To Örebro University

Periodontitis is a chronic infectious disease causing inflammation and destruction of tooth-supporting structures eventually leading to tooth loss. Periodontitis is induced by different subgingival microorganisms in combination with an excessive and dysregulated immune-inflammatory response against this microbial challenge. The pathogenicity of the periodontal biofilm is highly dependent upon some "keystone" species of which Porphyromonas gingivalis is considered to be one of the most important pathogens. P. gingivalis expresses a broad range of virulence factors including lipopolysaccharides (LPS), fimbriae, adhesins, hemagglutinins and an array of proteolytic enzymes. Among these factors, cysteine proteases (gingipains) are of special importance both for the bacterial survival/proliferation and for the pathological outcome. Gingipains consist of arginine-specific proteases (RgpA and RgpB) and lysine-specific protease (Kgp). 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, host lymphocytes were stimulated with P. gingivalis to develop antibodies in vitro and the anti-P. gingivalis antibodies were used for immunodetection in clinical samples. The in vitro method of antibody production developed during this study could be used for an efficient real-time detection of P. gingivalis infection and periodontitis, while the attenuating effects of the antibodies suggest a role in passive immunization to prevent periodontitis and its associated diseases. In study II, we have elucidated the properties and antimicrobial effects of different lactobacillus species and the two-peptide bacteriocin NC8 αβ on P. gingivalis. NC8 αβ was found to be efficient against P. gingivalis through bacterial binding followed by permeabilization of the membranes. Liposomal systems were acquired to verify membrane permeabilization by NC8 αβ. The antimicrobial activity of NC8 αβ was found to be rapid, potent and instant. In conclusion, soluble or immobilized NC8 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.

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.