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Infection and Immunity, December 2009, p. 5558-5563, Vol. 77, No. 12
0019-9567/09/$08.00+0     doi:10.1128/IAI.00648-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Saliva Enables the Antimicrobial Activity of LL-37 in the Presence of Proteases of Porphyromonas gingivalis ^,

Michal Gutner,^1, Stella Chaushu,^2, Daniela Balter,¹ and Gilad Bachrach^1*

Institute of Dental Sciences,¹ Department of Orthodontics, Hebrew University-Hadassah School of Dental Medicine, Jerusalem, Israel²

Received 8 June 2009/ Returned for modification 6 July 2009/ Accepted 19 September 2009

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Proteolysis is a common microbial virulence mechanism that enables the destruction of host tissue and evasion from host defense mechanisms. Antimicrobial peptides, also known as host defense peptides, are effector molecules of the innate immunity that demonstrate a broad range of antimicrobial and immunoregulatory activities. Deficiency of the human LL-37 antimicrobial peptide was previously correlated with severe periodontal disease. Porphyromonas gingivalis, the major pathogen associated with periodontitis, is highly proteolytic. In this study, P. gingivalis was found capable of degrading LL-37 by utilizing its arginine-specific gingipains. Saliva collected from volunteers with a healthy periodontium protected LL-37 from proteolysis by P. gingivalis. Salivary protection of LL-37 was heat resistant and specific and enabled LL-37 to inhibit growth of Escherichia coli in the presence of the P. gingivalis proteases. Previously, saliva and other body fluids have been shown to inhibit the antimicrobial activity of LL-37. Here we demonstrate that at a cost of a small reduction in the bactericidal activity of LL-37, saliva enables the antibacterial activity of LL-37 despite the presence of proteases secreted by the main periodontopathogen.

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Cationic antimicrobial peptides are components of the innate immunity that mediate a broad range of antimicrobial activity and play an important role in mucosal protection (48). In mammals, antimicrobial peptides also function as immunomodulators of the innate immune system that alter gene expression in host cells, induce or modulate chemokine and cytokine production, and elicit or inhibit proinflammatory responses (6, 24, 40, 46). Since the recognition of their immunoregulatory functions, antimicrobial peptides are often referred to as host defense peptides. The - and β-defensins, histatins, and LL-37 antimicrobial peptides play an important role in protection of the oral cavity (7, 11, 20, 36). LL-37, the only human host defense peptide of the cathelicidin family, is cleaved extracellularly from its 18-kDa human cationic antimicrobial protein (hCAP18) precursor into the biologically active 37-amino-acid antimicrobial peptide.

Periodontitis is a chronic inflammatory disease that leads to destruction of the attachment apparatus of the teeth. Deficiency of salivary LL-37 in patients with morbus Kostmann syndrome (36) or with Papillon-Lefevre syndrome (10) was previously correlated with severe periodontitis. Porphyromonas gingivalis is an oral anaerobe and the pathogen most associated with chronic periodontal disease (16, 18, 42). P. gingivalis has previously been found to be highly resistant to antimicrobial peptides (1, 33). The Arg-gingipains and Lys-gingipain cysteine proteases (cleaving after arginine and lysine, respectively) are among the major virulence factors expressed and secreted by P. gingivalis. Being positively charged, arginine and lysine are highly represented in host defense peptides, including LL-37 (which has five arginines and six lysines). Not surprisingly, P. gingivalis was found to be capable of degrading antimicrobial peptides in vitro and under ex vivo conditions with human serum (12) and of inactivating the antibacterial activity of LL-37 (2). Here, we report that saliva added under ex vivo conditions mimicking those in the oral environment protects the human LL-37 antimicrobial peptide from degradation and enables its antimicrobial activity in the presence of the proteases of P. gingivalis.

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Saliva. Whole saliva was collected from five healthy donors and clarified by centrifugation at 1,500 x g for 10 min followed by filtration (0.2 µm; Whatman Schleicher & Schuell, Germany). Saliva samples were pooled and kept in aliquots at –20°C. All donors provided an informed consent to a protocol that was approved by the institutional ethical committee.

Bacterial strains and growth conditions. P. gingivalis ATCC 33277 was grown in Wilkins broth (Oxoid, United Kingdom). P. gingivalis KDP133 (P. gingivalis ATCC 33277 with rgpA and rgpB inactivated) and P. gingivalis KDP129 (P. gingivalis ATCC 33277 with kgp inactivated) were generously supplied by Koji Nakayama (41) and grown in enriched brain heart infusion (BHI) broth (containing 37 g of BHI [Difco, MD], 5 g of yeast extract [Difco], 1 g of cysteine, 5 mg of hemin, and 1 mg of vitamin K₁ per liter) supplemented with erythromycin at 10 µg/ml and tetracycline at 0.7 µg/ml (for P. gingivalis KDP133) or with chloramphenicol at 20 µg/ml (for P. gingivalis KDP129). Treponema denticola ATCC 35404 was grown in GM-1 medium (5). T. denticola and P. gingivalis were grown in jars containing an anaerobic atmosphere generation system (Oxoid, United Kingdom). Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans Y4 (31), was cultured in 0.5% yeast extract, 1.5% Bacto tryptone, 0.75% D-glucose, 0.25% NaCl, 0.075% L-cysteine, 0.05% sodium trioglycolate, and 4% NaHCO₃. Streptococcus mutans ATCC 27351 was cultured in BHI broth. S. mutans and A. actinomycetemcomitans were grown at 37°C in an atmosphere enriched with 5% CO₂. Escherichia coli ATCC 25922 was grown in BHI broth under aerobic conditions. Bacterial purity was determined by microscopy and Gram staining.

LL-37 peptide, rhCAP18, and rHSP65. LL37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) was synthesized as described previously by the solid-phase method on a fully automated, programmable peptide synthesizer (model 433A; Applied Biosystems, Foster City, CA) (4). Peptide integrity and purity (higher than 95%) were determined by analytical high-performance liquid chromatography and mass spectrometry. rhCAP18, the recombinant cathelicidin domain of hCAP18 (lacking LL-37 at its C terminus), a kind gift of R. L. Gallo (47), was expressed in E. coli BL21(DE3) and purified under denaturating conditions using Ni-nitrilotriacetic acid columns (3). Recombinant 60-kDa heat shock protein (rHSP65) of Fusobacterium nucleatum was generated by amplifying the F. nucleatum ATCC 10953 hsp60 gene using specific PCR primers Fn605 (GGGGGATCCGCAAAAATTATAAATTTTAATGATG) and Fn606 (GGGAAGCTTCATCATTCCTGGCATCATTC) (containing BamHI and HindIII restriction sites [in bold], respectively) and cloning the amplification product into the BamHI and HindIII restriction sites of the E. coli pQE30 expression vector (Qiagen, Germany). The resulting plasmid, pQE65F, was transformed into E. coli strain SG13009, and rHSP65 was expressed and purified as described above.

Protein quantification. Protein concentrations of rhCAP18 and rHSP65 and in whole saliva were determined using the Bio-Rad protein assay.

Antibodies. Rabbit antisera were generated against the synthetic LL-37 peptide (4) and rhCAP18 (Harlan Biotech, Israel), as described previously (3), after receiving approval from the Animal Care and Use Committee of the Hebrew University of Jerusalem. LK2, an anti-HSP60 monoclonal antibody (StressGen) was used to detect rHSP65.

Degradation of LL-37 and rhCAP18 by P. gingivalis. P. gingivalis protease-containing conditioned culture supernatant was prepared from stationary-stage-grown P. gingivalis cultures by sedimentation of the bacterial cells at 1,500 x g for 20 min followed by filter sterilization (0.2 µm; Whatman Schleicher & Schuell, Germany). P. gingivalis conditioned medium (5 µl, when tested) was added to 30 ng LL-37 or rhCAP18 and brought to a total reaction volume of 20 µl with phosphate-buffered saline (PBS). Antiprotease cocktail (1 µl; Sigma-Aldrich) was added where indicated. Following incubation (as indicated in the figure legends), reaction mixtures were loaded on a 15% sodium dodecyl sulfate (SDS)-acrylamide gel, and remaining LL-37 or rhCAP18 was detected using Western immunodetection as described before (4).

Effect of saliva on degradation of LL-37, rhCAP18, or rHSP65 by P. gingivalis. P. gingivalis conditioned culture supernatant (5 µl, when tested) and saliva (13 µl, when tested, unless otherwise indicated) were added to LL-37, rhCAP18, or rHSP65 (2 µl, 15 ng/µl) and brought to a total volume of 20 µl with PBS. Incubation and detection of remaining LL-37, rhCAP18, or rHSP65 were performed as described above.

Effect of saliva on the Arg-gingipain activity of P. gingivalis. The effect of saliva on the trypsin-like activity of the P. gingivalis Arg-gingipains was determined using N-benzoyl-L-arginine 4-nitroanilide hydrochloride (BAPNA) (Sigma-Aldrich, Germany), an arginine protease chromogenic substrate. BAPNA solution was prepared by dissolving 1 mg BAPNA in 200 µl of dimethyl sulfoxide and adding 1.8 ml of BAPNA buffer (0.2 M Tris-HCl [pH 8.0], 0.1 M NaCl, 0.05 M CaCl₂, 0.05 M L-cysteine). Aliquots of 100 µl of BAPNA solution were added to wells of 96-well plates containing 20 µl of P. gingivalis culture supernatant (or BAPNA buffer when indicated) and 80 µl of one of the following: BAPNA buffer, saliva (native or boiled), or bovine serum albumin (BSA) (2 mg/ml in PBS). Plates were incubated at 37°C for 30 min, and optical density measured at 405 nm using a Thermo Max microplate spectrophotometer (Molecular Devices, CA).

Effect of saliva on the antibacterial activity of LL-37. LL-37 (10 µl, containing 400, 800, or 1,200 µg/ml), with or without P. gingivalis culture supernatant (10 µl) and in the presence or absence of saliva (30 µl), was brought to a total volume of 50 µl with PBS and added to 150 µl of E. coli ATCC 25922 (overnight culture, diluted 1:1,000 in BHI). The optical density (595 nm) was measured after 5 h of aerobic incubation at 37°C using a Thermo Max microplate spectrophotometer (Molecular Devices, CA).

Gel shift assays. LL-37 or rhCAP18 (2 µl, 15 ng/µl), preincubated or not with saliva (13 µl, for 3 h at 37°C), was brought to a total volume of 20 µl with PBS and subjected to native polyacrylamide electrophoresis as described above but at 4°C and omitting SDS (in all solutions), 2-mercaptoethanol, and sample boiling.

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LL-37 is degraded by P. gingivalis. Western blot immunodetections using anti-human LL-37 rabbit serum demonstrated the ability of conditioned culture supernatant prepared from P. gingivalis to degrade the LL-37 antimicrobial peptide (Fig. 1). Conditioned media prepared from the oral pathogens S. mutans and A. actinomycetemcomitans and from the highly proteolytic periodontal pathogen T. denticola did not affect the integrity of LL-37 under similar conditions (Fig. 1).

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FIG. 1. LL-37 degradation by P. gingivalis. LL-37 was incubated for 3 hours with conditioned medium prepared from T. denticola (A), A. actinomycetemcomitans (B), S. mutans (C), or P. gingivalis (D) or without culture supernatant (E) and subjected to SDS-polyacrylamide gel electrophoresis followed by Western immunodetection using anti-LL-37 rabbit serum.

LL-37 degradation was prevented if the P. gingivalis supernatant was boiled for 5 min prior to incubation with LL-37 or treated with protease inhibitors during incubation with LL-37 (not shown), confirming that the P. gingivalis proteases induced the disappearance of LL-37.

Saliva prevents digestion of LL-37, of the cathelicidin domain of the hCAP18 precursor of LL-37, and of salivary hCAP18 by P. gingivalis proteases. The ability of the P. gingivalis proteases to degrade LL-37 was tested under ex vivo conditions with saliva. As can be seen in Fig. 2A, whole saliva prevented LL-37 degradation by the P. gingivalis proteases in a dose-dependent manner. As mentioned above, LL-37 is processed from the 18-kDa hCAP18 precursor. Rabbit antiserum prepared against the recombinant cathelicidin domain of hCAP18 (rhCAP18, lacking LL-37 at its C terminus) revealed that saliva prevented rhCAP18 degradation by culture supernatants prepared from P. gingivalis (Fig. 2B) and from T. denticola (not shown). These results indicate that the inhibition of hCAP18 degradation by P. gingivalis is not LL-37 dependent. The endogenous full-length hCAP18 precursor of LL-37 that was detected in the added saliva by the LL-37 specific antibodies also remained mostly intact following overnight incubation with the P. gingivalis proteases (Fig. 2C).

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FIG. 2. Whole saliva prevents degradation of LL-37, rhCAP18, and hCAP18 by P. gingivalis. (A and B) Western immunodetection of LL-37 (A) or rhCAP18 (B) incubated for 3 hours with conditioned medium prepared from P. gingivalis in the absence or presence of saliva. (C) Intact salivary hCAP18 can be detected by Western immunodetection of LL-37 incubated overnight with conditioned medium prepared from P. gingivalis in the presence of 13 µl of saliva.

The ability of saliva to prevent LL-37 and rhCAP18 degradation is specific and heat resistant. The protein content of saliva used in our experiments was measured to be 1.3 mg/ml. As can be seen in Fig. 3A, BSA at a concentration higher than that of salivary proteins (2 mg/ml) did not inhibit LL-37 degradation by the P. gingivalis culture supernatant. This result excluded the possibility that saliva prevented proteolysis of LL-37, hCAP18, and rhCAP18 by P. gingivalis due to competition of nonspecific salivary proteins with LL-37 over the available proteases.

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FIG. 3. Saliva's ability to prevent degradation of LL-37 and rhCAP18 by P. gingivalis is specific. (A) Western immunodetection of LL-37 incubated for 3 hours in the presence of BSA (2 mg/ml) with or without conditioned medium prepared from P. gingivalis. (B) Western immunodetection of recombinant F. nucleatum hsp65 incubated overnight with native whole saliva (lane 1) or boiled whole saliva ((lane 2) or without saliva (lane 3), with or without P. gingivalis conditioned medium.

Specificity of the salivary ability to prevent LL-37, hCAP18, and rhCAP18 degradation by P. gingivalis was also demonstrated using a control protein. Degradation of the randomly chosen rHSP65 of F. nucleatum by the P. gingivalis conditioned supernatant was not prevented by saliva (Fig. 3B, lanes 1 and 2). Moreover, rather than being protected by saliva, rHSP65 was degraded by native saliva (Fig. 3B, lane 1). When incubated with boiled saliva, rHSP65 was cleaved only when conditioned medium prepared from P. gingivalis was added (Fig. 3B, lane 2). Heat treatment (boiling for 10 min) of saliva did not hamper the saliva's ability to prevent LL-37 degradation by the P. gingivalis proteases (data not shown).

LL-37 is degraded by the P. gingivalis Arg-gingipain. P. gingivalis mutants were used to identify the protease capable of degrading LL-37. In strain KDP129, the gene encoding the Lys-gingipain (kgp) is inactivated. This mutant, which expresses both Arg-gingipain A and Arg-gingipain B, degraded LL-37 (Fig. 4, lanes A and B). Mutant KDP133, defective in both Arg-gingipains (rgpA rgpB) failed to degrade LL-37 (Fig. 4, lanes C and D), indicating that LL-37, which contains five arginines and six lysines, is cleaved by the Arg-gingipains.

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FIG. 4. LL-37 is cleaved by Arg-gingipains. Western immunodetection of LL-37 incubated overnight with conditioned medium prepared from P. gingivalis strain KDP129 (ATCC 33277 with kgp inactivated, duplicate in lanes A and B), KDP133 (ATCC 33277 with both rgpA and rgpB inactivated, duplicate in lanes C and D), ATCC 33277 (wild type, duplicate in lanes E and F), and LL-37 incubated without conditioned medium (lane G) is shown.

Saliva does not inhibit the Arg-gingipains of P. gingivalis. Saliva can prevent degradation of human immunoeffectors such as LL-37 and its hCAP18 precursor by two mechanisms. The first is by inhibiting the P. gingivalis proteases, and the second is by blocking the restriction sites in the immunoeffectors and thus protecting them from proteolysis. In order to determine which of these mechanisms prevents LL-37 degradation, the effect of saliva on the trypsin-like activity of the P. gingivalis Arg-gingipains was determined using BAPNA, an arginine protease chromogenic substrate. Surprisingly, rather than the Arg-gingipain activity being inhibited, arginine proteolysis was increased (to 233%) when saliva was added to the P. gingivalis supernatant (Fig. 5). It appears that the added P. gingivalis proteases activated latent salivary proteases. Saliva without P. gingivalis supernatant produced only background levels of proteolysis (Fig. 5). Boiled saliva added to the P. gingivalis supernatant did not yield an increase in proteolytic activity. These results confirmed that the increase in proteolytic activity measured when saliva was incubated with the P. gingivalis proteases originated from activated salivary proteases. It has been suggested previously that P. gingivalis hydrolyzes salivary protease inhibitors, leading to the activation of salivary proteases (17). The fact that saliva did not inhibit the Arg-gingipains found capable of hydrolyzing LL-37 suggests again that saliva prevents degradation of LL-37, hCAP18, and rhCAP18 by P. gingivalis through specific protection rather than by inhibiting the gingipains' activities.

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FIG. 5. Saliva increases arginine specific trypsin-like activity of P. gingivalis supernatant. The protease activity of P. gingivalis conditioned medium coincubated with BSA, whole saliva, or boiled whole saliva is represented as the percentage of activity relative to that of reaction control (P. gingivalis conditioned medium [see Materials and Methods]). Open bars represent addition of P. gingivalis conditioned medium; hatched bars represent background signals obtained without P. gingivalis conditioned medium. Data represent means and standard deviations from one representative experiment out of three performed in triplicate.

Saliva alters the mobility of LL-37 and of rhCAP18 in native polyacrylamide electrophoresis. rhCAP18 and LL-37 were subjected to native polyacrylamide electrophoresis with or without preincubation with saliva. Incubation of rhCAP18 (Fig. 6) and of LL-37 (not shown) with saliva altered their mobility in native gels, suggesting that salivary components bound rhCAP18 and LL-37 or altered their conformation/charge. The change in mobility of rhCAP18 and of LL-37 following incubation with saliva supports once again the hypothesis that saliva prevents degradation of hCAP18 and of LL-37 by the P. gingivalis proteases by protecting them rather than by inhibiting the proteases.

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FIG. 6. Saliva alters the mobility of rhCAP18 in native polyacrylamide gels. Western immunodetection of rhCAP18 not preincubated (triplicate, lanes A to C) or preincubated (triplicate, lanes D to F) with saliva and subjected to native polyacrylamide gel electrophoresis is shown.

Saliva enables direct antibacterial activity of LL-37 in the presence of the proteases of P. gingivalis. As can be seen in Fig. 7, at concentrations of 20 µg/ml or higher, LL-37 inhibited the growth of an E. coli ATCC 25922 culture by 98%. Addition of conditioned medium prepared from P. gingivalis abolished this antimicrobial activity at all tested concentrations (up to 60 µg/ml). However, after addition of conditioned medium of P. gingivalis in the presence of saliva, the antimicrobial activity of LL-37 was retained at concentrations of 40 µg/ml or higher (99% growth inhibition) (Fig. 7). These results indicate that saliva protects LL-37's antimicrobial activity in the presence of P. gingivalis proteases.

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FIG. 7. Saliva enables antibacterial activity of LL-37 in the presence of the proteases of P. gingivalis. Growth inhibition of E. coli ATCC 25922 grown in the presence of LL-37 at the indicated final concentration, treated or untreated with P. gingivalis culture supernatant and in the presence or absence of saliva. Data represent means and standard deviations (smaller than the symbols) from one representative experiment out of three performed in triplicate.

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This study has demonstrated that P. gingivalis proteases can degrade LL-37 and rhCAP18 and thereby presumably shield P. gingivalis and neighboring dental plaque microorganisms from their host's protective activities. Saliva from healthy donors however, was found to prevent the degradation of LL-37 and of hCAP18 by P. gingivalis by means of a dose-dependent, heat-resistant mechanism. This mechanism was found to be specific, because saliva did not prevent the degradation of a randomly selected control protein (rHSP65).

Digestion of LL-37 by P. gingivalis was found to be executed by the Arg-gingipains. We have also shown that saliva did not inhibit the P. gingivalis Arg-gingipains; on the contrary, P. gingivalis proteases were found to activate salivary proteolytic activities that probably work in a destructive synergism with the P. gingivalis proteases.

Our results that show that the Arg-gingipains are not inhibited by saliva indicate that the salivary mechanism of preventing LL-37 and rhCAP18 degradation by P. gingivalis is by protection rather than by protease inhibition. These results therefore suggest the existence of a salivary factor(s) that protects LL-37 and hCAP18 from degradation by P. gingivalis. The existence of such a salivary protective factor was supported by our observation that saliva alters the mobilities of LL-37 and rhCAP18 in native polyacrylamide gels. Our results also demonstrated that while protecting LL-37 from degradation by the proteases of P. gingivalis, the salivary LL-37-protective factor enabled the direct bactericidal activity of LL-37 (as tested with E. coli).

Our observations so far suggest that saliva from orally healthy individuals is capable of protecting LL-37 and hCAP18 from proteolysis by P. gingivalis. This protective activity was found to be necessary to enable the direct bactericidal activity of LL-37 (as tested on E. coli) in the presence of virulence-associated proteases of P. gingivalis.

Similarly to LL-37, hCAP18 and rhCAP18 are positively charged (their theoretical pIs are 10.61, 9.48, and 8.39, respectively). Negatively charged proteoglycans have previously been shown to bind the positively charged antimicrobial peptides and to protect them from proteolysis by Pseudomonas aeruginosa, Streptococcus pyogenes, and Enterococcus faecalis (38). However, this protective binding was reported to abolish the bactericidal activity of the tested antimicrobial peptides (38). Furthermore, increasing the shedding of cell surface heparan sulfate proteoglycans has been shown to function as a virulence mechanism of P. aeruginosa that enhances its resistance to host antimicrobial peptides and results in increased mortality in lung-infected newborn mice (34). Here, salivary binding appears to be beneficial to the host because it enables the direct antimicrobial activity of LL-37 at low concentrations, despite the presence of proteases prevalent in periodontal disease.

Recent observations suggest that in higher evolutionary primates, direct microbiocidal activity is not the only role of antimicrobial peptides (49). Along with these findings, antimicrobial peptides are often referred to as host defense peptides (26, 39, 49) and alarmins (32). Apart from its protective microbiocidal activity (9), LL-37 and its murine homolog CRAMP (30) were found to trigger host tissue responses. These include binding and neutralizing adverse inflammatory effects of lipopolysaccharides (25, 37), wound healing by involvement in reepithelialization (19), angiogenic effects (21), and various host defense and immunomodulation activities, including cytokine induction (15, 24) and repression (29) and chemoattraction (45). It remains to be seen whether saliva enables such nonmicrobiocidal functions of LL-37 (and of other antimicrobial peptides) in the presence of pathogen-associated proteases.

Serum lipoproteins were found to bind and neutralize the antimicrobial activity of LL-37 (43, 44). It was suggested that the relatively high concentration of LL-37 in human serum might cause a toxic effect and therefore that LL-37 is blocked (a releasing mechanism has yet to be described). Unlike the circulatory system, the oral cavity can be considered an extracorporeal environment. Therefore, LL-37 neutralization might not be necessary. On the contrary, the oral cavity harbors a vast variety of true and opportunistic pathogens that are controlled by antimicrobial peptides. A recent article reported that saliva reduced the bactericidal activity of LL-37 and that mucins bound and inhibited the antimicrobial action of LL-37 (8). We also found that saliva slightly reduced the antimicrobial activity of LL-37. Saliva increased the minimal concentration of LL-37 required to inhibit growth of E. coli from 10 to 15 µg/ml (data not shown). At an LL-37 concentration of 20 µg/ml, adding saliva reduced growth inhibition of E. coli from 98% to 91% (Fig. 7). However, this relatively small reduction in LL-37 activity was found to enable the antimicrobial activity of LL-37 at concentrations of 40 to 60 µg/ml in the presence of proteases of P. gingivalis.

It is unlikely that saliva can penetrate the chronically inflamed periodontal pocket where P. gingivalis can be found in high numbers, which questions the biological relevance of the salivary protection of LL-37 from degradation by P. gingivalis. Low levels of P. gingivalis, however, are found in early-developing, saliva-exposed supraginigival plaque (13, 27), which is presumed to spread down into the gingival sulcus and form the subgingival-periodontitis-associated biofilm (23). Though gingipain levels might be negligible in saliva, gradient levels of gingipain are expected to reach physiological concentrations in proximity to supragingival P. gingivalis. A distance-dependent signal gradient in oral biofilms was previously suggested (14). We therefore suggest that in these early stages of P. gingivalis integration into the supragingival biofilm, salivary protection of LL-37 from gingipain-mediated proteolysis might have a meaningful role in periodontal health.

Previous experiments using a protease-resistant d enantiomer peptide, a protease-deficient P. gingivalis mutant, and a fluorescently labeled antimicrobial peptide revealed that resistance of P. gingivalis to direct killing by LL-37 is protease independent and results (at least partially) from the low affinity of antimicrobial peptides to P. gingivalis (2). Because proteolytic degradation of LL-37 is nonessential for survival of P. gingivalis, it was suggested that LL-37 proteolysis by P. gingivalis provides neighboring dental plaque species with resistance to LL-37, which in turn benefits P. gingivalis (2). Indeed, it has been demonstrated recently that F. nucleatum, which is sensitive to LL-37 (1), enables P. gingivalis growth in saliva (35). "Group protection," where a resistant species in the multispecies dental biofilm protects another species that is sensitive to a host factor, has been suggested previously (2, 22, 28). Here we suggest a host countermeasure for such phenomena.

 
We thank Koji Nakayama for generously providing P. gingivalis strains KDP133 and KDP129 and R. L. Gallo for generously supplying rhCAP18.

This work was supported by the Israel Science Foundation (grant 517/06). M. Gutner was supported in part by the Abisch-Frenkel Foundation award for young scientists.

 
* Corresponding author. Mailing address: Institute of Dental Sciences, Hebrew University-Hadassah School of Dental Medicine, Jerusalem, Israel. Phone: 972-2-6757117. Fax: 972-2-6758561. E-mail: giladba{at}ekmd.huji.ac.il

Published ahead of print on 5 October 2009.

The authors have paid a fee to allow immediate free access to this article.

Editor: A. Camilli

These authors contributed equally to the study.

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Infection and Immunity, December 2009, p. 5558-5563, Vol. 77, No. 12
0019-9567/09/$08.00+0     doi:10.1128/IAI.00648-09
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