Regulatory Peptides 194–195 (2014) 63–68
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Antimicrobial and immunomodulatory properties of PGLa-AM1, CPF-AM1, and magainin-AM1: Potent activity against oral pathogens Denise T.F. McLean a, Maelíosa T.C. McCrudden a, Gerard J. Linden b, Christopher R. Irwin c, J. Michael Conlon d, Fionnuala T. Lundy a,⁎ a
Centre for Infection & Immunity, School of Medicine, Dentistry and Biomedical Sciences, Queen's University, Belfast, Northern Ireland, UK Centre for Public Health, School of Medicine, Dentistry and Biomedical Sciences, Queen's University, Belfast, Northern Ireland, UK Centre for Dentistry, School of Medicine, Dentistry and Biomedical Sciences, Queen's University, Belfast, Northern Ireland, UK d Department of Biochemistry, College of Medicine and Health Sciences, United Arab Emirates University, 17666 Al-Ain, United Arab Emirates b c
a r t i c l e
i n f o
Article history: Received 7 March 2014 Received in revised form 29 October 2014 Accepted 5 November 2014 Available online 13 November 2014 Keywords: Host defence peptide Antimicrobial peptide Cytokine Lipopolysaccharide
a b s t r a c t Cationic amphipathic α-helical peptides are intensively studied classes of host defence peptides (HDPs). Three peptides, peptide glycine–leucine–amide (PGLa-AM1), caerulein-precursor fragment (CPF-AM1) and magaininAM1, originally isolated from norepinephrine-stimulated skin secretions of the African volcano frog Xenopus amieti (Pipidae), were studied for their antimicrobial and immunomodulatory activities against oral and respiratory pathogens. Minimal effective concentrations (MECs), determined by radial diffusion assay, were generally lower than minimal inhibitory concentrations (MICs) determined by microbroth dilution. PGLa-AM1 and CPFAM1 were particularly active against Streptococcus mutans and all three peptides were effective against Fusobacterium nucleatum, whereas Enterococcus faecalis and Candida albicans proved to be relatively resistant micro-organisms. A type strain of Pseudomonas aeruginosa was shown to be more susceptible than the clinical isolate studied. PGLa-AM1 displayed the greatest propensity to bind lipopolysaccharide (LPS) from Escherichia coli, P. aeruginosa and Porphyromonas gingivalis. All three peptides showed less binding to P. gingivalis LPS than to LPS from the other species studied. Oral fibroblast viability was unaffected by 50 μM peptide treatments. Production of the pro-inflammatory cytokine IL-8 by oral fibroblasts was significantly increased following treatment with 1 or 10 μM magainin-AM1 but not following treatment with PGLa-AM1 or CPF-AM1. In conclusion, as well as possessing potent antimicrobial actions, the X. amieti peptides bound to LPS from three human pathogens and had no effect on oral fibroblast viability. CPF-AM1 and PGLa-AM1 show promise as templates for the design of novel analogues for the treatment of oral and dental diseases associated with bacteria or fungi. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cationic amphipathic α-helical peptides represent one of the most widely studied classes of host defence peptides (HDPs). A large number of these peptides have been identified in and isolated from the skin secretions of certain species of Anura (frogs and toads), including frogs from the family Pipidae [reviewed in [1]]. To date, nine peptides have been identified in norepinephrine stimulated skin secretions of the African volcano frog Xenopus amieti (Pipidae) [2]. These peptides may be assigned to four peptide families on the basis of limited amino acid sequence similarity: peptide glycine–leucine–amide (PGLa-AM1), caeruleinprecursor fragment (CPF-AM1), xenopsin-precursor fragment (XPF) and magainin. PGLa-AM1, CPF-AM1, and magainin-AM1 are small cationic
⁎ Corresponding author at: Centre for Infection and Immunity, School of Medicine, Dentistry and Biomedical Sciences, Health Sciences Building, 97 Lisburn Road, Belfast BT9 7AE, UK. Tel.: +44 28 9097 5797; fax: +44 28 9097 2671. E-mail address: [email protected] (F.T. Lundy).
http://dx.doi.org/10.1016/j.regpep.2014.11.002 0167-0115/© 2014 Elsevier B.V. All rights reserved.
peptides with charges ranging between +2 and +5 at neutral pH. These peptides display a propensity to adopt an α-helical conformation on interactions with negatively charged polar head groups in bacterial membranes [2] and have antimicrobial activity against Escherichia coli ATCC25922 and Staphylococcus aureus ATCC25923 [2]. More recently PGLa-AM1 and CPF-AM1 were shown to display antimicrobial activities against the type strain of Acinetobacter baumannii [3]. In addition, the peptides were effective at inhibiting the growth of colistin-resistant clinical isolates of A. baumannii, as well as Acinetobacter nosocomialis [3]. Several frog skin peptides that were first identified as a result of their antimicrobial properties have subsequently been shown to possess complex cytokine-mediated immunomodulatory activities. Effects on the production of both pro-inflammatory and anti-inflammatory cytokines have been observed [reviewed in [4]]. The aim of the current study was to determine the antimicrobial activity and immunomodulatory abilities of PGLa-AM1, CPF-AM1 and magainin-AM1 against a panel of oral and respiratory pathogens. Antimicrobial activity was determined by both radial diffusion assay and microbroth dilution assay. Immunomodulatory activity was investigated by assessing the ability of
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each peptide to bind lipopolysaccharide (LPS) and determining the direct effects of the peptides on interleukin-8 (IL-8) production by primary oral fibroblasts in vitro. 2. Materials & methods 2.1. Peptide synthesis PGLa-AM1 (GMASKAGSVLGKVAKVALKAAL.NH2; molecular mass 2068.3), CPF-AM-1 (GLGSVLGKALKIG ANLL.NH2; molecular mass 1622.0), and magainin-AM1 (GIKEFAHSLGKFGKAFVGGILNQ; molecular mass 2417.3) were supplied in crude form by GL Biochem Ltd. (Shanghai, China). The peptides were purified by reversed-phase HPLC on a (2.2 cm × 25 cm) Vydac 218TP1022 (C-18; Grace, Deerfield, IL, USA) column equilibrated with acetonitrile/water/ trifluoroacetic acid (28.0/71.9/0.1, v/v/v) at a flow rate of 6.0 ml/min. The concentration of acetonitrile was raised to 56% (v/v) over 60 min using a linear gradient. Absorbance was measured at 214 and 280 nm, and the major peak in the chromatogram was collected manually. Identities of the peptides were confirmed by electrospray mass spectrometry, and the purity of all peptides tested was N98%. 2.2. Organisms and growth conditions Lactobacillus acidophilus NCTC 1723, Streptococcus milleri NCTC 11065, Enterococcus faecalis NCTC 12697, Streptococcus mutans ATCC 10449, Pseudomonas aeruginosa ATCC 27853 and Pseudomonas aeruginosa (PAO 239; Midlands UK), were maintained on blood agar plates and overnight cultures were prepared via subculture into 25 ml volumes of sterile Mueller Hinton broth (Oxoid) and subsequent incubation overnight at 37 °C. Candida albicans NCTC 3179 was maintained on Sabouraud plates, subcultured into 25 ml volume of sterile Sabouraud broth (Oxoid) and incubated overnight at 37 °C. Fusobacterium nucleatum NCTC10562 was grown on fastidious anaerobic agar plates (FAA; Southern Group Laboratories) under strict anaerobic conditions in a Whitley A35 anaerobic workstation (Don Whitley Scientific Ltd., Shipley, UK) at 37 °C and overnight cultures were prepared in 25 ml volumes of anaerobic basal broth (Oxoid) supplemented with 1% (v/v) menadione and 0.003% (v/v) haemin.
were then incubated under aerobic conditions for 3 h at 37 °C to allow peptide diffusion into the gel. A 1% (w/v) agar overlay gel (10 ml) containing medium specific for the organism being tested was then poured over the base to provide nutrients for microbial growth. The plates were incubated for 18 h at 37 °C and then stained with a dilute solution of Coomassie brilliant blue R-250 as previously described [5]. Zones of inhibition were measured using a jeweller's eye-piece (×8 magnification). The diameters of the zones of clearing were expressed in units (0.1 mm = 1 U) and were calculated after subtracting the diameter of the negative control well. The antimicrobial activities were expressed as minimal effective concentration values (MECs), determined as the x intercept obtained from the relationship between radial diffusion units versus log10 peptide concentration. The mean MECs were calculated from three replicate experiments (Supplementary materials).
2.4. Microbroth dilution assay Antimicrobial activities of PGLa-AM1, CPF-AM1 and magainin-AM1 (1.56–to 100 μM) against the oral and respiratory pathogens were also determined by microbroth dilution assay using the Clinical Laboratory and Standards Institute (CLSI) double dilution method [7] with polypropylene plates to limit peptide binding [8]. Organisms were grown aerobically in Mueller Hinton broth with the exception of F. nucelatum which was grown in a Whitley A35 anaerobic workstation (Don Whitley Scientific Ltd., Shipley, UK) at 37 °C in anaerobic basal broth (Oxoid) supplemented with 1% (v/v) menadione and 0.003% (v/v) haemin.
2.5. Biotinylation of lipopolysaccharide (LPS) LPS from Pseudomomas aeruginosa (Sigma-Aldrich, UK), Porphyromonas gingivalis (Source Biosciences, UK) and E. coli (Sigma-Aldrich) was biotinylated with biotin (long arm) hydrazide (Vector Laboratories, UK) using the methodology outlined by the manufacturer. The number of biotins incorporated per molecule of LPS was determined using the Quant*Tag Biotin Quantification Kit (Vector Laboratories, UK), as described by the manufacturer.
2.3. Radial diffusion assay 2.6. LPS binding assay A double layer radial diffusion assay [5,6] was performed to determine the antimicrobial activities of PGLa-AM1, CPF-AM1 and magaininAM1 against the oral and respiratory pathogens. This assay has previously been recommended [5,6] over microbroth dilution assays for the early stages of antimicrobial peptide discovery work since it uses much smaller quantities of synthetic peptide. Briefly, overnight cultures of aerobic organisms were grown in 25 ml of appropriate media overnight at 37 °C. F. nucleatum was grown in appropriate media overnight in a Whitley A35 anaerobic workstation under strict anaerobic conditions. Mid-logarithmic phase cultures were obtained by inoculating a 25 ml volume of the appropriate sterile broth with 100 μl of the requisite overnight culture and incubating for a further 3 h at 37 °C. Bacterial cultures were centrifuged at 1800 ×g for 10 min at 4 °C, washed three times with the same volume of 10 mM sodium phosphate buffer (pH 7.4) and resuspended in 10 ml of the same buffer. An underlay gel (10 ml) was prepared consisting of 1% (w/v) agarose containing approximately 4 × 105 yeast cells or 5 × 106 bacterial cells. The microorganisms were dispersed by vortexing; the underlay gel was then poured into a square Petri dish and allowed to solidify. A series of wells (2.5 mm in diameter) were punched in the agar and peptide solution (3 μl) was added to each well at concentrations ranging from 12 to 75 μM for PGLa-AM1 and CPFAM1 and 25–to 100 μM for magainin-AM1. A negative control well containing sterile water and a positive control well containing 100 μg ml−1 synthetic Cecropin A (Sigma, UK) were included on each plate. The plates
Binding of PGLa-AM1, CPF-AM1 and magainin-AM1 to biotinylated LPS was studied using a method adapted from [9], as described previously [10]. Briefly, the wells of a Greiner high binding plate (Sigma Aldrich, Dorset, UK) were coated with 100 μl/well of the relevant peptide (6.25 μM), prepared in Voller's buffer (26 mM Na2CO3, 23 mM, NaHCO3, pH 9.6) and incubated overnight at 37 °C in an unsealed plate. The following day, plates were washed three times with an excess of phosphate buffered saline (PBS) containing 0.05% (v/v) Tween-20 (PBST) and the wells of the plate (200 μl/well) were then blocked with PBST containing 1% (w/v) bovine serum albumin (BSA) for 1 h at room temperature. Plates were washed three times with PBST and 100 ng of biotinylated LPS (E. coli, P. aeruginosa or P. gingivalis) prepared in PBS was added to each well (100 μl/well). Plates were incubated at room temperature for 3 h and then washed three times with an excess of PBST. Horseradish peroxidase-conjugated streptavidin (Biolegend, UK) (diluted 1:2000 in PBST) was added to each well and incubated for 30 min at room temperature (100 μl/well). The wells were washed three times with PBST and peroxidase activity was detected following addition of the chromogenic substrate, 2,2-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]diammonium salt (ABTS) (Invitrogen, Paisley, UK) (100 μl/well). The absorbance measurements were determined at 405 nm on a Tecan Genios microtitre plate reader (Tecan, Reading, UK) using Magellan software.
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2.7. Haemolysis assay
3. Results
Peptides in the concentration range 6 to 200 μM were incubated with washed human erythrocytes from a healthy donor. After centrifugation (12,000 ×g for 15 s), the absorbance at 450 nm of the supernatant was measured. A parallel incubation in the presence of 1% v/v Tween-20 was carried out to determine the absorbance associated with 100% haemolysis. The LD50 value was taken as the mean concentration of peptide producing 50% haemolysis in three independent experiments.
3.1. Antimicrobial activity of PGLa-AM1, CPF-AM1 and magainin-AM1 against a range of oral and lung pathogens
2.8. Tissue culture Oral fibroblasts were derived by explant culture of dental pulp tissue removed from healthy teeth, with ethical approval, as previously described [11]. Oral fibroblasts were maintained in Dulbecco's Minimal Essential Medium (DMEM; Invitrogen) supplemented with 10% heat inactivated foetal bovine serum (FBS; Invitrogen), 2 mM L-glutamine (Invitrogen) and 1% (v/v) penicillin/streptomycin (PAA Laboratories GmbH, Austria). Cell lines were maintained at 37 °C in a humidified atmosphere of 5% CO2. Fibroblasts were grown to confluence before being harvested and subcultured. 2.9. MTT assay with oral fibroblast cells Oral fibroblasts were seeded into a 96-well culture plates at 1 × 105 cells/ml and maintained until they reached confluence. The medium was then replaced with DMEM without FBS overnight to induce quiescence. Peptide stock solutions in Muller Hinton broth were further diluted in DMEM to a final concentrations of 50 μM, with 100 μl per well added for cell treatments. Cells were then incubated at 37 °C for 24 h. Following this, 10 μl of thiazolyl blue tetrasodium bromide (MTT) reagent (Sigma Aldrich®, Dorset, UK) was added to each well. The plate was incubated at 37 °C for a further 2 h. Cell culture media were removed by aspiration and the wells of the plate were allowed to air dry in a laminar flow hood for 10 min. 200 μl dimethyl sulfoxide (DMSO) (Sigma Aldrich®, Dorset, UK) was added to each well and this was mixed by gentle agitation. The absorbance measurements were determined at 405 nm using a Tecan Genios microtitre plate reader (Tecan, Reading, UK) using Magellan software. Four replicates of each cell treatment were carried out. Vehicle controls were included in all experiments.
2.10. Measurement of interleukin-8 release Oral fibroblasts were seeded into a 24-well culture plate at a density of 1 × 105 cells/well until they achieved confluence. The cells were incubated in 1% (v/v) FBS for 24 h, and then PGLa-AM1, CPF-AM1 or magainin-AM-1 at concentrations of 1 or 10 μM, or medium (controls) were added. Cells were cultured for 21 h at 37 °C in a humidified atmosphere of 5% CO2 and the cell supernatants were collected and stored at −20 °C until required. IL-8 concentrations in the cell supernatants were measured by ELISA using the IL-8 DuoSet ELISA development kit (R & D Systems, Minneapolis, MN, USA). The absorbance was determined at 405 nm on a Tecan Genios microtitre plate reader (Tecan, Reading, UK) using Magellan software.
Variable MEC and MIC values were determined for PGLa-AM1, CPFAM1 and magainin-AM1 against a range of oral and lung pathogens (Table 1). MICs obtained by microbroth dilution assay were generally higher than MECs obtained by the radial diffusion assay. Our observations are consistent with those of Hancock [12] who reported that for a wide range of antimicrobial peptides, including magainin-2, MIC values measured by the broth dilution method are consistently much higher than the corresponding values measured by the radial diffusion assay method. PGLa-AM1 proved to be active against both Gram positive and Gram negative bacteria, with particularly good activity against S. mutans (MEC and MIC b 5 μM). CPF-AM1 was also active against both Gram positive and Gram negative bacteria and was also particularly effective against S. mutans (MEC and MIC b5 μM). Of the three frog peptides studied, magainin-AM1 had the least antimicrobial potency and was shown to be ineffective against the clinical isolate of P. aeruginosa (MEC and MIC N100 μM). The MIC values for PGLa-AM1 also showed that this peptide was less effective against a clinical isolate of P. aeruginosa compared with the type strain. Microbroth dilution assay results indicated that E. faecalis was resistant to all three peptides studied and also that MICs against C. albicans were relatively high for PGLa-AM1 (50 μM), CPF-AM1 (50 μM) and magainin-AM1 (100 μM). MEC values for the microaerophilic organisms S. milleri and L. acidophilus are presented using the radial diffusion assay as the microbroth dilution assay is reported to be suitable for aerobic organisms only [8]. However, modification of the CLSI protocol using supplemented anaerobic basal broth and anaerobic incubation allowed us to determine microbroth dilution MICs for PGLa-AM1 (12.5 μM), CPF-AM1 (6.25 μM) and magainin-AM1 (12.5 μM), showing the effectiveness of all three peptides against the oral anaerobe F. nucleatum. 3.2. LPS binding activities of PGLa-AM1, CPF-AM1 and magainin-AM1 PGLa-AM1, CPF-AM1 and magainin-AM1 bound with varying affinities to biotinylated E. coli LPS, with PGLa-AM1 displaying the greatest propensity to bind (Fig. 1A). PGLa-AM1, CPF-AM1 and magainin-AM1 bound to biotinylated P. aeruginosa LPS (Fig. 1B) with greater affinity than to E. coli LPS. PGLa-AM1 bound P. aeruginosa LPS significantly better than either CPF-AM1 or magainin-AM1. All three peptides showed less binding to P. gingivalis LPS (Fig. 1C) than to the other species studied. However, PGLa-AM1 showed a significantly higher level of binding to P. gingivalis LPS than either CPF-AM1 or magainin-AM1. 3.3. Haemolytic activity The LD50 (mean ± SEM) results for haemolytic activity against human erythrocytes were: CPF-AM1 = 155 ± 9 μM; PGLa-AM1 N 200 μM; and magainin-AM1 N 200 μM, suggesting that none of the peptides were toxic to human erythrocytes at any of the concentrations employed in our studies. 3.4. Cytotoxicity of PGLa-AM1, CPF-AM1 and magainin-AM1 to oral fibroblasts Oral fibroblast viability was unaffected by treatments with PGLaAM1, CPF-AM1 or magainin-AM1 at concentrations of 50 μM (Fig. 2).
2.11. Statistical analysis Statistical analysis was performed using commercially available software (Prism). All data were analysed by one way analysis of variance followed by Bonferroni's multiple comparison test. A p-value b 0.05, was considered statistically significant.
3.5. Cytokine production from oral fibroblast cells following treatment with PGLa-AM1, CPF-AM1 or magainin-AM1 Production of the pro-inflammatory cytokine IL-8 by unstimulated oral fibroblast cells was increased between 1 and 3 fold following
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Table 1 Minimal effective concentration (MEC) and minimal inhibitory concentration (MIC) values of PGLa-AM1, CPF-AM1 and magainin-AM1 against the aerobic oral microorganisms C. albicans, S. mutans, and E. faecalis; the microaerophilic oral microorganisms S. milleri and L. acidophilis; the anaerobic oral bacterium F. nucleatum and the lung pathogen P. aeruginosa using radial diffusion assay (mean of triplicate assays presented in (μM) +/− standard deviations in brackets) and microbroth dilution assay (duplicate assays). MEC (μM) determined by (A) radial diffusion assay and MIC (μM) determined by (B) microbroth dilution assay Microorganism
PGLa-AM1
CPF-AM1
Magainin-AM1
Candida albicans NCTC 3179
(A) 7.5 (+/−0.4) (B) 50 (A) 1.2 (+/−0.1) (B) 3.1 (A) 10.2 (+/−0.4) (B) N100 (A) 5.8 (+/−1.3) (B) ND (A) 6.7 (+/−0.4) (B) ND (A) 1.4 (+/−0.1) (B) 12.5 (A) 12.3 (+/−0.8) (B) 25 (A) 1.5 (+/−0.4) (B) 12.5
(A) 9.9 (+/−1.8) (B) 50 (A) 2.5 (+/−0.9) (B) 3.1 (A) 7.8 (+/−0.2) (B) N100 (A) 8.8 (+/−2.6) (B) ND (A) 2.5 (+/−0.5) (B) ND (A) 1.9 (+/−0.6) (B) 25 (A) 11.9 (+/−0.9) (B) 25 (A) 2.2 (+/−0.1) (B) 6.25
(A) (B) (A) (B) (A) (B) (A) (B) (A) (B) (A) (B) (A) (B) (A) (B)
Streptococcus mutans ATCC 10449 Enterococcus faecalis NCTC 12697 Streptococcus milleri NCTC 11065 Lactobacillus acidophilus NCTC 1723 Pseudomonas aeruginosa ATCC 27853 Pseudomonas aeruginosa PAO239 Fusobacterium nucleatum NCTC 10562a
24.3 (+/−4.4) 100 24 (+/−1.9) 50 22.9 (+/−0.1) N100 10.1 (+/−0.9) ND 27.2 (+/−1.3) ND 11.1 (+/−1.5) N100 N100 N100 9 (+/−3.8) 12.5
ND = not determined for microaerophilic microorganisms. a MECs and MICs determined under anaerobic conditions.
treatment with 1 or 10 μM PGLa-AM1, CPF-AM1 or magainin-AM1 (Fig. 3). Treatment with CPF-AM1 resulted in the lowest fold-increase in IL-8 at both 1 and 10 μM concentrations. Treatment of oral fibroblasts with 10 μM PGLa-AM1 or 10 μM magainin-AM1 increased production of IL-8 over that observed for 1 μM treatments. However, only the treatments with magainin-AM1 resulted in significant increases in IL-8 (both 1 μM and 10 μM treatments). 4. Discussion In the current study the amphipathic peptides PGLa-AM1, CPF-AM1 and magainin-AM1 were shown to have antimicrobial activity against a range of oral and respiratory pathogens. PGLa-AM1 and CPF-AM1 proved to be generally more effective, as measured by their MECs and MICs, than magainin-AM1. This may be due in part to the greater overall positive charge and hydrophobic ratio of PGLa-AM1 and CPF-AM1 compared with magainin-AM1. Both PGLa-AM1 and CPF-AM1 were equally effective against the Gram negative and Gram positive bacteria studied. Peptide cationicity contributes to the initial interaction of the peptide with the bacterial membrane [13] and the lower cationicity exhibited by magainin-AM1 may explain its weaker potency than PGLa-AM1 or CPF-AM1. Magainin-AM1 also has a lower cationicity than its magainin counterpart which could also explain its relatively weak potency compared with its X. laevis homolog [2]. Agar diffusion tests have found favour in the past for surface active antibiotics such as nisin or subtilin [14] and these assays have therefore also been employed in the testing of membrane targeting antimicrobial peptides. The generally higher MICs reported using the microbroth dilution assay versus the MECs reported using the radial diffusion assay may be related in part to differences in the methodologies, including less favourable growth conditions in the radial diffusion assay. It should be recognised that several factors including the antimicrobial activity of the test peptide, the bacterial growth rate and the density and metabolic activity of the inoculum may influence zone diameter [15]. Differential peptide diffusion rates also merit consideration in the radial diffusion assay and therefore comparison of results from peptide families with different physicochemical properties (for example hydrophobic versus amphipatic peptides) may be limited [16]. LPS is a major stimulus of inflammation, acting via Toll like receptors (TLRs) to enhance pro-inflammatory cytokine secretion. Thus, it was of interest to determine the ability of the peptides to bind LPS thereby limiting potential interactions of the LPS with TLRs thus negating their proinflammatory actions. In terms of ability to bind LPS, the cationicity of
the peptide is considered to play an important role. The higher net charge of PGLa-AM1 (+ 5) compared with CPF-AM1 (+ 3) or magainin-AM1 (+ 2) may contribute to significantly more effective binding of PGLA-AM1 to LPS from P. aeruginosa, E. coli and P. gingivalis. Studies on magainin from X. laevis have shown LPS binding at a 1:1 stoichiometry in solution [17,18]. However, magainin from X. laevis displays greater cationicity than magainin-AM1 from X. amieti. There have been no reports to date regarding the LPS binding abilities of either PGLa or CPF from X. laevis and no reports describing the ability of X. amieti homologs to bind to LPS from any bacterial species. PGLa-AM1 binding to LPS was shown to be strongest for P. aeruginosa followed by E. coli and P. gingivalis. Differences in binding activity between LPS from various bacteria may reflect differences in LPS composition [17]. Reduction in the overall anionic nature of the LPS may result in reduced potential electrostatic binding between the negatively charged LPS and cationic peptides. In P. aeruginosa, the lipid A moieties have been shown to display variable compositions representing different isoforms which have different potencies [19]. The lipid A moiety of E. coli LPS consists of a β-1,6-linked D-glucosamine (GlcN) disaccharide carrying two negatively charged phosphates and six saturated fatty acids in a defined asymmetric distribution, four at the GlcN II and two at the GlcN I, conferring an overall anionic charge of −6 [20]. Variation in E. coli lipid A moiety composition can reduce the number of acyl chains resulting in LPS with charges ranging from −4 to −2 in E. coli mutant strains [20,21]. The lipid A moiety of P. gingivalis comprises tetra-acylated, nonphosphorylated lipid A and tetra-acylated mono-phosphorylated lipid A. The non-phosphorylated lipid A moiety represents a significant proportion of the total lipid A species of P. gingivalis LPS. This LPS lipid A moiety composition results in a less anionic LPS thus reducing the potential for initial electrostatic interactions between the cationic HDPs and the anionic bacterial membrane and has been shown to be the important in P. gingivalis resistance to cationic, antimicrobial polymyxin B [22]. There was no evidence that any of the peptides investigated caused cytotoxicity, as determined by haemolytic assay with human erythrocytes and by MTT assay in primary oral fibroblasts at concentrations of 50 μM. It is known that antimicrobial peptides can modulate the innate immune response and in the context of the present study, only magainin-AM1 elicited a significant increase in IL-8 production by oral fibroblasts. IL-8 has an important role in LPS-driven inflammation, frequently seen in the oral cavity and respiratory tract. Although reduced IL-8 production may appear favourable in terms of limiting the inflammatory response, pro-inflammatory cytokines elicited by various
D.T.F. McLean et al. / Regulatory Peptides 194–195 (2014) 63–68
% Survival Relative to Control
ns
***
0.8 0.6 0.4
150 100 50
***
***
1.0 0.5
1 nAM
AM M
ag
ai ni
CP F-
PG
La -
AM
1
1
0.0
*** ns
***
0.4 0.3 0.2
in in -A M 1 ag a
0.1 *
400
1
IL-8 pg/ml
300 200 100 0
1
1 PF -
A
ai ni nA M
M
M A La -
ag M
peptides may paradoxically contribute to their protective role in host defence against infection [17] as long as the inflammatory response does not become dysregulated/chronic as a result of failure to eliminate the stimulus. Several naturally occurring frog skin peptides have been shown to increase production of pro-inflammatory cytokines [4]. For example, frenatin 2D from the Tyrrhenian painted frog Discoglossus
PG
C
on t
ro l
Fig. 1. PGLa-AM1, CPF-AM1 and magainin-AM1 binding to biotinylated (A) E. coli LPS, (B) P. aeruginosa LPS and (C) P. gingivalis LPS. Data were analysed by one way analysis of variance followed by Bonferroni's multiple comparison test (*** = p b 0.001; ns = non-significant).
1 M 10 M
*
in -A M in ag a M
C PF -A M 1
PG La -A M 1
0.0
C
Abs @ 405 nm
0.5
M
sardus potently stimulated production of TNF-α and IL-1β by mouse peritoneal macrophages and it was speculated that the peptide acts on macrophages in frog skin to produce a cytokine-mediated stimulation of the adaptive immune system in response to invasion by microorganisms [23]. A number of peptides have been exploited for therapeutic use such as Pexiganan, derived from magainin 2 from X. laevis, proposed as a topical antibiotic for use on diabetic foot ulcers [24]. The X. amieti peptides studied in the current work displayed potent antimicrobial activities against a wide range of oral and respiratory pathogens. As the oral cavity is easily accessible for the topical application of therapeutic agents, these peptides may prove to be templates for the development of novel antimicrobial agents for use in this site. PGLa-AM1 and CPFAM1 were particularly effective against the cariogenic pathogen S. mutans, whilst exhibiting no cytotoxicity against oral fibroblasts. Both peptides also displayed potent antimicrobial activity against F. nucleatum an important ‘bridging organism’ which facilitates coaggregation to incorporate late colonizers such as the Gram negative anaerobe P. gingivalis into the mature dental plaque biofilm. P. gingivalis has been identified as a keystone microorganism implicated in the development of periodontal disease [25]. The incorporation of novel peptides into formulations to be delivered directly to the periodontal pocket may provide an effective and targeted treatment for periodontal inflammation [26]. The synthetic peptide PAC-113, derived from histatins 3
1.5
(C)
C PF -A M 1
on t
A M 1 M ag ai ni n-
Fig. 2. Percentage cell survival of oral fibroblasts, relative to that of control, following a 24 hour exposure to 50 μM PGLA-AM1, CPF-AM1 or magainin-AM1. Cells were incubated in medium containing the vehicle only (Control) or vehicle and peptide (PGLA-AM1, CPFAM1 or magainin-AM1), (mean +/− SD, n = 4). Data were analysed by one way analysis of variance followed by Bonferroni's multiple comparison test, all comparisons were nonsignificant.
*** 2.0
50 µM Treatments
1
Abs @405 nm
(B)
C
PG
La -
PF -A
A
M
M
1
1
C
0.0
PG La -A M 1
0
0.2
ro l
Abs @ 405 nm
200
***
(A)
67
Fig. 3. Direct effects of PGLa-AM1, CPF-AM1 and magainin-AM1 on IL-8 production by oral fibroblasts. Control cells were treated with different volumes of vehicle corresponding to peptide dilution to working concentrations of 1 μM and 10 μM. Data were analysed by one way analysis of variance followed by Bonferroni's multiple comparison test (* = p b 0.05).
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and 5, has been incorporated into a mouthwash for the treatment of oral candidiasis [27]. CPF-AM1 and PGLa-AM1, which were shown to be very effective against S. mutans, could also be delivered in this way to the oral cavity. In conclusion, as well as possessing potent antimicrobial actions, the X. laevis peptides studied were shown to bind LPS from three human pathogens and to have no effect on the viability of oral fibroblasts. In general PGLa-AM1 had slightly greater potency than CPF-AM1. Magainin-AM1 displayed least antimicrobial and LPS-binding activity, and elicited higher levels of IL-8 production from oral fibroblasts. It is tempting to speculate that CPF-AM1 and PGLa-AM1 show promise as templates for the design of non-toxic, non-inflammatory analogues for the treatment of oral and dental diseases associated with bacteria or fungi. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgements This study was funded by the Department for Employment and Learning (Northern Ireland). We gratefully acknowledge the skilful technical assistance of Catherine Fulton. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.regpep.2014.11.002. References [1] Conlon JM. Structural diversity and species distribution of host-defense peptides in frog skin secretions. Cell Mol Life Sci 2011;68:2303–15. [2] Conlon JM, Al-Ghaferi N, Ahmed E, Meetani MA, Leprince J, Nielsen PF. Orthologs of magainin, PGLa, procaerulein-derived, and proxenopsin-derived peptides from skin secretions of the octoploid frog Xenopus amieti (Pipidae). Peptides 2010;31:989–94. [3] Conlon JM, Sonnevend A, Pál T, Vila-Farrés X. Efficacy of six frog skin-derived antimicrobial peptides against colistin-resistant strains of the Acinetobacter baumannii group. Int J Antimicrob Agents 2012;39:317–20. [4] Conlon JM, Mechkarska M, Lukic ML, Flatt PR. Potential therapeutic applications of multifunctional host-defense peptides from frog skin as anti-cancer, anti-viral, immunomodulatory, and anti-diabetic agents. Peptides 2014;57:67–77. [5] Lehrer RI, Rosenman M, Harwig SSSL, Jackson R, Eisenhauer P. Ultrasensitive assays for endogenous antimicrobial polypeptides. J Immunol Methods 1991;137:167–73. [6] Steinberg DA, Lehrer RI. Designer assays for antimicrobial peptides. Disputing the “one-size-fits-all” theory. Methods Mol Biol 1997;78:169–86.
[7] Laboratory Clinical, Institute Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, approved standard M07-A8. Wayne PA: CLSI; 2008. [8] Wiegand I, Hilpert K, Hancock REW. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc 2008;32:163–75. [9] Felgentreff K, Beisswenger C, Griese M, Gulder T, Bringmann G, Bals R. The antimicrobial peptide cathelicidin interacts with airway mucus. Peptides 2006;27:3100–6. [10] McLean DTF, Lundy FT, Timson DJ. IQ-motif peptides as novel anti-microbial agents. Biochimie 2013;95:875–80. [11] Killough SA, Lundy FT, Irwin CR. Dental pulp fibroblasts express neuropeptide Y Y1 receptor but not neuropeptide Y. Int Endod J 2010;43:835–42. [12] Hancock REW. Cationic peptides: effectors in innate immunity and novel antimicrobials. Lancet Infect Dis 2001;1:156–64. [13] Yin LM, Edwards MA, Li J, Yip CM, Deber CM. Roles of hydrophobicity and charge distribution of cationic antimicrobial peptides in peptide–membrane interactions. J Biol Chem 2012;287:7738–45. [14] Parisot J, Carey S, Breukink E, Chan WC, Narbad A, Bonev B. Molecular mechanism of target recognition by subtilin, a class 1 lanthionine antibiotic. Antimicrob Agents Chemother 2008;52:612–8. [15] Turnidge JD, Bell JM. Antimicrobial susceptibility on solid media. In: Lorian V, editor. Antibiotics in laboratory medicine. 5th ed. Philadelphia: Lippincott Williams & Wilkins; 2005. p. 8–59. [16] Bonev B, Hooper J, Parisot J. Principles of assessing bacterial susceptibility to antibiotics using the agar diffusion method. J Antimicrob Chemother 2008;61:1295–301. [17] Rosenfeld Y, Shai Y. Lipopolysaccharide (endotoxin)-host defense antibacterial peptides interactions: role in bacterial resistance and prevention of sepsis. Biochim Biophys Acta 2006;1758:1513–22. [18] Vorland LH, Ulvatne H, Rekdal O, Svendsen JS. Initial binding sites of antimicrobial peptides in Staphylococcus aureus and Escherichia coli. Scand J Infect Dis 1999;31: 467–73. [19] Pier GB. Pseudomonas aeruginosa lipopolysaccharide: a major virulence factor, initiator of inflammation and target for effective immunity. Int J Med Microbiol 2007; 297:277–95. [20] Kim S-H, Jia W, Parreira VR, Bishop RE, Gyles CL. Phosphoethanolamine substitution in the lipid A of Escherichia coli O157:H7 and its association with PmrC. Microbiology 2006;152:657–66. [21] Schromm AB, Brandenburg K, Loppnow H, Zahringer U, Rietschel ET, Carroll SF, et al. The charge of endotoxin molecules influences their conformation and IL-6-inducing capacity. J Immunol 1998;161:5464–71. [22] Coats SR, To TT, Jain S, Braham PH, Darveau RP. Porphyromonas gingivalis resistance to polymyxin B is determined by the lipid A 4′-phosphatase, PGN_0524. Int J Oral Sci 2009;1:126–35. [23] Conlon JM, Mechkarska M, Pantic JM, Lukic ML, Coquet L, Leprince J, et al. An immunomodulatory peptide related to frenatin 2 from skin secretions of the Tyrrhenian painted frog Discoglossus sardus (Alytidae). Peptides 2013;40:65–71. [24] Ge Y, MacDonald D, Henry MM, Hait HI, Nelson KA, Lipsky BA, et al. In vitro susceptibility to pexiganan of bacteria isolated from infected diabetic foot ulcers. Diagn Microbiol Infect Dis 1999;35:45–53. [25] Hajishengallis G, Liang S, Payne MA, Hashim A, Jotwani R, Eskan MA, et al. Lowabundance biofilm species orchestrates inflammatory periodontal disease through the commensal microbiota and complement. Cell Host Microbe 2011;10:497–506. [26] Nair SC, Anoop KR. Intraperiodontal pocket: an ideal route for local antimicrobial drug delivery. J Adv Pharm Technol Res 2012;3:9–15. [27] Van Dyke T, Paquette D, Grossi S, Braman V, Massaro J, D'Agostino R, et al. Clinical and microbial evaluation of a histatin-containing mouthrinse in humans with experimental gingivitis: a phase-2 multi-center study. J Clin Periodontol 2002; 29:168–76.
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Regulatory Peptides journal homepage: www.elsevier.com/locate/regpep
Antimicrobial and immunomodulatory properties of PGLa-AM1, CPF-AM1, and magainin-AM1: Potent activity against oral pathogens Denise T.F. McLean a, Maelíosa T.C. McCrudden a, Gerard J. Linden b, Christopher R. Irwin c, J. Michael Conlon d, Fionnuala T. Lundy a,⁎ a
Centre for Infection & Immunity, School of Medicine, Dentistry and Biomedical Sciences, Queen's University, Belfast, Northern Ireland, UK Centre for Public Health, School of Medicine, Dentistry and Biomedical Sciences, Queen's University, Belfast, Northern Ireland, UK Centre for Dentistry, School of Medicine, Dentistry and Biomedical Sciences, Queen's University, Belfast, Northern Ireland, UK d Department of Biochemistry, College of Medicine and Health Sciences, United Arab Emirates University, 17666 Al-Ain, United Arab Emirates b c
a r t i c l e
i n f o
Article history: Received 7 March 2014 Received in revised form 29 October 2014 Accepted 5 November 2014 Available online 13 November 2014 Keywords: Host defence peptide Antimicrobial peptide Cytokine Lipopolysaccharide
a b s t r a c t Cationic amphipathic α-helical peptides are intensively studied classes of host defence peptides (HDPs). Three peptides, peptide glycine–leucine–amide (PGLa-AM1), caerulein-precursor fragment (CPF-AM1) and magaininAM1, originally isolated from norepinephrine-stimulated skin secretions of the African volcano frog Xenopus amieti (Pipidae), were studied for their antimicrobial and immunomodulatory activities against oral and respiratory pathogens. Minimal effective concentrations (MECs), determined by radial diffusion assay, were generally lower than minimal inhibitory concentrations (MICs) determined by microbroth dilution. PGLa-AM1 and CPFAM1 were particularly active against Streptococcus mutans and all three peptides were effective against Fusobacterium nucleatum, whereas Enterococcus faecalis and Candida albicans proved to be relatively resistant micro-organisms. A type strain of Pseudomonas aeruginosa was shown to be more susceptible than the clinical isolate studied. PGLa-AM1 displayed the greatest propensity to bind lipopolysaccharide (LPS) from Escherichia coli, P. aeruginosa and Porphyromonas gingivalis. All three peptides showed less binding to P. gingivalis LPS than to LPS from the other species studied. Oral fibroblast viability was unaffected by 50 μM peptide treatments. Production of the pro-inflammatory cytokine IL-8 by oral fibroblasts was significantly increased following treatment with 1 or 10 μM magainin-AM1 but not following treatment with PGLa-AM1 or CPF-AM1. In conclusion, as well as possessing potent antimicrobial actions, the X. amieti peptides bound to LPS from three human pathogens and had no effect on oral fibroblast viability. CPF-AM1 and PGLa-AM1 show promise as templates for the design of novel analogues for the treatment of oral and dental diseases associated with bacteria or fungi. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cationic amphipathic α-helical peptides represent one of the most widely studied classes of host defence peptides (HDPs). A large number of these peptides have been identified in and isolated from the skin secretions of certain species of Anura (frogs and toads), including frogs from the family Pipidae [reviewed in [1]]. To date, nine peptides have been identified in norepinephrine stimulated skin secretions of the African volcano frog Xenopus amieti (Pipidae) [2]. These peptides may be assigned to four peptide families on the basis of limited amino acid sequence similarity: peptide glycine–leucine–amide (PGLa-AM1), caeruleinprecursor fragment (CPF-AM1), xenopsin-precursor fragment (XPF) and magainin. PGLa-AM1, CPF-AM1, and magainin-AM1 are small cationic
⁎ Corresponding author at: Centre for Infection and Immunity, School of Medicine, Dentistry and Biomedical Sciences, Health Sciences Building, 97 Lisburn Road, Belfast BT9 7AE, UK. Tel.: +44 28 9097 5797; fax: +44 28 9097 2671. E-mail address: [email protected] (F.T. Lundy).
http://dx.doi.org/10.1016/j.regpep.2014.11.002 0167-0115/© 2014 Elsevier B.V. All rights reserved.
peptides with charges ranging between +2 and +5 at neutral pH. These peptides display a propensity to adopt an α-helical conformation on interactions with negatively charged polar head groups in bacterial membranes [2] and have antimicrobial activity against Escherichia coli ATCC25922 and Staphylococcus aureus ATCC25923 [2]. More recently PGLa-AM1 and CPF-AM1 were shown to display antimicrobial activities against the type strain of Acinetobacter baumannii [3]. In addition, the peptides were effective at inhibiting the growth of colistin-resistant clinical isolates of A. baumannii, as well as Acinetobacter nosocomialis [3]. Several frog skin peptides that were first identified as a result of their antimicrobial properties have subsequently been shown to possess complex cytokine-mediated immunomodulatory activities. Effects on the production of both pro-inflammatory and anti-inflammatory cytokines have been observed [reviewed in [4]]. The aim of the current study was to determine the antimicrobial activity and immunomodulatory abilities of PGLa-AM1, CPF-AM1 and magainin-AM1 against a panel of oral and respiratory pathogens. Antimicrobial activity was determined by both radial diffusion assay and microbroth dilution assay. Immunomodulatory activity was investigated by assessing the ability of
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each peptide to bind lipopolysaccharide (LPS) and determining the direct effects of the peptides on interleukin-8 (IL-8) production by primary oral fibroblasts in vitro. 2. Materials & methods 2.1. Peptide synthesis PGLa-AM1 (GMASKAGSVLGKVAKVALKAAL.NH2; molecular mass 2068.3), CPF-AM-1 (GLGSVLGKALKIG ANLL.NH2; molecular mass 1622.0), and magainin-AM1 (GIKEFAHSLGKFGKAFVGGILNQ; molecular mass 2417.3) were supplied in crude form by GL Biochem Ltd. (Shanghai, China). The peptides were purified by reversed-phase HPLC on a (2.2 cm × 25 cm) Vydac 218TP1022 (C-18; Grace, Deerfield, IL, USA) column equilibrated with acetonitrile/water/ trifluoroacetic acid (28.0/71.9/0.1, v/v/v) at a flow rate of 6.0 ml/min. The concentration of acetonitrile was raised to 56% (v/v) over 60 min using a linear gradient. Absorbance was measured at 214 and 280 nm, and the major peak in the chromatogram was collected manually. Identities of the peptides were confirmed by electrospray mass spectrometry, and the purity of all peptides tested was N98%. 2.2. Organisms and growth conditions Lactobacillus acidophilus NCTC 1723, Streptococcus milleri NCTC 11065, Enterococcus faecalis NCTC 12697, Streptococcus mutans ATCC 10449, Pseudomonas aeruginosa ATCC 27853 and Pseudomonas aeruginosa (PAO 239; Midlands UK), were maintained on blood agar plates and overnight cultures were prepared via subculture into 25 ml volumes of sterile Mueller Hinton broth (Oxoid) and subsequent incubation overnight at 37 °C. Candida albicans NCTC 3179 was maintained on Sabouraud plates, subcultured into 25 ml volume of sterile Sabouraud broth (Oxoid) and incubated overnight at 37 °C. Fusobacterium nucleatum NCTC10562 was grown on fastidious anaerobic agar plates (FAA; Southern Group Laboratories) under strict anaerobic conditions in a Whitley A35 anaerobic workstation (Don Whitley Scientific Ltd., Shipley, UK) at 37 °C and overnight cultures were prepared in 25 ml volumes of anaerobic basal broth (Oxoid) supplemented with 1% (v/v) menadione and 0.003% (v/v) haemin.
were then incubated under aerobic conditions for 3 h at 37 °C to allow peptide diffusion into the gel. A 1% (w/v) agar overlay gel (10 ml) containing medium specific for the organism being tested was then poured over the base to provide nutrients for microbial growth. The plates were incubated for 18 h at 37 °C and then stained with a dilute solution of Coomassie brilliant blue R-250 as previously described [5]. Zones of inhibition were measured using a jeweller's eye-piece (×8 magnification). The diameters of the zones of clearing were expressed in units (0.1 mm = 1 U) and were calculated after subtracting the diameter of the negative control well. The antimicrobial activities were expressed as minimal effective concentration values (MECs), determined as the x intercept obtained from the relationship between radial diffusion units versus log10 peptide concentration. The mean MECs were calculated from three replicate experiments (Supplementary materials).
2.4. Microbroth dilution assay Antimicrobial activities of PGLa-AM1, CPF-AM1 and magainin-AM1 (1.56–to 100 μM) against the oral and respiratory pathogens were also determined by microbroth dilution assay using the Clinical Laboratory and Standards Institute (CLSI) double dilution method [7] with polypropylene plates to limit peptide binding [8]. Organisms were grown aerobically in Mueller Hinton broth with the exception of F. nucelatum which was grown in a Whitley A35 anaerobic workstation (Don Whitley Scientific Ltd., Shipley, UK) at 37 °C in anaerobic basal broth (Oxoid) supplemented with 1% (v/v) menadione and 0.003% (v/v) haemin.
2.5. Biotinylation of lipopolysaccharide (LPS) LPS from Pseudomomas aeruginosa (Sigma-Aldrich, UK), Porphyromonas gingivalis (Source Biosciences, UK) and E. coli (Sigma-Aldrich) was biotinylated with biotin (long arm) hydrazide (Vector Laboratories, UK) using the methodology outlined by the manufacturer. The number of biotins incorporated per molecule of LPS was determined using the Quant*Tag Biotin Quantification Kit (Vector Laboratories, UK), as described by the manufacturer.
2.3. Radial diffusion assay 2.6. LPS binding assay A double layer radial diffusion assay [5,6] was performed to determine the antimicrobial activities of PGLa-AM1, CPF-AM1 and magaininAM1 against the oral and respiratory pathogens. This assay has previously been recommended [5,6] over microbroth dilution assays for the early stages of antimicrobial peptide discovery work since it uses much smaller quantities of synthetic peptide. Briefly, overnight cultures of aerobic organisms were grown in 25 ml of appropriate media overnight at 37 °C. F. nucleatum was grown in appropriate media overnight in a Whitley A35 anaerobic workstation under strict anaerobic conditions. Mid-logarithmic phase cultures were obtained by inoculating a 25 ml volume of the appropriate sterile broth with 100 μl of the requisite overnight culture and incubating for a further 3 h at 37 °C. Bacterial cultures were centrifuged at 1800 ×g for 10 min at 4 °C, washed three times with the same volume of 10 mM sodium phosphate buffer (pH 7.4) and resuspended in 10 ml of the same buffer. An underlay gel (10 ml) was prepared consisting of 1% (w/v) agarose containing approximately 4 × 105 yeast cells or 5 × 106 bacterial cells. The microorganisms were dispersed by vortexing; the underlay gel was then poured into a square Petri dish and allowed to solidify. A series of wells (2.5 mm in diameter) were punched in the agar and peptide solution (3 μl) was added to each well at concentrations ranging from 12 to 75 μM for PGLa-AM1 and CPFAM1 and 25–to 100 μM for magainin-AM1. A negative control well containing sterile water and a positive control well containing 100 μg ml−1 synthetic Cecropin A (Sigma, UK) were included on each plate. The plates
Binding of PGLa-AM1, CPF-AM1 and magainin-AM1 to biotinylated LPS was studied using a method adapted from [9], as described previously [10]. Briefly, the wells of a Greiner high binding plate (Sigma Aldrich, Dorset, UK) were coated with 100 μl/well of the relevant peptide (6.25 μM), prepared in Voller's buffer (26 mM Na2CO3, 23 mM, NaHCO3, pH 9.6) and incubated overnight at 37 °C in an unsealed plate. The following day, plates were washed three times with an excess of phosphate buffered saline (PBS) containing 0.05% (v/v) Tween-20 (PBST) and the wells of the plate (200 μl/well) were then blocked with PBST containing 1% (w/v) bovine serum albumin (BSA) for 1 h at room temperature. Plates were washed three times with PBST and 100 ng of biotinylated LPS (E. coli, P. aeruginosa or P. gingivalis) prepared in PBS was added to each well (100 μl/well). Plates were incubated at room temperature for 3 h and then washed three times with an excess of PBST. Horseradish peroxidase-conjugated streptavidin (Biolegend, UK) (diluted 1:2000 in PBST) was added to each well and incubated for 30 min at room temperature (100 μl/well). The wells were washed three times with PBST and peroxidase activity was detected following addition of the chromogenic substrate, 2,2-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]diammonium salt (ABTS) (Invitrogen, Paisley, UK) (100 μl/well). The absorbance measurements were determined at 405 nm on a Tecan Genios microtitre plate reader (Tecan, Reading, UK) using Magellan software.
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2.7. Haemolysis assay
3. Results
Peptides in the concentration range 6 to 200 μM were incubated with washed human erythrocytes from a healthy donor. After centrifugation (12,000 ×g for 15 s), the absorbance at 450 nm of the supernatant was measured. A parallel incubation in the presence of 1% v/v Tween-20 was carried out to determine the absorbance associated with 100% haemolysis. The LD50 value was taken as the mean concentration of peptide producing 50% haemolysis in three independent experiments.
3.1. Antimicrobial activity of PGLa-AM1, CPF-AM1 and magainin-AM1 against a range of oral and lung pathogens
2.8. Tissue culture Oral fibroblasts were derived by explant culture of dental pulp tissue removed from healthy teeth, with ethical approval, as previously described [11]. Oral fibroblasts were maintained in Dulbecco's Minimal Essential Medium (DMEM; Invitrogen) supplemented with 10% heat inactivated foetal bovine serum (FBS; Invitrogen), 2 mM L-glutamine (Invitrogen) and 1% (v/v) penicillin/streptomycin (PAA Laboratories GmbH, Austria). Cell lines were maintained at 37 °C in a humidified atmosphere of 5% CO2. Fibroblasts were grown to confluence before being harvested and subcultured. 2.9. MTT assay with oral fibroblast cells Oral fibroblasts were seeded into a 96-well culture plates at 1 × 105 cells/ml and maintained until they reached confluence. The medium was then replaced with DMEM without FBS overnight to induce quiescence. Peptide stock solutions in Muller Hinton broth were further diluted in DMEM to a final concentrations of 50 μM, with 100 μl per well added for cell treatments. Cells were then incubated at 37 °C for 24 h. Following this, 10 μl of thiazolyl blue tetrasodium bromide (MTT) reagent (Sigma Aldrich®, Dorset, UK) was added to each well. The plate was incubated at 37 °C for a further 2 h. Cell culture media were removed by aspiration and the wells of the plate were allowed to air dry in a laminar flow hood for 10 min. 200 μl dimethyl sulfoxide (DMSO) (Sigma Aldrich®, Dorset, UK) was added to each well and this was mixed by gentle agitation. The absorbance measurements were determined at 405 nm using a Tecan Genios microtitre plate reader (Tecan, Reading, UK) using Magellan software. Four replicates of each cell treatment were carried out. Vehicle controls were included in all experiments.
2.10. Measurement of interleukin-8 release Oral fibroblasts were seeded into a 24-well culture plate at a density of 1 × 105 cells/well until they achieved confluence. The cells were incubated in 1% (v/v) FBS for 24 h, and then PGLa-AM1, CPF-AM1 or magainin-AM-1 at concentrations of 1 or 10 μM, or medium (controls) were added. Cells were cultured for 21 h at 37 °C in a humidified atmosphere of 5% CO2 and the cell supernatants were collected and stored at −20 °C until required. IL-8 concentrations in the cell supernatants were measured by ELISA using the IL-8 DuoSet ELISA development kit (R & D Systems, Minneapolis, MN, USA). The absorbance was determined at 405 nm on a Tecan Genios microtitre plate reader (Tecan, Reading, UK) using Magellan software.
Variable MEC and MIC values were determined for PGLa-AM1, CPFAM1 and magainin-AM1 against a range of oral and lung pathogens (Table 1). MICs obtained by microbroth dilution assay were generally higher than MECs obtained by the radial diffusion assay. Our observations are consistent with those of Hancock [12] who reported that for a wide range of antimicrobial peptides, including magainin-2, MIC values measured by the broth dilution method are consistently much higher than the corresponding values measured by the radial diffusion assay method. PGLa-AM1 proved to be active against both Gram positive and Gram negative bacteria, with particularly good activity against S. mutans (MEC and MIC b 5 μM). CPF-AM1 was also active against both Gram positive and Gram negative bacteria and was also particularly effective against S. mutans (MEC and MIC b5 μM). Of the three frog peptides studied, magainin-AM1 had the least antimicrobial potency and was shown to be ineffective against the clinical isolate of P. aeruginosa (MEC and MIC N100 μM). The MIC values for PGLa-AM1 also showed that this peptide was less effective against a clinical isolate of P. aeruginosa compared with the type strain. Microbroth dilution assay results indicated that E. faecalis was resistant to all three peptides studied and also that MICs against C. albicans were relatively high for PGLa-AM1 (50 μM), CPF-AM1 (50 μM) and magainin-AM1 (100 μM). MEC values for the microaerophilic organisms S. milleri and L. acidophilus are presented using the radial diffusion assay as the microbroth dilution assay is reported to be suitable for aerobic organisms only [8]. However, modification of the CLSI protocol using supplemented anaerobic basal broth and anaerobic incubation allowed us to determine microbroth dilution MICs for PGLa-AM1 (12.5 μM), CPF-AM1 (6.25 μM) and magainin-AM1 (12.5 μM), showing the effectiveness of all three peptides against the oral anaerobe F. nucleatum. 3.2. LPS binding activities of PGLa-AM1, CPF-AM1 and magainin-AM1 PGLa-AM1, CPF-AM1 and magainin-AM1 bound with varying affinities to biotinylated E. coli LPS, with PGLa-AM1 displaying the greatest propensity to bind (Fig. 1A). PGLa-AM1, CPF-AM1 and magainin-AM1 bound to biotinylated P. aeruginosa LPS (Fig. 1B) with greater affinity than to E. coli LPS. PGLa-AM1 bound P. aeruginosa LPS significantly better than either CPF-AM1 or magainin-AM1. All three peptides showed less binding to P. gingivalis LPS (Fig. 1C) than to the other species studied. However, PGLa-AM1 showed a significantly higher level of binding to P. gingivalis LPS than either CPF-AM1 or magainin-AM1. 3.3. Haemolytic activity The LD50 (mean ± SEM) results for haemolytic activity against human erythrocytes were: CPF-AM1 = 155 ± 9 μM; PGLa-AM1 N 200 μM; and magainin-AM1 N 200 μM, suggesting that none of the peptides were toxic to human erythrocytes at any of the concentrations employed in our studies. 3.4. Cytotoxicity of PGLa-AM1, CPF-AM1 and magainin-AM1 to oral fibroblasts Oral fibroblast viability was unaffected by treatments with PGLaAM1, CPF-AM1 or magainin-AM1 at concentrations of 50 μM (Fig. 2).
2.11. Statistical analysis Statistical analysis was performed using commercially available software (Prism). All data were analysed by one way analysis of variance followed by Bonferroni's multiple comparison test. A p-value b 0.05, was considered statistically significant.
3.5. Cytokine production from oral fibroblast cells following treatment with PGLa-AM1, CPF-AM1 or magainin-AM1 Production of the pro-inflammatory cytokine IL-8 by unstimulated oral fibroblast cells was increased between 1 and 3 fold following
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Table 1 Minimal effective concentration (MEC) and minimal inhibitory concentration (MIC) values of PGLa-AM1, CPF-AM1 and magainin-AM1 against the aerobic oral microorganisms C. albicans, S. mutans, and E. faecalis; the microaerophilic oral microorganisms S. milleri and L. acidophilis; the anaerobic oral bacterium F. nucleatum and the lung pathogen P. aeruginosa using radial diffusion assay (mean of triplicate assays presented in (μM) +/− standard deviations in brackets) and microbroth dilution assay (duplicate assays). MEC (μM) determined by (A) radial diffusion assay and MIC (μM) determined by (B) microbroth dilution assay Microorganism
PGLa-AM1
CPF-AM1
Magainin-AM1
Candida albicans NCTC 3179
(A) 7.5 (+/−0.4) (B) 50 (A) 1.2 (+/−0.1) (B) 3.1 (A) 10.2 (+/−0.4) (B) N100 (A) 5.8 (+/−1.3) (B) ND (A) 6.7 (+/−0.4) (B) ND (A) 1.4 (+/−0.1) (B) 12.5 (A) 12.3 (+/−0.8) (B) 25 (A) 1.5 (+/−0.4) (B) 12.5
(A) 9.9 (+/−1.8) (B) 50 (A) 2.5 (+/−0.9) (B) 3.1 (A) 7.8 (+/−0.2) (B) N100 (A) 8.8 (+/−2.6) (B) ND (A) 2.5 (+/−0.5) (B) ND (A) 1.9 (+/−0.6) (B) 25 (A) 11.9 (+/−0.9) (B) 25 (A) 2.2 (+/−0.1) (B) 6.25
(A) (B) (A) (B) (A) (B) (A) (B) (A) (B) (A) (B) (A) (B) (A) (B)
Streptococcus mutans ATCC 10449 Enterococcus faecalis NCTC 12697 Streptococcus milleri NCTC 11065 Lactobacillus acidophilus NCTC 1723 Pseudomonas aeruginosa ATCC 27853 Pseudomonas aeruginosa PAO239 Fusobacterium nucleatum NCTC 10562a
24.3 (+/−4.4) 100 24 (+/−1.9) 50 22.9 (+/−0.1) N100 10.1 (+/−0.9) ND 27.2 (+/−1.3) ND 11.1 (+/−1.5) N100 N100 N100 9 (+/−3.8) 12.5
ND = not determined for microaerophilic microorganisms. a MECs and MICs determined under anaerobic conditions.
treatment with 1 or 10 μM PGLa-AM1, CPF-AM1 or magainin-AM1 (Fig. 3). Treatment with CPF-AM1 resulted in the lowest fold-increase in IL-8 at both 1 and 10 μM concentrations. Treatment of oral fibroblasts with 10 μM PGLa-AM1 or 10 μM magainin-AM1 increased production of IL-8 over that observed for 1 μM treatments. However, only the treatments with magainin-AM1 resulted in significant increases in IL-8 (both 1 μM and 10 μM treatments). 4. Discussion In the current study the amphipathic peptides PGLa-AM1, CPF-AM1 and magainin-AM1 were shown to have antimicrobial activity against a range of oral and respiratory pathogens. PGLa-AM1 and CPF-AM1 proved to be generally more effective, as measured by their MECs and MICs, than magainin-AM1. This may be due in part to the greater overall positive charge and hydrophobic ratio of PGLa-AM1 and CPF-AM1 compared with magainin-AM1. Both PGLa-AM1 and CPF-AM1 were equally effective against the Gram negative and Gram positive bacteria studied. Peptide cationicity contributes to the initial interaction of the peptide with the bacterial membrane [13] and the lower cationicity exhibited by magainin-AM1 may explain its weaker potency than PGLa-AM1 or CPF-AM1. Magainin-AM1 also has a lower cationicity than its magainin counterpart which could also explain its relatively weak potency compared with its X. laevis homolog [2]. Agar diffusion tests have found favour in the past for surface active antibiotics such as nisin or subtilin [14] and these assays have therefore also been employed in the testing of membrane targeting antimicrobial peptides. The generally higher MICs reported using the microbroth dilution assay versus the MECs reported using the radial diffusion assay may be related in part to differences in the methodologies, including less favourable growth conditions in the radial diffusion assay. It should be recognised that several factors including the antimicrobial activity of the test peptide, the bacterial growth rate and the density and metabolic activity of the inoculum may influence zone diameter [15]. Differential peptide diffusion rates also merit consideration in the radial diffusion assay and therefore comparison of results from peptide families with different physicochemical properties (for example hydrophobic versus amphipatic peptides) may be limited [16]. LPS is a major stimulus of inflammation, acting via Toll like receptors (TLRs) to enhance pro-inflammatory cytokine secretion. Thus, it was of interest to determine the ability of the peptides to bind LPS thereby limiting potential interactions of the LPS with TLRs thus negating their proinflammatory actions. In terms of ability to bind LPS, the cationicity of
the peptide is considered to play an important role. The higher net charge of PGLa-AM1 (+ 5) compared with CPF-AM1 (+ 3) or magainin-AM1 (+ 2) may contribute to significantly more effective binding of PGLA-AM1 to LPS from P. aeruginosa, E. coli and P. gingivalis. Studies on magainin from X. laevis have shown LPS binding at a 1:1 stoichiometry in solution [17,18]. However, magainin from X. laevis displays greater cationicity than magainin-AM1 from X. amieti. There have been no reports to date regarding the LPS binding abilities of either PGLa or CPF from X. laevis and no reports describing the ability of X. amieti homologs to bind to LPS from any bacterial species. PGLa-AM1 binding to LPS was shown to be strongest for P. aeruginosa followed by E. coli and P. gingivalis. Differences in binding activity between LPS from various bacteria may reflect differences in LPS composition [17]. Reduction in the overall anionic nature of the LPS may result in reduced potential electrostatic binding between the negatively charged LPS and cationic peptides. In P. aeruginosa, the lipid A moieties have been shown to display variable compositions representing different isoforms which have different potencies [19]. The lipid A moiety of E. coli LPS consists of a β-1,6-linked D-glucosamine (GlcN) disaccharide carrying two negatively charged phosphates and six saturated fatty acids in a defined asymmetric distribution, four at the GlcN II and two at the GlcN I, conferring an overall anionic charge of −6 [20]. Variation in E. coli lipid A moiety composition can reduce the number of acyl chains resulting in LPS with charges ranging from −4 to −2 in E. coli mutant strains [20,21]. The lipid A moiety of P. gingivalis comprises tetra-acylated, nonphosphorylated lipid A and tetra-acylated mono-phosphorylated lipid A. The non-phosphorylated lipid A moiety represents a significant proportion of the total lipid A species of P. gingivalis LPS. This LPS lipid A moiety composition results in a less anionic LPS thus reducing the potential for initial electrostatic interactions between the cationic HDPs and the anionic bacterial membrane and has been shown to be the important in P. gingivalis resistance to cationic, antimicrobial polymyxin B [22]. There was no evidence that any of the peptides investigated caused cytotoxicity, as determined by haemolytic assay with human erythrocytes and by MTT assay in primary oral fibroblasts at concentrations of 50 μM. It is known that antimicrobial peptides can modulate the innate immune response and in the context of the present study, only magainin-AM1 elicited a significant increase in IL-8 production by oral fibroblasts. IL-8 has an important role in LPS-driven inflammation, frequently seen in the oral cavity and respiratory tract. Although reduced IL-8 production may appear favourable in terms of limiting the inflammatory response, pro-inflammatory cytokines elicited by various
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% Survival Relative to Control
ns
***
0.8 0.6 0.4
150 100 50
***
***
1.0 0.5
1 nAM
AM M
ag
ai ni
CP F-
PG
La -
AM
1
1
0.0
*** ns
***
0.4 0.3 0.2
in in -A M 1 ag a
0.1 *
400
1
IL-8 pg/ml
300 200 100 0
1
1 PF -
A
ai ni nA M
M
M A La -
ag M
peptides may paradoxically contribute to their protective role in host defence against infection [17] as long as the inflammatory response does not become dysregulated/chronic as a result of failure to eliminate the stimulus. Several naturally occurring frog skin peptides have been shown to increase production of pro-inflammatory cytokines [4]. For example, frenatin 2D from the Tyrrhenian painted frog Discoglossus
PG
C
on t
ro l
Fig. 1. PGLa-AM1, CPF-AM1 and magainin-AM1 binding to biotinylated (A) E. coli LPS, (B) P. aeruginosa LPS and (C) P. gingivalis LPS. Data were analysed by one way analysis of variance followed by Bonferroni's multiple comparison test (*** = p b 0.001; ns = non-significant).
1 M 10 M
*
in -A M in ag a M
C PF -A M 1
PG La -A M 1
0.0
C
Abs @ 405 nm
0.5
M
sardus potently stimulated production of TNF-α and IL-1β by mouse peritoneal macrophages and it was speculated that the peptide acts on macrophages in frog skin to produce a cytokine-mediated stimulation of the adaptive immune system in response to invasion by microorganisms [23]. A number of peptides have been exploited for therapeutic use such as Pexiganan, derived from magainin 2 from X. laevis, proposed as a topical antibiotic for use on diabetic foot ulcers [24]. The X. amieti peptides studied in the current work displayed potent antimicrobial activities against a wide range of oral and respiratory pathogens. As the oral cavity is easily accessible for the topical application of therapeutic agents, these peptides may prove to be templates for the development of novel antimicrobial agents for use in this site. PGLa-AM1 and CPFAM1 were particularly effective against the cariogenic pathogen S. mutans, whilst exhibiting no cytotoxicity against oral fibroblasts. Both peptides also displayed potent antimicrobial activity against F. nucleatum an important ‘bridging organism’ which facilitates coaggregation to incorporate late colonizers such as the Gram negative anaerobe P. gingivalis into the mature dental plaque biofilm. P. gingivalis has been identified as a keystone microorganism implicated in the development of periodontal disease [25]. The incorporation of novel peptides into formulations to be delivered directly to the periodontal pocket may provide an effective and targeted treatment for periodontal inflammation [26]. The synthetic peptide PAC-113, derived from histatins 3
1.5
(C)
C PF -A M 1
on t
A M 1 M ag ai ni n-
Fig. 2. Percentage cell survival of oral fibroblasts, relative to that of control, following a 24 hour exposure to 50 μM PGLA-AM1, CPF-AM1 or magainin-AM1. Cells were incubated in medium containing the vehicle only (Control) or vehicle and peptide (PGLA-AM1, CPFAM1 or magainin-AM1), (mean +/− SD, n = 4). Data were analysed by one way analysis of variance followed by Bonferroni's multiple comparison test, all comparisons were nonsignificant.
*** 2.0
50 µM Treatments
1
Abs @405 nm
(B)
C
PG
La -
PF -A
A
M
M
1
1
C
0.0
PG La -A M 1
0
0.2
ro l
Abs @ 405 nm
200
***
(A)
67
Fig. 3. Direct effects of PGLa-AM1, CPF-AM1 and magainin-AM1 on IL-8 production by oral fibroblasts. Control cells were treated with different volumes of vehicle corresponding to peptide dilution to working concentrations of 1 μM and 10 μM. Data were analysed by one way analysis of variance followed by Bonferroni's multiple comparison test (* = p b 0.05).
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and 5, has been incorporated into a mouthwash for the treatment of oral candidiasis [27]. CPF-AM1 and PGLa-AM1, which were shown to be very effective against S. mutans, could also be delivered in this way to the oral cavity. In conclusion, as well as possessing potent antimicrobial actions, the X. laevis peptides studied were shown to bind LPS from three human pathogens and to have no effect on the viability of oral fibroblasts. In general PGLa-AM1 had slightly greater potency than CPF-AM1. Magainin-AM1 displayed least antimicrobial and LPS-binding activity, and elicited higher levels of IL-8 production from oral fibroblasts. It is tempting to speculate that CPF-AM1 and PGLa-AM1 show promise as templates for the design of non-toxic, non-inflammatory analogues for the treatment of oral and dental diseases associated with bacteria or fungi. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgements This study was funded by the Department for Employment and Learning (Northern Ireland). We gratefully acknowledge the skilful technical assistance of Catherine Fulton. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.regpep.2014.11.002. References [1] Conlon JM. Structural diversity and species distribution of host-defense peptides in frog skin secretions. Cell Mol Life Sci 2011;68:2303–15. [2] Conlon JM, Al-Ghaferi N, Ahmed E, Meetani MA, Leprince J, Nielsen PF. Orthologs of magainin, PGLa, procaerulein-derived, and proxenopsin-derived peptides from skin secretions of the octoploid frog Xenopus amieti (Pipidae). Peptides 2010;31:989–94. [3] Conlon JM, Sonnevend A, Pál T, Vila-Farrés X. Efficacy of six frog skin-derived antimicrobial peptides against colistin-resistant strains of the Acinetobacter baumannii group. Int J Antimicrob Agents 2012;39:317–20. [4] Conlon JM, Mechkarska M, Lukic ML, Flatt PR. Potential therapeutic applications of multifunctional host-defense peptides from frog skin as anti-cancer, anti-viral, immunomodulatory, and anti-diabetic agents. Peptides 2014;57:67–77. [5] Lehrer RI, Rosenman M, Harwig SSSL, Jackson R, Eisenhauer P. Ultrasensitive assays for endogenous antimicrobial polypeptides. J Immunol Methods 1991;137:167–73. [6] Steinberg DA, Lehrer RI. Designer assays for antimicrobial peptides. Disputing the “one-size-fits-all” theory. Methods Mol Biol 1997;78:169–86.
[7] Laboratory Clinical, Institute Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, approved standard M07-A8. Wayne PA: CLSI; 2008. [8] Wiegand I, Hilpert K, Hancock REW. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc 2008;32:163–75. [9] Felgentreff K, Beisswenger C, Griese M, Gulder T, Bringmann G, Bals R. The antimicrobial peptide cathelicidin interacts with airway mucus. Peptides 2006;27:3100–6. [10] McLean DTF, Lundy FT, Timson DJ. IQ-motif peptides as novel anti-microbial agents. Biochimie 2013;95:875–80. [11] Killough SA, Lundy FT, Irwin CR. Dental pulp fibroblasts express neuropeptide Y Y1 receptor but not neuropeptide Y. Int Endod J 2010;43:835–42. [12] Hancock REW. Cationic peptides: effectors in innate immunity and novel antimicrobials. Lancet Infect Dis 2001;1:156–64. [13] Yin LM, Edwards MA, Li J, Yip CM, Deber CM. Roles of hydrophobicity and charge distribution of cationic antimicrobial peptides in peptide–membrane interactions. J Biol Chem 2012;287:7738–45. [14] Parisot J, Carey S, Breukink E, Chan WC, Narbad A, Bonev B. Molecular mechanism of target recognition by subtilin, a class 1 lanthionine antibiotic. Antimicrob Agents Chemother 2008;52:612–8. [15] Turnidge JD, Bell JM. Antimicrobial susceptibility on solid media. In: Lorian V, editor. Antibiotics in laboratory medicine. 5th ed. Philadelphia: Lippincott Williams & Wilkins; 2005. p. 8–59. [16] Bonev B, Hooper J, Parisot J. Principles of assessing bacterial susceptibility to antibiotics using the agar diffusion method. J Antimicrob Chemother 2008;61:1295–301. [17] Rosenfeld Y, Shai Y. Lipopolysaccharide (endotoxin)-host defense antibacterial peptides interactions: role in bacterial resistance and prevention of sepsis. Biochim Biophys Acta 2006;1758:1513–22. [18] Vorland LH, Ulvatne H, Rekdal O, Svendsen JS. Initial binding sites of antimicrobial peptides in Staphylococcus aureus and Escherichia coli. Scand J Infect Dis 1999;31: 467–73. [19] Pier GB. Pseudomonas aeruginosa lipopolysaccharide: a major virulence factor, initiator of inflammation and target for effective immunity. Int J Med Microbiol 2007; 297:277–95. [20] Kim S-H, Jia W, Parreira VR, Bishop RE, Gyles CL. Phosphoethanolamine substitution in the lipid A of Escherichia coli O157:H7 and its association with PmrC. Microbiology 2006;152:657–66. [21] Schromm AB, Brandenburg K, Loppnow H, Zahringer U, Rietschel ET, Carroll SF, et al. The charge of endotoxin molecules influences their conformation and IL-6-inducing capacity. J Immunol 1998;161:5464–71. [22] Coats SR, To TT, Jain S, Braham PH, Darveau RP. Porphyromonas gingivalis resistance to polymyxin B is determined by the lipid A 4′-phosphatase, PGN_0524. Int J Oral Sci 2009;1:126–35. [23] Conlon JM, Mechkarska M, Pantic JM, Lukic ML, Coquet L, Leprince J, et al. An immunomodulatory peptide related to frenatin 2 from skin secretions of the Tyrrhenian painted frog Discoglossus sardus (Alytidae). Peptides 2013;40:65–71. [24] Ge Y, MacDonald D, Henry MM, Hait HI, Nelson KA, Lipsky BA, et al. In vitro susceptibility to pexiganan of bacteria isolated from infected diabetic foot ulcers. Diagn Microbiol Infect Dis 1999;35:45–53. [25] Hajishengallis G, Liang S, Payne MA, Hashim A, Jotwani R, Eskan MA, et al. Lowabundance biofilm species orchestrates inflammatory periodontal disease through the commensal microbiota and complement. Cell Host Microbe 2011;10:497–506. [26] Nair SC, Anoop KR. Intraperiodontal pocket: an ideal route for local antimicrobial drug delivery. J Adv Pharm Technol Res 2012;3:9–15. [27] Van Dyke T, Paquette D, Grossi S, Braman V, Massaro J, D'Agostino R, et al. Clinical and microbial evaluation of a histatin-containing mouthrinse in humans with experimental gingivitis: a phase-2 multi-center study. J Clin Periodontol 2002; 29:168–76.
