Skip Navigation



JAC Advance Access published online on May 1, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn176
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
62/2/329    most recent
dkn176v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Bucki, R.
Right arrow Articles by Janmey, P. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bucki, R.
Right arrow Articles by Janmey, P. A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Original research

Salivary mucins inhibit antibacterial activity of the cathelicidin-derived LL-37 peptide but not the cationic steroid CSA-13

Robert Bucki1,*, Dorota B. Namiot2, Zbigniew Namiot3, Paul B. Savage4 and Paul A. Janmey1

1 Department of Physiology, Institute for Medicine and Engineering, University of Pennsylvania, 1010 Vagelos Research Laboratories, 3340 Smith Walk, Philadelphia, PA 19104, USA 2 Department of Prosthetic Dentistry, Medical University of Bialystok, 15-230 Bialystok, Poland 3 Department of Physiology, Medical University of Bialystok, 15-230 Bialystok, Poland 4 Department of Chemistry and Biochemistry, Brigham Young University, C-I00 BNSN, Provo, UT 84602, USA

* Corresponding author. Tel: +1-215-573-9787; Fax: +1-215-573-7227; E-mail: buckirob{at}mail.med.upenn.edu

Received 29 August 2007; returned 2 January 2008; revised 20 February 2008; accepted 29 March 2008


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 Transparency declarations
 References
 
Objectives: Cationic antimicrobial peptides (CAPs) are the effector molecules of innate immunity, similar in potency to classic antibiotics that function in the first-line of defence against infectious agents. The purpose of this study was to investigate the effects of negatively charged mucins on the antibacterial activity of the positively charged cathelicidin LL-37 peptide, its synthetic analogue WLBU2 and the antimicrobial cationic steroid CSA-13.

Methods: Mucin, DNA, F-actin and hCAP-18/LL-37 in saliva samples were evaluated by microscopy or immunoblotting. Bacterial killing assays and determination of MICs were used to determine bactericidal activity. Binding of rhodamine-B-labelled LL-37 peptide to mucin was fluorimetrically assessed.

Results: Microscopic evaluation of saliva after addition of rhodamine-B-labelled LL-37 showed localization similar to that observed after the addition of a specific mucin-binding lectin. Immunoblotting confirmed the presence of hCAP-18/LL-37 in saliva samples and LL-37 peptide bound to isolated submaxillary gland mucin-coated plates. Mucin/LL-37 binding was partially prevented by treatment of mucin with neuraminidase, indicating involvement of sialic acid moieties. Decreased LL-37 and WLBU2 antibacterial activity was observed in the presence of mucin or dialysed human saliva, whereas CSA-13 antibacterial activity was significantly resistant to inhibition by mucins.

Conclusions: This study shows that the antibacterial LL-37 peptide and its synthetic analogue WLBU2 are inhibited by salivary mucin and that the cationic steroid CSA-13 retains most of its function in the presence of an equal amount of mucin or saliva.

Key Words: neuraminidase , hCAP-18 , antibacterial peptides , WLBU2


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 Transparency declarations
 References
 
Cationic antimicrobial peptides (CAPs) are widely expressed in the skin, mouth, airways, digestive tract and genitourinary system as an important component of complex secretions, acting synergistically to prevent infection and to control resident microbial populations.1–5 Saliva contains a range of CAPs including {alpha}- and β-defensins, the cathelicidin-derived LL-37 peptide, histatins and secretory leukocyte protease inhibitor. Most of these agents are synthesized in salivary glands and duct cells or are released by neutrophils.6–9 LL-37 peptide is proteolytically processed from human cathelicidin, hCAP-18, which is secreted following cell stimulation and cleaved extracellularly by proteinase-3 or neutrophil elastase.10 LL-37 acts directly as an antimicrobial11 against periodontopathogenic and cariogenic bacteria,12 functions as an immunomodulatory molecule,13 neutralizes lipopolysaccharide (LPS) bioactivity14 and acts as a cell membrane receptor agonist stimulating epithelial cell proliferation, angiogenesis, wound healing and cytokine release.1,15 At mucosal surfaces of healthy individuals, LL-37 can be detected at concentrations of 2–5 µg/mL, but during infection, its concentration increases 400% to 500%.16 The importance of salivary CAPs becomes apparent in patients with severe congenital neutropenia (‘Morbus Kostmann’) suffering from deficiency of saliva LL-37 and other neutrophil-derived molecules that result in a high occurrence of periodontal disease.17,18 Similarly, significantly lower levels of {alpha}-defensins were observed in children with caries,2 suggesting that CAPs are important factors controlling the growth of microorganisms in the oral cavity.19

Among the salivary proteins, mucins are primarily responsible for the lubricating and film-forming properties of saliva.20,21 They are complex glycoproteins synthesized in epithelial cells and are typically characterized by large molecular weight (2–20 x 105 Da). Terminal carboxyl groups of sialic acid and sulphates on galactose or N-acetylglucosamine moieties impart negative charges and confer rigidity to mucins.22 Upon bacterial neuraminidase and sulphatase action, sialic acids and sulphate residues can be removed from mucin unmasking underlying sugars to further enzymatic attack, promoting mucin degradation. This process can cause dysfunction of the mucosal barrier and represents an important step during bacterial invasion. Epithelial cells subjected to the bacterial wall products, LPS or lipoteichoic acid, increase the expression of hCAP-18/LL-37 and mucin,23 indicating that at sites of infection, inducible CAPs may function in the presence of increasing mucin concentrations. Charge-based interactions between CAPs and negatively charged polyelectrolytes such as DNA, F-actin, mucins and exopolysaccharides were previously shown to interfere with CAPs' bacterial killing activities.24–26 The purpose of this study was to investigate the influence of negatively charged mucins on the antibacterial activities of positively charged LL-37 and WLBU2 peptides, and the ceragenin CSA-13. In vitro assays show that LL-37 is present in saliva and interacts with salivary mucin. This interaction inhibits the antibacterial function of LL-37 and is reversed by treatment of mucin with neuraminidase. Ceragenin CSA-13 antibacterial activity is more resistant to inhibition by mucins when compared with LL-37 and WLBU2 peptides.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 Transparency declarations
 References
 
Antibacterial agents

LL-37, rhodamine-B-labelled LL-37 (RhB-LL-37) and WLBU2 were purchased from Bachem (King of Prussia, PA, USA). CSA-13 was prepared as described previously27 (Table 1). Stock solutions of antibacterial molecules were prepared in PBS.


View this table:
[in this window]
[in a new window]

  		Table 1. Characteristics of antibacterial agents

 
Saliva collection

Unstimulated whole saliva was collected by the spitting method from healthy donors aged 20–28 years, between 8 and 11 AM. All subjects had refrained from eating or drinking for 2 h prior to collection. Samples used for the killing assay were centrifuged at 1000 g for 5 min at 4°C, and the resulting clarified supernatant fluid was dialysed against PBS using Slide-A-Lyzer dialysis cassettes (20000 MWCO, Pierce, Rockford, IL, USA) to eliminate endogenous low molecular weight saliva factors with antibacterial activity. The study was approved by Medical University of Bialystok Ethics Committee for Research on Humans and Animals, and written consent was obtained from all subjects.

Optical microscopy

Saliva samples were viewed using a Leica microscope (Leica Microsystems Inc., Bannockburn, IL, USA) with a 40x objective. Images were acquired using a Cool SNAP (HQ) camera (Trenton, NJ, USA). Mucin was labelled using Texas Red-conjugated Ulex europaeus lectin (Sigma, St Louis, MO, USA), as described previously.28 DNA was labelled with YOYO-1 (Molecular Probes, Boulder, CO, USA) and F-actin was labelled with rhodamine (TRITC) phalloidin (Sigma). In addition to visualizing stained DNA and F-actin, a pattern of fluorescence was also acquired after incubation of saliva samples with RhB-LL-37 (1 µM) for 5 min.

Immunoblot analysis

Samples of saliva were added to gel sample buffer (62.5 mM Tris–HCl, pH 6.8; 2% SDS, 25% glycerol, 0.01% Bromophenol Blue, protease inhibitor cocktail 150 µL/10 mL), boiled for 10 min and subjected to electrophoresis on a 16.5% Tris-Tricine SDS–PAGE peptide analysis gel from Bio-Rad (Philadelphia, PA, USA) for LL-37 peptide analyses. After electrophoresis, proteins were transferred to nitrocellulose membranes (Immobilon-NC, Millipore) that were blocked by incubation in 5% (w/v) non-fat dry milk dissolved in TTBS (150 mM NaCl, 50 mM Tris, 0.05% Tween 20, pH 7.4). After transfer to the membrane, proteins were probed for 1 h with a monoclonal anti-LL-37/hCAP-18 antibody (clone 1-1C12, 1:250 dilution, HyCult Biotechnology, Canton, MA, USA) in TTBS. horseradish peroxidase (HRP)-conjugated secondary antibodies were used at a 1:20 000 dilution in TTBS. Immunoblots were developed with the Kodak BioMax MR film using an HRP-targeted chemiluminescent substrate. The relative amount of LL-37 peptide and hCAP-18 protein in each lane was determined by gel densitometry, followed by image analysis with ImageQuant software.

Binding of rhodamine-B-labelled LL-37 peptide to mucin

To evaluate whether mucin binds LL-37, we performed a binding assay based on previously described techniques.29 Flat-bottomed multi-well polystyrene plates were coated with: (i) mucins; (ii) mucins treated with neuraminidase type V from Clostridium perfingens; or (iii) neuraminidase by itself. Treatment of mucins with neuraminidase was performed by incubation of 0.2 mg of mucin and 10 U of neuraminidase mixed in 1 mL of 15 mM phosphate buffer, pH 5.0 for 5 h at 37°C under agitation. After protein attachment (1 h at 37°C), plates were washed with PBS/0.1% Tween 20 and blocked with 0.1% BSA, and the RhB-LL-37 peptide (5 µL of 1 mM stock solution) was added to each well alone or with LPS from Escherichia coli (E. coli) serotype O26:B6 (5 µL of 2 mg/mL solution/well). After 1 h of incubation, plates were washed and the binding of RhB-LL-37 peptide was detected fluorimetrically (Iex = 544/Iem = 590), using a multiple plate reader (Fluoroskan Ascent FL, Labsystems Inc., MA, USA).

Antimicrobial activity

The bactericidal activities of LL-37, RhB-LL-37 and WLBU2 peptides and ceragenin CSA-13 in the presence of mucin from bovine submaxillary glands (Sigma) and/or human saliva were measured as described previously.30 Kanamycin-resistant Pseudomonas aeruginosa (PAO1) and E. coli MG1655 were grown to mid-log phase at 37°C (controlled by the evaluation of optical density at 600 nm) and resuspended in PBS buffer. The bacterial suspensions were then diluted 10 times in 100 µL of solutions containing antibacterial agents by themselves or with mucin (1–1000 µg/mL) or with saliva (20% or 50%). After a 1 h incubation at 37°C, the suspensions were placed on ice and diluted 10- to 1000-fold; 10 µL aliquots of each dilution were spotted on Pseudomonas isolation agar containing 25 µg/mL kanamycin or Luria-Bertani broth (LB) agar plates for overnight culture at 37°C. The number of colonies at each dilution was counted the following morning. The cfu/mL of the individual samples were determined from the dilution factor. To evaluate the presence of bacteria in saliva samples that may potentially grow on agar used in this assay and interfere with evaluated cfu, saliva after centrifugation was spotted on Pseudomonas isolation agar containing kanamycin or LB agar. Those controls revealed a lack of any bacterial growth.

Evaluation of MICs

The MIC of antibacterial agents was determined by a microbroth dilution method with Mueller–Hinton broth (MHB),31 with and without the addition of mucin, mucins+neuraminidase or neuraminidase by itself. Bacteria were grown to mid-log phase at 37°C (an optical density of 0.35 at 600 nm) and used at a final concentration of 106 cfu/mL (10 µL of bacteria inoculated into 10 mL of MHB). A series of 2-fold dilutions of LL-37, WLBU2 or CSA-13 was prepared from a stock solution and placed in 96-well plates. Dilutions of P. aeruginosa PAO1 or E. coli MG1655 bacteria were then added. After incubation for 18 h at 37°C, the bacterial concentration was measured as optical density at 595 nm, and the MIC was read as the lowest concentration resulting in the inhibition of detectable bacterial growth.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 Transparency declarations
 References
 
Visualization of mucin, DNA and F-actin in saliva

The addition of Texas-Red-conjugated lectin or RhB-LL-37 to saliva samples produced high-intensity labelling without any detectable cellular structures. The similarity of rhodamine B fluorescence patterns with those of lectin labelling indicates the ability of the LL-37 peptide to interact with mucin networks. Labelling with YOYO-1 or fluorescent phalloidin that binds DNA and F-actin, respectively, confirmed the presence of both polymers in saliva, but likely at much lower concentrations. DNA and F-actin are present as point-like structures that are more dense and larger in the case of DNA (Figure 1). The fluorescence intensity of lectin labelling indicates higher mucin concentrations in saliva when compared with DNA and F-actin.


Figure 1
View larger version (88K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1. Microscopic images of whole saliva labelled with Texas Red-conjugated lectin and after addition of RhB-LL-37 peptide reveal similar patterns. F-actin and DNA were visualized by Alexa-Fluor phalloidin and YOYO-1 labelling, respectively. The morphology of corresponding samples is shown by phase contrast microscopy (left-hand column). Scale bar = 20 µm. Data shown are representative of three experiments.

 
Immunoblotting analysis of LL-37 in saliva

Figure 2 shows results from immunoblotting analyses of three different human saliva samples with anti-LL-37/hCAP-18 antibodies. The analysis revealed that hCAP-18 protein and LL-37 peptide are constituents of saliva, as reported previously.7 Densitometry of the blots with ImageQuant software showed that LL-37 levels in saliva are <10% of the level of hCAP-18 protein. In equal volumes of saliva, a detectable heterogeneity of hCAP-18 and LL-37 peptide concentrations exists among different subjects (data not shown).


Figure 2
View larger version (87K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2. Presence of hCAP-18 and LL-37 peptide in saliva detected using the immunoblotting analysis with monoclonal antibodies to hCAP-18/LL-37. Sample 1 (left-hand lane) represents synthetic LL-37 peptide (25 ng). Samples 2–4 (right-hand lanes) represent saliva obtained from three different donors (15 µL of whole saliva samples mixed with 5 µL of 4x sample buffer in each lane).

 
Mucin binds to LL-37 peptide

Measurements of RhB-LL-37 bound to multi-well plates coated with mucin indicate that mucins may selectively bind LL-37 peptides (Figure 3). Treatment of mucin with neuraminidase to hydrolyse N-acetyl-neuraminic acid residues from glycoproteins decreased LL-37 peptide binding. A previous study in which neuraminidase-treated mucin, derived from the bovine submaxillary gland, was found to lose its sialic-acid-mediated ability to bind a Clostridial toxin32 is consistent with the current finding that the negative charge of sialic acid moieties linked to mucin determines mucin's ability to interact with the cationic LL-37 peptide. Binding of LL-37 to mucin was also inhibited by bacterial endotoxin, indicating a possible competition between mucins and LPS for binding LL-37.


Figure 3
View larger version (8K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3. LL-37 peptide binds mucin. Fluorescence measurement of RhB-LL-37 peptide after its addition to multi-well plates (bar A), coated with mucin (bar B), neuraminidase (bar C), mucin pre-treated with neuraminidase (bar D) or to mucin when LL-37 peptide was added to the wells in the presence of bacterial LPS (bar E). Data shown are means ± SD of four experiments. Differences between mean data in this experiment were evaluated by Student's t-test, with P < 0.05 being taken as the level of significance (*statistically significant).

 
Mucins inhibit antibacterial activity of LL-37, WLBU2 and CSA-13 to different extents

The antibacterial activities of LL-37, WLBU2 and CSA-13 against P. aeruginosa PAO1 and E. coli MG1655 were compromised when tested using a bacterial killing assay in the presence of mucin from bovine submaxillary glands, but the magnitude of the effect was significantly less for CSA-13 (Figure 4). At the same mucin concentration: antibacterial agent ratio, the bacterial killing activity of CSA-13 was more resistant to inhibition by mucin compared with LL-37 and WLBU2 peptides. The ability of mucin to inhibit bactericidal activity was also decreased by neuraminidase (Table 2). LL-37 peptide labelled with rhodamine B at the N-terminus has activity equal to that of non-labelled LL-37.33 Consistent with previous results, we observed similar antibacterial activity of rhodamine-B-labelled and non-labelled LL37 peptides against P. aeruginosa PAO1 and E. coli MG1655, and both of them were inhibited by mucin (1 mg/mL) to the same extent (data not shown).


Figure 4
View larger version (8K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 4. Antibacterial activity of LL-37 (squares), WLBU2 peptide (diamonds) and ceragenin CSA-13 (triangles) against P. aeruginosa PAO1 (a) and E. coli MG1655 (b) in the presence of increasing concentrations of mucin from bovine submaxillary glands. Concentration of antibacterial agent was constant (20 µg/mL). Mucin by itself has no effect on the growth of tested bacteria (circles in a and b). Data shown are means ± SD of four experiments.

 


View this table:
[in this window]
[in a new window]

  		Table 2. Activity of antibacterial agents tested against P. aeruginosa PAO1 and E. coli MG1655 bacteria; MIC (mg/L)

 
Antibacterial activity of LL-37, WLBU2 and CSA-13 in the presence of human saliva

Human saliva contains multiple antibacterial factors, including lysozyme (~14 kDa), secretory leucocyte protease inhibitor (~12 kDa), {alpha}- and β-defensins (~10 kDa), LL-37 peptide (~4 kDa) and histatins (~3 kDa), that kill different microorganisms, and their effect may be potentiated by synergistic and additive interaction.12,34 As the presence of these molecules may interfere with the evaluation of saliva's effect on the antibacterial activity of synthetic LL-37, WLBU2 and CSA-13, saliva samples used to perform this evaluation were dialysed to remove endogenous antibacterial agents. As shown in Figure 5, increasing concentrations of human saliva compromised bacterial killing activity of antibacterial agents, but the inhibitory effect decreased in the following order LL37>WLBU2>CSA-13.


Figure 5
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 5. Antibacterial activity of LL-37, WLBU2 and CSA-13 against P. aeruginosa PAO1 (a–c) and E. coli MG1655 (d–f) by themselves (open squares) and in the presence of human saliva 20% (filled squares) and 50% (filled circles). Data from one representative experiment are shown. Two other experiments revealed similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 Transparency declarations
 References
 
Decreased LL-37 antibacterial activity in the presence of saliva and host mucins isolated from submaxillary glands indicate that in the oral cavity and other mucin-containing environments, CAPs' bacterial killing activity will not correlate with their concentration. From a functional point of view, both mucins and CAPs are members of the immune system that appear to influence the activity of each other.25 At physiological pH, CAPs and mucins have opposite charges that promote their electrostatic interaction.35 This interaction can concentrate antibacterial factors in the thin layer containing mucins, but within that layer, the antibacterial activity is reduced with respect to the antibacterial potency in simple solutions. A crucial factor that determines the strength of counterions (such as LL-37) interaction with polyelectrolytes (such as mucins) is the spacing of charges within the multivalent ion. A recent study showed that as univalent positive-charged groups were increasingly separated from each other by placing methylene spacers between multiple amino groups, the divalent cations eventually lost their ability to aggregate polyelectrolytes and functioned more like multiple monovalent ions.36 The importance of spacing charges over a polyvalent counterion to avoid condensation of linear polyelectrolytes was the rationale for designing antibacterial molecules that enhance resistance to inactivation by F-actin and DNA. Accordingly, the antibacterial activity of the non-peptide antibacterial molecule CSA-13, a member of the ceragenin family37 that has a smaller net charge and unique distribution of this charge over a hydrophobic scaffold when compared with natural CAPs, was found to be less inhibited by polyelectrolytes.38 Similarly, in the current study, the inhibitory effect of mucin on CSA-13 bactericidal activity was less pronounced when compared with its effect on LL-37 or WLBU2 peptides. The ability of CSA-13 to maintain its antibacterial function in the presence of mucin in addition to DNA/F-actin38 and its broad-spectrum activities against multidrug-resistant bacteria such as clinical isolates of vancomycin-resistant Staphylococcus aureus37 and clinical isolates of P. aeruginosa, including multidrug-resistant P. aeruginosa,39 suggest its potential in treating a range of infections.

The extrinsic mucosal barrier composed of mucins, immunoglobulins and various antibacterial molecules prevents host colonization by a number of microorganisms. In order to breach this barrier, bacteria produce a range of enzymes that, by synergistic action, are capable of partial or complete mucin degradation.40,41 Some pathogens express sialic acid units on their surfaces, mimicking the sialyl-rich mucin layer coating epithelial cells and glycoconjugates present on host cell surfaces and serum proteins that function as anti-recognition molecules to escape host immune mechanisms. The presence of sialic acid on bacterial surfaces may significantly impact host–pathogen interaction, leading to immune tolerance.42 Our finding that inhibition of the antibacterial activity of LL-37 by mucins was reversed upon neuraminidase treatment suggests some important features of the mucosal barrier. First, the ability of mucin to bind CAPs would increase their concentration at the mucosal barrier, a location where these molecules should be present to effectively prevent bacterial invasion. Second, although mucin–LL-37 binding results in the partial loss of LL-37’s bacterial killing activity, the release of LL-37 trapped in mucin networks upon bacterial enzyme digestion of mucin would restore the antimicrobial activity of LL37. Third, even if the antibacterial activity of LL-37 peptide decreases when bound to mucins, it is possible that its hormonal activity as a growth factor43,44 is maintained. Fourth, during bacterial invasion, cathelicidins and/or LL-37 concentrated within a thin layer at the mucin surface can be processed to shorter forms with enhanced activity and decreased binding, distinct from the LL-37 peptide.45,46 These points underline the complexity of mechanisms in the mucosal barrier that hinder microorganism attack. Previous studies that support these speculations include immunohistochemical analysis of LL-37 in human gastric mucosa that showed staining throughout the gastric tubular units, generative zone and mucins,47 and evidence of {alpha}-helical structure of LL-37 upon interaction with mucins,25 a process previously observed when LL-37 interacts with bacterial wall moieties.48

In conclusion, the present data shows that the interaction of three cationic antibacterial agents with mucin results in various degrees of inhibition of their antibacterial activity, an effect that was prevented by removing sialic acid from mucin with neuraminidase. The ceragenin CSA-13 is more resistant to inhibition by either purified mucins or whole saliva when compared with the peptides LL-37 and WLBU2, suggesting its possible application in the treatment of oral infection.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
Transparency declarations
 References
 
This work was supported by NIH grants R01 HL67286 and AR38910.


   Transparency declarations
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 Transparency declarations
References
 
Brigham Young University has licensed ceragenin technology to Ceragenix Pharmaceuticals. P. B. S. is a paid consultant for Ceragenix Pharmaceuticals, Innate Immune Inc. and WittyCell. R. B. obtained a reimbursement from Ceragenix for attending a 46th Interscience Conference on Antimicrobial Agents and Chemotherapy. None of the research reported in this paper was supported by Ceragenix or by any other corporate entity. Other authors: none to declare.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 Transparency declarations
 References
 
1 . Jenssen H, Hamill P, Hancock RE. Peptide antimicrobial agents. Clin Microbiol Rev (2006) 19:491–511.[Abstract/Free Full Text]

2 . Dale BA, Tao R, Kimball JR, et al. Oral antimicrobial peptides and biological control of caries. BMC Oral Health (2006) 6(Suppl_1):S13.[CrossRef][Medline]

3 . Bucki R, Levental I, Janmey PA. Antibacterial peptides—a bright future or a false hope. Anti-Infective Agents Med Chem (2007) 6:175–84.

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

5 . Chromek M, Slamova Z, Bergman P, et al. The antimicrobial peptide cathelicidin protects the urinary tract against invasive bacterial infection. Nat Med (2006) 12:636–41.[CrossRef][Web of Science][Medline]

6 . Bals R, Wilson JM. Cathelicidins—a family of multifunctional antimicrobial peptides. Cell Mol Life Sci (2003) 60:711–20.[CrossRef][Web of Science][Medline]

7 . Murakami M, Ohtake T, Dorschner RA, et al. Cathelicidin antimicrobial peptides are expressed in salivary glands and saliva. J Dent Res (2002) 81:845–50.[Abstract/Free Full Text]

8 . Oppenheim FG, Xu T, McMillian FM, et al. Histatins, a novel family of histidine-rich proteins in human parotid secretion: isolation, characterization, primary structure, and fungistatic effects on Candida albicans. J Biol Chem (1988) 263:7472–7.[Abstract/Free Full Text]

9 . Ganz T, Selsted ME, Szklarek D, et al. Defensins: natural peptide antibiotics of human neutrophils. J Clin Invest (1985) 76:1427–35.[Web of Science][Medline]

10 . Sorensen OE, Follin P, Johnsen AH, et al. Human cathelicidin, hCAP-18, is processed to the antimicrobial peptide LL-37 by extracellular cleavage with proteinase 3. Blood (2001) 97:3951–9.[Abstract/Free Full Text]

11 . Devine DA. Antimicrobial peptides in defence of the oral and respiratory tracts. Mol Immunol (2003) 40:431–43.[CrossRef][Web of Science][Medline]

12 . Ouhara K, Komatsuzawa H, Yamada S, et al. Susceptibilities of periodontopathogenic and cariogenic bacteria to antibacterial peptides, β-defensins and LL37, produced by human epithelial cells. J Antimicrob Chemother (2005) 55:888–96.[Abstract/Free Full Text]

13 . Tjabringa GS, Aarbiou J, Ninaber DK, et al. The antimicrobial peptide LL-37 activates innate immunity at the airway epithelial surface by transactivation of the epidermal growth factor receptor. J Immunol (2003) 171:6690–6.[Abstract/Free Full Text]

14 . Nell MJ, Tjabringa GS, Wafelman AR, et al. Development of novel LL-37 derived antimicrobial peptides with LPS and LTA neutralizing and antimicrobial activities for therapeutic application. Peptides (2006) 27:649–60.[CrossRef][Web of Science][Medline]

15 . Elssner A, Duncan M, Gavrilin M, et al. A novel P2X7 receptor activator, the human cathelicidin-derived peptide LL37, induces IL-1β processing and release. J Immunol (2004) 172:4987–94.[Abstract/Free Full Text]

16 . Schaller-Bals S, Schulze A, Bals R. Increased levels of antimicrobial peptides in tracheal aspirates of newborn infants during infection. Am J Respir Crit Care Med (2002) 165:992–5.[Abstract/Free Full Text]

17 . Putsep K, Carlsson G, Boman HG, et al. Deficiency of antibacterial peptides in patients with morbus Kostmann: an observation study. Lancet (2002) 360:1144–9.[CrossRef][Web of Science][Medline]

18 . Carlsson G, Wahlin YB, Johansson A, et al. Periodontal disease in patients from the original Kostmann family with severe congenital neutropenia. J Periodontol (2006) 77:744–51.[CrossRef][Web of Science][Medline]

19 . Dommett R, Zilbauer M, George JT, et al. Innate immune defence in the human gastrointestinal tract. Mol Immunol (2005) 42:903–12.[CrossRef][Web of Science][Medline]

20 . Tao R, Jurevic RJ, Coulton KK, et al. Salivary antimicrobial peptide expression and dental caries experience in children. Antimicrob Agents Chemother (2005) 49:3883–8.[Abstract/Free Full Text]

21 . Dodds MW, Johnson DA, Yeh CK. Health benefits of saliva: a review. J Dent (2005) 33:223–33.[CrossRef][Web of Science][Medline]

22 . Rose MC, Voynow JA. Respiratory tract mucin genes and mucin glycoproteins in health and disease. Physiol Rev (2006) 86:245–78.[Abstract/Free Full Text]

23 . Nell MJ, Tjabringa G, Vonk MJ, et al. Bacterial products increase expression of the human cathelicidin hCAP-18/LL-37 in cultured human sinus epithelial cells. FEMS Immunol Med Microbiol (2004) 42:225–31.[CrossRef][Web of Science][Medline]

24 . Weiner DJ, Bucki R, Janmey PA. The antimicrobial activity of the cathelicidin LL37 is inhibited by F-actin bundles and restored by gelsolin. Am J Respir Cell Mol Biol (2003) 28:738–45.[Abstract/Free Full Text]

25 . Felgentreff K, Beisswenger C, Griese M, et al. The antimicrobial peptide cathelicidin interacts with airway mucus. Peptides (2006) 27:3100–6.[CrossRef][Web of Science][Medline]

26 . Herasimenka Y, Benincasa M, Mattiuzzo M, et al. Interaction of antimicrobial peptides with bacterial polysaccharides from lung pathogens. Peptides (2005) 26:1127–32.[CrossRef][Web of Science][Medline]

27 . Ding B, Guan Q, Walsh JP, et al. Correlation of the antibacterial activities of cationic peptide antibiotics and cationic steroid antibiotics. J Med Chem (2002) 45:663–9.[CrossRef][Web of Science][Medline]

28 . Henke MO, Renner A, Huber RM, et al. MUC5AC and MUC5B mucins are decreased in cystic fibrosis airway secretions. Am J Respir Cell Mol Biol (2004) 31:86–91.[Abstract/Free Full Text]

29 . Arana Mde J, Vallespi MG, Chinea G, et al. Inhibition of LPS-responses by synthetic peptides derived from LBP associates with the ability of the peptides to block LBP–LPS interaction. J Endotoxin Res (2003) 9:281–91.[Medline]

30 . Bucki R, Pastore JJ, Randhawa P, et al. Antibacterial activities of rhodamine B-conjugated gelsolin-derived peptides compared to those of the antimicrobial peptides cathelicidin LL37, magainin II, and melittin. Antimicrob Agents Chemother (2004) 48:1526–33.[Abstract/Free Full Text]

31 . Giacometti A, Cirioni O, Greganti G, et al. In vitro activities of membrane-active peptides against Gram-positive and Gram-negative aerobic bacteria. Antimicrob Agents Chemother (1998) 42:3320–4.[Abstract/Free Full Text]

32 . Nakamura T, Takada N, Tonozuka T, et al. Binding properties of Clostridium botulinum type C progenitor toxin to mucins. Biochim Biophys Acta (2007) 1770:551–5.[Medline]

33 . Oren Z, Lerman JC, Gudmundsson GH, et al. Structure and organization of the human antimicrobial peptide LL-37 in phospholipid membranes: relevance to the molecular basis for its non-cell-selective activity. Biochem J (1999) 341:501–13.[CrossRef][Web of Science][Medline]

34 . Shugars DC. Endogenous mucosal antiviral factors of the oral cavity. J Infect Dis (1999) 179(Suppl 3):S431–5.[CrossRef][Web of Science][Medline]

35 . Vitorino R, Lobo MJ, Duarte JA, et al. Analysis of salivary peptides using HPLC-electrospray mass spectrometry. Biomed Chromatogr (2004) 18:570–5.[CrossRef][Web of Science][Medline]

36 . Butler JC, Angelini T, Tang JX, et al. Ion multivalence and like-charge polyelectrolyte attraction. Phys Rev Lett (2003) 91:283–301.

37 . Chin JN, Rybak MJ, Cheung CM, et al. Antimicrobial activities of ceragenins against clinical isolates of resistant Staphylococcus aureus. Antimicrob Agents Chemother (2007) 51:1268–73.[Abstract/Free Full Text]

38 . Bucki R, Sostarecz AG, Byfield FJ, et al. Resistance of the antibacterial agent ceragenin CSA-13 to inactivation by DNA or F-actin and its activity in cystic fibrosis sputum. J Antimicrob Chemother (2007) 60:535–45.[Abstract/Free Full Text]

39 . Chin JN, Jones RN, Sader HS, et al. Potential synergy activity of the novel ceragenin, CSA-13, against clinical isolates of Pseudomonas aeruginosa, including multidrug-resistant P. aeruginosa. J Antimicrob Chemother (2008) 61:365–70.[Abstract/Free Full Text]

40 . Corfield AP, Carroll D, Myerscough N, et al. Mucins in the gastrointestinal tract in health and disease. Front Biosci (2001) 6:D1321–57.[Web of Science][Medline]

41 . Wiggins R, Hicks SJ, Soothill PW, et al. Mucinases and sialidases: their role in the pathogenesis of sexually transmitted infections in the female genital tract. Sex Transm Infect (2001) 77:402–8.[Abstract/Free Full Text]

42 . Vimr E, Lichtensteiger C. To sialylate, or not to sialylate: that is the question. Trends Microbiol (2002) 10:254–7.[CrossRef][Web of Science][Medline]

43 . Koczulla R, von Degenfeld G, Kupatt C, et al. An angiogenic role for the human peptide antibiotic LL-37/hCAP-18. J Clin Invest (2003) 111:1665–72.[CrossRef][Web of Science][Medline]

44 . Malmsten M, Davoudi M, Walse B. Antimicrobial peptides derived from growth factors. Growth Factors (2007) 25:60–70.[CrossRef][Web of Science][Medline]

45 . Murakami M, Lopez-Garcia B, Braff M, et al. Postsecretory processing generates multiple cathelicidins for enhanced topical antimicrobial defense. J Immunol (2004) 172:3070–7.[Abstract/Free Full Text]

46 . Li X, Li Y, Han H, et al. Solution structures of human LL-37 fragments and NMR-based identification of a minimal membrane-targeting antimicrobial and anticancer region. J Am Chem Soc (2006) 128:5776–85.[CrossRef][Web of Science][Medline]

47 . Hase K, Murakami M, Iimura M, et al. Expression of LL-37 by human gastric epithelial cells as a potential host defense mechanism against Helicobacter pylori. Gastroenterology (2003) 125:1613–25.[CrossRef][Web of Science][Medline]

48 . Travis SM, Anderson NN, Forsyth WR, et al. Bactericidal activity of mammalian cathelicidin-derived peptides. Infect Immun (2000) 68:2748–55.[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Infect. Immun.Home page
M. Gutner, S. Chaushu, D. Balter, and G. Bachrach
Saliva Enables the Antimicrobial Activity of LL-37 in the Presence of Proteases of Porphyromonas gingivalis
Infect. Immun., December 1, 2009; 77(12): 5558 - 5563.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
62/2/329    most recent
dkn176v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Bucki, R.
Right arrow Articles by Janmey, P. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bucki, R.
Right arrow Articles by Janmey, P. A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?