Molecular identification of a Fusobacterium nucleatum beta defensin inducer

We previously reported that, by using biochemical proteomic approaches, we identified a fraction from the cell wall of F. nucleatum (ATCC 25586) containing four proteins that stimulated the production of human beta defensin 2 and 3 (hBD-2 and hBD3) in human oral epithelial cells (HOECs) (Scott et al, J. Dent. Res. 81:A-199, Abstract #1475, 2002). These are FadA (Han et al. 2005), FN1527, FN1529 and FN1792 (Kapatral et al. 2002; Accession NC_003454). Recombinant FadA failed to stimulate hBD production in HOECs. Objective: To identify which of the other three F. nucleatum cell wall associated proteins induce hBD-2 and -3 production in HOECs. Methods: We used standard recombinant methods to generate E.coli expression plasmids containing DNA sequences encoding the three proteins, respectively. HOEC monolayers were exposed to these recombinant proteins, followed by qPCR and ELISA (Ghosh et al, 2007) to determine hBD-2 and -3 transcript and peptide levels. For ease of purification and identification, we tagged FN1527 with c-myc on the C-terminal end. The peptide generated in E.coli was purified to near homogeneity by using the c-myc tag. Results: The E.coli clone expressing FN1527 induced hBD-2 and -3 mRNA significantly above baseline, followed by FN1792. FN1529 did not induce either hBD-2 or hBD-3 to any significant degree. Purified recombinant FN1527 induced hBD-2 and 3 mRNA and peptide significantly above baseline when applied to HOEC monolayers. Conclusion: FN1527 induces hBD expression in oral epithelial cells. It or its derivates may one day be used to enhance antimicrobial activity in human mucosal barriers. Supported by NIH/NIDCR 1R01DE016334; 1R01DE18276.