The effect of autoinducer 2 quorum sensing inhibitors against biofilm formation of periodontopathogens

Abstract

Objectives Bacterial behaviors which are critical for survival such as virulence factor secretion, biofilm formation are performed effectively through quorum sensing, to which bacteria respond to control gene expression in response to changes in cell density and species complexity. Quorum sensing involves the production, release, and subsequent detection of chemical signal molecules called autoinducers. Autoinducer 2 (AI-2) is an universal quorum sensing molecule that mediates intergeneric signaling in multispecies bacterial communities. Especially, AI-2 of F. nucleatum plays an important role in formation of subgingival biofilm composed mostly of Gram-negative anaerobic bacteria in oral environment. The aim of this study was to design and synthesize new quorum sensing inhibitors (QSIs) and evaluate their inhibitory effect on AI-2 activity and thus reduce biofilm formation of pathogenic oral bacteria.

Methods D-Galactose and newly synthesized brominated furanone analogs were used as quorum sensing inhibitors (QSIs). Also, reported QSIs including D-ribose and (Z-)-4-Bromo-5-bromomethylene-2(5H)-furanone compound were used as reference compounds to compare the relative efficacy of the QSIs. To evaluate the effect on AI-2 activity, AI-2 reporter strain Vibrio harveyi BB170 and semi-purified Fusobacterium nucleatum AI-2 was used and the bioluminescence of Vibrio harveyi BB170 was assessed. The effect on biofilm formation of periodontopathogens was evaluated by crystal violet staining and confocal laser scanning microscopy after culturing each bacteria with F. nucleatum AI-2 and the QSIs. As QSIs should attenuate bacterial pathogenicity rather than bacterial growth, the bacterial growth was monitored in the presence of the each QSI without F. nucleatum AI-2. After that, to determine whether the QSIs are toxic or induce inflammatory response in host cell, the effect of the QSIs on host cells response were assessed by cytotoxicity test and detection of gene expression level of pro-inflammatory cytokines using real time RT-PCR.

Results Designed QSIs remarkably inhibited AI-2 activity of F. nucleatum and biofilm formation of major periodontopathogens (F. nucleatum, Porphyromonas gingivalis, and Tannerella forsythia) which was induced by F. nucleatum AI-2 without bacteriocidal effect. Furthermore, the synthesized QSIs did not have cytotoxicity and induce expression of host inflammatory cytokines in host cells (THP-1, HGFs and HOK-16B).

Conclusion Our results demonstrate that newly designed QSIs may be used as a preventive agent against biofilm formation of pathogenic bacteria by targeting QS signaling. Especially, the widespread use of antibiotics has resulted in bacterial resistance such as multiple drug resistance, to control the pathogens by QS inhibition appears to be a promising strategy to prevent periodontitis and other bacterial infectious diseases caused by biofilms.