TLR2-mediated epithelial cell activation by the Tannerella forsythia BspA protein

OBJECTIVES: Tannerella forsythia is a gram-negative anaerobe strongly associated with pathogenesis of human periodontitis. One of the putative virulence factors of this bacterium is the BspA protein, a cell surface-associated or secreted protein belonging to the leucine-rich repeat (LRR) protein family. BspA has been shown to interact with a variety of host cells through Toll-like receptor (TLR) signaling. Since gingival epithelium forms a barrier against periodontal pathogens, this study was undertaken to determine if gingival epithelial cells respond to BspA challenge and if TLRs play any role in BspA recognition. The study was also directed towards identifying the BspA domains responsible for cellular activation. To determine the role of TLRs in BspA-mediated activation of epithelial cells leading to chemokine secretion and to identify active domains/epitopes of BspA involved in TLR activation.

METHODS: Recombinant BspA protein (rBspA) and its truncated mutant derivatives were utilized to stimulate HEK293 cells transiently expressing TLRs alone or in combination. Cellular activation was determined by NF-κB-induced luciferase reporter assay as well as IL-8 ELISA. Alternatively, primary cells from human gingival epithelium were stimulated with BspA and TLR function blocking antibodies were used to identify the receptors involved. To evaluate direct binding of BspA to TLR2, we utilized ligand-receptor binding assay with biotin-labeled BspA as ligand and TLR2-Fc fusion protein coated on microtiter plates as receptor.

RESULTS: BspA binds to TLR2 leading to release of the chemokine IL-8 from human gingival epithelial cells. Furthermore, the LRR domain of BspA is involved in activation of TLR2, while TLR1 serves as a signaling partner.

CONCLUSION: BspA is an important modulator of host innate immune responses through activation of TLR2 in cooperation with TLR1. Further investigations are in progress to identify the peptide epitopes of BspA involved in TLR2 activation. This study was supported by a grant DE014749 (NIDCR).