Production of Indole that Increases Biofilm Formation of Fusobacterium nucleatum

Objectives: Indole, which is produced by degradation of L-tryptophan, has been proposed to be one of extracellular signal molecules that influence biofilm formation in a range of bacteria. The aim of this study was to gain insights about mechanisms to produce indole in F. nucleatum and about effects of tryptophan and indole on biofilms formed by the microorganism. Methods: The transcript of tnaA encoding tryptophanase and the flanking regions was analyzed using RT-PCR and RLM-RACE. Real-time PCR was performed to analyze tnaA expression in F. nucleatum cells incubated with or without tryptophan or indole. The enzymatic properties of the purified TnaA of F. nucleatum were determined using a spectrophotometrical assay and HPLC analysis. Biofilm formation in the presence of tryptophan or indole was examined using crystal violet assay and confocal laser scanning microscopy. Result: The amino acid sequence of TnaA in F. nucleatum was 28% identical with that in Escherichia coli. Transcriptional analyses showed that tnaA was cotranscribed with the downstream gene and that the transcript started at positions–68 and –153 from the first nucleotide of tnaA. HPLC analysis demonstrated that the purified TnaA of F. nucleatum degraded L-tryptophan to produce of indole. The K _m and k _cat values of the enzyme were 0.26±0.03 mM and 0.74±0.04 S^–1, respectively. Real-time PCR analysis showed that tryptophan at any concentrations tested did not affect tnaA expression, whereas indole at a high concentration decreased the tnaA expression. Biofilms formed by F. nucleatum were increased by tryptophan and exogenous indole added in the cell culture in a dose-dependent manner. Conclusion: Indole produced by degradation of L-tryptophan function as a cell- to- cell signaling to increase biofilms formed by F. nucleatum.