1 Department of Bacteriology, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima 734-8553, Japan; 2 Department of Periodontal Medicine, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima 734-8553, Japan; 3 Department of Microbiology, Kawasaki Medical School, Matsushima Kurashiki, Okayama 701-0192, Japan; 4 Department of Dermatology, Ehime University School of Medicine, Onsen-gun, Ehime 791-0295, Japan
Received 9 December 2004; returned 2 February 2005; revised 14 February 2005; accepted 22 February 2005
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Abstract |
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Methods: Synthetic antimicrobial peptides of human ß-defensin-1 (hBD1), hBD2, hBD3 and LL37 (CAP18) were evaluated for their antimicrobial activity against oral bacteria. They included Actinobacillus actinomycetemcomitans (20 strains), Porphyromonas gingivalis (6), Prevotella intermedia (7), Fusobacterium nucleatum (7), Streptococcus mutans (5), Streptococcus sobrinus (5), Streptococcus salivarius (5), Streptococcus sanguis (4), Streptococcus mitis (2) and Lactobacillus casei (1).
Results: Although the four peptides had bactericidal activity against all bacteria tested, the degree of antibacterial activity was variable against the different strains and species. The antibacterial activity of hBD1 was lower than that of the other peptides. Among the bacteria tested in this study, F. nucleatum was highly susceptible to hBD3 and LL37, and S. mutans was highly susceptible to hBD3. We measured the Zeta-potential, representing the net charge of whole bacteria, to study the relationship between susceptibility to cationic peptide and the net charge of the bacteria. Although we found some correlation in A. actinomycetemcomitans strains, we did not find a definite correlation with all the bacterial species.
Conclusions: These results indicate that ß-defensins and LL37 have versatile antibacterial activity against oral bacteria.
Keywords: oral bacteria , defensins , cathelicidins
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Introduction |
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Tooth decay (dental caries) and periodontal diseases are caused by bacterial infection.2024 Cariogenic bacteria such as Streptococcus mutans and Streptococcus sobrinus, and also periodontopathogenic bacteria such as Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans, have been identified as causative agents. These Gram-positive and -negative bacteria tend to aggregate and coexist in dental plaque. Dental plaque is the pathogenic source for dental caries and periodontitis. Many antimicrobial agents, such as histatin, lactoferrin and lysozyme, are known to be produced in the oral cavity. The production of these agents is considered to be one of the roles of innate immunity against bacterial infection.3,25 Gingival epithelial cells are also reported to produce antimicrobial peptides, such as ß-defensins and calprotectin.16,17,26,27 Gingival epithelial cells, especially non-keratinized cells at the bottom of the periodontal pocket, are considered to produce these antimicrobial peptides in contact with bacteria in the dental plaque. It has been demonstrated that Fusobacterium nucleatum induced hBD2 production through the mitogen-activated protein (MAP) kinase pathway.16 However, little is known about the mechanism of the interaction between these peptides and bacteria. Several reports concerning the activity of antimicrobial peptides, especially cathelicidins,2830 have been published, but a detailed investigation has not been conducted so far. Therefore, an investigation into the susceptibility of cariogenic or periodontal bacteria to these peptides is of great interest for understanding the potential role of innate immunity in dental diseases.
In this study, we have investigated the antimicrobial activity of hBD1-3 and LL37 against four periodontopathogenic, five oral streptococci and one Lactobacillus sp. containing clinical isolates. Also, an electron microscopic observation of A. actinomycetemcomitans exposed to these peptides was performed. Finally, we also assessed the net charge of bacteria to investigate whether the bacterial charge is associated with susceptibility to these cationic peptides.
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Materials and methods |
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Bacterial strains used in this study were A. actinomycetemcomitans (20 strains), P. gingivalis (6), P. intermedia (7), F. nucleatum (7), S. mutans (5), S. sobrinus (5), Streptococcus sanguis (4), Streptococcus salivarius (5), Streptococcus mitis (2) and Lactobacilllus casei (1). Three A. actinomycetemcomitans strains (Y4, IDH781, SUNYaB75), two P. gingivalis (WA83, WA50), one F. nucleatum (ATCC 25586), one S. sobrinus (OMZ176) and one L. casei (IFO3983) were standard strains, and other strains were clinically isolated. A. actinomycetemcomitans was cultured in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, MD, USA) supplemented with 1% (w/v) yeast extract (TSBYE) in a 5% CO2 atmosphere. P. gingivalis, P. intermedia and F. nucleatum were cultured in TSB supplemented with 1% yeast extract, haemin (5 mg/L), vitamin K3 (1 mg/L) and 5% sheep blood (TSBYE-B) in an anaerobic atmosphere using an Anaero Pack system (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan). Streptococci and L. casei were grown aerobically in brain heart infusion broth (BHI; Difco Laboratory, Detroit, MI, USA).
Synthetic peptides
Synthetic peptides used are listed in Table 1. We constructed hBD13 as mature forms and LL37 as a C-terminally truncated form (34 amino acids), which has previously shown antibacterial activity.31
Peptides were synthesized in a Shimazu peptide synthesizer. Purification of peptides was performed by the method described previously.31
In brief, peptides were purified by reversed phase high performance liquid chromatography with an octadecyl-4PW column (Tosoh, Tokyo, Japan). Separation was performed with a linear gradient, from aqueous 0.05% trifluoroacetic acid (TFA) to 100% acetonitrile containing 0.05% TFA at a flow rate of 1 mL/min for 30 min. Major peak fractions (absorbance at 230 nm) were collected and lyophilized to completely remove the organic solvent. To confirm the purity and the quality of the peptides, mass spectrometry using MALDI/TOF-MS was performed using Voyager (PerSeptive Biosystems, MA, USA). TOF/MS analysis revealed that the masses of hBD1, hBD2, hBD3 and LL37 were 4533.6, 4228.7, 5152.3 and 4174.1 Da, respectively (Table 1). The mass of synthetic LL37 was identical to that calculated from the primary sequence, whereas the masses of each of the ß-defensins (hBD1, hBD2, hBD3) were 6 Da less than expected from the primary sequence, respectively. Native ß-defensins have three disulphide bonds using six cysteine residues that form three ß-sheets and one -helix.32,33
Our mass spectrometry data suggested that each synthetic ß-defensin possesses three disulphide bonds, respectively. We also measured the antimicrobacterial activity of ß-defensins using synthetic peptides (Peptide Institute, Inc., Osaka, Japan), which were shown to be structurally similar to the native peptides, by our antimicrobial assay, and confirmed that our synthetic peptides showed comparable antimicrobial activity against S. mutans, Staphylococcus aureus and Escherichia coli.
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Two methods were used for the antibacterial assay. One has been described elsewhere.31 Briefly, for this first method, overnight cultures of bacterial strains were harvested, washed with Dulbecco's phosphate-buffered saline (PBS) and suspended with 10 mM sodium phosphate buffer (PB) (pH 6.8). The bacterial suspension was diluted to 107 cells/mL with PB (pH 6.8), and 10 µL of bacterial suspension (105 cells) was inoculated into 200 µL of PB with or without various concentrations of antibacterial peptides (final concentration: 0.5, 1, 5, 10, 20, 50 mg/L) and incubated anaerobically for 2 h at 37°C. An appropriate dilution of the reaction mixture in PB (100 µL) was plated on an appropriate agar plate for each species (TSBYE agar for A. actinomycetemcomitans, TSBYE-B agar for P. gingivalis, P. intermedia and F. nucleatum, BHI agar for streptococci and Lactobacillus), and then incubated at 37°C overnight. Inoculum density (cfu/mL) was calculated from the number of colonies on each plate. The antibacterial effect was estimated as the rate of cells surviving against the total number of cells used. To evaluate the effect of NaCl, 10 mM PB (pH 6.8) containing 10 mg/L of antimicrobial peptides with two different concentrations of NaCl (100 and 500 mM) were used in the antibacterial assay described above. Also, to evaluate the effect of saliva, we used artificial saliva instead of PB to determine the antibacterial activity. Artificial saliva contained 1.2 g of KCl, 0.844 g of NaCl, 0.34 g of K2PO4, 0.15 g of CaCl2 and 0.05 g of MgCl2 per 1 L of water (pH 7.0). We measured the antibacterial activity of 10 mg/L of antimicrobial peptides in the presence of artificial saliva.
A slightly modified method of that described by Wu and Hancock34 was the second method used to the monitor the antibacterial effect. Series of two-fold dilutions of the antibacterial peptides in the range 20001.95 mg/L were prepared in 0.2% bovine serum albumin, 0.01% acetic acid buffer in polypropylene microtubes. Each dilution (10 µL) was pipetted into the wells of a 96-well microtitre plate. Overnight cultures of bacterial strains were diluted to 106 bacterial cells per mL in half-strength of an appropriate medium for each bacterial species and 90 µL was pipetted into each well. The final concentration of each peptide was from 2000.195 mg/L. The plate was incubated at 37°C overnight in an anaerobic or aerobic condition for each species. The MIC was measured as the lowest concentration that prevented visible growth.
Electron microscopy
Thin-section electron microscopy was performed to observe the influence of each antimicrobial peptide on cultured A. actinomycetemcomitans. An overnight culture of the Y4 strain was harvested, washed with 10 mM sodium PB (pH 6.8) and suspended in the same buffer. About 109 cfu/mL of bacteria were reacted with the antimicrobial peptides at a final concentration of 100 mg/L, and incubated for 2 h at 37°C. Cells were washed with PBS and then were doubly fixed with 2.5% glutaraldehyde. The samples were dehydrated in a series of ethanol concentrations and then embedded in Spurr's Epon. Thin sections were cut on an ultramicrotome with a diamond knife and examined in a JEOL JEM-2000 EX II electron microscope at 80 kV.
Measurement of the Zeta-potential
The Zeta-potential of bacterial cells was measured by particle micro-electrophoresis using the Zeta-potential analyser Zeecom (Microtec, Nition, Funabashi, Japan). Overnight cultures of the bacterial strains were harvested, washed with 10 mM PB (pH 6.8), then resuspended with the same buffer to give a final concentration of 109 cfu/mL. Five microlitres of cell suspension was added to 10 mL of PB (pH 6.8) and the bacterial suspension applied to the apparatus for measurement of the Zeta-potential under a voltage of 100 V. The electrophoresis mobility of 100 particles of each strain was automatically measured, and the Zeta-potential calculated from the electrophoresis mobility using the Smoluchowski equation as described elsewhere.35
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Results |
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Four Gram-negative periodontopathogenic and six Gram-positive cariogenic strains were analysed (Figure 1). Compared with Gram-positive bacteria, Gram-negative bacteriaexcept F. nucleatumtended to show low susceptibility to antimicrobial peptides. The strain F. nucleatum 21 had a remarkable susceptibility to hBD3 and LL37, having 100% susceptibility in the presence of 1 mg/L of the peptides. A. actinomycetemcomitans Y4, P. gingivalis WA83 and P. intermedia 163 showed an almost similar susceptibility pattern to the peptides; hBD1 and hBD2 were less effective than hBD3 and LL37. Six Gram-positive bacteria, oral streptococci and L. casei, showed an almost similar susceptibility pattern to the peptides. Except for hBD1, all peptides demonstrated nearly 100% bactericidal activity with concentrations > 10 mg/L of the peptides.
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In the presence of 100 mM NaCl, antibacterial activities of hBD3 and LL37 on A. actinomycetemcomitans and S. mutans were not influenced, whereas that of hBD1 or hBD2 on these two strains was reduced to 50 and 80%, or 80 and 85%, respectively (Figure 2). In the presence of 500 mM NaCl, 2055% inhibition of antibacterial activity was observed with all peptides against A. actinomycetemcomitans and S. mutans.
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Susceptibility of clinical isolates to hBD3 and LL37
Forty strains of Gram-negative bacteria, including 20 of A. actinimycetemcomitans, seven of P. intermedia, six of P. gingivalis and seven of F. nucleatum were analysed (Figure 3). In Figure 3, the percentage ratio of the bacterial survival is shown when hBD3 (1 mg/L) or LL37 (1 mg/L) was used. The susceptibility of all F. nucleatum strains to hBD3 and LL37 was higher than those of other species. P. intermedia and P. gingivalis strains showed low susceptibility to hBD3, while A. actinomycetemcomitans strains showed variable susceptibility to hBD3. The four species showed a variable response to LL37 antimicrobial activity. There was no significant correlation between susceptibility to hBD3 and LL37 in each strain; some strains were highly susceptible to both peptides, whereas others were highly susceptible to only one of them.
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Electron microscopic features
Electron microscopic observations of A. actinomycetemcomitans treated with the four antimicrobial peptides revealed common morphological changes: the cytoplasmic content was released to the outside of the bacterial cells, and only cell walls lacking the inner content were observed (Figure 4). In some bacterial cells, the perforation of the peripheral cell wall shown by arrowheads was observed. The electron microscopic features were no different among bacterial specimens treated with different peptides.
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The distribution of the Zeta-potential in 100 particles of various bacterial strains is shown in Figure 5. The mean values of the Zeta-potential were variable among the different species, and it was difficult to see any correlation between the Zeta-potential and susceptibility to the peptides among the tested bacterial species (Table 2). We found some correlation in a limited number of A. actinomycetemcomitans strains. In A. actinomycetemcomitans strains, the strains (29523, IDH781, 2267) having a higher value (more negative charge) exhibited higher susceptibility to hBD3 and LL37, compared with the strains (129, SUNYaB75) having a lower value of the Zeta-potential. However, the Y4 strain was an exception. Other strains showed various values of the Zeta-potential, and we saw no correlation with the susceptibility to hBD3 and LL37.
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Discussion |
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Electron microscopic observations of A. actinomycetemcomitans cells exposed to these peptides revealed the disintegration of the outer and inner membranes, resulting in the perforation of the cell membrane. The target of ß-defensins and LL37 is thought to be the bacterial membrane and lipopolysaccharide (LPS).19,39,40 It has been reported that Treponema denticola showed resistance to antimicrobial peptides due to the lack of LPS.36 Also, the mprF S. aureus mutant that had an altered, more negative membrane charge, showed a remarkable increase in susceptibility to antimicrobial peptides.37 Consequently, the chemical composition of LPS and/or membrane in F. nucleatum may contribute to a higher susceptibility to these peptides. Other species of periodontopathogenic bacteria showed variable susceptibility to hBD3 and LL37, implying that the factors affecting the susceptibility to these peptides were different among species and strains.
Compared with Gram-negative bacteria, Gram-positive oral bacteria showed relatively high susceptibility to these peptides. Among oral streptococci, S. mutans had the highest susceptibility to hBD3, although the susceptibility to LL37 was not as high when compared with other streptococci. The proportion of S. mutants and S. sobrinus in oral streptococci in saliva and buccal mucosae is very low, and other streptococci, especially S. salivarius, S. sanguis and S. mitis, are dominant.41,42 Salivary glands and oral epithelia in gums and mucosae were reported to produce ß-defensins, LL37, carprotectin and lactoferrin.16,25,26,27,43 We demonstrated the antibacterial effect of ß-defensins and LL37 on oral bacteria in the presence of saliva (Figure 2), indicating that these peptides are active in the presence of saliva. Therefore, antimicrobial peptides in saliva may affect the composition of oral bacteria. In contrast, S. mutans and/or S. sobrinus in dental plaque are present as aggregates together with other bacterial species. Thus, they are protected by forming a biofilm producing exopolysaccharide, which might prevent exposure to antimicrobial peptides. Therefore, antimicrobial peptides could be one of the selective pressures that bacterial cells need to overcome in order to colonize specific loci in the oral environment, such as dental plaque and saliva.
Since some reports have demonstrated that the bacterial charge affected the susceptibility to these cationic antimicrobial peptides,37,44 we measured the net charge of whole live bacteria. We have shown that the tested strains possess various levels of negative charge even in the same species. In some A. actinomycetemcomitans strains we saw a correlation between the negative charge of strains and the susceptibility to antimicrobial peptides. In S. aureus, the dlt mutant has a strong negative net charge due to the lack of D-alanine esters in its teichoic acids and showed an increased susceptibility to antimicrobial peptides.44 These results suggest that the strains with a highly negative charge are more susceptible to antimicrobial peptides among the same bacterial species. However, some strains among the species were highly susceptible to these peptides although their net charge was low, implying that factors other than net charge are involved. The mechanism of antibacterial activity of ß-defensins and LL37 seems not to be completely identical because the degree of susceptibility to ß-defensin did not always correlate with the degree of LL37 susceptibility (Figure 3). Also, LL37 has been implicated as the LPS neutralizing factor, although few reports were made about the interaction between ß-defensins and LPS.39,40 Although, structural differences between ß-defensins and LL37 may result in a difference in bactericidal effect, the microscopic features of the bacterial cells exposed to ß-defensins and LL37 are similar.
In conclusion, although we found that synthetic peptides of hBD1-3 and LL37 had antimicrobial activity against oral bacteria, the activity of these peptides is different among species and strains. The net charge of bacterial cells may be one of the factors affecting the susceptibility to these peptides, but involvement of other factors should be considered. These peptides may contribute to the selective colonization of bacterial cells in the oral cavity.
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Acknowledgements |
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