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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 J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01729 • Publication Date (Web): 12 Apr 2018 Downloaded from http://pubs.acs.org on April 12, 2018

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Journal of Medicinal Chemistry

Combating drug-resistant fungi with novel imperfectly amphipathic palindromic peptides Jiajun Wang, Shuli Chou, Zhanyi Yang, Yang Yang, Zhihua Wang, Jing Song, Xiujing Dou, Anshan Shan* Institute of Animal Nutrition, Northeast Agricultural University, Harbin 150030, P. R. China

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 6.62µM. Of such peptides, 13 (I6) (RRIRIIIRIRR-NH2) that was Ile rich in its hydrophobic face had the highest antifungal activity (GMfungi =1.64µM), meanwhile 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.

*

Corresponding author. Tel.: +86 451 55190685; Fax: +86 451 5510-3336; E-mail address: asshan@ neau.edu.cn

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INTRODUCTION 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 2

.Compared 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 meanwhile with 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, 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 sub-classified as antifungal peptides (AFPs) have also been developed as surface-based strategies to prevent


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Candida albicans from forming a fungal biofilm on the surface of urinary catheters to reduce the incidence of fungal catheter-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 agents12. However, AFPs have had limited success in clinical applications, primarily due to their low clinical antifungal activity, high systemic toxicity towards 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 well-suited as a new generation of antifungal drugs. Currently, alpha-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 alpha-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 exist

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 distribution 21, and found that centrosymmetric peptides with 3 units (containing 7 net charges, due to the amidation) had the greatest antimicrobial



Page 4 of 62

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 re-designed an imperfectly amphipathic palindromic structure by adding 1~2 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. The 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.

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 shown in results, the


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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 helix-stabilizing 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 environment24. The spectra of the engineered peptides were not characteristic of alpha-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 evidenced typical helical structure propensity, while phenylalanine-containing peptides

displayed

unusual

alpha-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 alpha-peptide melittin, all engineered peptides had very significantly lower hemolytic activity (P3 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)/ (c.l.n). 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-diphenyltetrazoliumbbromideb (MTT) dye reduction assay and the last 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 24h at 37°C in 5% CO2. Then, 50 µL MTT were 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 1, 000 g for 5 min, and the supernatants were discarded. The formazan crystals were dissolved with 150 µL DMSO, and the OD at 570 nm was measured using a microplate reader (TECAN GENios F129004; TECAN, Austria). A 1 mL fresh hRBCs was collected and diluted 10-fold 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 hour at 37°C. The plates were centrifuged at 1000g for 10min, 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


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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) / (At − A0)] ×100 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 CFUmL−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 plate. After incubation for 24h 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, 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 CFUmL−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 48h 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 48h 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 means of three independent assays. Drug resistance was induced by treating fungal cells repeatedly with antimicrobial agents63. We choose Fluconazole-resistant C. albicans 56452 as the model microorganisms. 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 protocol64. The results were from three independent assays. Antimicrobial mechanism studies Cell wall permeabilization


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Peptide

cell

wall

permeabilization

was

analyzed

by

the

uptake

1-N-phenylnaphthylamine (NPN, Sigma-Aldrich, China) as previously described

of

12, 65

.

Briefly, logarithmic growing microbial cells were collected and diluted to an 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 30min. 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 raised. 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: NPN uptake (%) = (Fobs − F0)/ (F100 − F0) × 100%. 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 with microbial cells upon addition of 10µg/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 previously22.

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 membrane mimicking the fungal membrane erythrocyte cell membrane

67

66

), PC/PE/PI/ ergosterol (5:4:1:2, w/w/w/w,

) and PC/cholesterol (10:1,w/w, mimicking the human

66

) phospholipid was 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 Stewart 68 and diluted to a final concentration of 100 µM



Page 24 of 62

in Tris-HCl buffer. Subsequently, the peptides were serially diluted in Tris-HCl 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 (%) = (Fobs - F0) / (F100 - F0) ×100%. Where F0 is the fluorescence intensity of liposomes (background), 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

(Sigma-Aldrich), as previously described

64, 69

fluorescent

dye,

DiSC3-5

. Briefly, logarithmic growing microbial cells

were harvested and diluted to an OD600 = 0.05 in 5 mM HEPES buffer (pH 7.4, containing 20 mM glucose). The cell suspension was further incubated with 0.4µM DiSC3-5 and 100 mM K+ until no further fluorescence reduced. 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 800s (excitation λ=622nm, 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 15min. Then, the mixture was centrifuged and washed three times with PBS buffer at 1,000×g. The cells were resuspended and incubated with 10 µg mL−1 PI in PBS buffer for 15 min at 4 °C, free PI


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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 nm 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 an OD600 = 0.2 in 10 mM PBS. Then, the cell suspension was incubated with the peptides at 1 × MBC/MFC for 60min at 37°C, harvested by centrifugation and fixed with 2.5% (w/v) glutaraldehyde at 4°C overnight. The samples were continuous dehydrated for 10 min in different concentrations of ethanol (50%, 70%, 90%, and 100%) 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 pre-fixation with 2.5% glutaraldehyde at 4°C overnight, the samples were post-fixed with osmium tetroxide for 70 min and washed twice with PBS (pH 7.2). Subsequently, the samples were continuous 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 0.5-32 µM for 60 min at 37°C, and then stained with 10µM 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-TR-cadaverine (BC Sigma, USA) displacement assay70,

71

. 50µg/ml LPS from E. coli O111:B4 was

incubated with 5µg/ml BODIPY-TR-cadaverine in Tris buffer (50 mM, pH 7.4) for 4h at room temperature. Subsequently, the peptides were serially diluted in Tris buffer and incubated with equal volumes of the LPS-probe mixture for 1h 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 %∆F (AU) using the following equation: %∆F (AU) = [(Fobs - F0) / (F100-F0)] ×100. 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 25.6µM 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


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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 stimulating the murine macrophage cell line RAW264.7 by using LPS were conducted to evaluate the LPS neutralizing properties of the peptides48. Briefly, 1.0-2.0×105 cells/well RAW264.7 cells were plated in 96-well plates and stimulated 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 control. 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, Illinois, USA). Continuous variables are expressed as the mean ± standard error (SE), and. P < 0.01 is considered statistically very significant.

ANCILLARY INFORMATION Supporting Information Availability GM values comparison between the engineered peptides---net charges; GM values comparison between the engineered peptides---imperfectly/perfectly amphipathicity; The toxicity of 17 against hRBCs, RAW 264.7 cells and HEK 293T cells; The 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.



Page 28 of 62

AUTHOR INFORMATION Corresponding Author Information: *E-mail: [email protected]. Tel: +86 451 55190685. Fax: +86 451 55103336.

Author contributions J.J.W and S.L.C. contributed equally to this work, and they are both co-first authors. J.J.W and A.S.S designed and conceived the experiments; J.J.W. and S.L.C. conducted the main experiments assay; Z.Y.Y. conducted the membrane permeability assay and TEM assay. Y.Y performed the antifungal activity assay. Z.H.W conducted the endotoxin neutralization Assay. J.S conducted the SEM assay and DNA-binding assay. J.J.W wrote the main manuscript text. X.J.D and A.S.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 Competing financial interests: The authors declare no competing financial interests.

ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (31272453, 31472104, 31672434), the China Agriculture Research System (CARS-36) and the Program for Universities in Heilongjiang Province (1254CGZH22).

ABBREVIATIONS USED AMPs, antimicrobial peptides; hRBCs, human red blood cells; LPS, lipopolysaccharides; 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-2-yl)-2,5-diphenyltetrazolium

bromide;

IFI,

invasive

fungal

infections; AFPs, antifungal peptides ; CAUTI, catheter-associated urinary tract infections;


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CD, circular dichroism; SEM, scanning electron microscopy; AFM, atomic force microscopy; TEM, transmission electron microscopy; PI, propidium iodide ; DCFH-DA, 2’, 7’-dichlorofluorescin;

MALDI-TOF

MS, matrix-assisted

laser

desorption/ionization

time-of-flight mass spectrometry; MOPS, morpholinepropanesulfonic acid; LUVs, large unilamellar vesicle; SPR, Surface Plasmon Resonance; PC, Phosphatidylcholine; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; CL, cholesterol, cardiolipin; PI, phosphatidylinositol; MBC, the minimum bactericidal concentrations; MFC, the minimum fungicidal concentrations

TOC Graphic

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Ciociola, T.; Giovati, L.; Conti, S.; Magliani, W.; Santinoli, C.; Polonelli, L. Natural and

synthetic peptides with antifungal activity. Future Med Chem 2016, 8, 1413-1433. 4.

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Page 34 of 62

1859, 467-474. 68. Stewart,

J.

C.

Colorimetric

determination

of

phospholipids

with

ammonium

ferrothiocyanate. Anal Biochem 1980, 104, 10-14. 69. Qi, X.; Zhou, C.; Li, P.; Xu, W.; Cao, Y.; Ling, H.; Ning Chen, W.; Ming Li, C.; Xu, R.; Lamrani, M.; Mu, Y.; Leong, S. S.; Wook Chang, M.; Chan-Park, M. B. Novel short antibacterial and antifungal peptides with low cytotoxicity: efficacy and action mechanisms. Biochem Biophys Res Commun 2010, 398, 594-600. 70. Sautrey, G.; Zimmermann, L.; Deleu, M.; Delbar, A.; Souza Machado, L.; Jeannot, K.; Van Bambeke, F.; Buyck, J. M.; Decout, J. L.; Mingeot-Leclercq, M. P. New amphiphilic neamine derivatives active against resistant Pseudomonas aeruginosa and their interactions with lipopolysaccharides. Antimicrob Agents Chemother 2014, 58, 4420-4430. 71. Ma, Z.; Wei, D.; Yan, P.; Zhu, X.; Shan, A.; Bi, Z. Characterization of cell selectivity, physiological stability and endotoxin neutralization capabilities of alpha-helix-based peptide amphiphiles. Biomaterials 2015, 52, 517-530. 72. Wang, J.; Li, Y.; Wang, X.; Chen, W.; Sun, H.; Wang, J. Lipopolysaccharide induces amyloid formation of antimicrobial peptide HAL-2. Biochim Biophys Acta 2014, 1838, 2910-2918.

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.

Figure 2. The 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


Page 35 of 62 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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.

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

Figure 5. (A)The 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) The 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) The 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 means ± SEM of three independent experiments. P** 64)

64(>64)

>64(>64)

7

4(4)

2(4)

4(4)

2(8)

4(16)

2(2)

4(8)

4(8)

8

8(32)

4(8)

8(16)

8(16)

4(16)

4(4)

4(8)

4(8)

9

4(4)

4(4)

4(4)

2(8)

16(16)

8(16)

4(4)

4(8)

10

4(16)

4(4)

8(16)

8(32)

4(16)

8(16)

4(4)

8(16)

11

4(4)

4(4)

4(8)

2(4)

4(8)

4(8)

4(4)

8(16)

12

8(8)

8(8)

4(8)

4(8)

8(16)

4(8)

4(4)

4(8)

13

4(4)

4(4)

4(4)

2(4)

4(8)

4(4)

4(4)

4(8)

14

16(32)

8(8)

16(32)

16(64)

64(>64)

16(16)

16(32)

32(64)

15

4(4)

4(4)

4(8)

2(4)

2(4)

4(8)

4(8)

4(8)

16

16(16)

8(8)

8(32)

4(8)

4(8)

4(8)

4(8)

8(16)

17

4(4)

8(16)

4(8)

4(4)

4(4)

4(4)

2(4)

8(8)

Polymyxin B

2(4)

1(2)

2(2)

1(2)

64(64)

64(64)

32(32)

32(64)

Melittin

2(2)

2(2)

1(2)

2(2)

8(8)

1(1)

0.5(1)

4(4)

Gentamicin

1(2)

0.5(1)

2(4)

1(2)

1(1)

8(8)

0.5(1)

1(1)

Ciprofloxacin

2(4)

2(4)

8(16)

2(4)

4(4)

1(1)

2(4)

8(8)


Page 55 of 62 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


a

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 b

independent experiments. Minimum bactericidal concentrations (MBC, µM) were determined as the lowest peptide concentration that killed greater than 99.9% of the bacterial c

cells. The data were derived from representative value of three independent experimental trials. Methicillin-resistant S. aureus.


Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 56 of 62

Table 3. The MICsa (MFCsb) (µM) of the engineered peptides against fungi C.

C. C.

C.

C.

C.

C. albicans

C.

albicans Peptides

C.

C.

C.

albicans

albicans

albicans

58288

14936

17546

C. albicans albicans

albicans

albicans

albicans

isolated from

albicans

SP3931

SP3903

SP3902

SP3876

Alveolar fluid

56452

cgmcc

C. krusei tropicalis

56214

C. parapsilosis cgmcc

cgmcc

cgmcc 2.3989 2.1857

2.1975

2.2086

1

8(16)

8(16)

8(8)

4(8)

4(4)

8(16)

8(8)

8(16)

8(8)

4(4)

16(16)

2(4)

8(8)

>64(>64)

2

32(64)

32(32)

16(16)

>64(>64)

32(64)

64(>64)

32(32)

32(32)

64(64)

32(>64)

64(>64)

4(8)

8(8)

>64(>64)

3

8(16)

8(16)

4(4)

4(4)

8(8)

8(16)

4(4)

8(16)

8(8)

8(8)

4(4)

2(4)

4(4)

16(32)

4

16(32)

16(32)

16(16)

32(32)

16(32)

16(32)

16(16)

16(32)

16(32)

16(32)

4(8)

4(8)

4(4)

>64(>64)

5

4(8)

4(8)

2(8)

2(2)

1(1)

4(8)

4(4)

2(4)

2(2)

2(2)

2(2)

2(4)

2(4)

8(16)

6

64(64)

>64(>64)

>64(>64)

>64(>64)

>64(>64)

64(>64)

64(64)

64(64)

>64(>64)

>64(>64)

>64(>64)

4(8)

16(16)

>64(>64)

7

8(16)

8(16)

8(8)

8(16)

4(4)

8(16)

4(4)

4(8)

4(4)

8(8)

1(2)

2(2)

4(8)

16(32)

8

8(16)

16(32)

4(4)

8(8)

4(4)

8(16)

8(8)

4(8)

8(8)

8(8)

2(4)

4(8)

4(4)

32(64)

9

8(16)

8(16)

2(2)

4(4)

4(4)

4(8)

4(8)

4(8)

8(8)

8(8)

8(16)

2(4)

4(8)

64(64)

10

8(16)

8(8)

4(4)

4(8)

4(8)

8(16)

8(8)

8(16)

4(4)

8(8)

8(16)

2(4)

4(8)

8(16)

11

4(16)

4(8)

2(2)

4(4)

4(4)

4(8)

4(4)

4(8)

2(2)

4(4)

2(2)

1(2)

2(2)

16(32)

12

8(16)

8(16)

4(4)

8(8)

8(8)

8(16)

8(8)

8(16)

4(4)

8(16)

4(8)

2(4)

2(4)

>64(>64)

13

2(2)

2(4)

1(1)

2(2)

1(1)

2(4)

1(1)

1(2)

2(2)

2(2)

2(2)

1(2)

2(2)

4(8)

14

16(64)

16(32)

4(4)

16(16)

8(16)

16(32)

8(8)

8(16)

8(16)

8(8)

16(16)

1(2)

4(8)

>64(>64)

15

4(8)

4(8)

4(4)

4(4)

2(4)

4(8)

4(4)

4(8)

2(4)

8(8)

4(4)

2(4)

2(2)

8(16)

16

4(8)

8(16)

2(2)

4(4)

2(2)

4(8)

8(8)

4(8)

2(4)

4(4)

4(4)

2(4)

2(4)

16(32)

17

2(2)

4(4)

2(2)

2(2)

2(2)

4(4)

2(2)

2(4)

4(4)

2(2)

2(2)

4(8)

2(2)

4(8)

Melittin

4(8)

8(8)

4(4)

2(2)

4(4)

4(4)

8(8)

4(4)

4(4)

4(8)

8(8)

4(8)

2(4)

2(2)

Fluconazole

1(2)

1(64)

16(64)

16(>64)

>64(>64)

2(8)

>64(>64)

>64(>64)

8(>64)

32(>64)

>64(>64)

4(32)

64(64)

4(8)

Amphotericin B

1(1)

0.25(1)

0.125(1)

0.25(1)

0.25(0.5)

1(2)

1(2)

1(2)

0.5(1)

0.25(0.5)

0.25(1)

1(2)

1(1)

2(2)

Ketoconazole

0.25(1)

>64(>64)

>64(>64)

>64(>64)

>64(>64)

0.5(2)

64(>64)

64(>64)

1(>64)

>64(>64)

32(64)

0.25(1)

1(1)

0.25(1)


Page 57 of 62 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


a

Minimum inhibitory concentration (MIC) was determined as the lowest concentration of peptide that inhibited 95% of the fungal growth. Data are representative of three b

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.



Page 58 of 62

Table 4. The MHC, GM and TI values of the engineered peptides. GMb

a

Peptide

MHC

1

>128

2

>128

3

>128

4

>128

5

>128

6

>128

7

>128

8

>128

9

>128

10

>128

11

>128

12

>128

13

>128

14

>128

15

>128

16

>128

17

>128

Melittin

0.25

Bacteria

Fungi

TIc ALL

4.76 8.00 6.62 19.03 33.62 27.34 3.67 5.94 4.99 6.73 14.49 10.96 4.36 2.56 3.11 49.35 70.66 62.02 3.08 5.12 4.26 5.19 6.56 6.02 4.76 5.66 5.31 5.66 5.66 5.66 4.00 3.28 3.53 5.19 6.90 6.22 3.67 1.64 2.20 19.03 9.75 12.44 3.36 3.62 3.53 6.17 3.81 4.54 4.36 2.56 3.11 1.83 4.00 3.01

Bacteria

Fungi

ALL

53.82 32.00 38.66 13.45 7.61 9.36 69.79 43.07 51.33 38.05 17.67 23.35 58.69 99.93 82.35 5.19 3.62 4.13 83.00 49.97 60.09 49.35 39.01 42.49 53.82 45.25 48.20 45.25 45.25 45.25 64.00 78.02 72.60 49.35 37.12 41.17 69.79 156.10 116.36 13.45 26.25 20.59 76.11 70.66 72.60 41.50 67.25 56.42 58.72 100 82.32 0.14 0.08 0.06

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 b

detectable hemolytic activity was observed at 128µM, a value of 256µM was used to calculate the therapeutic index. The geometric mean (GM) of the peptide MICs against C

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. TI is calculated as MHC/GM. Larger values indicate greater cell selectivity.


Page 59 of 62 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


Table 5. The MIC values of the engineered peptides against E.coil ATCC 25922 in the presence of physiological salts. Peptide

Controla

NaCla

KCla

NH4Cla

MgCl2a

CaCl2a

ZnCl2a

FeCl3a

1

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

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

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

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

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

8 64 4 32 16 64 4 32 4 8 4 8 8 32

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

4 16 4 8 8 64 4 4 8 16 4 8 4 16

4 16 2 2 1 2

4 16 4 8 2 2

4 16 2 2 1 2

4 16 2 2 2 2

2 8 4 4

8 32 8 2 1 8

4 16 2 2 2 2

4 16 2 2 1 4

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Melittin Polymyxin B Gentamicin Ciprofloxacin a

1

8

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.



Page 60 of 62

Table 6. The 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

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

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

Fluconazole Amphotericin B Ketoconazole 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.


Page 61 of 62 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


Table 7. The MIC values of the peptide 13 against E.coil and C.albicans in the presence of serum Peptide

Control

E.coli ATCC 25922 13 4 Melittin 2 Polymyxin B 2 Gentamicin 1 Ciprofloxacin 2 C.albicans cgmcc 2.2086 13 2 Melittin 4 Fluconazole 1 Amphotericin B 1 Ketoconazole 0.25

12.5%

25%

50%

4 32 2 1 2

4 64 2 1 2

8 64 4 2 4

2 32 1 1 0.25

4 32 1 1 0.25

8 64 2 1 0.5




Page 62 of 62

Combating drug-resistant fungi with novel ... - ACS Publications

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