Molecular structure and immunological function of lipoteichoic acid purified from Enterococcus faecalis

Abstract

Enterococcus faecalis is a Gram-positive commensal bacterium that is frequently found in mucosal tissues of the oral cavity, gastrointestinal tract, and genital tract in humans. E. faecalis is one of the major opportunistic pathogens causing inflammatory diseases such as bacteremia and refractory apical periodontitis. Unlike Gram-negative bacteria that express lipopolysaccharide (LPS), a major etiologic component that causes inflammatory responses, Gram-positive bacteria express lipoteichoic acid (LTA) that is considered as the counterpart of LPS. Despite the importance of LTA, its molecular structure and immunological functions have been poorly characterized because the earlier LTA preparations were often contaminated and/or structurally damaged due to improper purification processes. Recently, a novel method has been introduced for purification of LTA using the sequential application of mild organic solvent extraction, hydrophobic-interaction column chromatography and ion-exchange column chromatography. The aims of the present study were (1) to purify highly pure and structurally intact LTA from E. faecalis, (2) to investigate its structural and functional relationship in the modulation of innate immune responses, and (3) to elucidate the action mechanism of endodontic medicament against E. faecalis and its LTA. LTAs from E. faecalis, Staphylococcus aureus, Bacillus anthracis, and Lactobacillus plantarum were purified by butanol extraction followed by chromatography with the hydrophobic-interaction and ion-exchange columns using octyl-sepharose and diethlyaminoethyl (DEAE)-sepharose, respectively. Endotoxin level in the LTA preparations was determined by Limulus amebocyte lysate (LAL) assay. The contamination of LTA preparations with nucleic acid was examined by using spectrophotometry. The contamination of LTA preparations with proteins was determined via coomassie blue and silver stainings. A murine macrophage cell-line (RAW 264.7) and bone marrow-derived macrophage (BMM) from wild-type or Toll-like receptor (TLR) 2-deficient C57/BL6 mice were used to determine the abilities of the purified LTAs to induce the production of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, interferon gamma-inducible protein 10 (IP-10), macrophage inflammatory protein-1 alpha (MIP-1α), and monocyte chemoattractant protein-1 (MCP-1). To determine the production of proinflammatory mediators in macrophages, RAW 264.7 cells and BMMs were stimulated with purified LTAs in the presence or absence of recombinant mouse interferon-gamma (IFN-γ) and the production of TNF-α, IL-6, IP-10, MIP-1α, and MCP-1 in the culture supernatant was measured by enzyme-linked immunosorbent assay (ELISA). The nitric oxide (NO) production in LTA-stimulated macrophages was determined by reacting with an equal volume of Griess reagent. The ability to activate TLR2 or TLR4 by purified LTAs was determined by flow cytometry with nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) reporter cell lines, CHO/CD14/TLR2 and CHO/CD14/TLR4, respectively. DNA-binding activity of NF-κB was determined by electrophoretic mobility shift assay (EMSA). Backbone structure of LTAs was determined by Western blot analysis. The production of TNF-α in macrophages upon stimulation with heat-killed or calcium hydroxide-killed E. faecalis was determined by ELISA. The immunostimulating abilities of intact E. faecalis LTA or calcium hydroxide-treated E. faecalis LTA to induce the production of NO, IP-10, and MIP-1α were determined by ELISA. TLR2-stimulating activity of intact E. faecalis LTA or calcium hydroxide-treated E. faecalis LTA was determined by flow cytometry using CHO/CD14/TLR2. The structure of glycolipid in E. faecalis LTA or in calcium hydroxide-treated E. faecalis LTA was analyzed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and thin layer chromatography (TLC). Upon exposure to E. faecalis LTA, RAW 264.7 cells significantly produced TNF-α in a concentration-dependent manner. Although E. faecalis LTA weakly induced the production of NO in RAW 264.7 cells, its ability to induce NO was remarkably increased in the presence of IFN-γ under the same condition. E. faecalis LTA preferentially activated TLR2 cells rather than TLR4. Concomitantly, E. faecalis LTA enhanced the DNA-binding activity of NF-κB, which plays an important role in the transcriptional activation of genes encoding inflammatory mediators. E. faecalis LTA was not able to induce TNF-α in macrophages lacking TLR2 while significantly induced TNF-α in wild-type macrophages. Compared with LTAs purified from other Gram-positive bacteria such as S. aureus, L. plantarum, and B. anthracis, E. faecalis LTA showed a moderate potential in the activation of TLR2 and the induction of inflammatory mediators. E. faecalis LTA weakly interacted with anti-polyglycerophosphate antibody compared with other polyglycerophosphate-type LTAs such as S. aureus LTA, L. plantarum LTA, and B. anthracis LTA, implying that E. faecalis LTA might have a subtle structural difference in its polyglycerophosphate backbone compared with other polyglycerophosphate-type LTAs. The structural analysis using MALDI-TOF demonstrated that the glycolipids from E. faecalis LTA consist of dihexosyl diacylglycerol harboring fatty acids ranging from C16 to C22. Interestingly, unlike most LTAs which likely possess saturated and mono-unsaturated acyl chains, E. faecalis LTA contained mono- and di-unsaturated fatty acids. Heat-killed E. faecalis potently induced the production of TNF-α in RAW 264.7 cells while such induction was significantly dampened by calcium hydroxide-killed E. faecalis under the same condition. Treatment of E. faecalis LTA with calcium hydroxide remarkably abrogated its ability to induce the production of proinflammatory mediators such as NO, TNF-α, IP-10, and MIP-1α in RAW 264.7 cells. Furthermore, calcium hydroxide-treated E. faecalis LTA was not able to activate TLR2 signaling pathway. Structural analysis using MALDI-TOF and TLC showed that calcium hydroxide deacylated the glycolipid moiety of E. faecalis LTA, implying that the lipid moiety of E. faecalis LTA is crucial for its inflammatory potential. A novel purification method using mild organic solvent extraction, hydrophobic-interaction and ion-exchange chromatography provided a highly-pure and structurally-intact E. faecalis LTA preparation. E. faecalis LTA is one of the major immunostimulating components in the Gram-positive bacterial cell wall capable of activating the TLR2 signaling pathway. LTAs of E. faecalis have moderate inflammatory potential compared to LTAs purified from different bacterial species. E. faecalis LTA is a polyglycerophosphate-type LTA with subtle structural differences of substituent in repeating units and fatty acid composition. The lipid moiety of E. faecalis LTA plays a crucial role in determining its ability to induce the production of TNF-α, IL-6, IP-10, MIP-1α, and MCP-1 in macrophages by affecting TLR2 activation. Treatment with calcium hydroxide abrogates the inflammatory potential of E. faecalis LTA by inducing deacylation of the lipid moiety of LTA. Thus, development of medicament targeting either the lipid moiety of LTA or TLR2 signaling would be a good therapeutic candidate. Collectively, E. faecalis LTA is an important virulence factor of E. faecalis for inducing inflammatory responses through TLR2 signaling pathway and its lipid moiety is essential for its inflammatory activity.