Human salivary proteins with affinity to lipoteichoic acid of Enterococcus faecalis

Enterococcus faecalis is associated with refractory apical periodontitis and its lipoteichoic acid (Ef.LTA) is considered as a major virulence factor. Although the binding proteins of Ef.LTA may play an important role for mediating infection and immunity in the oral cavity, little is known about Ef.LTA-binding proteins (Ef.LTA-BPs) in saliva. In this study, we identified salivary Ef.LTA-BPs with biotinylated Ef.LTA (Ef.LTA-biotin) through mass spectrometry. The biotinylation of Ef.LTA was confirmed by binding capacity with streptavidin-FITC on CHO/CD14/TLR2 cells. The biological activity of Ef.LTA-biotin was determined based on the induction of nitric oxide and macrophage inflammatory protein-1α in a macrophage cell-line, RAW 264.7. To identify salivary Ef.LTA-BPs, the Ef.LTA-biotin was mixed with a pool of human saliva obtained from nine healthy subjects followed by precipitation with a streptavidin-coated bead. Ef.LTA-BPs were then separated with 12% SDS-PAGE and subjected to the mass spectrometry. Six human salivary Ef.LTA-BPs including short palate lung and nasal epithelium carcinoma-associated protein 2, zymogen granule protein 16 homolog B, hemoglobin subunit α and β, apolipoprotein A-I, and lipocalin-1 were identified with statistical significance (P < 0.05). Ef.LTA-BPs were validated with lipocalin-1 using pull-down assay. Hemoglobin inhibited the biofilm formation of E. faecalis whereas lipocalin-1 did not show such effect. Collectively, the identified Ef.LTA-BPs could provide clues for our understanding of the pathogenesis of E. faecalis and host immunity in oral cavity.