Immunological properties of lipoteichoic acid isolated from Streptococcus mutans and its binding proteins in human saliva

Abstract

Streptococcus mutans is a pathogenic Gram-positive bacterium that is closely associated with dental caries and subsequent pulpal inflammation. Although lipoteichoic acid (LTA) is considered a major virulence factor of Gram-positive bacteria, little is known about the innate immunity to S. mutans LTA and its binding proteins in human saliva. In this study, LTA was purified from S. mutans (Sm.LTA) through n-butanol extraction, hydrophobic-interaction column chromatography, and ion-exchange column chromatography to investigate its immunological properties using murine macrophages. Furthermore, its binding proteins (Sm.LTA-BPs) were identified in the saliva from caries-free and caries-positive human subjects using Sm.LTA-conjugated beads and LTQ-Orbitrap hybrid Fourier transform mass spectrometry. The Sm.LTA preparation had no detectable contamination with endotoxins, proteins, or nucleic acids. Upon exposure to Sm.LTA, the murine macrophage cell-line RAW 264.7 cells produced tumor necrosis factor-alpha (TNF-α) and nitric oxide (NO) in a dose-dependent manner. Sm.LTA preferentially bound to and activated CHO/CD14/TLR2 cells rather than CHO/CD14/TLR4 cells, which are stable transfectants expressing CD14 together with TLR2 or TLR4. Sm.LTA could not induce TNF-α or NO production in macrophages derived from TLR2-deficient mice whereas it dose-dependently induced those inflammatory mediators in wild-type macrophages. Furthermore, Sm.LTA deacylated by alkaline hydrolysis neither stimulated TLR2 nor induced TNF-α or NO production, suggesting that lipid moieties are crucial for the immuno-stimulatory activity of Sm.LTA. Unlike Staphylococcus aureus LTA, which has a potent immuno-stimulating activity, Sm.LTA showed a modest induction of NO production comparable to LTAs of Enterococcus faecalis and Lactobacillus plantarum. To further identify the binding proteins in saliva from caries-free and caries-positive human subjects, Sm.LTA was conjugated onto N-hydroxysuccinimidyl-Sepharose® 4 Fast Flow beads (Sm.LTA-beads). Sm.LTA retained its biological properties during conjugation as determined by the expression of NO and interferon gamma-inducible protein 10 (IP-10) in RAW 264.7 cells and activation of TLR2 in CHO/CD14/TLR2 cells. Then, Sm.LTA-BPs were isolated from the salivary pools prepared from each of ten caries-free and caries-positive human subjects followed by electrophoresis to separate distinctively-expressed proteins for further identification using the high-resolution mass spectrometry. A total of 9 and 12 LTA-BPs were identified with statistical significance in each salivary pool from caries-free and caries- positive human subjects, respectively. Sm.LTA-BPs found in caries-free saliva included histone H4, profilin-1, and neutrophil defensin-1 and those in caries- positive saliva included cystatin-C, cystatin-SN, cystain-S, cystain-D, lysozyme C, calmodulin-like protein 3, and actin. Sm.LTA-BPs found in both groups were hemoglobins, prolactin-inducible protein, protein S100-A9, and SPLUNC2. In conclusion, the present study suggest that the Sm.LTA contributes to the inflammatory responses induced by S. mutans in which its lipid moiety is essential for TLR2 binding and activation.