antimicrobial activity against bacteria and fungi (Supporting Information Table S2). It appeared that imperfect amphipathicity was necessary to facilitate the formation of membrane pores and increased the antimicrobial activity of this structure.20 Remarkably, 9 (L6) and 10 (L6pf) had the same GM for fungi, while an individual analysis of the MIC of each strain indicated that the antimicrobial activity of 9 against most of the strains was similar or better than 10, except for C. parapsilosis cgmcc 23989, which obviously compromised the total GM values of 9. Thus, this result suggested that imperfect amphipathicity could have a negative effect on the antimicrobial activity of an AMP for certain strains in some sequences. Adequate cationic residue is a necessary condition for electrostatic attraction of a peptide to anionic wall components such as lipopolysacchrides, mannoproteins, or anionic phospholipids in microbial membranes such as phosphatidylglycerol or phosphatidylinositol in bacteria and fungi, respectively.40 In contrast, excessive charge results in higher cytotoxicity and lower cell selectivity and even inhibits a peptide from aggregation on the membrane surface and penetrating the membrane.41 Thus, reports indicate that the relationship between charge and biological activity is not linear and an intermediate charge of +4 to +6 appears to be optimal. Our antimicrobial activity assay results showed that increasing cationic residues to six (+7 net charges) improved the antifungal activity of the engineered peptides but had weaker influence on the antibacterial activity except for the corresponding peptides with four cationic residues (+5 net charges) with low activity (Supporting Information Table S1). Thus, six cationic residues were necessary for the engineered peptides to enhance the interaction between the peptide and the thick fungal cell wall and improve antifungal activity,42 but four cationic residues in this structure were sufficient for the antibacterial activity of the most peptides, suggesting that fungi were more sensitive to a highly cationic peptide than bacteria. Hydrophobicity is another important parameter responsible for the antimicrobial activity of an AMP. Studies have shown that the antibacterial activity of a peptide tends to increase with increasing hydrophobicity to a certain extent, which was consistent with our results that the imperfectly amphipathic 3901



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effect on cell viability, indicating its suitability as novel LPSneutralizing agent.

this binding reduced the effective concentration of peptides available to combat microorganism.50 It is widely accepted that AMPs exert antimicrobial activity by membrane permeabilization.51 Peptides first aggregate on the microbial surface and cross the barrier cell wall before interacting with the cytoplasmic membrane. After the local peptide concentration reaches a threshold that enables productive action, the peptide conformation changes, and it is then inserted into the cytoplasmic membrane, leading to the membrane potential disturbance, disruption of the membrane integrity, or formation of pore/ion channels, finally resulting in cytoplasmic leakage that facilitates cell death.52 We demonstrated that in E. coli and C. albicans, 13 could permeate the cell wall barrier in a concentration-dependent manner. It simultaneously perturbed the cytoplasmic membrane potential, and the LUVs results further indicated that 13 disrupted the membrane by the formation of pore/ion channels, thereby leading to the leakage of the cytoplasmic contents. The FITClabeled peptides localization demonstrated that 13 acted on the cell membrane and compromised the membrane integrity. The SEM, AFM, and TEM results visually determined that 13 killed pathogens via membrane destruction and content leakage. Moreover, some peptides also exert antimicrobial activity by production of high level reactive oxygen species (ROS)29,53 which has a destructive effect on many intracellular molecules including nucleic acids, proteins, and lipids.30 Thus, in our study, we also demonstrated that the antimicrobial activity of 13 was associated with accumulation of ROS. In summary, 13 could readily permeabilize the cell wall and damage the integrated cell cytoplasmic membrane, inducing membrane surface atrophy and fractures due to the massive outflow of the intracellular contents, and it also could induce production of ROS, finally leading to cell death. Thus, compared to the conventional antibacterial and antifungal agents that only target specific pathogen molecular receptors,54 13 relies on its membrane-disruptive mechanism and accumulation of reactive oxygen species (ROS) to overcome the bacterial and fungal resistance. Lipopolysaccharide (LPS; endotoxin) released from the outer membrane of Gram-negative bacteria is a strong immunostimulants55,56 and can stimulate the immunocytes (e.g., macrophages, monocytes, and neutrophils) to release inflammatory cytokines (e.g., TNF-α) via formation of LPSCD14 complexes.57 However, excessive inflammatory cytokines may cause a serious shock syndrome, even resulting in death.58 Previous studies have demonstrated that AMPs have a potent endotoxin-neutralizing capacity by binding directly to LPS or blocking the binding of LPS to LPS-binding protein (LBP).59 They bind to the anionic amphiphilic lipid A domain of LPS via electrostatic adsorption and dissociate LPS aggregates via hydrophobic interactions between the alkyl chains of LPS and nonpolar amino acid side chains of AMPs.60 Thus, a sufficient number of positive charges is a prerequisite for AMPs to inactivate LPS, as well appropriate hydrophobicity to allow localized insertion of the peptide into the lipid A domain of the LPS.61 Given that 13 had a high positive charge and moderate hydrophobicity, it was not difficult to conclude that 13 significantly decreased NO and TNF-α production compared to the control at the low concentration of 2 μM, indicating potent LPS-neutralizing activity. We further evaluated the cytotoxicity against the RAW 264.7 cell and found that the cell viability exceeded 80% at 32 μM for 13. Therefore, the LPSneutralizing property of the peptide 13 was independent of its

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CONCLUSION In this study, we designed an imperfectly amphipathic palindromic structure based on a centrosymmetric α-helical template. A series of imperfectly amphipathic α-helical peptides was synthesized and tested for their antibacterial, antifungal, and antiendotoxin capacities. The corresponding perfectly amphipathic peptides with the same amino acid composition but with a different amino acid distribution were also synthesized to study the effect of perfect/imperfect amphipathicity on the biological activity. In this system, imperfectly amphiphilic peptides showed enhanced antimicrobial activity against most of the microbes tested, but the toxicity of the engineered peptides was independent of the type of amphipathicity, which was a greatly different result from in previous studies. Hydrophobicity and cationicity had a relatively greater effect on the antifungal activity of the engineered peptides than the antibacterial activity. Of the engineered peptides, 13 had the greatest activity and selectivity against fungi and bacteria, specifically against drug-resistant fungi, with rapid sterilization efficiency, low likelihood of resistance development, and high salt and serum tolerance. 13 exerted its fungicidal and bactericidal action by targeting the cell membrane surfaces, damaging the membrane integrity, and inducing the formation of intracellular ROS, eventual leading to cell death; this mechanism may reduce probability of resistant development in microorganisms. Moreover, 13 also showed noticeable LPS-neutralizing properties. Thus, 13 has potential as novel antifungal/antibacterial agent. Altogether, these findings provided a rationale for peptide design and optimization and laid the foundation for the development of peptide-based antimicrobial agents. However, the study only evaluated the activity of peptides in vitro. Further in vivo assessments will help to improve the potency of biotechnological and clinical applications of AMPs.

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EXPERIMENTAL SECTION

Peptide Synthesis, Purification, and Sequence Analysis. The peptides designed and the fluorescent-labeled peptides in this study were synthesized by the Synpeptide Co. Ltd. (Nanjing, China), and their actual molecular weights were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; Linear Scientific Inc., USA). Peptide purity (95%) was assessed by reverse-phase high-performance liquid chromatography (HPLC) with a column of SHIMADZU Inertsil ODS-SP 4.6 mm × 250 mm × 5 μm, 214 nm, 20 μL column using a nonlinear water/acetonitrile gradient containing 0.1% trifluoroacetic at a flow rate of 1.0 mL/min. The primary structural parameters were calculated online using the HeliQuest analysis Web site (http://heliquest.ipmc.cnrs.fr/cgi-bin/ ComputParamsV2.py). The helical wheel projection was performed online using the helical wheel projection (http://rzlab.ucr.Edu/ scripts/wheel/wheel.cgi). The three-dimensional structure projection was predicted online with I-TASSER (http://zhanglab.ccmb.med. umich.edu/I-TASSER/). Bacterial Strains. Escherichia coli (E. coli) ATCC25922, Salmonella pullorum (S. pullorum) ATCC 7913, Salmonella typhimurium (S. typhimurium) ATCC14028, Staphylococcus aureus (S. aureus) ATCC 29213, S. aureus ATCC25923, Staphylococcus epidermidis (S. epidermidis) ATCC12228, and methicillin-resistant S. aureus ATCC 43300 were obtained from the College of Veterinary Medicine, Northeast Agricultural University (Harbin, China), and E. coli UB1005 was kindly provided by the State Key Laboratory of Microbial Technology (Shandong University, China). C. albicans cgmcc 2.2086, 3902



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plate. After incubation for 24 h at 37 °C, the minimum inhibitory concentrations (MICs) were determined by the absorbance at 492 nm with a microplate reader as the lowest peptide concentration that inhibited 95% of the bacterial growth. Subsequently, 50 μL of each incubation mixture was further transferred to agar plates and incubated overnight, and the minimum bactericidal concentrations (MBCs) were determined as the lowest peptide concentration that killed greater than 99.9% of the bacterial cells. Each test was reproduced at least 3 times. The peptide antifungal activity was determined according to a standardized broth microdilution method (Clinical and Laboratory Standards Institute (CLSI) document M27-A2) with modifications.62 Briefly, yeast colonies were picked and diluted in RPMI 1640 growth medium buffered with morpholinepropanesulfonic acid (MOPS) to give a final concentration of (0.5−1) × 103 CFU mL−1. Subsequently, the peptides were serially diluted in 0.2% BSA solution and then mixed with equal volumes of fungal solution in 96-well plates. After incubation for 48 h at 28 °C, the minimum inhibitory concentrations (MICs) were determined by the absorbance at 492 nm with a microplate reader as the lowest peptide concentration that inhibited 95% of the fungal growth. 50 μL samples of each well were further removed and plated on YM agar plates, incubated for 48 h at 28 °C. The minimum fungicidal concentrations (MFCs) were determined as the lowest peptide concentration that killed at least 99.9% of the fungal cells. Each experiment was performed in triplicate with three biological replicates. The time-kill kinetics of a peptide for E. coli ATCC 25922 and C. albicans cgmcc 2.2086 was further assessed. The microbial cells were treated with peptides at 1× MBC/MFC concentration, and at various subsequent times, aliquots of a microbial suspension was diluted and spread on solid medium plates (Mueller−Hinton agar, MHA and yeast peptone dextrose agar, YPDA). Bacterial colonies were counted after 24 h incubation at 37 °C (fungal colonies were incubated for 48 h at 28 °C). The results were the mean values of three independent assays. Drug resistance was induced by treating fungal cells repeatedly with antimicrobial agents.63 We choose fluconazole-resistant C. albicans 56452 as the model microorganism. Overnight cultures of C. albicans cells were serially passaged by 100-fold dilution in 2 mL batch cultures every 24 h in RPMI 1640 containing the sub-MBC/MFC concentration of the peptides. The MBC/MFC of the peptides against each passage’s cells was tested. As a control, MBCs/MFCs were also obtained using cells serially passaged in fresh RPMI 1640 alone. Salt and Serum Sensitivity Assays. The MICs of the peptides against E. coli ATCC 25922 and C. albicans cgmcc 2.2086 were determined in MHB with different concentrations of physiological salts (150 mM NaCl, 4.5 mM KCl, 6 mM NH4Cl, 8 mM ZnCl2, 1 mM MgCl2, 2 mM CaCl2, and 4 mM FeCl3) or human heat-inactivated serum (12.5%, 25%, and 50%), according to our previous protocol.64 The results were from three independent assays. Antimicrobial Mechanism Studies. Cell Wall Permeabilization. Peptide cell wall permeabilization was analyzed by the uptake of 1-N-phenylnaphthylamine (NPN, Sigma-Aldrich, China) as previously described.12,65 Briefly, logarithmic growing microbial cells were collected and diluted to OD600 = 0.2 in 5 mM HEPES buffer (pH 7.4, containing 5 mM glucose). The cell suspension was further incubated with 10 μM NPN for 30 min. Subsequently, the different concentrations of the peptides were added to 2 mL of cell suspension in a 1 cm quartz cuvette. The fluorescence was detected (excitation λ = 350 nm, emission λ = 420 nm) with an F-4500 fluorescence spectrophotometer (Hitachi, Japan) until no further fluorescence increase; the background fluorescence was also recorded. Each test was performed independently in triplicate, and the results were converted to percent NPN uptake using the equation

C. tropocalis cgmcc 2.1975, C. krusei cgmcc 21857, C. parapsilosis cgmcc 23989 were purchased from the China General Microbiological Culture Collection Center (CGMCC, Beijing, China). Clinical isolated C. albicans SP3931, C. albicans SP3902, C. albicans SP3903, C. albicans SP3876, and C. albicans isolated from alveolar fluid were provided by the Medical College of Nanchang University. Fluconazole-resistant C. albicans 56452, fluconazole-resistant C. albicans 56214, fluconazoleresistant C. albicans 17546, C. albicans 58288, and C. albicans 14936 were provided by the Zhongshan Hospital of Fudan University. CD Measurements. The CD spectra (λ190−250nm) of each peptide at a final concentration of 150 μM were recorded in 10 mM PBS, 30 mM sodium dodecyl sulfate (SDS) micelles, and 50% trifluoroethyl alcohol (TFE) on a J-820 spectropolarimeter (Jasco, Tokyo, Japan) using a quartz cell with 1.0 mm path length. An average of >3 runs was made for each sample. The acquired CD spectra were then converted to the mean residue ellipticity by using the following equation:

θM =

θobs 1000 cln

where θM is residue ellipticity (deg cm2 dmol−1), θobs is the observed ellipticity corrected for the buffer at a given wavelength (mdeg), c is the peptide concentration (mM), l is the path length (mm), and n is the number of amino acids. Cytotoxicity Assays. The cytotoxicity of the peptides was determined with three cell types, including the murine macrophage cell line RAW264.7, human embryonic kidney (HEK) 293T cells, and fresh, healthy human red blood cells (hRBCs), via modified standard microtiter dilution methods. The first two cell types were tested via the 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay, and the last was tested with a previously described hemolysis assay.22 Briefly for the MTT assay, (1.0−2.0) × 105 cells/well were plated in 96-well plates and then treated with various concentration of peptides for 24 h at 37 °C in 5% CO2. Then, an amount of 50 μL of MTT was added to the cell cultures at a final concentration of 0.5 mg/mL and the mixture was further incubated for 4 h at 37 °C, centrifuged at 1000g for 5 min, and the supernatants were discarded. The formazan crystals were dissolved with 150 μL of DMSO, and the OD at 570 nm was measured using a microplate reader (TECAN GENios F129004; TECAN, Austria). An amount of 1 mL of fresh hRBCs was collected and diluted 10fold with PBS (pH 7.4). Subsequently, an equal volume of hRBCs solution and peptide solution with various concentrations were mixed in 96-well plates and incubated for 1 h at 37 °C. The plates were centrifuged at 1000g for 10 min, and the supernatant was transferred to a new 96-well plate. The hRBCs were incubated with PBS alone and 0.1% Triton X-100 alone and were regarded as the negative control and the positive control, respectively. The release of hemoglobin was monitored by the absorbance at 576 nm with a microplate reader (TECAN GENios F129004; TECAN, Austria). The peptide concentration that caused 10% hemolysis was considered as the minimal hemolysis concentration (MHC). The percent hemolysis was calculated using the following formula:

percent hemolysis =

A − A0 × 100 At − A0

where A represents the absorbance of the peptide sample at 576 nm and A0 and At represent 0% and 100% hemolysis determined in 10 mM PBS and 0.1% Triton X-100, respectively. A minimum of three independent experiments were conducted for the assay, and three technical replicates were used in each experiment. Antimicrobial Assays. The antibacterial activity of the peptides was measured using a method adopted from the National Committee for Clinical Laboratory Standards (NCCLS), with modifications.12 The specific method for these tests was previously described. In brief, bacterial cells were grown to mid logarithmic phase and diluted in MHB to a final concentration of (0.5−1) × 105 CFU mL−1. Subsequently, the peptides were serially diluted in 0.2% BSA solution and then mixed with equal volumes of bacterial solution in a 96-well

NPN uptake (%) =

Fobs − F0 × 100 F100 − F0

where Fobs is the observed fluorescence at a given peptide concentration, F0 is the initial fluorescence of NPN with microbial cells in the absence of peptide, and F100 is the fluorescence of NPN 3903



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with microbial cells upon addition of 10 μg/mL polymyxin B (Sigma) (for bacteria)/0.1% Triton X-100 (for fungi) as a positive control. Liposome Leakage. Calcein-entrapped large unilamellar vesicles (LUVs) were prepared as described previously.22 Phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), cholesterol, cardiolipin (CL), phosphatidylinositol (PI), and ergosterol were purchased from Sigma-Aldrich, Shanghai, China. PG/CL/PE (2:1:7, w/w/w, mimicking the E. coli membrane66), PC/PE/PI/ ergosterol (5:4:1:2, w/w/w/w, mimicking the fungal membrane67), and PC/cholesterol (10:1,w/w, mimicking the human erythrocyte cell membrane66) phospholipid were dissolved in chloroform, dried with nitrogen, and resuspended in dye buffer solution (70 mM calcein, 10 mM Tris, 150 mM NaCl, 0.1 mM EDTA, pH 7.4). Each mixture was repeatedly frozen and thawed 20 times in liquid nitrogen and extruded 20 times through a polycarbonate filter (two stacked 100 nm pore size filters) with a LiposoFast extruder (Avestin, Inc., Canada). Unencapsulated calcein was sieved with gel filtration on a Sephadex G-50 column. The lipid concentration was determined by the method of Stewart68 and diluted to a final concentration of 100 μM in TrisHCl buffer. Subsequently, the peptides were serially diluted in TrisHCl buffer and then mixed with equal volumes of the lipid suspension in a 96-well plate. After incubation for 15 min, the fluorescence produced by the leakage of calcein from an LUV was detected (excitation λ = 490 nm, emission λ = 520 nm) with a spectrofluorophotometer (the Infinite 200 pro, Tecan, China). A 0.1% Triton X-100 solution was used to determine 100% dye leakage. The percentage of dye leakage caused by a peptide was calculated using the following formula: dye release (%) =

and for 15 min in 100% ethanol, then transferred to a mixture (v:v = 1:1) of 100% ethanol and tert-butanol and absolute tert-butanol for 15 min. The specimens were lyophilized, coated with gold−palladium, and observed using a Hitachi S-4800 SEM. For AFM sample preparation, microbial suspension was initially prepared in the same way as for SEM; 10 μL of microbial suspension was smeared on the slide, followed by air-drying. The images were obtained using a Bioscope atomic force microscope (Bruker, USA). TEM sample preparation was consistent with SEM. Followed by prefixation with 2.5% glutaraldehyde at 4 °C overnight, the samples were postfixed with osmium tetroxide for 70 min and washed twice with PBS (pH 7.2). Subsequently, the samples were continuously dehydrated for 8 min in different concentrations of ethanol (50%, 70%, 90%, and 100%) and for 10 min in 100% ethanol, then transferred to a mixture (v:v = 1:1) of 100% ethanol and acetone and absolute acetone for 10 min. The samples were further embedded in 1:1 mixtures of absolute acetone and epoxy resin for 30 min and absolute epoxy resin overnight. Finally, the specimens were sectioned with an ultramicrotome, stained with uranyl acetate and lead citrate, and observed using a Hitachi H-7650 TEM. ROS Production. The intracellular generation of ROS was measured by 2′,7′-dichlorofluorescin diacetate (DCFH-DA) as described earlier.5,29 Briefly, the cells (OD600 = 0.6) were incubated with different concentrations of peptides ranging from 0.5 to 32 μM for 60 min at 37 °C and then stained with 10 μM DCFH-DA for 1 h at 37 °C, following the manufacturer’s instructions. The fluorescence intensities were recorded (excitation λ = 488 nm, emission λ = 525 nm) with a spectrofluorophotometer (the Infinite 200 pro, Tecan, China). A minimum of three independent experiments were conducted for the assay, and three technical replicates were used in each experiment. Peptide Interaction with LPS. LPS Binding Assay. The peptide binding affinity to LPS was examined using the BODIPY-TRcadaverine (BC Sigma, USA) displacement assay.70,71 50 μg/mL LPS from E. coli O111:B4 was incubated with 5 μg/mL BODIPY-TRcadaverine in Tris buffer (50 mM, pH 7.4) for 4 h at room temperature. Subsequently, the peptides were serially diluted in Tris buffer and incubated with equal volumes of the LPS-probe mixture for 1 h in a 96-well black plate. The fluorescence was measured (excitation λ = 580 nm, emission λ = 620 nm) on a spectrofluorophotometer (the Infinite 200 pro, Tecan, China). Each test was performed independently in triplicate. The values were converted to % ΔF (AU) using the following equation:

Fobs − F0 × 100 F100 − F0

where F0 is the fluorescence intensity of liposomes (background) and Fobs and F100 are the respective intensities of the fluorescence of the peptide and the Triton X-100. Each experiment was performed in triplicate with three biological replicates. Cytoplasmic Membrane Electrical Potential Measurement. The cytoplasmic membrane electrical potential change induced by the peptides was determined with the membrane potential-sensitive fluorescent dye, DiSC3-5 (Sigma-Aldrich), as previously described.64,69 Briefly, logarithmic growing microbial cells were harvested and diluted to OD600 = 0.05 in 5 mM HEPES buffer (pH 7.4, containing 20 mM glucose). The cell suspension was further incubated with 0.4 μM DiSC3-5 and 100 mM K+ until no further reduction of fluorescence. Subsequently, 2 mL of cell suspension was added to a 1 cm quartz cuvette and mixed with the peptides at their 0.5×, 1×, and 2× MBCs/ MFCs. The fluorescence was continuous detected for 800 s (excitation λ = 622 nm, emission λ = 670 nm) with an F-4500 fluorescence spectrophotometer (Hitachi, Japan). The background fluorescence was also recorded. Localization of FITC-Labeled Peptides. The action site of the peptides was further determined by using FITC-labeled peptides and propidium iodide (PI) and observed by confocal laser scanning microscopy and 3D-SIM super-resolution microscopy.29 Microbial cells (OD600 = 0.2) were incubated with FITC-labeled peptides at 1× MBC/MFC at 37 °C for 15 min. Then, the mixture was centrifuged and washed three times with PBS buffer at 1000g. The cells were resuspended and incubated with 10 μg mL−1 PI in PBS buffer for 15 min at 4 °C, and free PI dye was removed by centrifugation. A smear was made, and the images were observed using a Leica TCS SP2 confocal laser scanning microscope and Deltavision OMX system with a 488 and 535 nm band-pass filter for FITC and PI excitation, respectively. Cells without peptides served as control. SEM, AFM, and TEM Characterization. For SEM sample preparation, logarithmic growing microbial cells were collected and diluted to OD600 = 0.2 in 10 mM PBS. Then, the cell suspension was incubated with the peptides at 1× MBC/MFC for 60 min at 37 °C, harvested by centrifugation, and fixed with 2.5% (w/v) glutaraldehyde at 4 °C overnight. The samples were continuously dehydrated for 10 min in different concentrations of ethanol (50%, 70%, 90%, and 100%)

% ΔF (AU) =

Fobs − F0 × 100 F100 − F0

where Fobs is the observed fluorescence at a given peptide concentration, F0 is the initial fluorescence of BC with LPS in the absence of peptides, and F100 is the BC fluorescence with LPS cells upon the addition of 10 μg mL−1 polymyxin B, a prototype LPS binder that was the positive control. Surface Plasmon Resonance (SPR) Experiments. The real-time binding interaction between the peptides and LPS was measured by SPR using a Biacore 3000 instrument (GE Healthcare)72 at 25 °C. The peptides were covalently immobilized on a certified grade CM5 sensor chip at a concentration of 25.6 μM in 10 mM PBS buffer (pH 6.0). Nearly 1200 resonance units (RUs) of the peptide (13) were immobilized by using an amine coupling kit according to the manufacturer’s instructions. Unreacted surface moieties were blocked with ethanolamine. LPS in running buffer (20 mM Tris, 100 mM NaCl, pH 7.4) was flowed over the surface at 3.125−25 μg/mL with a flow rate of 10 μL/min. After each injection, the surface was regenerated with 50 mM NaOH containing 0.05% (w/v) SDS. Each test was performed independently in triplicate. Endotoxin Neutralization Assay. The NO and TNF-α production through stimulation of the murine macrophage cell line RAW264.7 by using LPS was conducted to evaluate the LPS neutralizing properties of the peptides.48 Briefly, (1.0−2.0) × 105 cells/well RAW264.7 cells were plated in 96-well plates and stimulated 3904


Journal of Medicinal Chemistry with LPS (50 ng/mL) in the absence or presence of the peptides (2− 64 μM) for 18 h at 37 °C. Untreated cells and LPS-alone treated cells served as the respective negative and positive controls. The supernatant was collected for analyzing NO and TNF-α production using the Griess reagent (Promega, USA) and an ELISA (Boster, China) according to the manufacturer’s protocol, respectively. Each experiment was performed in triplicate with three biological replicates. Statistical Analysis. All data were subjected to a one-way analysis of variance (ANOVA), and significant differences between the means were evaluated by Tukey’s test for multiple comparisons. The data were analyzed using the Social Sciences (SPSS) version 20.0 (Chicago, IL, USA). Continuous variables are expressed as the mean ± standard error (SE), and P < 0.01 is considered statistically very significant.

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ABBREVIATIONS USED

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REFERENCES

AMP, antimicrobial peptide; hRBC, human red blood cell; LPS, lipopolysaccharide; BC, BODIPY-TR-cadaverine; SDS, sodium dodecyl sulfate; TFE, trifluoroethyl alcohol; GM, geometric mean; TI, therapeutic index; TLR4, Toll-like receptor-4; LBP, LPS binding protein; RP-HPLC, reverse-phase high-performance liquid chromatography; MTT, 3-(4,5-dimethylthiozol-2yl)-2,5-diphenyltetrazolium bromide; IFI, invasive fungal infection; AFP, antifungal peptide; CAUTI, catheter-associated urinary tract infection; CD, circular dichroism; SEM, scanning electron microscopy; AFM, atomic force microscopy; TEM, transmission electron microscopy; PI, propidium iodide; DCFH-DA, 2′,7′-dichlorofluorescein; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MOPS, morpholinepropanesulfonic acid; LUV, large unilamellar vesicle; SPR, surface plasmon resonance; PC, phosphatidylcholine; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; CL, cholesterol, cardiolipin; PI, phosphatidylinositol; MBC, minimum bactericidal concentration; MFC, minimum fungicidal concentration

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01729. GM values comparison between the engineered peptides (net charges); GM values comparison between the engineered peptides (imperfect/perfect amphipathicity); toxicity of 17 against hRBCs, RAW 264.7 cells, and HEK 293T cells; cytoplasmic membrane potential variation of E. coli (A) and C. albicans (B) treated with the peptide 13; correlation between the GM values and H values of the imperfectly amphipathic peptides; HPLC spectra of the synthetic peptides; MALDI-TOF MS of the synthetic peptides; HPLC spectra and MALDI-TOF MS of the fluorescein-labeled 17 (PDF) 17 presenting green fluorescent signal around E. coli cell surface (AVI) 17 presenting green fluorescent signal around C. albicans cell surface (AVI)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +86 451 55190685. Fax: +86 451 55103336. ORCID

Jiajun Wang: 0000-0002-2086-2105 Author Contributions

J.W. and S.C. contributed equally to this work, and they are both co-first-authors. J.W. and A.S. designed and conceived the experiments. J.W. and S.C. conducted the main experiments assay. Z.Y. conducted the membrane permeability assay and TEM assay. Y.Y. performed the antifungal activity assay. Z.W. conducted the endotoxin neutralization assay. J.S. conducted the SEM assay and DNA-binding assay. J.W. wrote the main manuscript text. X.D. and A.S. supervised the work and revised the final version of the manuscript. All of the authors have read and approved the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grants 31272453, 31472104, 31672434), the China Agriculture Research System (CARS36), and the Program for Universities in Heilongjiang Province (Grant 1254CGZH22). 3905



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Combating Drug-Resistant Fungi with Novel Imperfectly Amphipathic

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