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A designed tryptophan and lysine/arginine-rich antimicrobial peptide with therapeutic potential for clinical antibiotic-resistance Candida albicans vaginitis Lin Jin, Xuewei Bai, Ning Luan, Huimin Yao, Zhiye Zhang, Weihui Liu, Yan Chen, Xiuwen Yan, mingqiang rong, Ren Lai, and Qiumin Lu J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01264 • Publication Date (Web): 16 Feb 2016 Downloaded from http://pubs.acs.org on February 20, 2016

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Journal of Medicinal Chemistry

A designed tryptophan and lysine/arginine-rich antimicrobial peptide with therapeutic potential for clinical antibiotic-resistance Candida albicans vaginitis

Lin Jin†, 1, Xuewei Bai†, 1, Ning Luan†, Huimin Yao†, Zhiye Zhang§, ¶, Weihui Liu†, Yan †

†

Chen , Xiuwen Yan , Mingqiang Rong§, ¶, Ren Lai†, §, ¶, *, and Qiumin Lu§, ¶, *

†

Life Sciences College of Nanjing Agricultural University, Nanjing 210095, Jiangsu,

China §

Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese

Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Kunming 650223, Yunnan, China ¶

Joint Laboratory of Natural peptide, University of Science and Technology of China

and Kunming Institute of Zoology, Chinese Academy of Sciences

1

These authors contributed equally to this work.

*

Correspondence: Life Sciences College of Nanjing Agricultural University, Nanjing

210095, Jiangsu, China. E-mail: R.L., [email protected]; Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Kunming 650223, Yunnan, China. E-mail: Q.L., [email protected].

Running title: Artificial antimicrobial peptide treats C. albicans vaginitis

1

ACS Paragon Plus Environment


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ABSTRACT: New therapeutic agents for Candida albicans vaginitis are urgently awaiting to be developed because of the increasing antibiotic resistance of C. albicans. Antimicrobial peptides (AMPs) are one of the most promising choices for next-generation antibiotics. In this study, novel peptides were designed based on snake venom antimicrobial peptide cathelicidin-BF to promote anti-C. albicans activity and decrease side-effect. The designing strategies include substitutions of non-charged polar amino acid residues with charged or hydrophobic residues to promote antimicrobial activity and insertion of a hydrophobic residue in the hydrophilic side of the helix structure to reduce hemolysis. A designed tryptophan and lysine/arginine-rich cationic peptide 4 (ZY13) (VKRWKKWRWKWKKWV-NH2) exhibited excellent antimicrobial activity against either common strain or clinical isolates of antibiotic-resistance C. albicans with little hemolysis. Peptide 4 showed significant therapeutic effects on vaginitis in mice induced by the infection of clinical antibiotic-resistance C. albicans. The approaches herein might be useful for designing of AMPs.

KEY WORDS: Candida albicans; Vaginitis; Anti-inflammation; Antimicrobial peptide

2


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■ INTRODUCTION The incidence of Candida infections is increasing with a large number of antibiotic resistant strains in recent years 1. C. albicans vaginitis caused by a polymorphic fungus C. albicans is a common gynecological infection in women around the word 2. Recurrent infection usually occurs even after C. albicans vaginitis patients are treated with antifungal drugs 3. New therapeutic agents for C. albicans vaginitis are urgently awaiting for development because of the antibiotic resistance of clinical isolated C. albicans strains. AMPs exist widely in nearly all organisms and take part in the innate immune 4, 5. They are one of the most promising choices for next-generation antibiotics because their antimicrobial activities are rapid and broad spectrum. AMPs are usually short cationic peptides with 10 to 50 amino acid residues. They contain about 50% hydrophobic amino acid residues which form an amphipathic structure

1, 2

. Though

sequence and structural diversities of AMPs are very rich, active antimicrobial peptides have a number of common physicochemical characteristics. The principal factors for AMPs to be active include cationic charges and high hydrophobicity

5-9

.

The cationic residues of AMPs initially contact with the negatively charged bacterial surface through electrostatic interactions to facilitate subsequent actions 10-12. With much deeper researches on AMPs, our choices for fighting the increasing risk of antibiotic-resistance microorganism are greatly extended over the past decades 6

. Many works have devoted to artificial design, modification and chemical synthesis

of AMPs and these efforts broadened the sources of AMPs 7. The aim of modification of these peptides is to increase their potency and several are in clinical trials. For example, pexiganan derived from frog skin AMP is now in phase III clinical trials for diabetic foot ulcers 8. Several hurdles including the stability in serum, hemolysis and synthesizing cost have to be overcome before new peptide drugs can come out to market 9. Various strategies for designing novel synthetic AMP analogues have been described

9, 10

. One of the widely used method is based on sequences of AMPs, for

antimicrobial activity is often correlate with the presence of specific amino acid at 3



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specific

position.

Several

physicochemical

parameters

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including

charge,

hydrophobicity and hydrophobic moment are generally involved in the design of AMPs 10. Our previous work indicated that cathelicidin-BF, an antimicrobial peptide from snake venoms of Bungarus fasciatus, had strong and rapid antimicrobial activities against many microorganisms such as Gram-negative, Gram-positive bacteria and fungi, including some clinically isolated drug resistance microorganisms 11. However, it is not stable in human serum since its antimicrobial activities could not be detected after 3-h incubation in human serum. In this work, we designed several peptides based on cathelicidin-BF. After screening of antimicrobial activity and hemolytic activity, we found peptide 4 (ZY13) exhibited excellent activity towards many microbial, especially to C. albicans, low hemolysis to human red blood cell and high stability in human serum. Its potential therapeutic use for clinical antibiotic-resistance C. albicans vaginitis was also evaluated. ■ RESULTS Functional screening of the designed peptides. The sequences and physicochemical properties of the designed peptides were listed in Table 1. All the peptides (range from 15-17 residues) were positively charged with 8 net charges. The ratios of polar residues to nonpolar residues were about 50%. Among them, cathelicidin-BF15 had the lowest hydrophobicity of 0.101 and peptide 6 had the highest hydrophobicity of 0.602. However, the hydrophobic moment of peptide 6 was very low. The predicted helix structures of the peptides were shown in Fig. 1. All the peptides could form helixes and most of them have an amphipathic structure forming a hydrophilic and hydrophobic side, except for peptides 5 and 6 (Fig. 1). The antimicrobial activities of the designed peptides to E. coli, S. aureus, B. subtilis and C. albicans were tested (Table 2). The activities of peptides 1, 2, 5 and 6 were very low to S. aureus with MICs higher than 100 µg/mL. peptides 5 and 6 had no effect on all the four strains. Peptide 4 showed high activity to all the tested strains. 4


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Its MICs for E. coli, S. aureus and B. subtilis were 9.38, 1.17 and 1.17 µg/mL, respectively. Especially, its activity to C. albicans was very high with MIC as low as 0.59 µg/mL. Hemolysis is one of toxic effects of some AMPs and greatly hinders their application. Hemolytic activities of the designed peptides are shown in Table 3. All the peptides did not induce 50% of hemolysis at the highest concentration tested (320 µg/mL). Peptide 3 induced 10% of hemolysis at a concentration of 51.7 µg/mL and 27.6% hemolysis at a concentration of 320 µg/mL, which is the strongest in all the designed peptides. Though the antimicrobial activities of peptides 5 and 6 were very low, they showed strong hemolysis activities with Hmax of 17.6 and 30.8, respectively (Table 3). Peptide 4 showed the lowest hemolysis. It induced only 3.1% of hemolysis at the concentration of 320 µg/mL. Among them, peptide 4 is the most promising candidate for further development.

Antimicrobial activities of peptide 4 to antibiotic-resistance C. albicans. Seven C. albicans strains including 6 clinically isolated antibiotic-resistance strains were used in this experiment. Peptide 4 showed antimicrobial activity against these antibiotic-resistance strains with MICs from 1.40 to 4.69 µg/mL (Table 4). However, fluconazole, a clinical drug applied for fungus infection had effect only on standard strain C. albicans ATCC2002 with a MIC of 18.7 µg/mL and its activity on the 6 antibiotic-resistance strains was not detectable (Table 4).

Hemolytic and cytotoxic assays. Peptide 4 showed little hemolytic activity (no more than 3.1%) on human red blood cells even at very high concentrations up to 320 µg/mL which was 542 times higher than its MIC to standard strain C. albicans ATCC2002 (Supplemental Fig. S3A). In addition, no significant cytotoxicity against human HEK293 embryonic kidney cells was observed after treatment by peptide 4 at various concentrations from 0.39 to 100 µg/mL (Supplemental Fig. S3B).

C. albicans-Killing Kinetics. Anti-C. albicans effect of peptide 4 was tested by 5



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a colony counting assay. Peptide 4 rapidly killed C. albicans effect in 1 ×, 5 ×, and 10 × MICs (Table 5). Peptide 4 killed all the C. albicans 08030809 at 1× MIC in 30 min. At the concentration of 5 × and 10 × MIC, Peptide 4 killed the fungi in less than 20 and 10 min, respectively. The anti-C. albicans activity was lethal for C. albicans 08030809 since it could not resume growth on agar plates (data not shown). On the contrary, the antibiotics clotrimazole could not completely kill the fungi at 10 mg/mL. Though the clone count was less than the control at each time point, it increased dramatically from 50 to 2674 in 6 h, indicating this clinically isolated strain is resistant to clotrimazole which is widely used to treat fungus infections. Even when the incubation time was prolonged to 24 h, no colony of C. albicans 08030809 was observed in the peptide 4 groups. However, the clone count increased to 57425 in the clotrimazole group.

Effects of human plasma and salt ion on the antimicrobial activity of peptide 4. We used antimicrobial zone assay to check the stability of peptide 4 in human plasma because the absorbance of the plasma itself had great influence on the MIC determination. Peptide 4 maintained its antimicrobial activity even after incubation with human plasma for 6 h (Supplemental Fig. S4), though the size of antimicrobial zone reduced. In addition, different concentrations of salt ions didn’t affect the antimicrobial activity of peptide 4 on the four tested microorganisms (Table 6).

Membrane morphology of peptide 4-treated C. albicans. The morphology difference of peptide 4-treated and -untreated C. albicans was observed by SEM. The untreated C. albicans has an intact cell wall and cytoplasmic membrane, and the outer membranes were smooth (Fig. 2 A, C). After peptide 4 treatment, the membrane was full of perforations and intracellular inclusions showed efflux (Fig. 2 B, D). In addition, the cell swelled obviously. These indicated that the damages might target the plasma membranes of C. albicans. 6


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SYTOX Green assay. In Sytox Green assay (Fig. 3), once treated with peptide 4 at 1× MIC, the intensity of green fluorescence was markedly increased within twenty minutes, which was directly proportional to the damage of bacterium membrane. However, PBS and clotrimazole didn’t affect the intensity of fluorescence. SYTOX Green is unable to enter intact bacterium. When the membrane of C. albicans became permeabilized to SYTOX Green stain, nucleic acids within it were subsequently labeled, resulting in an increase of fluorescence. Considering the fluorescence increased so fast within 20 minutes, peptide 4 might kill C. albicans by forming small pores, either permanent or transient. Combining the result of SEM (Fig. 2), peptide 4 treatment evidently causes the membrane perforation of C. albicans.

The effect of peptide 4 on mice C. albicans vaginitis model. The colony count of C. albicans 08030809 in mouse vagina began to decrease in the mice treated with 50 µg peptide 4 on day 1 by 28%. The inhibition of peptide 4 was dose-dependent. The inhibitory rate of 50 µg peptide 4 on day 6, 11 and 16 was 48, 66, and 83%, respectively; the corresponding inhibitory rate for 20 µg peptide 4 was 22, 37, and 71%, respectively. However, the therapeutic effect of clotrimazole was significant only on day 16 and the inhibitory rate was 38%. The colony count on day 16 of clotrimazole group (1.2 × 104) was much higher than that of 20 µg peptide 4 group (5.4 × 103) and 50 µg peptide 4 group (2.9 × 103) (Fig. 4). The microabscess number in mouse vaginal tissue section represents the severity of inflammatory and the therapeutic effect. As illustrated in Fig. 5A, peptide 4 treatment significantly reduced the microabscess number. The microabscess number of blank control was 61, while those of clotrimazole-, 20 µg peptide 4- and 50 µg peptide 4-treated groups were 18, 16, and 7 on day 6, respectively. After 16-day treatment, the microabscess number in 50 µg peptide 4 group was almost undetectable and it was less than 3 in 20 µg peptide 4 group, while there still were about 10 microabscess in the clotrimazole group (Fig. 5B).

Inhibition of peptide 4 on inflammatory cytokines secretion in mouse 7



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vaginal tissues. ELISA was applied to evaluate the anti-inflammatory effect of peptide 4 in mouse vaginal tissues. The production of inflammatory cytokines including TNF-α, IL-6, IL-1β and IL-10 was significantly inhibited by peptide 4 in a dose-dependent manner (Fig. 6). At the concentration of 20 µg, peptide 4 reduced the secretion of TNF-α, IL-6, IL-1β, and IL-10 by 32, 46 ， 44, and 39%. The corresponding reduction by 50 µg peptide 4 was 53, 57, 63, and 45%, respectively. The positive control clotrimazole only had slight effects on inflammatory cytokine secretions (Fig. 6).

■ DISCUSSION AND CONCLUSION Vaginitis which causes vaginal mucosa and inflammation of submucous connective tissue is one of the most common gynecological inflammations in adult women

13

.

According to the surveys, about three-quarters of women get at least once vaginitis 14. Fungus is a large microbial group and causes many human diseases including vaginitis. Among them, C. albicans causes most of the diagnosed vulvovaginal candidiasis cases (80–90%)

15

. It is known that recurrent vulvovaginal candidiasis 15

affect 5–8% of the menarchal women

. Women are suffering from such a high

incidence of persistent and recurrent vaginal yeast infection. Vaginal infection caused by C. albicans is common in medical care, but there is no specific drug to cure at present

16

. With the wide spread of antibiotics, the

incidence of C. albicans infection is increasing, accompanied by a large number of resistant strains. Antibiotic treatment for C. albicans infections such as C. albicans vaginitis is often futile because of antibiotic resistance

3, 16

. Therefore, developing

new therapeutic agents without inducing resistance will make for the treatment of C. albicans vaginitis. Many organisms produce AMPs as a part of immune system to withstand harmful microorganisms

17, 18

. Their potential to induce drug resistance of microorganisms is

very low. Among the known AMPs, a specific subset, tryptophan (Trp), lysine (Lys) 8


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or arginine (Arg) rich AMPs, has attracted increasing interest

19, 20

. Some specific

chemical properties of these residues make the AMPs more effective and the molecular mechanisms of the AMPs are well studied. The hydrophobic Trp prefers the interfacial region of lipid bilayers, while positively changed Lys and Arg residues facilitate the interaction of the AMPs with the anionic components of the bacterial membrane 21. In recent years, designed AMPs using various approaches have been confirmed to have strong antimicrobial activity and are promising as new antibiotic candidates 10. For example, acid-amide substitution is used to endow net positive charge to natural non-antimicrobial sequences which are structurally distinct from known AMPs

22

;

several of the synthetic AMPs designed based on physicochemical parameters including charge, hydrophobicity, hydrophobic moment, and polar angle exhibited improved selectivity against specific bacterial 23; two lead peptides from unrelated classes of AMPs were hybridized into hybrid peptides with conserved N- and C-termini, allowing sequence bridging of two highly dissimilar AMPs and sequence-activity relationship to be studied at single amino acid level. However, many designed antimicrobial peptides show side-effects such as hemolysis 24. In the present study, a previously reported antimicrobial peptide cathelicidin-BF was used as the template to design a series of peptide, aimed to promote its properties 11

. Among the designed peptides, 6 does not take an amphipathic helix structure. The

hydrophilic residues and hydrophobic residues in it are distributed at regular intervals (Fig. 1), and its antimicrobial activities were very low. Similarly, 5 takes a ‘four side’ helix structure with two hydrophilic sides and two hydrophobic sides (Fig. 1), and its antimicrobial activities are also very low. Other peptides with strong antimicrobial activity have a common feature that they form one hydrophilic side and one hydrophobic side along the helix. These results confirm that active AMPs generally have an amphipathic helix structure. There are two peptides (1 and 2) with a Ser at the C-terminus of their sequences (Table 1). Their activity is relatively low compared with peptides 3 and 4 which have a Trp C-terminus. This suggests that introduction of an aromatic amino acid at the 9



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C-terminus might be a way to promote the antimicrobial activities of AMPs if the substitution does not change the amphipathic helix structure. Phe8, Arg9 and Trp15 of peptide 3 were substituted with Arg8, Trp9 and Val15 to generate peptide 4 (Table 1). These changes did not alter the net charge but reduced the hydrophobic moment by about 30%, resulting in significant difference in hemolytic activities. The hemolytic activity of peptide 3 is about 9-fold higher than that of peptide 4 (Table 3), which is possibly due to the different helix structure between peptide 3 and peptide 4. A Trp residue is distributed in the hydrophilic side of peptide 4, while only charged residues are in the correspond region of peptide 3 (Fig. 1). This pattern did not affect the antimicrobial activity much and may reduce the interaction of the hydrophilic or the hydrophobic regions of AMPs with red cells, thus alleviating the hemolysis. Because of the high antimicrobial activity against C. albicans and low hemolytic activity, peptide 4 is the most promising peptide among the designed peptides for development. There are rare reports of topical application of antimicrobial peptides against C. albicans vaginitis. It is reported that a cationic antimicrobial peptide purified from haemocytes of the spider Acanthoscurria gomesiana was effective on vaginal candidiasis with low toxicity, though its mechanism is not clear 25. The results of SEM and SYTOX Green assay showed that peptide 4 treatment evidently cause perforation of the membrane of C. albicans (Fig. 2 & Fig. 3). The in vivo anti-C. albicans ability of peptide 4 was investigated in mouse C. albicans vaginitis model. The results showed that the clearance of C. albicans in the mouse vagina by peptide 4 was quick (Fig. 4 & 5). In addition, peptide 4 significantly inhibited C. albicans infection-induced vagina inflammation by decreasing production of inflammatory cytokines as illustrated in Fig. 6. The microabscess number on the vaginal tissue sections of mouse represents the severity of inflammatory and the therapeutic effect. With the time after drug delivery, the microabscess number significantly reduced by peptide 4 treatment. On day 11 of treatment for C. albicans vaginitis with peptide 4, some typical inflammatory cytokines including TNF-α, IL-6, IL-1β, and IL-10 were significantly inhibited (Fig. 6). It suggested that peptide 4 suppressed the 10


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inflammation in C. albicans vaginitis by inhibiting the production of inflammatory cytokines as did by some other AMPs 26. Hemolysis, cytotoxicity and stability in plasma greatly hamper the application of antimicrobial peptides. Peptide 4 showed little hemolytic activity and cytotoxicity even at high concentrations (Supplemental Fig. S3). AMPs may be degraded by various proteases in plasma 9, which causes problems in application of AMPs. Peptide 4 is very stable in human plasma since its antimicrobial activity didn’t lose after incubation with human plasma for 6 h at 37°C (Supplemental Fig. S4). Furthermore, the antibacterial activity of peptide 4 didn’t change in different concentrations of salt ions (Table 6). The good stability under various conditions ensures peptide 4 with promising in vivo application. In conclusion, the designed AMP 4 showed high antimicrobial abilities against clinical antibiotic-resistance C. albicans in vitro and therapeutic potential for inflammatory vaginitis induced by C. albicans in vivo in a mouse model. Combined its simple structure, little hemolytic activity and cytotoxicity and high stability in physiological environment, it might be an excellent candidate or template for the development of therapeutic agent to treat clinical antibiotic-resistance C. albicans vaginitis. In addition, the design strategies reported here may provide guides for AMP modification.

■ EXPERIMENTAL SECTION Peptides design and synthesis. Six peptides (1, 2, 3, 4, 5 and 6) were designed based on two previously reported AMPs, cathelicidin-BF

11

and cathelicidin-BF15 12.

Leu11 of cathelicidin-BF15 (VKRFKKFFRKLKKSV) was substituted with Phe11 to generate 1 (VKRFKKFFRKLKKSV); Phe4, Phe7, Phe8 of cathelicidin-BF15 were substituted by Trp4, Trp7, Trp8 to generate 2 (VKRWKKWWRKLKKSV); Phe4, Phe7, Leu11, Ser14 and Val15 of cathelicidin-BF15 were substituted with Trp4, Trp7, Trp11, Trp14 and Trp15 to generate 3 (VKRWKKFWRKWKKWW); Phe8, Arg9 and Trp 15 of 3 were substituted with Arg8, Trp9 and Val15 to generate 4 11



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(VKRWKKWRWKWKKWV); A Trp3 was inserted after Lys2 of 4, and Lys13 and Trp14

were

substituted

by

Trp15

and

Lys16

to

generate

5

(VKWRWKKWRWKWKWKV); A Trp7 was inserted after Lys6 of 5 to generate 6 (VKWRWKWKWRWKWKWKV). C-terminals of all the peptides are amidated (-NH2). All the designed peptides were synthesized by GL Biochem (Shanghai) Ltd. (Shanghai, China) and analyzed by reversed phase high performance liquid chromatography (RP-HPLC) and mass spectrometry to confirm their purity to be greater than 98% (Supplemental Table S1 and Supplemental Fig. S1).

Bioinformatics analysis. Physical and chemical parameters of the designed peptides

were

analyzed

through

ExPASy

Bioinformatics

Resource

Portal

(http://www.expasy.org/tools/). The helix structures of the peptides were predicted by HeliQuest (http://heliquest.ipmc.cnrs.fr/).

Microorganism strains and growth conditions. Seven strains of C. albicans (ATCC2002 and six clinically isolated antibiotic resistance strains), Escherichia coli (ATCC25922), Bacillus subtilis and Staphylococcus aureus (ATCC2592) were obtained from Kunming Medical University. The strains of C. albicans were cultured with shaking at 28°C in salouraud liquid medium (1% peptone, 4% glucose, 0.01% chloramphenicol). E. coli (ATCC25922), B. subtilis and S. aureus (ATCC2592) were grown in LB (Luria-Bertani) broth as our previous report 27.

In vitro antimicrobial testing. MIC (minimal inhibitory concentration) of peptide 4 was determined using broth dilution determination as our previous methods 28

. Ampicillin (100 µg/mL) and clotrimazole (10 mg/mL), which have long been used

clinically in vaginal infection treatment

29, 30

were used as controls. Briefly, samples

were prepared as a stock solution in 0.9% sodium chloride solution at a series of concentrations. 90 µL of broth (salouraud liquid medium broth for C. albicans, LB broth for E. coli, B. subtilis and S. aureus), 100 µL bacterial suspension (2 × 105 CFU/mL) and 10 µL test sample were put together in 96-well plates and incubated at 12


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28°C for 16 h for C. albicans and at 37°C for 16 h for E. coli, B. subtilis and S. aureus. An absorbance at 600 nm was measured by a microplate reader to estimate bacterial growth. The MIC was defined as the lowest concentration of tested sample completely inhibiting the growth of microorganisms.

Assays of hemolysis and cytotoxicity. Hemolysis assay was undertaken using human red blood cells in liquid medium as our previous reports

12, 31

. Serial dilutions

of testing sample were incubated with human red cells at 37°C. After incubation for 30 min, the cells were centrifuged and the absorbance of the supernatant was measured at 540 nm. Maximum hemolysis was determined by adding 1% Triton X-100 to a sample of cells. Hemolysis of testing samples was calculated as the percentage of Triton X-100. Results were pooled from three independent experiments. HC10 and HC50 were defined as the peptide concentrations causing 10% and 50% hemolysis on human erythrocytes, respectively. Hmax was the percentage hemolysis observed at the maximum concentration as defined throughout this study (all peptides 320 µg/mL). Human HEK293 embryonic kidney cells (1 × 105 cells/mL, obtained from Cell Bank of Kunming Institute of Zoology, Chinese Academy of Sciences) were cultured in 96-well plates with Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 10% fetal calf serum and penicillin (100 U/mL)-streptomycin (100 µg/mL) at 37 °C in a humidified 5% CO2 atmosphere. Cell viability was evaluated by conventional 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) reduction assays. After a 24 h treatment by testing sample, 20 µL of MTT (5 mg/mL) was added to each well. The MTT solution was then removed and 200 µL dimethyl sulfoxide (DMSO) was added to solubilize the MTT-formazan crystals in living cells. The absorbance at 570 nm of the resulting solution was measured. The experiments were performed in triplicate.

Effects of human plasma and salt ions on peptide 4’s antimicrobial activity. Peptide 4 was mixed with human plasma (final concentration 100 µM) for 0, 1, 2, 3, 6, 13



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8, 10, 12, and 24 h at 37°C, respectively. Residual antimicrobial activity was evaluated by using 10 µL aliquot of the mixture to measure inhibitory zone on microorganism growth on salouraud liquid medium containing 0.7% agar as previously described

28

. The diameters of inhibition zones were recorded after a

36-hour incubation for C. albicans. Peptide 4 (2 mg/mL) was in sterilized ddH2O, 150 mM NaCl solution and phosphate-buffered saline (PBS, 150 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8 mM K2HPO4, pH 7.2) and then the anti-C. albicans activity of peptide 4 was detected.

Scanning electron microscopy (SEM). According to the previous method 32, C. albicans 08030809 was grown to the exponential-phase in salouraud liquid medium and then centrifuged at 2,000 rpm for 10 min. The pellet was then washed with PBS twice and re-suspended in salouraud liquid medium. Peptide 4 (1 × MIC or 5 × MIC) was incubated with C. albicans 08030809 at 37°C for 30 min and then centrifuged at 2,000 rpm for 10 min. Negative control group received the same volume of vehicle. The pellet was then washed with PBS twice and re-suspended in 2.5% glutaraldehyde solution at 4°C for 2 h. Then the C. albicans were centrifuged (2,000 rpm for 10 min) and washed with PBS. The pellet was then fixed in 1% osmium tetroxide in PBS for 1 h. Cells were rinsed with same buffer and dehydrated in a grade series of ethanol, and then were frozen in liquid nitrogen cooled tertbutylalcohol and vacuum dried overnight. The samples were mounted onto aluminum stubs. After sputter-coating with gold, they were analyzed by FEI quauta200 SEM.

SYTOX Green assay. The method for SYTOX Green assay 33, 34 was adapted to analyze the membrane integrity of C. albicans during peptide 4 treatment. Briefly, the antibiotic-resistance C. albicans 08030809 was grown to the exponential-phase in salouraud liquid medium and then centrifuged at 1000 g for 10 min. The bacterium pellet was then washed twice and re-suspended in PBS (10 mM, pH 7.4) to a cell 14


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density of 106 CFU/mL and incubated with SYTOX Green nucleic acid stain (Thermo Fisher Scientific, USA) at a final concentration of 0.1 µM at 37°C for 15 min on a shaking table. SYTOX Green-treated C. albicans were loaded into a FlexStation microplate reader (Molecular Devices, Sunnyvale, CA) and tested samples were added robotically. Monitoring was initiated and the fluorescence of each well was recorded every 2 minutes using an excitation of 488 nm and emission of 530 nm. Results were determined as relative fluorescence units (RFU).

In vivo anti-C. albicans experiments. As described previously

35

, the

antibiotic-resistance C. albicans 08030809 was grown to the exponential-phase in salouraud liquid medium and then centrifuged at 1, 000 g for 10 min. The bacterium pellet was then washed twice and re-suspended in 30% glycerol (107 CFU/mL). In order to induce a pseudo-estrus condition, mice received weekly subcutaneous injection of 0.5 mg of estradiol valerate dissolved in 0.1 mL of vegetable oil, 72 h prior to C. albicans inoculation throughout the experimental period. Infection was induced by intra-vaginally inoculation of 20 µL of a 107 CFU/mL of C. albicans suspension. Groups of 25 mice (body weight 20 ± 1 g) were used for each drug, peptide 4, clotrimazole or vehicle treatment. Treatments were given by intravaginal instillation of 20 µL of peptide 4 (20 µg/mouse, 50 µg/mouse) or clotrimazole (10 mg/mL, 20 µL/mouse) for 16 days. On day 1, 6, 11, and 16 during treatment, 5 mice were sacrificed, and a vaginal lavage was performed with 20 µL of 0.9% NaCl. The lavage fluids were serially diluted and cultured on sabouraud’s agar medium with chloramphenicol at 37°C for 28 h to determine CFU. All the experimental protocols to use animals were approved by the Animal Care and Use Committee of Kunming Institute of Zoology, Chinese Academy of Sciences.

15



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Histological check. Following vaginal lavage, the mice were sacrificed immediately by cervical dislocation, vaginal canals were excised and fixed in 10% buffered formalin solution and embedded in paraffin by routine procedures. The slides were cut at approximately 0.5 µm and stained routinely with haematoxylin and eosin (HE). Images were obtained using a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan).

Cytokine measurement. Vaginal canals of mice were inoculated with live C. albicans as described above. The vaginal canals were excised on day 10 during treatment, and then homogenized in 0.9% NaCl with a glass homogenizer. The supernatant was collected after the centrifugation at 2,000 g for 15 min and then was used for cytokine measurement. The concentration of TNF-α, IL-6, IL-1β and IL-10 was measured by using enzyme-linked immunosorbent assay kits (Dakewe, Beijing, China).

Statistical analysis. Data were assessed for statistical significance using Student’s (unpaired) t-test. Results were reported as mean ± SD or SE with significance accepted at P < 0.05.

16


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■ AUTHOR INFORMATION Corresponding Author *

For

R.L.,

phone,

+86-25-84396849;

fax,

+86-25-84396542;

E-mail,

[email protected]. For Q.L., phone, +86-871-65199086; fax, +86-871-65199086; E-mail, [email protected]. Author Contributions 1

These authors contributed equally to this paper. The manuscript was written through

contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS This work was supported by the Ministry of Science and Technology (2013CB911300), Chinese National Natural Science Foundation (31025025, 31260208, U1132601, 31200590), Chinese Academy of Sciences (SAJC201308, Y302B01021) and Yunnan Provincial Science and Technology Department (2011CI139, 2012BC009).

■ ABBREVIATIONS USED AMPs, antimicrobial peptides; TNF, tumor necrosis factor; IL, interleukin; RP-HPLC, reversed phase high performance liquid chromatography; MIC, minimal inhibitory concentration; CFU, colony-forming unit; DMEM, Dulbecco’s modified Eagle’s

17



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medium; MTT, 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide; DMSO, dimethyl sulfoxide; HE, haematoxylin and eosin; SEM, scanning electron microscopy; PBS, phosphate-buffered saline; RFU, relative fluorescence units.

■ ASSOCIATED CONTENT Supporting Information. Supplemental Table S1. Structural information of the peptides; Supplemental Figure S1. HPLC spectrum of the synthetic peptides; Supplemental Figure S2. The linear structure and the antimicrobial activities of peptide 4; Supplemental Figure S3. Peptide 4 showed low hemolytic activities and cell toxicity; Supplemental Figure S4. The stability of peptide 4 in human plasma.

18


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Cathelicidin-BF, a snake cathelicidin-derived antimicrobial peptide,

could be an excellent therapeutic agent for acne vulgaris. PLoS ONE 2011, 6, e22120. (12) Chen, W.; Yang, B.; Zhou, H.; Sun L.; Dou, J.; Qian, H.; Huang, W.; Mei, Y.; Han, J. (2011) Structure-activity relationships of a snake cathelicidin-related peptide, BF-15. Peptides 2011, 32, 2497-2503. (13) Kent, H. L. Epidemiology of vaginitis. Am. J. Obstet. Gynecol. 1991, 165, 1168-1175. (14) Cabezas, J.; Albaina, O.; Montañez, D.; Sevilla, M. J.; Moragues, M. D.; Pontón,

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susceptibility testing with SYTOX green nucleic acid stain. Appl. Environ. Microbiol. 1997, 63, 2421-2431. (34) Sochacki, K. A.; Barns, K. J.; Bucki, R.; Weisshaar, J.C. Real-time attack on single Escherichia coli cells by the human antimicrobial peptide LL-37. Proc. Natl. Acad. Sci. U.S. A. 2011, 108, E77-E81. (35) Fidel, P. L.; Cutright, J. L.; Tait, L.; Sobel, J. D. A murine model of Candida glabrata vaginitis. J. Infect. Dis. 1996, 173, 425-431.

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Figure legends

Figure 1. Designed peptides with antimicrobial activities take an amphipathic helix structure. cathelicidin-BF15, 1, 2, 3, and 4 are active, while 5 and 6 have no antimicrobial activity. The hydrophobic residues are yellow; positively charged hydrophilic residues are blue; negatively changed hydrophilic residues are red. The non-charged

polar

residues

are

purple.

BF-30,

cathelicidin-BF;

BF-15,

cathelicidin-BF15.

Figure 2. Effect of peptide 4 on the morphologies of C. albicans. Compared with control (A, C), 1 × MIC peptide 4 (B) or 2.5 × MIC peptide 4 (D) caused cell wall/membrane breakages. The arrows indicate the typical damage to the plasma membranes of C. albicans. Data are typical results of three separate experiments.

Figure 3. Peptide 4 induced increasing of the intensity of green fluorescence of C. albicans in SYTOX Green assay. 4, 1× MIC; clotrimazole, 20 µg/mL. RFU: relative fluorescence units. Data are mean values ± SE of five independent experiments.

Figure 4. Peptide 4 reduced colony counts of C. albicans in lavage liquid of mouse vagina. The results represent mean values ± SE of five independent experiments. *P < 0.05, **P < 0.01 vs. control.

Figure 5. Peptide 4 alleviated the microabscess in vaginal tissue of mouse vaginitis model. A) HE staining of the vaginal tissue sections of mouse. B) The microabscess numbers of per high power field were significant difference between peptide 4 (20 µg or 50 µg/mouse), clotrimazole (200 µg/mouse) and vehicle. **P < 0.01 vs. control. Average and standard error of the mean were derived from three independent experiments and 6 different fields each section.

24


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Figure 6. Peptide 4 inhibited inflammatory cytokine secretion in mouse vaginal tissues of mouse vaginitis model. 20 µg of peptide 4, 50 µg of peptide 4 or 200 µg of clotrimazole inhibited the secretion of TNF-α (A), IL-6 (B), IL-1β (C), and IL-10 (D). *P < 0.05 vs. control. Values are expressed as mean ± SE of three independent experiments.

25



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Page 26 of 39

Tables

Table 1．Physicochemical properties of the designed peptides. Peptide

CN

KFFRKLKKSVKKRAKEFFKKP-

cathelicidin-BF

RVIGVSIPFa VKRFKKFFRKLKKSVa

cathelicidin-BF15 FS-15

Sequence

1

VKRFKKFFRKFKKSVa a

L

NC

H

HM

PR/n%

NPR/n%

30

+11

0.233

0.427

16/53.33

14/46.67

15

+8

0.101

0.780

9/60.00

6/40.00

15

+8

0.107

0.786

9/60.00

6/40.00

WS-15

2

VKRWKKWWRKLKKSV

15

+8

0.193

0.850

9/60.00

6/40.00

ZY8

3

VKRWKKFWRKWKKWWa

15

+8

0.420

0.969

8/53.33

7/46.67

ZY13

4

VKRWKKWRWKWKKWVa

15

+8

0.382

0.606

8/53.33

7/46.67

16

+8

0.499

0.126

8/50.00

8/50.00

17

+8

0.602

0.078

8/47.06

9/52.94

a

ZY15

5

VKWRWKKWRWKWKWKV

ZY16

6

VKWRWKWKWRWKWKWKVa

a

C-terminal amidation (-NH2). Lys (K) and Arg (R) were assigned with +1 charge.

Asp (D) and Glu (E) were assigned with -1 charge. CN: compound number; L: length; NC: Net charge; H: Hydrophobicity; HM: Hydrophobic moment; PR: Polar residues; NPR: Nonpolar residues.

26


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Table 2. Antimicrobial activity comparison of the designed peptides. MIC（（µg/mL））

Peptide E. coli

S. aureus

B. subtilis

C. albicans

cathelicidin-BF

2.34

>100

9.38

4.70

cathelicidin-BF15

18.75

>100

75.0

18.75

1

37.50

>100

>100

9.38

2

18.75

>100

>100

9.38

3

18.75

4.69

4.69

2.34

4

9.38

1.17

1.17

0.59

5

>100

>100

>100

>100

6

>100

>100

>100

>100

MIC: minimal inhibitory concentration. These concentrations represent mean values of three independent experiments performed in duplicates.

27



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Table 3．Hemolytic activity of designed peptides on human erythrocytes. Peptide

Hemolytic activity (µ µg/mL) ± SD

Percent hemolysis ± SD

HC10

HC50

Hmax (100%)

cathelicidin-BF

>320

>320

7.4 ± 2.4

cathelicidin-BF15

>320

>320

9.7 ± 2.1

1

>320

>320

7.5 ± 2.7

2

>320

>320

8.7 ± 1.9

3

51.7 ± 5.3

>320

27.6 ± 5.7

4

>320

>320

3.1 ± 1.2

5

178.3 ± 11.5

>320

17.6 ± 3.5

6

62.8 ± 5.8

>320

30.8 ± 4.6

HC10 and HC50 were the concentrations of peptide causing 10% and 50% hemolysis on human erythrocytes, respectively. Hmax was the percentage (%) hemolysis at the highest peptide concentration tested (320 µg/mL).

28


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Table 4. The MIC (µg/mL) of peptide 4 compared with ampicillin and fluconazole against several strains of microorganism. Microorganism strains

4

Ampicillin

Fluconazole

B. subtilis

1.17

0.02

-

S. aureus ATCC2592

1.17

0.15

-

E. coli ATCC25922

9.38

18.7

-

C. albicans ATCC2002

0.59

-

18.7

C. albicans 08032815*

2.79

-

ND


4.69

-

ND


4.69

-

ND


3.13

-

ND


3.13

-

ND


1.40

-

ND

*clinical antibiotic-resistance strain; ND: not detectable; -: not determined. Values are expressed as mean of three independent experiments.

29



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Table 5. Bacteria killing kinetics of peptide 4 against C. albicans 08030809. NaCl Samples

Clotrimazole 1 × MIC

5 × MIC

10 × MIC

(0.15 M)

(10 mg/mL)

0 min

50

50

50

50

50

1min

62 ± 7

18 ± 4

10 ± 3

6±2

42 ± 6

10 min

102 ± 11

15 ± 3

9±3

4±1

64 ± 8

30 min

156 ± 16

0

0

0

106 ± 10

1h

228 ± 23

0

0

0

178 ± 19

3h

3687 ± 543

0

0

0

533 ± 68

6h

14536 ± 2467

0

0

0

2674 ± 424

12 h

54153 ± 8769

0

0

0

15789 ± 2786

24 h

>105

0

0

0

57425 ± 8543

Values are CFU counts and expressed as mean ± SD of three independent experiments.

30


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Table 6. The MIC (µg/mL) of peptide 4 in different solutions. Microorganism

ddH2O

150 mM NaCl

150 mM PBS

C. albicans 08032821

2.65

2.65

2.65


2.79

2.79

2.79


2.79

2.79

2.79


1.40

1.40

1.40

31



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TABLE OF CONTENTS GRAPHIC

32


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Figure 1. Designed peptides with antimicrobial activities take an amphipathic helix structure. cathelicidinBF15, 1, 2, 3, and 4 are active, while 5 and 6 have no antimicrobial activity. The hydrophobic residues are yellow; positively charged hydrophilic residues are blue; negatively changed hydrophilic residues are red. The non-charged polar residues are purple. BF-30, cathelicidin-BF; BF-15, cathelicidin-BF15. 257x130mm (150 x 150 DPI)



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

Figure 2. Effect of peptide 4 on the morphologies of C. albicans. Compared with control (A, C), 1 × MIC peptide 4 (B) or 2.5 × MIC peptide 4 (D) caused cell wall/membrane breakages. The arrows indicate the typical damage to the plasma membranes of C. albicans. Data are typical results of three separate experiments. 192x145mm (150 x 150 DPI)


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Figure 3. Peptide 4 induced increasing of the intensity of green fluorescence of C. albicans in SYTOX Green assay. 4, 1× MIC; clotrimazole, 20 µg/mL. RFU: relative fluorescence units. Data are mean values ± SE of five independent experiments. 133x73mm (300 x 300 DPI)



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

Figure 4. Peptide 4 reduced colony counts of C. albicans in lavage liquid of mouse vagina. The results represent mean values ± SE of five independent experiments. *P < 0.05, **P < 0.01 vs. control. 225x121mm (138 x 138 DPI)


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Figure 5. Peptide 4 alleviated the microabscess in vaginal tissue of mouse vaginitis model. A) HE staining of the vaginal tissue sections of mouse. B) The microabscess numbers of per high power field were significant difference between peptide 4 (20 µg or 50 µg/mouse), clotrimazole (200 µg/mouse) and vehicle. **P < 0.01 vs. control. Average and standard error of the mean were derived from three independent experiments and 6 different fields each section. 187x229mm (150 x 150 DPI)



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

Figure 6. Peptide 4 inhibited inflammatory cytokine secretion in mouse vaginal tissues of mouse vaginitis model. 20 µg of peptide 4, 50 µg of peptide 4 or 200 µg of clotrimazole inhibited the secretion of TNF-α (A), IL-6 (B), IL-1β (C), and IL-10 (D). *P < 0.05 vs. control. Values are expressed as mean ± SE of three independent experiments. 235x151mm (150 x 150 DPI)


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Table of Content Graphic 90x59mm (300 x 300 DPI)


Arginine-Rich ... - ACS Publications

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