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Development of novel tools for prevention and diagnosis of Porphyromonas gingivalis infection and periodontitis
Örebro University, School of Medical Sciences.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Periodontitis is a chronic inflammatory disease caused by exaggerated host immune responses to dysregulated microbiota in dental biofilms leading to degradation of tissues and alveolar bone loss. Porphyromonas gingivalis is a major periodontal pathogen and expresses several potent virulence factors. Among these factors, arginine and lysine gingipains are of special importance, both for the bacterial survival/proliferation and the pathological outcome. The major aim of this thesis was to develop and test novel methods for diagnosis and prevention of P. gingivalis infection and periodontitis. In study I, anti-P. gingivalis antibodies were developed in vitro for immunodetection of bacteria in clinical samples using a surface plasmon resonance (SPR)-based biosensor. Specific binding of the antibodies to P. gingivalis was demonstrated in samples of patients with periodontitis and the results were validated using real-time PCR and DNA-DNA checkerboard analysis. In study II, we elucidated the properties and antimicrobial effects of different lactobacillus species and the two-peptide bacteriocin PLNC8 αβ on P. gingivalis. L. plantarum NC8 and 44048 effectively inhibited P. gingivalis growth and pure PLNC8 αβ induced bacterial lysis by damaging P. gingivalis membrane. In study III, we demonstrated that PLNC8 αβ dose-dependently induces proliferation and release of growth factors in gingival epithelial cells (GECs). Furthermore, PLNC8 αβ decreased P. gingivalis-induced cytotoxic effects in GECs but did not alter the effect of gingipains on cytokine expression. In study IV, we elucidated the effects of anti-P. gingivalis antibodies and PLNC8 αβ in regulating cellular responses during P. gingivalis infection. Both antibodies and PLNC8 αβ modulated P. gingivalis-induced expression of growth factors in GECs, however, their effects were diminished when used in combination. The results of this thesis demonstrate a possible role of anti-P. gingivalis antibodies and PLNC8 αβ in prevention and treatment of P. gingivalis infection and periodontitis with no cytotoxic effects on human cells.

Place, publisher, year, edition, pages
Örebro: Örebro university , 2016. , 57 p.
Series
Örebro Studies in Medicine, ISSN 1652-4063 ; 151
Keyword [en]
Periodontitis, Porphyromonas gingivalis, anti-P. gingivalis antibodies, surface plasmon resonance, PLNC8 αβ, proliferation, growth factors.
National Category
Other Basic Medicine
Research subject
Biomedicine
Identifiers
URN: urn:nbn:se:oru:diva-52056ISBN: 978-91-7668-162-8 OAI: oai:DiVA.org:oru-52056DiVA: diva2:967367
Public defence
2016-10-28, Campus USÖ, hörsal C1, Södra Grev Rosengatan 30, Örebro, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2016-09-08 Created: 2016-09-08 Last updated: 2016-10-06Bibliographically approved
List of papers
1. Antibodies produced in vitro in the detection of periodontal bacteria by using surface plasmon resonance analysis
Open this publication in new window or tab >>Antibodies produced in vitro in the detection of periodontal bacteria by using surface plasmon resonance analysis
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2015 (English)In: Clinical and Experimental Dental Research, ISSN 2057-4347, Vol. 1, no 1, 32-44 p.Article in journal (Refereed) Published
Abstract [en]

Porphyromonas gingivalis (P. gingivalis) is a major etiological agent associated with periodontitis. This study aims to develop antibodies to P. gingivalis in vitro for real-time detection of bacteria in clinical samples. Lymphocytes were isolated from whole blood of patient treated for periodontitis and were stimulated with P. gingivalis ATCC 33277. B-cell maturation to long-living antibody secreting-plasma cells was studied using flow cytometry and immunofluorescence staining. The antibodies developed in vitro were immobilized onto a CM-5 sensor chip of a biosensor to detect the presence of P. gingivalis in the gingival crevicular fluid of patients with periodontitis compared to periodontally healthy controls (n = 30). Surface plasmon resonance (SPR) analysis was performed to evaluate specific interactions of bacteria in samples with the immobilized antibodies. The results of SPR analysis were compared to the detection of P. gingivalis in the samples using DNA–DNA checkerboard hybridization technique. A clear and distinct change in lymphocyte morphology upon stimulation with P. gingivalis was observed. Anti-P. gingivalis antibodies secreted by CD38+ plasma cells showed the presence of all the four IgG subclasses. The results of DNA–DNA checkerboard analysis were in agreement with that of SPR analysis for the detection of P. gingivalis in patient samples. Furthermore, incubation with anti-P. gingivalis attenuated the bacterial response in SPR. The in vitro method for antibody production developed during this study could be used for an efficient real-time detection of periodontitis, and the attenuating effects of in vitro antibodies suggest their role in passive immunization to prevent periodontitis and their associated risk factors.

Place, publisher, year, edition, pages
Wiley-Blackwell, 2015
Keyword
Antibodies, B-cell, plasma cell, periodontitis, P. gingivalis, surface plasmon resonance
National Category
Microbiology in the medical area
Research subject
Biomedicine
Identifiers
urn:nbn:se:oru:diva-46728 (URN)10.1002/cre2.6 (DOI)
Available from: 2015-11-24 Created: 2015-11-23 Last updated: 2016-10-06Bibliographically approved
2. Antibacterial effects of Lactobacillus and bacteriocin PLNC8 αβ on the periodontal pathogen Porphyromonas gingivalis
Open this publication in new window or tab >>Antibacterial effects of Lactobacillus and bacteriocin PLNC8 αβ on the periodontal pathogen Porphyromonas gingivalis
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2016 (English)In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 16, no 1, 188Article in journal (Refereed) Published
Abstract [en]

Background: The complications in healthcare systems associated with antibiotic-resistant microorganisms have resulted in an intense search for new effective antimicrobials. Attractive substances from which novel antibiotics may be developed are the bacteriocins. These naturally occurring peptides are generally considered to be safe and efficient at eliminating pathogenic bacteria. Among specific keystone pathogens in periodontitis, Porphyromonas gingivalis is considered to be the most important pathogen in the development and progression of chronic inflammatory disease. The aim of the present study was to investigate the antimicrobial effects of different Lactobacillus species and the two-peptide bacteriocin PLNC8 αβ on P. gingivalis.

Results: Growth inhibition of P. gingivalis was obtained by viable Lactobacillus and culture media from L. plantarum NC8 and 44048, but not L. brevis 30670. The two-peptide bacteriocin from L. plantarum NC8 (PLNC8 αβ) was found to be efficient against P. gingivalis through binding followed by permeabilization of the membranes, using Surface plasmon resonance analysis and DNA staining with Sytox Green. Liposomal systems were acquired to verify membrane permeabilization by PLNC8 αβ. The antimicrobial activity of PLNC8 αβ was found to be rapid (1 min) and visualized by TEM to cause cellular distortion through detachment of the outer membrane and bacterial lysis.

Conclusion: Soluble or immobilized PLNC8 αβ bacteriocins may be used to prevent P. gingivalis colonization and subsequent pathogenicity, and thus supplement the host immune system against invading pathogens associated with periodontitis.

Place, publisher, year, edition, pages
London, United Kingdom: BioMed Central, 2016
Keyword
Periodontitis, P. gingivalis, Lactobacillus, Bacteriocin, PLNC8
National Category
Microbiology
Identifiers
urn:nbn:se:oru:diva-51749 (URN)10.1186/s12866-016-0810-8 (DOI)000383422500001 ()27538539 (PubMedID)2-s2.0-84982308370 (Scopus ID)
Funder
Swedish Heart Lung FoundationKnowledge FoundationMagnus Bergvall Foundation
Note

Funding Agency:

Foundation of Olle Engkvist

Available from: 2016-08-23 Created: 2016-08-23 Last updated: 2017-06-07Bibliographically approved
3. Bacteriocin plantaricin NC8 αβ antagonizes Porphyromonas gingivalis infection and induces proliferation of gingival epithelial cells
Open this publication in new window or tab >>Bacteriocin plantaricin NC8 αβ antagonizes Porphyromonas gingivalis infection and induces proliferation of gingival epithelial cells
(English)Manuscript (preprint) (Other academic)
National Category
Other Basic Medicine
Research subject
Biomedicine
Identifiers
urn:nbn:se:oru:diva-52858 (URN)
Available from: 2016-10-06 Created: 2016-10-06 Last updated: 2016-10-06Bibliographically approved
4. Effects of plantaricin NC8 αβ and antibodies on gingival epithelial cells infected by Porphyromonas gingivalis
Open this publication in new window or tab >>Effects of plantaricin NC8 αβ and antibodies on gingival epithelial cells infected by Porphyromonas gingivalis
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(English)Manuscript (preprint) (Other academic)
National Category
Other Basic Medicine
Research subject
Biomedicine
Identifiers
urn:nbn:se:oru:diva-52859 (URN)
Available from: 2016-10-06 Created: 2016-10-06 Last updated: 2016-10-06Bibliographically approved

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