The long term goal of this research is to determine whether specific bacterial oral pathogens utilize quorum-sensing as a mechanism to regulate the expression of virulence products involved in the initiation and progression of periodontal disease. Quorum-sensing systems make use of a regulatory protein, termed the R-protein, and a small apparently freely-diffusible autoinducer (AI) molecule which act in concert to stimulate the expression of various genes. In addition, bacterial populations utilize AI as a signal molecule with which to monitor their own cell density. Quorum sensing as a regulatory mechanism appears to be almost ubiquitous among Gram negative organisms. In some organisms, such as the pathogen Pseudomonas aeruginosa, the genes involved in the quorum sensing mechanism and their target genes are fairly well identified, while in others, the function of this regulatory system has yet to be elucidated. The involvement of specific gram negative bacteria such as Prevotella intermedia, Porphyromonas gingivalis, Treponema denticola, and Actinobacillus actinomycetemcomitans, and the role of some of the specific virulence factors they produce in the progression of periodontitis has been well described. Given our previous experience in working with these compounds, and by making use of the fact that all Gram negative AIs thus far identified are structurally similar, we propose: 1) To determine whether periodontal pathogens produce AI compound(s) by determining whether untreated or ethyl acetate-extracted culture supernatants of these pathogens can stimulate expression of various quorum-sensing responsive reporter systems. 2) To chemically characterize the AI molecule through the use of HPLC to isolate the active fraction present within the supernatant. HPLC-purified material will be further tested for its ability to act as an AI in reporter systems. Identification of an AI in periodontal pathogens will be unique as no AI has been reported from oral bacteria or anaerobic organisms. Demonstration of AI production will provide insight into the regulation of virulence factor production by these organisms. In addition, the presence of a quorum sensing mechanism may suggest novel areas for research into interactions between oral bacteria. Most exciting is the potential for new therapeutic approaches for periodontal disease.
