(The) role of autoinducer 2 quorum sensing in biofilm formation by oral bacteria

다국어 초록 (Multilingual Abstract)

The role of autoinduer 2 quorum sensing of oral bacteria in biofilm formation
Yunji Jang
Department of Dental Science, Major of Immunology and Molecular Microbiology in Dentistry, Graduate School, Seoul National University
(Directed by Professor Bong-Kyu Choi,Ph.D. )

Objectives
Periodontitis is initiated by bacteria in subgingival biofilms, which are composed of multi-species oral bacteria that are commensal bacteria (yellow complex) or periodontopathogens (red complex). Streptococci are early colonizers of oral bioflm. Streptococci colonized tooth surfaces and then biomass is increases with cell division. Fusobacterium attach to the early colonizers, and periodontopathogens join this developing biofilm communities then the mature biofilm is formed. Autoinduer-2(AI-2) is a universal quorum sensing (QS) molecule that mediates intergeneric signaling in multi species bacterial communities and may induce biofilm formation. As Fusobacterium nucleatum is the major coaggregation bridge organism that links early colonizers and late pathogenic colonizers in dental biofilm, I hypothesized that AI-2 of F. nucleatum contributes to this inter species interaction, and interruption of this signaling can result in the inhibition of biofilm formation of early and late colonizers.

Methods
The AI-2 activity of the culture supernatants of F. nucleatum in different growth phase was evaluated by a bioluminescence assay using V. harveyi BB170 (an AI-2 reporter strain). AI-2 was partially purified from the culture supernatants at 20 hours which showed highet bioluminescence. The purification method was as follows: The culture supernatant was
filtered using 0.2 μm-syringe-filter, then separated under 3 kDa molecules using YM-3 3 kDa-Centricon. The hydrophilic molecules that contained AI-2 were purified using a Sep-pak C18 column. The bioluminescence of the partially purified AI-2 was compared with those of positive control (the AI-2 of Vibrio harveyi BB152) and negative control (the culture supernatant Pseudomonas aeruginosa). The inhibition of bioluminescence effect of the purified AI-2 by QSIs, Furanone and D-Ribose was confirmed. To investigate effect of AI-2
on single-species biofilm formation, dual-species biofilm formation, and coaggregation between F. nucleatum and other oral bacteria including so-called ‘yellow complex’(Streptococcus gordonii and Streptococcus oralis), ‘red complex’ (Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia). The effect of two QSIs, (5Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone (furanone compound) and D-ribose, on AI-2-induced biofilm formation and coaggregation was tested. The effect of AI-2 and QSIs on the bacterial growth was evaluated. To verify the regulatory effect of AI-2 on the expression of virulence factors of periodotopathogens, the level of gene expression of virulence factors in AI-2-treated periodontopathogens was analyzed by real-time RT-PCR.

Results
The AI-2 of F. nucleatum remarkably induced the growth of single- and dual-species biofilm and coaggregation between F. nucleatum and species of the ‘yellow and red complex’ except S. oralis. The biofilm formation and coaggregation were inhibited by the QSIs. However, the biofilm fotmation and coaggregation were inhibited by AI-2 of F. nucleatum and increased by the QSIs. F. nucleatum AI-2 induced the expression of the representative adhesion molecules of bacteria, which were inhibited by the QSIs. F. nucleatum AI-2 and the QSIs did not affect the growth of bacteria.

Conclusion
Our results demonstrate that F. nucleatum AI-2 plays an important role in inter- and intra-species interactions of oral bacteria. These results show the possibility of AI-2 can be a target for the inhibition of dental biofilm formation by periodonyopathogens.

목차 (Table of Contents)

• Abstract
• Ⅰ. INTRODECTION
• Ⅱ. MATREIAL AND METHODS
• 1) Bacteria culture
• 2) Partial purification of Fusobacterium nucleatum autoinducer 2 (AI-2)
• 3) Quorum sensing inhibitor (QSIs)
• 4) Determination of AI-2-mediated bioluminescence
• 5) Biofilm formation assay
• 6) Coaggregation assay
• 7) Turbidity test
• 8) Analysis of expression of bacterial adhesion molecules by real-time RT-PCR
• 9) Cytotoxicity test of QSIs
• 10) Bacterial growth test
• 11) Statistical analysis
• Ⅲ. RESULTS
• 1) F. nucleatum AI-2 activity according to the growth phase of F. nucleatum
• 2) The activity of partially purified F. nucleatum AI-2
• 3) Effect of QSIs on AI-2 activity
• 4) Effect of QSIs on biofilm formation of periodontopathogens and streptococci by F. nucleatum AI-2
• 5) Effect of QSIs on coaggregation of periodontopathogens and streptococci by F. nucleatum AI-2
• 6) Modulation of the bacterial adhesion expression in periodontopathogens by F. nucleatum AI-2 and QSIs
• 7) Effects of F. nucleatum AI-2 on bacterial growth
• Ⅳ. DISCUSSION
• Ⅴ. CONCLUSION
• Ⅵ. REFERENCE
• 국문초록