Susceptibilities of periodontopathogenic and cariogenic bacteria to antibacterial peptides, ß-defensins and LL37, produced by human epithelial cells

Kazuhisa Ouhara1,2, Hitoshi Komatsuzawa1,*, Sakuo Yamada3, Hideki Shiba2, Tamaki Fujiwara1, Masaru Ohara1, Koji Sayama4, Koji Hashimoto4, Hidemi Kurihara2 and Motoyuki Sugai1

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


* Correspondence address. Department of Bacteriology, Hiroshima University Graduate School of Biomedical Sciences, Kasumi 1-2-3, Minami-ku, Hiroshima City Hiroshima 734-8553, Japan. Tel: +81-82-257-5637; Fax: +81-82-257-5639; Email: hkomatsu{at}hiroshima-u.ac.jp

Received 9 December 2004; returned 2 February 2005; revised 14 February 2005; accepted 22 February 2005


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: Antimicrobial peptides are one of the factors involved in innate immunity. The susceptibility of periodontopathogenic and cariogenic bacteria to the major antimicrobial peptides produced by epithelia was investigated.

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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mammalian cells produce several kinds of antimicrobial peptides, such as {alpha}-defensin in neutrophils, ß-defensins in epithelia, histatins in saliva and cathelicidin (CAP18 or LL37) in neutrophils and epithelia.17 These peptides have been reported to function as antimicrobial agents against Gram-negative and Gram-positive bacteria, fungi and viruses.2,4 Some of them are also implicated as mediators for inflammation (chemotactic factor).7,8 Therefore, these peptides may be an important component of the innate immune system. Among these peptides, ß-defensins are thought to be the first barrier against bacterial infection because epithelial cells in the skin and mucosae produce them.9 Four human ß-defensins (hBD1–4) have been identified in several organs.1015 It is well accepted that hBD1 is constitutively expressed, and that other peptides show inducible expression by bacterial contact. hBD1 and hBD2 are salt-sensitive, and both act mainly on Gram-negative bacteria,1317 whereas hBD3 is salt-insensitive, and effective on Gram-positive and -negative bacteria.12 CAP18 is an 18 kDa protein that is a member of the cathelicidin family of antimicrobial peptides. It has been reported that CAP18 is processed by a protease, and the last 37 amino acid residues at the C-terminus (LL37) are active against bacteria.1,18,19 LL37 has been identified in several tissues, such as neutrophils and epithelium.1,19

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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains and culture conditions

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 hBD1–3 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 {alpha}-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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Table 1. Synthetic peptides

 
Antibacterial assay

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 2000–1.95 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 200–0.195 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


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibacterial activity of hBD1, 2, 3 and LL37 against periodontopathogenic and cariogenic bacteria

Four Gram-negative periodontopathogenic and six Gram-positive cariogenic strains were analysed (Figure 1). Compared with Gram-positive bacteria, Gram-negative bacteria—except F. nucleatum—tended 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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Figure 1. Antibacterial activity of hBD1–3 and LL37 against periodontopathogenic and cariogenic bacterial species. Each peptide was incubated for 2 h at 37°C in 200 µL of 10 mM sodium PB (pH 6.8) containing 105 bacterial cells. Serial dilutions were then plated on Trypticase soy agar (TSA), and colony counts were performed after 24 h of incubation at 37°C. The ratio of bacterial survival as a percentage of survival in the presence of peptides compared with that without peptides is represented in the longitudinal axis. The results represent the means ± SD from three independent experiments. Symbols: squares, hBD1; circles, hBD2; diamonds, hBD3; triangles, LL37.

 
Table 2 shows the MICs determined by the microdilution method using the bacterial medium. Compared with the results of the assay using the PB, the antibacterial effect was weak. The MICs of hBD3 and LL37 for almost all P. gingivalis, P. intermedia and A. actinomycetemcomitans strains were 100 or 200 mg/L, whereas the MICs of the peptides for F. nucleatum showed low values (12.5 or 25 mg/L). As for Gram-positive bacteria, the MICs of the peptides were relatively lower than those for Gram-negative bacteria except for F. nucleatum. Comparison among the strains of the antibacterial effect of growing (microdilution method) and non-growing (PB) conditions revealed that there was no difference in terms of antimicrobial activity.


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Table 2. Zeta-potential and susceptibility to hBD3 and LL37

 
Effect of NaCl and saliva on the susceptibility of bacteria to the antimicrobial peptides

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, 20–55% inhibition of antibacterial activity was observed with all peptides against A. actinomycetemcomitans and S. mutans.



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Figure 2. Effect of NaCl and saliva on the susceptibility to hBD1–3 and LL37. Activities of hBD3 and LL37 against A. actinomycetemcomitans Y4 (grey bars) and S. mutans LM-7 (white bars) were measured in the presence of 100 mM NaCl, 500 mM NaCl and synthetic saliva. Each peptide (10 mg/L) was reacted with bacterial cells by the method described in the Materials and methods section. The y-axis indicates the percentage of bactericidal activity compared with that using 10 mM phosphate buffer (pH 6.8). The results represent the means ± SD from three independent experiments.

 
In the presence of saliva, antibacterial activity on A. actinomycetemcomitans showed 54% reduction when incubating with hBD1 and hBD2, 20% with hBD3 and 30% with LL37. The antibacterial activity on S. mutans showed 23% reduction with hBD1, 11% with hBD2 and no reduction with hBD3 and LL37.

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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Figure 3. Antibacterial activity of hBD3 and LL37 to various clinically isolated strains. Antibacterial activities of hBD3 (1 mg/L) and LL37 (1 mg/L) against Gram-negative (upper panels) and Gram-positive bacteria (lower panels) were analysed with the method described in the Materials and methods section. The results represent the means ± SD from three independent experiments.

 
S. mutans, S. salivarius and S. sobrinus strains tested in this study were more susceptible to hBD3 than were other Gram-positive species. The susceptibility of Gram-positive bacteria to LL37 was variable among species.

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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Figure 4. Thin sections of A. actinomycetemcomitans Y4 exposed to antimicrobial peptides. A. actinomycetemcomitans cells were exposed to PBS, or to 200 mg/L of hBD1, hBD2, hBD3 or LL37. Typical membrane perforation is shown by arrowheads. Bars, 100 nm.

 
Measurement of the Zeta-potential of whole bacteria

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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Figure 5. Measurement of the Zeta-potential of various bacterial strains. About 100 particles of bacterial cells were measured with the method described in the Materials and methods section. The distribution for each value of the particles is represented. Longitudinal axis and horizontal axis represent the number of particles and the Zeta-potential (–mV), respectively. The dashed line represents the mean value of the Zeta-potential.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have demonstrated the antimicrobial activities of hBD1–3 and LL37 against oral bacteria. The synthetic peptides used in this study all had varied antibacterial activity (Figure 1 and Table 2). Previously, two methods using growing or non-growing conditions have been used to measure the antibacterial activities of peptides.3,19,28,31,34,36,37 In the method using growing conditions, the MIC value after 24 h of incubation was determined, whereas in the method using non-growing conditions, the killing rate after 1–2 h of incubation was determined. We evaluated the antibacterial effect using these two methods. Although we found that there was no obvious difference in terms of antibacterial activity among strains, the concentration of the inhibitory effect was different in both conditions (Table 2). The peptides hBD1 and hBD2 have been reported to be less effective against Gram-positive bacteria.13,15 Although we detected the antimicrobial activities of hBD1 and hBD2 against Gram-positive bacteria, streptococci and L. casei, the antimicrobial effect of hBD3 and LL37 was stronger than that of hBD1 and hBD2, showing a similar tendency with previous results.13,15 Among ß-defensins, hBD3 had the strongest antibacterial activity. This is because hBD3 is the most basic and positively charged peptide among those tested.38 hBD3 and LL37, due to a strong charge, had less influence with the change in salt concentration. However, the effect of NaCl varied among species, showing a strong effect on S. aureus31 and a weak effect on S. mutans and A. actinomycetemcomitans. Therefore, NaCl does not affect the peptide itself, but is likely to affect the interaction between the peptides and the bacteria. Among periodontopathogenic bacteria, all F. nucleatum strains tested in this study showed the highest sensitivity to hBD3 and LL37 when compared with those of other bacteria. However, the net charge (negative charge) of F. nucleatum was not so strong compared with those of other Gram-negative bacteria. Therefore, the high susceptibility of F. nucleatum is not only due to the net charge, but also involves other factors.

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.


    Acknowledgements
 
We are grateful to Neil Ledger for editorial assistance. Part of this study was carried out in the Research Center for Molecular Medicine, Faculty of Medicine, Hiroshima University. This work was supported by a grant-in-aid for scientific research of Health and Labor Sciences Research Grants from the Ministry of Health and Welfare of Japan.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Bals, R., Weiner, D. J., Moscioni, A. D. et al. (1999). Augmentation of innate host defense by expression of a cathelicidin antimicrobial peptide. Infection and Immunity 67, 6084–9.[Abstract/Free Full Text]

2 . Ganz, T., Selsted, M. E. D., Szklarek, D. et al. (1985). Defensins. Natural peptide antibiotics of human neutrophils. Journal of Clinical Investigation 76, 1427–35.[ISI][Medline]

3 . Gyurko, C., Lendenmann, U., Troxler, R. F. et al. (2000). Candida albicans mutants deficient in respiration are resistant to the small cationic salivary antimicrobial peptide histatin 5. Antimicrobial Agents and Chemotherapy 44, 348–54.[Abstract/Free Full Text]

4 . Lehrer, R. I. & Ganz, T. (1999). Antimicrobial peptides in mammalian and insect host defense. Current Opinion in Immunology 11, 23–7.[CrossRef][ISI][Medline]

5 . Selsted, M. E., Harwig, S. S., Ganz, T. et al. (1985). Primary structures of three human neutrophil defensins. Journal of Clinical Investigation 76, 1436–9.[ISI][Medline]

6 . Xu, Y., Ambudkar, I., Yamagishi, H. et al. (1999). Histatin 3-mediated killing of Candida albicans: effect of extracellular salt concentration on binding and internalization. Antimicrobial Agents and Chemotherapy 43, 2256–62.[Abstract/Free Full Text]

7 . Yang, D., Chertov, O. & Oppenheim, J. J. (2001). Participation of mammalian defensins and cathelicidins in antimicrobial immunity: receptors and activities of human defensins and cathelicidin (LL-37). Journal of Leukocyte Biology 69, 691–7.[Abstract/Free Full Text]

8 . Yang, D., Chertov, O., Bykovskaia, S. N. et al. (1999). ß-Defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science 286, 525–8.[Abstract/Free Full Text]

9 . Bals, R. (2000). Epithelial antimicrobial peptides in host defense against infection. Respiratory Research 1, 141–50.[CrossRef][Medline]

10 . Diamond, G., Kaiser, V., Rhodes, J. et al. (2000). Transcriptional regulation of ß-defensin gene expression in tracheal epithelial cells. Infection and Immunity 68, 113–9.[Abstract/Free Full Text]

11 . Garcia, J. R., Krause, A., Schulz, S. et al. (2001). Human ß-defensin 4: a novel inducible peptide with a specific salt-sensitive spectrum of antimicrobial activity. FASEB Journal 15, 1819–21.[Abstract/Free Full Text]

12 . Harder, J., Bartels, J., Christophers, E. et al. (2001). Isolation and characterization of human ß-defensin-3, a novel human inducible peptide antibiotic. Journal of Biological Chemistry 276, 5707–13.[Abstract/Free Full Text]

13 . Harder, J., Bartels, J., Christophers, E. et al. (1997). A peptide antibiotic from human skin. Nature 387, 861.[CrossRef][ISI][Medline]

14 . Schroder, J.-M. & Harder, J. (1999). Human ß-defensin-2. International Journal of Biochemistry & Cell Biology 31, 645–51.[CrossRef][ISI][Medline]

15 . Valore, E. V., Park, C. H., Quayle, A. J. et al. (1998). Human ß-defensin-1: an antimicrobial peptide of urogenital tissues. Journal of Clinical Investigation 101, 1633–42.[Abstract/Free Full Text]

16 . Krisanaprakornkit, S., Kimball, J. R., Weinberg, A. et al. (2000). Inducible expression of human ß-defensin 2 by Fusobacterium nucleatum in oral epithelial cells: multiple signaling pathways and role of commensal bacteria in innate immunity and the epithelial barrier. Infection and Immunity 68, 2907–15.[Abstract/Free Full Text]

17 . Krisanaprakornkit, S., Weinberg, A., Perez, C. N. et al. (1998). Expression of the peptide antibiotic human ß-defensin 1 in cultured gingival epithelial cells and gingival tissue. Infection and Immunity 66, 4222–8.[Abstract/Free Full Text]

18 . Frohm, M., Agerberth, B., Ahangari, G. et al. (1997). The expression of the gene coding for the antibacterial peptide LL-37 is induced in human keratinocytes during inflammatory disorders. Journal of Biological Chemistry 272, 15258–63.[Abstract/Free Full Text]

19 . Larrick, J. W., Hirata, M., Balint, R. F. et al. (1995). Human CAP18: a novel antimicrobial lipopolysaccharide-binding protein. Infection and Immunity 63, 1291–7.[Abstract]

20 . van Houte, J. (1994). Role of micro-organisms in caries etiology. Journal of Dental Research 73, 672–81.[Abstract/Free Full Text]

21 . Kuramitsu, H. K. (1993). Virulence factors of mutans streptococci: role of molecular genetics. Critical Reviews in Oral Biology and Medicine 4, 159–76.

22 . Mayer, D. H. & Fives-Taylor, P. M. (1997). The role of Actinobacillus actinomycetemcomitans in the pathogenesis of periodontal desease. Trends in Microbiology 5, 224–8.[CrossRef][ISI][Medline]

23 . Slots, J. N., Bragd, L., Wikström, M. et al. (1986). The occurence of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, and Bacteroides intermedius in destructive periodontal desease in adults. Journal of Clinical Periodontology 13, 570–77.[ISI][Medline]

24 . Zambon, J. J., Reynolds, H., Fischer, J. G. et al. (1988). Microbiological and immunological studies of adult periodontitis in patients with noninsulin-dependent diabetes mellitus. Journal of Periodontology 5, 23–31.

25 . Tenovuo, J., Lumikari, M. & Soukka, T. (1991). Salivary lysozyme, lactoferrin and peroxidases: antibacterial effects on cariogenic bacteria and clinical applications in preventive dentistry. Proceedings of The Finnish Dental Society 87, 197–208.

26 . Dunsche, A., Acil, Y., Dommisch, H. et al. (2002). The novel human ß-defensin-3 is widely expressed in oral tissues. European Journal of Oral Sciences 109, 121–4.[CrossRef]

27 . Ross, K. F. & Herzberg, M. C. (2001). Calprotectin expression by gingival epithelial cells. Infection and Immunity 69, 3248–54.[Abstract/Free Full Text]

28 . Guthmiller, J. M., Vargas, K. G., Srikantha, R. et al. (2001). Suceptibilities of oral bacteria and yeast to mammalian cathelicidins. Antimicrobial Agents and Chemotherapy 45, 3216–9.[Abstract/Free Full Text]

29 . Larrick, J. W., Hirata, M., Shimomura, Y. et al. (1993). Antimicrobial activity of rabbit CAP18-derived peptides. Antimicrobial Agents and Chemotherapy 37, 2534–9.[Abstract]

30 . Travis, S. M., Anderson, N. N., Forsyth, W. R. et al. (2000). Bactericidal activity of mammalian cathelicidin-derived peptides. Infection and Immunity 68, 2748–55.[Abstract/Free Full Text]

31 . Midorikawa, K., Ouhara, K., Komatsuzawa, H. et al. (2003). Staphylococcus aureus susceptibility to innate antimicrobial peptides, ß-defensins and CAP18, expressed by human keratinocytes. Infection and Immunity 71, 3730–9.[Abstract/Free Full Text]

32 . Hoover, D. M., Chertov, O. & Lubkowski, J. (2001). The structure of human ß-defensin-1. Journal of Biological Chemistry 276, 39021–6.[Abstract/Free Full Text]

33 . Hoover, D. M., Rajashankar, K. R., Blumenthal, R. et al. (2000). The structure of human ß-defensin-2 shows evidence of higher order oligomerization. Journal of Biological Chemistry 275, 32911–8.[Abstract/Free Full Text]

34 . Wu, M. & Hancock, R. E. W. (1999). Interaction of the cyclic antimicrobial cationic peptide bactenecin with the outer and cytoplasmic membrane. Journal of Biological Chemistry 274, 29–35.[Abstract/Free Full Text]

35 . Miyake, Y., Tsunoda, T., Minagi, S. et al. (1990). Antifungal drugs affect adherence of Candida albicans to acrylic surfaces by changing the zeta-potential of fungal cells. FEMS Microbiology Letters 69, 211–4.[CrossRef][ISI]

36 . Brissette, C. A. & Lukehart, S. A. (2002). Treponema denticola is resistant to human ß-defensins. Infection and Immunity 70, 3982–4.[Abstract/Free Full Text]

37 . Peschel, A., Jack, R. W., Otto, M. et al. (2001). Staphylococcus aureus resistance to human defensins and evasion of neutrophil killing via the novel virulence factor mprF is based on modification of membrane lipids with L-lysine. Journal of Experimental Medicine 193, 1067–76.[Abstract/Free Full Text]

38 . Schibli, D. J., Hunter, H. N., Aseyev, V. et al. (2002). The solution structure of the human ß-defensins lead to a better understanding of the potent bactericidal activity of HBD3 against Staphylococcus aureus. Journal of Biological Chemistry 277, 8279–89.[Abstract/Free Full Text]

39 . Scott, M. G., Gold, M. R. & Hancock, R. E. W. (1999). Interaction of cationic peptides with lipoteichoic acid and gram-positive bacteria. Infection and Immunity 67, 6445–53.[Abstract/Free Full Text]

40 . Scott, M. G., Vreugdenhil, A. C. E., Buurman, W. A. et al. (2000). Cationic antimicrobial peptides block the binding of lipopolysaccharide (LPS) to LPS binding protein. Journal of Immunology 164, 549–53.[Abstract/Free Full Text]

41 . Nyvad, B. & Kilian, M. (1990). Comparison of the initial streptococcal microflora on dental enamel in caries-active and in caries-inactive individuals. Caries Research 24, 267–72.[ISI][Medline]

42 . Sheehy, E. C., Beighton, D. & Roberts, G. J. (2000). The oral microbiota of children undergoing liver transplantation. Oral Microbiology and Immunology 15, 203–10.[CrossRef][ISI][Medline]

43 . Mathews, M., Jia, H. P., Guthmiller, J. M. et al. (1999). Production of ß-defensin antimicrobial peptides by the oral mucosa and salivary glands. Infection and Immunity 67, 2740–5.[Abstract/Free Full Text]

44 . Peschel, A., Otto, M., Jack, R. W. et al. (1999). Inactivation of the dlt operon in Staphylococcus aureus confers sensitivity to defensins, protegrins, and other antimicrobial peptides. Journal of Biological Chemistry 274, 8405–10.[Abstract/Free Full Text]