De novo synthetic short antimicrobial peptides against cariogenic bacteria

Accepted Manuscript Title: De novo synthetic short antimicrobial peptides against cariogenic bacteria Authors: Yufei Wang, Yingying Fan, Zhengli Zhou, Huanxin Tu, Qian Ren, Xiuqing Wang, Longjiang Ding, Xuedong Zhou, Linglin Zhang PII: DOI: Reference:

S0003-9969(17)30096-1 http://dx.doi.org/doi:10.1016/j.archoralbio.2017.03.017 AOB 3836

To appear in:

Archives of Oral Biology

Received date: Revised date: Accepted date:

20-9-2016 17-2-2017 23-3-2017

Please cite this article as: Wang Yufei, Fan Yingying, Zhou Zhengli, Tu Huanxin, Ren Qian, Wang Xiuqing, Ding Longjiang, Zhou Xuedong, Zhang Linglin.De novo synthetic short antimicrobial peptides against cariogenic bacteria.Archives of Oral Biology http://dx.doi.org/10.1016/j.archoralbio.2017.03.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

De novo synthetic short antimicrobial peptides against cariogenic bacteria

Yufei Wanga,c, Yingying Fana,c, Zhengli Zhoub, Huanxin Tua, Qian Rena, Xiuqing Wanga, Longjiang Dinga, Xuedong Zhoua and Linglin Zhanga,*

a

State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China b The c

First People's Hospital of Yunnan Province, Kunming, China

Yufei Wang and Yingying Fan are co-first authors; they contributed equally to the work.

* Corresponding author at State

Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, No.14, Section 3 of Renmin Road South, Chengdu, China; Tel: 028-85503470, Fax: 028-85501436;

E-mail address: [email protected]

Highlights:



A series of novel antimicrobial peptides, GH8, GH12, and GH16 are designed.



GH12 owns the most balanced structural parameters, and a high content of αhelix.



GH12 has negligible cytotoxicity, good antimicrobial and antibiofilm activity.



GH12 causes cell lysis and pore formation on cytomembranes.

1

Abstract Objective: Antimicrobial peptides (AMPs) have shown the ability to inhibit planktonic bacteria and biofilms. The objectives of this study were to de novo design and synthesize a series of cationic, amphipathic α-helical AMPs that would be shorter, less cytotoxic, and more potent than existing AMPs against cariogenic bacteria. Design: Three short AMPs (GH8, GLLWHLLH-NH2; GH12, GLLWHLLHHLLH-NH2; and GH16, GLLWHLLHHLLHLLHH-NH2) were designed, synthesized and characterized structurally. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against eight major cariogenic bacteria were tested to select the most promising peptide. Scanning electron microscopy (SEM) was used to observe the bacterial membrane after treatment with selected peptides. The bactericidal kinetics, effects on biofilm and cytotoxity were further investigated. Results: Of the three AMPs, GH12 had the most balanced structural parameters and a high content of α-helical structure. GH12 had a MIC of 4.0-8.0 μg/mL and MBC of 8.0-32.0 μg/mL. The corresponding values for the other two AMPs were 2- to 64- fold higher. In time-kill assays, GH12 killed all bacterial strains within 60 min at 4- fold MBC. SEM observed lysis and pore formation of the cytomembrane after treatment with GH12. 8.0 μg/mL GH12 inhibited Streptococcus mutans biofilm formation. Confocal laser scanning microscopy showed that GH12 effectively reduced the biomass of 1-day-old S. mutans biofilm. Cytotoxicity assays indicated that GH12 showed little toxic effect on the viability of human gingival fibroblasts. Conclusion: These results indicate that GH12 shows antimicrobial activity against cariogenic bacteria and biofilms in vitro.

Keywords Antimicrobial peptide; Dental caries; Streptococcus mutans; Antimicrobial activity; Biofilms

1. Introduction Dental caries is the localized destruction of susceptible dental hard tissues resulting from exposure to acidic by-products produced by cariogenic bacteria, including Streptococci species, Lactobacilli species and Actinomyces species (Bowden, Ekstrand, McNaughton, & Challacombe, 1990; Gross et al., 2012; Selwitz, Ismail, & Pitts, 2007). Streptococcus sanguinis, Streptococcus gordonii and Actinomyces spp. are involved in early plaque development (Rozen, Bachrach, Bronshteyn, Gedalia, & Steinberg, 2001; Ruby, Li, Luo, & Caufield, 2002). Streptococcus mutans and Lactobacilli spp., capable of acidogenicity and aciduricity, then colonize and cause enamel demineralization (Marsh, 2006). Particularly, S. mutans, with strong ability to form biofilms, is still considered as a primary etiologic agent of dental caries (Gross et al., 2012). Effectively controlling these cariogenic bacteria and biofilm is key to caries prevention and treatment. Antimicrobial peptides (AMPs), especially cationic amphipathic α-helical peptides, have shown the ability to directly inhibit microbial growth, suppress biofilm formation and induce the dissolution of existing biofilms (Sullivan et al., 2011). So far, the effects of many natural AMPs against cariogenic bacteria have been studied, including α-Defensin, β-Defensin, Cathelicidin LL37, Histatin 5, Human Lactoferrin, Nisin and Pleurocidin (da Silva et al., 2012; Kreling et al., 2016; 2

Tao et al., 2011; Tong, Ni, & Ling, 2014). However, most natural AMPs show several disadvantages for clinical use: their large size which makes them difficult and costly to be prepared in purified form, high cytotoxicity, and poor pharmaceutical and pharmacokinetic properties (Fjell, Hiss, Hancock, & Schneider, 2012; Zaiou, 2007). To circumvent these problems, several groups have experimented with synthetic analogs or fragments of natural AMPs, some of which have shown promise against cariogenic bacteria (Kreling et al., 2016; Taniguchi et al., 2015; M. Zhang et al., 2016). But this screening process is random and inefficient. Moreover, there is an increasing argument that clinical use of AMPs with sequences that are too close to those of human AMPs would inevitably compromise own natural defenses, possibly posing threat to public health (Bell & Gouyon, 2003). In this view, de novo synthetic AMPs were developed to expand arsenals of AMPs. The classic de novo synthetic decapeptide KSL is shorter, but it shows low potency against cariogenic bacteria, with a minimal inhibitory concentration of 62.5 μg/mL against S. mutans and L. acidophilus (Liu et al., 2011). This poor efficacy may be related to the fact that KSL does not adopt the optimal structure of cationic, amphipathic α-helical AMPs. Thus, our strategy is to design and synthesize de novo a series of cationic, amphipathic α-helical AMPs that would be shorter, less cytotoxic, and more potent than existing AMPs against cariogenic bacteria. We designed the peptides using a combination of the “template-assisted”, “minimalist” and “sequence modification” approaches to AMP design (Zelezetsky & Tossi, 2006). We started from the sequence (XXYY)n based on the principles of -helix folding, where X refers to a hydrophobic residue, Y to a hydrophilic residue, and n to the number of repeats (Wiradharma et al., 2011). Following the minimalist approach, we limited the residues to the alkaline residue histidine (His, H) and the hydrophobic residue leucine (Leu, L) to create an amphipathic α-helix, and the size to 8- 16 residues to minimize helix length. At the same time, we included a glycine (Gly, G) in the first position, where it appears to act as a good N-capping residue for the α-helix in natural AMPs (Tossi, Tarantino, & Romeo, 1997). We also included tryptophan (Trp, W), since it has substantial affinity for the interface between lipid bilayer and aqueous medium (Braun & von Heijne, 1999), and helps anchor peptides to the lipid bilayer surface (Schiffer, Chang, & Stevens, 1992). Studies suggest that it should be in the fourth position which is the amphipathic interface between the hydrophilic and hydrophobic faces of the helix (Won et al., 2006). Therefore we tested sequences of the form GLLW+ (HLLH)1-3. All peptides were C-terminally amidated to add the terminal positive charge and increase antimicrobial activity, given that many natural AMPs are amidated in vivo (Patrzykat, Gallant, Seo, Pytyck, & Douglas, 2003). In this study, we integrated the sequence modification, minimalist and template-assisted approaches to design and synthesize three cationic, amphipathic α-helical AMPs named GH8, GH12 and GH16. We characterized them structurally in terms of structural parameters, helical wheel diagrams and circular dichroism spectroscopy, and we investigated their antimicrobial activity against eight major cariogenic bacteria. The most promising AMP (GH12) was further examined by measuring bactericidal kinetics, effects on biofilms and cytotoxicity on human gingival fibroblasts (HGFs). We also carried out preliminary mechanistic studies to explain the observed antimicrobial effects of GH12.

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2. Materials and methods 2.1. Peptide synthesis, purification and preparation The following peptides were designed: GH8, GLLWHLLH-NH2; GH12, GLLWHLLHHLLHNH2; and GH16, GLLWHLLHHLLHLLHH-NH2. Peptides were synthesized by GL Biochem (Shanghai, China) using standard Fmoc-solid phase peptide chemistry. Peptide sequence and integrity were confirmed by electrospray ionization mass spectrometry (ESI-MS; Shimadzu, Kyoto, Japan) and reverse-phase high-performance liquid chromatography (RP-HPLC; CHTH Sci and Tech, Beijing, China). Peptide purity was determined to be >98.0% using RP-HPLC. Purified peptides were dissolved in sterile deionized water (5120.0 μg/mL) and stored at −20 °C. 2.2. Sequence analysis Propensity for amphipathic helix formation was evaluated based on helical wheel diagrams and three major structural parameters of amphipathic helix (Giangaspero, Sandri, & Tossi, 2001): the hydrophobic residue composition, the net positive charge, and the relative amphipathicity (μH/μHmax). Hydrophobicity values were based on the ‘consensus’ scale (Eisenberg, Weiss, & Terwilliger, 1984). The hydrophobic moment (μH) was determined by the Eisenberg equation (Eisenberg, Weiss, & Terwilliger, 1982): 𝑁

2

𝑁

2

𝜇𝐻 = √(∑ 𝐻𝑛 × 𝑠𝑖𝑛 𝛿𝑛) + (∑ 𝐻𝑛 × 𝑐𝑜𝑠 𝛿𝑛) 𝑛=1

𝑛=1

where Hn is the hydrophobicity index value of residue n, and δ = 100° for peptides in α-helical conformation. To facilitate comparisons, the amphipathicity of peptides was expressed relative to the maximum possible value (μHmax) resulting from a perfectly amphipathic 18-residue peptide, composed only of isoleucine and aspartic acid, which would thus be assigned an μH/μHmax value of 1 (Giangaspero et al., 2001). 2.3. Circular dichroism (CD) spectroscopy Peptides were dissolved to a final concentration of 200.0 μg/mL in 20 mM sodium phosphate buffer (PBS, pH 7.0) containing either 50% (v/v) trifluoroethanol (TFE) or 25 mM sodium dodecyl sulfate (SDS) micelles (Ahn et al., 2006). These solvents were designed to simulate a lipid membrane environment. The CD spectra of the peptide solutions were recorded at room temperature using a CD Spectrometer (J-1500, JASCO, Tokyo, Japan) and a quartz cell with 1.0-mm path length. Wavelength was scanned from 190 to 240 nm at 100 nm/min. An average of 10 scans were taken for each sample. CD spectra were expressed as the mean residue ellipticity [θM] (deg·M-1·m-1). Spectra were analyzed using CDPro (http://lamar.colostate.edu/~sreeram/CDPro/main.html). Fractions of different types of secondary structure were calculated using the programs CONTIN-LL based on the SDP48 reference set (Sreerama & Woody, 2000).

4

2.4. Bacterial strains and growth media Streptococcus mutans UA159, Streptococcus gordonii DL1, Streptococcus sanguinis ATCC10556, Lactobacillus acidophilus ATCC14931, Lactobacillus casei ATCC393, Lactobacillus fermentium ATCC9338, Actinomyces viscosus ATCC15987, and Actinomyces naeslundii ATCC12104 were obtained from the State Key Laboratory of Oral Diseases at Sichuan University (Chengdu, China). Strains were grown in brain-heart infusion broth (BHI; Oxoid, Basingstoke, Hampshire, UK) anaerobically (85% N2, 10% H2 and 5% CO2) at 37 °C (Tao et al., 2011). 2.5. Bacterial susceptibility assay Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of peptides were determined using a modified broth microdilution method (Joycharat et al., 2013). Two-fold serial dilutions of three peptides were prepared from 5120.0 to 5.0 μg/mL in 96-well Ubottom microtiter plates (20.0 μL of each well). Then peptide dilutions were mixed with BHI broth (80.0 μL) and bacterial culture (100.0 μL) containing 2.0 × 106 CFU/mL. Final peptide concentrations ranged from 0.5 to 512.0 μg/mL. The final bacterial concentration was approximately 1.0 × 106 CFU/mL. Positive controls were incubated with chlorhexidine (CHX; Sigma-Aldrich, Steinheim, Germany) instead of peptide, at concentrations from 0.5 to 512.0 μg/mL. Negative and blank controls were incubated, respectively, with sterile deionized water or only BHI broth. Incubation steps complied with the CLSI guideline (CLSI, 2012). Microtiter plates were incubated at 37 °C for 46-48 h under the anaerobic conditions mentioned above. OD600 was measured using a microplate spectrophotometer (Multiskan GO, Thermo Scientific, USA). MIC was recorded as the endpoint where no difference of OD600 could be detected with respect to the blank BHI broth (Song, Choi, Jin, Yoon, & Choi, 2012). For MBC assays, aliquots (100.0 μL) were taken from wells in the MIC assays described above that showed no visual bacterial growth, and spread on BHI agar. These cultures were incubated anaerobically at 37 °C for 48 h. MBC was defined as the lowest peptide concentration that completely prevented bacterial growth (Isogai, Isogai, Takahashi, Okumura, & Savage, 2009). MIC and MBC assays were performed three times for all strains. Results from these assays were used to identify the peptide showing the highest antimicrobial activity, which was then evaluated further in cell morphology observation, in time-kill assays, in biofilm assays and in cytotoxity assays. 2.6. Scanning electron microscopy (SEM) observation of bacterial membrane S. mutans, L. acidophilus and A. viscosus were selected as representative strains for analyzing the effects of the selected peptide on the planktonic bacterial membrane. Bacterial cultures in the mid-log phase of growth were treated with the peptide at a final concentration of 256.0 μg/mL and incubated anaerobically at 37 °C for 24 h. Cells were collected by centrifugation for 5 min at 4500 g, washed twice and resuspended in PBS. Bacterial suspension (5.0 μL) was deposited on clean, sterile glass slides and air-dried at 37 °C. Samples were fixed using 2.5% glutaraldehyde solution for 2 h, and dehydrated with increasing ethanol percentages (35%, 50%, 75%, 2×90%, and 2×100%) for 30 min in each solution (Weber, Delben, Bromage, & Duarte, 2014). Samples were dried to a critical point, gold sputter-coated and observed on a scanning electron microscope (Inspect F, FEI, 5

Eindhoven, The Netherlands) at 20.0 kV. 2.7. Time-kill assay The bactericidal kinetics of the selected peptide against S. mutans, L. acidophilus and A. viscosus were assessed using time-kill assay as described(F. Li, Weir, Fouad, & Xu, 2013; L. Li, Shi, Cheserek, Su, & Le, 2013). Briefly, the selected peptide was added to bacterial cultures at a final peptide concentration equal to 1- fold, 2- fold and 4- fold MBC followed by anaerobic incubation at 37 °C. Sterile deionized water was used as negative control. At 0, 1, 5, 10, 20, 30, 60, 120, 240 and 360 min, aliquots of suspension (10.0 μL) were withdrawn and serially diluted 10- fold. Aliquots of the dilutions (10.0 μL) were plated on BHI agar and incubated anaerobically at 37 °C for 48 h. Assays were performed in triplicate on three different days. Time-kill kinetic curves were constructed by plotting lg(CFU/mL) versus incubation time over 6 h. 2.8. Biofilm susceptibility assay The following two models were used: (1) Polystyrene tissue culture plate. The effects of the selected peptide on S. mutans biofilm formation were assessed using the modified microdilution method (Costa et al., 2014; Stepanovic, Vukovic, Dakic, Savic, & SvabicVlahovic, 2000). Briefly, two-fold serial dilutions of the peptide were prepared to final concentration ranging from 0.5 to 512.0 μg/mL. Culture of S. mutans was diluted with BHI broth supplemented with 1% sucrose (BHIS) to a final concentration of 1.0 × 106 CFU/mL. After anaerobic incubation (24 h, 37 °C), culture supernatants were decanted and planktonic cells were removed by washing with PBS. The biofilm was fixed with methanol for 15 min, then stained with 0.1% (w/v) crystal violet (Sigma) for 5 min and gently rinsed with water to remove excess dye. Subsequently, the dye bound to the cell was resolubilized with 160.0 μL of 33% (v/v) glacial acetic acid. Absorbance at 595 nm (A595) was measured using a microplate spectrophotometer (Multiskan GO) to quantify biofilm formation. Inhibition rate was calculated using the equation [1− (A595 of the test/A595 of non-treated control)] × 100%. MBIC50 was defined as the lowest peptide concentration that show 50% or more inhibition of biofilm formation.(Wei, Campagna, & Bobek, 2006) To examine the reduction effects of the selected peptide on 1-day-old biofilm, the biofilm of S. mutans cells were inoculated into a flat 48-well microtiter plate, and grown at 37 °C for 24 h. The culture supernatant was decanted and planktonic cells were removed by washing with PBS. BHIS containing 0.5-512.0 μg/mL peptides, prepared in another microtitre plate, was then transferred to the 1-day-old biofilm plate, and the plates were further incubated at 37 °C for 24 h. The cell growth was then assessed by measuring the absorbance at 595 nm, and the biofilm was fixed, stained and quantified as described above. MBRC50 was defined as the lowest peptide concentration that showed reduction of biofilm by 50% or more (Liu et al., 2011; Wei et al., 2006). (2) Hydroxyapatite discs To further examine the inhibition effects of selected peptide on S. mutans biofilm, the pattern of the biofilms formed on hydroxyapatite (HA) discs were observed by SEM. HA discs were 6

deposited in the wells of a flat 48-well microtiter plate. S. mutans (1.0 × 106 CFU/mL) in 0.5 mL of BHIS was inoculated to each well of the plate with or without the selected peptide at a concentration of MBIC50. After anaerobic incubation at 37 °C for 24 h, the HA discs were removed and rinsed once with PBS for SEM analysis. The fixation, ethanol dehydrations, desiccation, gold sputter coating and observation were conducted as described in 2.6. 2.9. Confocal laser scanning microscopy (CLSM) Sterile glass slides were transferred into 24-well microplate with 1.99 mL BHIS, and then 10 μL S. mutans culture was added to form biofilms. After anaerobic incubation at 37 °C for 24 h, slides were washed three times with PBS and were challenged in the peptide (at concentrations of MBRC50) for 24 h. PBS was used as control. After treatment, biofilms were stained using the LIVE/DEAD® BacLightTM Bacterial Viability Kit (L-7012, Molecular ProbesTM, Invitrogen, Carlsbad, CA, USA) (Zhou et al., 2013) following the manufacturer’s instruction (Invitrogen). The labeled biofilms were imaged with a confocal laser scanning microscope (DMIRE2, Leica, Wetzlar, Germany) equipped with a 60× oil immersion objective lens (Zheng et al., 2013). All 3-dimensional reconstructions of the biofilms were performed with Imaris 7.0.0 (Bitplane, Zürich, Switzerland), and the quantification of biomass was performed with COMSTAT (http://www.imageanalysis.dk) (K. Zhang et al., 2015). 2.10.

In vitro Cytotoxicity assay

Healthy gingival samples were harvested from gingiva overlying the third molar teeth of adult humans, after obtaining informed consent. Primary HGFs were isolated and grown in Dulbecco's modified Eagle's medium (DMEM; GibcoTM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 1% Penicillin–Streptomycin solution (Invitrogen). HGFs were cultured at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air. Passage 4 cultures were used for experimental studies. HGFs were seeded at the concentration of 0.5 × 105 cells per well in 96-well plates. Then cells were exposed for 5, 60 and 120 min to the selected peptide at concentrations from 4.0 to 128.0 μg/mL. Control cells were exposed to medium alone. After treatment, the supernatant was discarded, wells were washed with PBS, and fresh medium (200.0 μL) was added. Cells were cultured for 24 h and their viability was quantitated using the SunBio Am-Blue Cell Viability Assay Kit (SBO Bio, Shanghai, China) according to the manufacturer's instructions. Results were obtained by determining optical density at 570 nm on a microplate reader. 2.11.

Statistical analysis

Inter-group differences were assessed for significance using ANOVA, while intra-group differences were assessed using Tukey HSD tests. Statistical analyses were performed using SPSS 20.0 (IBM, Chicago, IL, USA) at a significance level of 0.05.

3. Results 3.1. Structural characterization of peptides Sequences and structural parameters of the three peptides are shown in Fig. 1A. The peptides 7

varied in content of hydrophobic residues from 56.25% for GH16 to 62.5% for GH8, in net charge from +2 for GH8 to +6 for GH16, and in relative amphipathicity from 0.29 for GH8 to 0.98 for GH16. For all three peptides, the helical wheel diagrams indicated the hydrophilic sector was clearly separate from the hydrophobic sector (Fig. 1B). GH8 did not show strong α-helical structure in solution (Fig. 2A), as its spectrum did not show typical double minima at 208 and 222 nm (Chen, Yang, & Martinez, 1972). In contrast, strong αhelical signals were observed with GH12 (Fig. 2B) and GH16 (Fig. 2C), and a high α-helical content was confirmed by CDPro analysis (Fig. 2D). The minimum originally at 222 nm in the CD spectrum of GH12 shifted slightly to longer wavelengths, which may indicate that GH12 simultaneously adopts α-helix and a few coils. CDPro quantitated the percent of coil structure as 7.70% in SDS and 12.0% in TFE. 3.2. Peptide antimicrobial activity against cariogenic bacteria MICs and MBCs of the three peptides were determined against cariogenic bacteria (Table 1). GH12 showed the most potent inhibiting and killing activity against all bacterial strains, with MICs of 4.0-8.0 μg/mL and MBCs of 8.0-32.0 μg/mL. The MICs and MBCs of GH8 were 16- to 32-times higher than those of GH12. GH16 showed antimicrobial activity only against Lactobacillus species, and even then MIC and MBC were higher than the values for GH12. Since GH12 showed the most desirable secondary structure and most potent antimicrobial activity of the three peptides, it was selected for further evaluation in cell morphology observation, time-kill assays, biofilm assays and cytotoxity assays. 3.3. Morphological observation of bacterial membrane The structural effects of GH12 on the bacterial membrane are presented in Fig. 3. Untreated bacteria displayed a smooth surface (Fig. 3A, C and E), with no apparent cell lysis or cellular debris. Treatment with 256.0 μg/mL GH12 caused substantial changes in the bacterial cell membrane: GH12-treated S. mutans (Fig. 3B), L. acidophilus (Fig. 3D) and A. viscosus (Fig. 3F) displayed rough, shrunken and collapsed cells and obvious membrane rupture, with several small membrane pores visible. 3.4. Time-kill assay Time-killing results for GH12 against three bacterial strains are plotted in Fig. 4. Higher GH12 concentrations led to more rapid reduction in viable bacteria. For S. mutans (Fig. 4A), at 1- fold MBC, bacterial number decreased by approximately 3 orders of magnitude within 60 min. At 2fold MBC, S. mutans were all killed within 60min. At 4- fold MBC, all S. mutans were killed in 20min. The kill efficacy of GH12 against L. acidophilus (Fig. 4B) was similar, due to the same MBC. However, A. viscosus required a GH12 concentration of 128.0 μg/mL to be killed in 60 min (Fig. 4C). 3.5. Biofilm inhibition As showed in Fig. 5A, GH12 exhibited a good inhibitory effect on biofilm formation of S. mutans, with MBIC50 values of 8.0 μg/mL. In addition, GH12 exhibited effect of reduction on 8

biomass of 1-day-old S. mutans biofilm in a dose-dependent manner, with MBRC50 values of 256.0 μg/mL (Fig. 5B). At low concentrations of 0.5 to 16.0 μg/mL, GH12 did not cause significant reduction of biomass (P> 0.05). But at the concentration of 32.0 μg/mL, it started to show significant effects of reduction (P< 0.05), with the reduction rate of 38%. These results indicated that GH12 had to kill S. mutans within the biofilm to eradicate the existing biofilm. SEM photographs of biofilm formed on HA discs are shown in Figure 5C- F. For non-treated controls, S. mutans biofilm was nearly uniform and thick (Fig. 5C- D). The formation of the biofilm on HA discs was greatly reduced with the presence of GH12 at the concentration of MBIC50 (Fig. 5E-F). 3.6.

CLSM

As shown in Fig.6A, live bacteria were stained green, and dead bacteria were stained red. Yellow color appeared when live and dead bacteria were very near. In the control group, S. mutans biofilm was compact and mostly green. However, in GH12 group, S. mutans biofilm was unconsolidated, the green area is diminished, and the red area is increased. The calculation by COMSTAT substantiated results of CLSM micrographs (Fig. 6B), signifying remarkable decrease in live bacteria and increase in dead bacteria. 3.7. Viability of GH12-treated HGFs GH12 at 128.0 μg/mL slightly inhibited HGFs proliferation relative to the control group after 5 min treatment (P< 0.05, Fig. 7), but no significant differences were observed at longer treatment times or other peptide concentrations (P> 0.05).

4. Discussion In this paper, we exploited existing design strategies to create a series of novel AMPs showing antimicrobial activity against cariogenic bacteria. After screening the series for the hydrophobiccationic balance and amphipathicity, the desired α-helical secondary structure and antimicrobial activity, we identified GH12 as the most promising AMP, and we showed that this peptide has negligible cytotoxicity on HGFs, good killing kinetics against cariogenic bacteria and good inhibitory activity on biofilms. Our design was initially inspired by the formula (XXYY)n proposed by Wiradharma et al (Wiradharma et al., 2011) for novel α-helical AMPs. They synthesized the α-helical AMPs (FFRR)n, (LLRR)n, and (LLKK)n, which showed different antibacterial efficacies. Leu and lysine occur often in synthetic AMPs (Béven, Castano, Dufourcq, Wieslander, & Wróblewski, 2003; Blondelle & Houghten, 1992). Since the alkaline amino acid His has not yet been used together with Leu in reported AMP sequences, we chose to start our AMP design from the sequence (LLHH)n. Expanding this formula to N-LLHHLLHHLLHH… suggested that we could work with repeats of (LHHL) n, (HHLL)n or (HLLH)n. Positive residues in helices are more likely to occur at the C-terminus (Cook, 1967; Ptitsyn, 1969), probably because they help neutralize the partial negative charge there due to the net helix dipole (Branden & Tooze, 1999). We reasoned that His near the C-terminus of our AMP helix might act as a “protective plug” and stabilize the entire α-helical structure. Therefore we focused on the repeats (LLHH)n and (HLLH)n for designing our AMPs. In order to balance the 9

desire for Gly at position 1 with the need for a consistent arrangement of hydrophilic and hydrophobic residues, we ruled out (LLHH)n and adjusted the model to GLLW+ (HLLH)n-1. We focused on repeat numbers (n) of 2-4 to give peptides ranging in length from 8 residues (GH8) to 16 residues (GH16). This was intended to ensure relatively straightforward and cost-effective synthesis and purification on a large scale. We changed the last 4 residues of GH16 from (-HLLH) to (-LLHH) to ensure well-defined and balanced hydrophilic and hydrophobic sectors. Finally, we amidated the C-terminus of all peptides. Our SEM studies showed clear evidence that GH12 caused cell lysis and pore formation on cytomembranes, suggesting that GH12 is likely to function via the currently accepted mechanism of antimicrobial activity of amphipathic α-helical AMPs (Brogden, 2005; Melo, Ferre, & Castanho, 2009). This mechanism involves two steps. First, the peptides accumulate at the membrane of bacteria via electrostatic interactions with the negatively charged groups on the cell surface. Second, the hydrophobic moieties of AMPs insert into lipid bilayers, to mediate direct membrane disruptions via the barrel-stave pore, toroidal pore, disordered toroidal pore and/or carpet mechanisms. This results in depolarization, leakage of essential metabolites and loss of specific membrane composition. Based on this mechanism of action, amphipathic α-helical peptides appear to require two features to be effective: net positive charge, which attracts them to the anionic microbial surface; and ability to assume an amphipathic structure, which favors insertion into microbial membranes. Most natural AMPs have a net positive charge ranging from +4 to +6, 40-60% content of hydrophobic residues and relative amphipathicity from 0.3 to 0.6 (Giangaspero et al., 2001). Based on these reference values, the experimentally determined structural parameters of synthetic AMPs can be used to predict and rationalize their antimicrobial potency. The structural parameters of GH12 fell within the optimal ranges (Fig. 1A), suggesting that it would show the highest antimicrobial activity among the three AMPs we designed. AMP secondary structure was characterized by CD spectroscopy, which showed that GH12 and GH16 adopted helical structures in membrane-mimetic solvents (25 mM SDS and 50% TFE). Percent helical composition >80% has been proposed to correlate with antimicrobial potency (Deslouches et al., 2004). Indeed, GH12, which was 88.9% α-helical in SDS and 83.9% in TFE, showed good antibacterial activity in susceptibility assays. Despite showing >95% α-helical content, GH16 showed potent antimicrobial activity against only Lactobacillus species, and even then it was not superior to GH12. Similarly, Wiradharma et al (Wiradharma et al., 2011) identified three-repeat AMPs [e.g. (LLKK)3] the most promising. In summary, these results suggest that potency depends not only on stronger inclination to form an α-helical conformation, but also on proper hydrophobiccationic balance as well as optimal amphipathicity. Time-kill assays confirmed that GH12 showed rapid bactericidal action against cariogenic bacteria. At 16.0 μg/mL, GH12 killed 99% of S. mutans and L. acidophilus within 5 min. At 32.0 μg/mL, GH12 killed 90% of S. mutans and L. acidophilus within 1 min. These results suggest that GH12 has the potential to be used in mouthwash or chewing gum, assuming that its stability and antibacterial activity in saliva can be confirmed. The resistance of A. viscosus to GH12 is consistent with its resistance to other antibacterial agents (Fang, Chen, Xu, Yang, & Hildebrand, 2006; Xiao 10

et al., 2008), and may reflect characteristics of its cellular structure. Future studies should be carried out to investigate how to optimize AMPs in order to render them more lethal to this strain. Biofilms are complex microbial communities. Bacteria in biofilms are encapsulated in an organic matrix of polysaccharides, proteins and DNA secreted by the cells. The extracellular matrix provides protection from desiccation, host defenses and provides enhanced resistance to antimicrobial agents (Fejerskov & Kidd, 2008; Scheie & Petersen, 2004; Selwitz et al., 2007). In the present study, GH12 inhibited the formation of S. mutans biofilm at low concentrations, whose MBIC50 was the same with its MIC. This inhibition of biofilm formation may largely due to inhibition of bacteria growth since the observed reduction in biofilm biomass accompanied with the reduction of planktonic cells. Furthermore, GH12 significantly reduced the biomass of 1-day-old S. mutans biofilm at concentrations over 32.0 μg/mL. Meanwhile, by using CLSM, it was observed that the structure of GH12-treated biofilm was incompact and disordered, and there was a remarkable increased number of .dead bacterial cells that emitted red fluorescence within the GH12treated S. mutans biofilm. These results demonstrate that GH12 at the concentration of MBRC50 could enter small channels in the biofilm, penetrate the thick extracellular matrix, kill bacteria, and destroy the structure of S. mutans biofilm. However, the limitations of such in vitro assays should be taken into consideration. It is noteworthy that human dental plaque is a highly diverse resident community of microorganisms, including about 500 species of oral bacteria (Paster et al., 2001; Rasiah, Wong, Anderson, & Sissons, 2005; Wong & Sissons, 2001). Moreover, in an analysis of the microflora associated with dental caries, around 100 species of bacteria were found inhabiting dentin lesions (Munson, Banerjee, Watson, & Wade, 2004). Meanwhile, dental plaque is formed in various fluctuating oral environments, and hydrodynamic shear forces in oral environment keep changing the strength of biofilm (Paramonova, Kalmykowa, van der Mei, Busscher, & Sharma, 2009). Therefore, some researchers suggest studying caries using plaque biofilms containing multiple species of appropriate bacteria (Shu, Wong, Miller, & Sissons, 2000), and in a controlled environment that mimics the in vivo oral niches and habitats of the human mouth (Tang, Yip, Cutress, & Samaranayake, 2003). Eight common cariogenic planktonic bacteria and one monospecies static biofilm had been tested in this study. It still can not be compared to the natural biofilm causing caries. Future studies should examine the effects of GH12 in a more sophisticated caries model with multiple-species biofilm and a biomimic environment. Besides, with an eye toward clinical applications for the treatment and prevention of dental caries, the effects of GH12 on caries in vivo and molecular mechanism of action of GH12 also should be futher clarified. In conclusion, GH12 was the most promising of the three synthetic AMPs in our study. It showed proper hydrophobic-cationic balance and amphipathicity, strong capacity to form an α-helix, low cytotoxicity, excellent bactericidal efficiency, and good antibiofilm activity in vitro.

Acknowledgments The authors declare no potential conflicts of interest with respect to the authorship and/or 11

publication of this article. This work was supported by the National Natural Science Foundation of China [No. 81271128, No. 81470734]. There are no human or animal studies in this work. Cytotoxity assay in this work was approved by the medical ehics committee of West China Hospital of Stomatology, Sichuan University [NO.WCHSIRBST2015030]. The authors are thankful to Arne Heydorn (Technical University of Denmark) during biofilm image analysis using COMSTAT.

Ethical Approval

There are no human or animal studies in this work. Cytotoxity assay in this work was approved by the medical ehics committee of West China Hospital of Stomatology, Sichuan University [NO.WCHSIRBST2015030] Declarations

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Please state any conflict of interests. A conflict of interest exists when an author or the author's institution has financial or personal relationships with other people or organisations that inappropriately influence (bias) his or her actions. Financial relationships are easily identifiable, but conflicts can also occur because of personal relationships, academic competition, or intellectual passion. A conflict can be actual or potential, and full disclosure to The Editor is the safest course.

There are no human or animal studies in this work. Cytotoxity assay in this work was approved by the medical ehics committee of West China Hospital of Stomatology, Sichuan University [NO.WCHSIRBST2015030]

sources of funding

This work was supported by the National Natural Science Foundation of China [No. 81271128, No. 81470734]

12

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Fig. 1 Sequence, structural parameters (A) and helical diagrams (B) of GH8, GH12 and GH16. Hydrophobic residue composition (% hydrophobic residues), net positive charge, and relative amphipaticity (μH relative) were plotted for each peptide. Grey circles refer to hydrophilic residues, and white circles refer to hydrophobic residues.

Fig. 2 Circular dichroism of designed peptides in different environments. The spectra for GH8 (A), GH12 (B), and GH16 (C) were measured in the presence of 25 mM SDS or 50% TFE. The α-helical content (D) of the three peptides in different solvents was estimated by CDpro.

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Fig. 3 Scanning electron microscopy observation of bacterial membrane of S. mutans (A and B), L. acidophilus (C and D) and A. viscosus (E and F) after treatment with GH12. Images A, C and E are non-treated controls. White arrows indicate pores in the cytomembrane.

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Fig. 4 Time-kill curves for GH12 against S. mutans (A), L. acidophilus (B), and A. viscosus (C). Results are expressed as lg(CFU/mL). Data are presented as mean ±standard deviation from at least three independent experiments.

Fig. 5 Effects of GH12 on S. mutans biofilm and Scanning electron microscopy of S. mutans biofilm. The effects of GH12 on inhibition of biofilm formation (A) and reduction of 1-day-old biofilm (B) are expressed as the absorbance at 595 nm (A595). Data are presented as mean ±standard deviation from at least three independent experiments. *P < 0.05. Images E and F show the reduction of biofilm formation with the presence of GH12, compared with non-treated control (C and D).

Fig. 6 The antibacterial effect of GH12 on S. mutans biofilms. (A) The 3-dimensional reconstruction of S. mutans biofilms (live bacteria, stained green; dead cells, stained red); (B) The biomass of live and dead bacteria, calculated according to 5 random sights of S. mutans biofilms by COMSTAT. Data are presented as mean ±standard deviation. *P < 0.05.

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Fig. 7 Effects of GH12 on viability of human gingival fibroblast cells. Results are expressed as the optical density (OD) at 570 nm. Data are presented as mean ±standard deviation from at least three independent experiments. *P< 0.05.

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Table 1. In vitro Susceptibility of Oral Cariogenic Bacteria to GH8, GH12, GH16 AMPs MIC(μg/mL) Strain

MBC(μg/mL)

Source GH8

GH12

GH16

CHX

GH8

GH12

GH16

CHX

Streptococcus mutans

UA159

256

8

>512

1

256

8

>512

4

Streptococcus gordonii

DL1

>512

8

128

4

>512

16

>512

8

Streptococcus sanguinis

ATCC10556

>512

8

128

2

>512

16

>512

8

Lactobacillus acidophilus

ATCC14931

128

4

8

2

256

8

8

4

Lactobacillus casei

ATCC393

64

4

8

2

512

16

64

8

Lactobacillus fermentium

ATCC9338

128

4

32

2

>512

8

128

4

Actinomyces viscosus

ATCC15987

32

8

>512

2

64

32

>512

4

Actinomyces naeslundii

ATCC12104

128

4

>512

2

>512

32

>512

8

22