Abstract
Biofilms formed by nontypeable Haemophilus influenzae (NTHI) are associated with multiple chronic infections of the airway, including otitis media. Extracellular DNA (eDNA) is part of the biofilm matrix and serves as a structural component. Human β-defensin-3 (hBD-3) is a cationic antimicrobial host defense protein (AMP) critical to the protection of the middle ear. We hypothesized that anionic eDNA could interact with and bind hBD-3 and thus shield NTHI in biofilms from its antimicrobial activity. We demonstrated that recombinant hBD-3 [(r)hBD-3] bound eDNA in vitro and that eDNA in biofilms produced by NTHI in the chinchilla middle ear co-localized with the orthologue of this AMP. Incubation of physiological concentrations of (r)hBD-3 with NTHI genomic DNA abrogated the ability of this innate immune effector to prevent NTHI from forming robust biofilms in vitro. Establishment of NTHI biofilms in the presence of both DNase I and (r)hBD-3 resulted in a marked reduction in the overall height and thickness of the biofilms and rescued the antimicrobial activity of the AMP. Our results demonstrated that eDNA in NTHI biofilms sequestered hBD-3 and thus diminished the biological activity of an important effector of innate immunity. Our observations have important implications for chronicity of NTHI-induced diseases.
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References
1.
Infante-Rivard C, Fernandez A: Otitis media in children: frequency, risk factors, and research avenues. Epidemiol Rev 1993;15:444–465.
[PubMed]
2.
Agrawal A, Murphy TF: Haemophilus influenzae infections in the H. influenzae type b conjugate vaccine era. J Clin Microbiol 2011;49:3728–3732.
[PubMed]
3.
Foxwell AR, Kyd JM, Cripps AW: Nontypeable Haemophilus influenzae: pathogenesis and prevention. Microbiol Mol Biol Rev 1998;62:294–308.
[PubMed]
4.
Erwin AL, Smith AL: Nontypeable Haemophilus influenzae: understanding virulence and commensal behavior. Trends Microbiol 2007;15:355–362.
[PubMed]
5.
Alsarraf R, Jung CJ, Perkins J, Crowley C, Alsarraf NW, Gates GA: Measuring the indirect and direct costs of acute otitis media. Arch Otolaryngol Head Neck Surg 1999;125:12–18.
[PubMed]
6.
Kaplan B, Wandstrat TL, Cunningham JR: Overall cost in the treatment of otitis media. Pediatr Infect Dis J 1997;16:S9–S11.
[PubMed]
7.
Woodfield G, Dugdale A: Evidence behind the WHO guidelines: hospital care for children: what is the most effective antibiotic regime for chronic suppurative otitis media in children? J Trop Pediatr 2008;54:151–156.
[PubMed]
8.
Leach AJ, Morris PS, Mathews JD: Compared to placebo, long-term antibiotics resolve otitis media with effusion (OME) and prevent acute otitis media with perforation (AOMwiP) in a high-risk population: a randomized controlled trial. BMC Pediatr 2008;8:23.
[PubMed]
9.
World Health Organization: Chronic Suppurative Otitis Media: Burden of Illness and Management Options. Geneva, WHO, 2004.
10.
Murphy TF, Kirkham C: Biofilm formation by nontypeable Haemophilus influenzae: strain variability, outer membrane antigen expression and role of pili. BMC Microbiol 2002;2:7.
[PubMed]
11.
Jurcisek JA, Bakaletz LO: Biofilms formed by nontypeable Haemophilus influenzae in vivo contain both double-stranded DNA and type IV pilin protein. J Bacteriol 2007;189:3868–3875.
[PubMed]
12.
Hall-Stoodley L, Hu FZ, Gieseke A, Nistico L, Nguyen D, Hayes J, Forbes M, Greenberg DP, Dice B, Burrows A, Wackym PA, Stoodley P, Post JC, Ehrlich GD, Kerschner JE: Direct detection of bacterial biofilms on the middle-ear mucosa of children with chronic otitis media. JAMA 2006;296:202–211.
[PubMed]
13.
Flemming HC, Wingender J: The biofilm matrix. Nat Rev Microbiol 2010;8:623–633.
[PubMed]
14.
Webster P, Wu S, Gomez G, Apicella M, Plaut AG, St Geme JW 3rd: Distribution of bacterial proteins in biofilms formed by non-typeable Haemophilus influenzae. J Histochem Cytochem 2006;54:829–842.
[PubMed]
15.
Jurcisek J, Greiner L, Watanabe H, Zaleski A, Apicella MA, Bakaletz LO: Role of sialic acid and complex carbohydrate biosynthesis in biofilm formation by nontypeable Haemophilus influenzae in the chinchilla middle ear. Infect Immun 2005;73:3210–3218.
[PubMed]
16.
Goodman SD, Obergfell KP, Jurcisek JA, Novotny LA, Downey JS, Ayala EA, Tjokro N, Li B, Justice SS, Bakaletz LO: Biofilms can be dispersed by focusing the immune system on a common family of bacterial nucleoid-associated proteins. Mucosal Immunol 2011;4:625–637.
[PubMed]
17.
Das T, Sharma PK, Busscher HJ, van der Mei HC, Krom BP: Role of extracellular DNA in initial bacterial adhesion and surface aggregation. Appl Environ Microbiol 2010;76:3405–3408.
[PubMed]
18.
Spoering AL, Gilmore MS: Quorum sensing and DNA release in bacterial biofilms. Curr Opin Microbiol 2006;9:133–137.
[PubMed]
19.
Palchevskiy V, Finkel SE: Escherichia coli competence gene homologs are essential for competitive fitness and the use of DNA as a nutrient. J Bacteriol 2006;188:3902–3910.
[PubMed]
20.
Slinger R, Chan F, Ferris W, Yeung SW, St Denis M, Gaboury I, Aaron SD: Multiple combination antibiotic susceptibility testing of nontypeable Haemophilus influenzae biofilms. Diagn Microbiol Infect Dis 2006;56:247–253.
[PubMed]
21.
Costerton JW, Stewart PS, Greenberg EP: Bacterial biofilms: a common cause of persistent infections. Science 1999;284:1318–1322.
[PubMed]
22.
Foreman A, Wormald PJ: Different biofilms, different disease? A clinical outcomes study. Laryngoscope 2010;120:1701–1706.
[PubMed]
23.
Ganz T: Defensins: antimicrobial peptides of innate immunity. Nat Rev Immunol 2003;3:710–720.
[PubMed]
24.
Ganz T: Antimicrobial polypeptides in host defense of the respiratory tract. J Clin Invest 2002;109:693–697.
[PubMed]
25.
Singh PK, Jia HP, Wiles K, Hesselberth J, Liu L, Conway BA, Greenberg EP, Valore EV, Welsh MJ, Ganz T, Tack BF, McCray PB Jr: Production of beta-defensins by human airway epithelia. Proc Natl Acad Sci USA 1998;95:14961–14966.
[PubMed]
26.
Yang D, Chertov O, Bykovskaia SN, Chen Q, Buffo MJ, Shogan J, Anderson M, Schroder JM, Wang JM, Howard OM, Oppenheim JJ: Beta-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science 1999;286:525–528.
[PubMed]
27.
Salzman NH, Hung K, Haribhai D, Chu H, Karlsson-Sjoberg J, Amir E, Teggatz P, Barman M, Hayward M, Eastwood D, Stoel M, Zhou Y, Sodergren E, Weinstock GM, Bevins CL, Williams CB, Bos NA: Enteric defensins are essential regulators of intestinal microbial ecology. Nat Immunol 2010;11:76–83.
[PubMed]
28.
Dhople V, Krukemeyer A, Ramamoorthy A: The human beta-defensin-3, an antibacterial peptide with multiple biological functions. Biochim Biophys Acta 2006;1758:1499–1512.
[PubMed]
29.
McGillivary G, Mason KM, Jurcisek JA, Peeples ME, Bakaletz LO: Respiratory syncytial virus-induced dysregulation of expression of a mucosal beta-defensin augments colonization of the upper airway by non-typeable Haemophilus influenzae. Cell Microbiol 2009;11:1399–1408.
[PubMed]
30.
Song JJ, Chae SW, Woo JS, Lee HM, Jung HH, Hwang SJ: Differential expression of human beta defensin 2 and human beta defensin 3 in human middle ear cholesteatoma. Ann Otol Rhinol Laryngol 2007;116:235–240.
[PubMed]
31.
Mason KM, Raffel FK, Ray WC, Bakaletz LO: Heme utilization by nontypeable Haemophilus influenzae is essential and dependent on Sap transporter function. J Bacteriol 2011;193:2527–2535.
[PubMed]
32.
Kuo HH, Chan C, Burrows LL, Deber CM: Hydrophobic interactions in complexes of antimicrobial peptides with bacterial polysaccharides. Chem Biol Drug Des 2007;69:405–412.
[PubMed]
33.
Remijsen Q, Kuijpers TW, Wirawan E, Lippens S, Vandenabeele P, Vanden Berghe T: Dying for a cause: NETosis, mechanisms behind an antimicrobial cell death modality. Cell Death Differ 2011;18:581–588.
[PubMed]
34.
Juneau RA, Pang B, Weimer KE, Armbruster CE, Swords WE: Nontypeable Haemophilus influenzae initiates formation of neutrophil extracellular traps. Infect Immun 2011;79:431–438.
[PubMed]
35.
Beckloff N, Laube D, Castro T, Furgang D, Park S, Perlin D, Clements D, Tang H, Scott RW, Tew GN, Diamond G: Activity of an antimicrobial peptide mimetic against planktonic and biofilm cultures of oral pathogens. Antimicrob Agents Chemother 2007;51:4125–4132.
[PubMed]
36.
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;28:738–745.
[PubMed]
37.
Yin L, Chino T, Horst OV, Hacker BM, Clark EA, Dale BA, Chung WO: Differential and coordinated expression of defensins and cytokines by gingival epithelial cells and dendritic cells in response to oral bacteria. BMC Immunol 2010;11:37.
[PubMed]
38.
Bakaletz LO, Baker BD, Jurcisek JA, Harrison A, Novotny LA, Bookwalter JE, Mungur R, Munson RS Jr: Demonstration of type IV pilus expression and a twitching phenotype by Haemophilus influenzae. Infect Immun 2005;73:1635–1643.
[PubMed]
39.
Harris RH, Wilk D, Bevins CL, Munson RS Jr, Bakaletz LO: Identification and characterization of a mucosal antimicrobial peptide expressed by the chinchilla (Chinchilla lanigera) airway. J Biol Chem 2004;279:20250–20256.
[PubMed]
40.
Martinez LR, Casadevall A: Cryptococcus neoformans cells in biofilms are less susceptible than planktonic cells to antimicrobial molecules produced by the innate immune system. Infect Immun 2006;74:6118–6123.
[PubMed]
41.
Lande R, Gregorio J, Facchinetti V, Chatterjee B, Wang YH, Homey B, Cao W, Su B, Nestle FO, Zal T, Mellman I, Schroder JM, Liu YJ, Gilliet M: Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide. Nature 2007;449:564–569.
[PubMed]
42.
Bucki R, Namiot DB, Namiot Z, Savage PB, Janmey PA: Salivary mucins inhibit antibacterial activity of the cathelicidin-derived LL-37 peptide but not the cationic steroid CSA-13. J Antimicrob Chemother 2008;62:329–335.
[PubMed]
43.
Mulcahy H, Charron-Mazenod L, Lewenza S: Extracellular DNA chelates cations and induces antibiotic resistance in Pseudomonas aeruginosa biofilms. PLoS Pathog 2008;4:e1000213.
[PubMed]
44.
Llobet E, Tomas JM, Bengoechea JA: Capsule polysaccharide is a bacterial decoy for antimicrobial peptides. Microbiology 2008;154:3877–3886.
[PubMed]
45.
Jono H, Xu H, Kai H, Lim DJ, Kim YS, Feng XH, Li JD: Transforming growth factor-beta-Smad signaling pathway negatively regulates nontypeable Haemophilus influenzae-induced MUC5AC mucin transcription via mitogen-activated protein kinase (MAPK) phosphatase-1-dependent inhibition of p38 MAPK. J Biol Chem 2003;278:27811–27819.
[PubMed]
46.
Chen R, Lim JH, Jono H, Gu XX, Kim YS, Basbaum CB, Murphy TF, Li JD: Nontypeable Haemophilus influenzae lipoprotein P6 induces MUC5AC mucin transcription via TLR2-TAK1-dependent p38 MAPK-AP1 and IKKβ-IĸBα-NF-ĸB signaling pathways. Biochem Biophys Res Commun 2004;324:1087–1094.
[PubMed]
47.
Kubiet M, Ramphal R, Weber A, Smith A: Pilus-mediated adherence of Haemophilus influenzae to human respiratory mucins. Infect Immun 2000;68:3362–3367.
[PubMed]
48.
Davies J, Carlstedt I, Nilsson AK, Hakansson A, Sabharwal H, van Alphen L, van Ham M, Svanborg C: Binding of Haemophilus influenzae to purified mucins from the human respiratory tract. Infect Immun 1995;63:2485–2492.
[PubMed]
49.
Mason KM, Bruggeman ME, Munson RS, Bakaletz LO: The non-typeable Haemophilus influenzae Sap transporter provides a mechanism of antimicrobial peptide resistance and SapD-dependent potassium acquisition. Mol Microbiol 2006;62:1357–1372.
[PubMed]
50.
Mason KM, Munson RS Jr, Bakaletz LO: A mutation in the Sap operon attenuates survival of nontypeable Haemophilus influenzae in a chinchilla model of otitis media. Infect Immun 2005;73:599–608.
[PubMed]
51.
McGillivary G, Bakaletz LO: The multifunctional host defense peptide SPLUNC1 is critical for homeostasis of the mammalian upper airway. PLoS One 2010;5:e13224.
[PubMed]
52.
Johnson RW, McGillivary G, Denoel P, Poolman J, Bakaletz LO: Abrogation of nontypeable Haemophilus influenzae protein D function reduces phosphorylcholine decoration, adherence to airway epithelial cells, and fitness in a chinchilla model of otitis media. Vaccine 2011;29:1211–1221.
[PubMed]
53.
Bucki R, Byfield FJ, Janmey PA: Release of the antimicrobial peptide LL-37 from DNA/F-actin bundles in cystic fibrosis sputum. Eur Respir J 2007;29:624–632.
[PubMed]
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