Biological properties of the native and synthetic lipid A of Porphyromonas gingivalis lipopolysaccharide.
ABSTRACT A pentaacyl and diphosphoryl lipid A molecule found in the lipid A isolated from Porphyromonas gingivalis lipopolysaccharide (LPS) was chemically synthesized, and its characteristics were evaluated to reconfirm its interesting bioactivities including low endotoxicity and activity against LPS-unresponsive C3H/HeJ mouse cells.
The synthesized P. gingivalis lipid A (synthetic Pg-LA) exhibited strong activities almost equivalent to those of Escherichia coli-type synthetic lipid A (compound 506) in all assays on LPS-responsive mice, and cells. LPS and native lipid A of P. gingivalis displayed overall endotoxic activities, but its potency was reduced in comparison to the synthetic analogs. In the assays using C3H/HeJ mouse cells, the LPS and native lipid A significantly stimulated splenocytes to cause mitosis, and peritoneal macrophages to induce tumor necrosis factor-alpha and interleukin-6 production. However, synthetic Pg-LA and compound 506 showed no activity on the LPS-unresponsive cells. Inhibition assays using some inhibitors including anti-human Toll-like receptor 2 (TLR2) and TLR4/MD-2 complex monoclonal antibodies showed that the biological activity of synthetic Pg-LA was mediated only through the TLR4 signaling pathway, which might act as a receptor for LPS, whereas TLR2, possibly together with CD14, was associated with the signaling cascade for LPS and native lipid A of P. gingivalis, in addition to the TLR4 pathway.
These results suggested that the moderated and reduced biological activity of P. gingivalis LPS and native lipid A, including their activity on C3H/HeJ mouse cells via the TLR2-mediated pathway, may be mediated by bioactive contaminants or low acylated molecules present in the native preparations having multiple lipid A moieties.
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ABSTRACT: Capnocytophaga canimorsus is a usual member of dog's mouths flora that causes rare but dramatic human infections after dog bites. We determined the structure of C. canimorsus lipid A. The main features are that it is penta-acylated and composed of a "hybrid backbone" lacking the 4' phosphate and having a 1 phosphoethanolamine (P-Etn) at 2-amino-2-deoxy-d-glucose (GlcN). C. canimorsus LPS was 100 fold less endotoxic than Escherichia coli LPS. Surprisingly, C. canimorsus lipid A was 20,000 fold less endotoxic than the C. canimorsus lipid A-core. This represents the first example in which the core-oligosaccharide dramatically increases endotoxicity of a low endotoxic lipid A. The binding to human myeloid differentiation factor 2 (MD-2) was dramatically increased upon presence of the LPS core on the lipid A, explaining the difference in endotoxicity. Interaction of MD-2, cluster of differentiation antigen 14 (CD14) or LPS-binding protein (LBP) with the negative charge in the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) of the core might be needed to form the MD-2 - lipid A complex in case the 4' phosphate is not present.PLoS Pathogens 05/2012; 8(5):e1002667. · 8.14 Impact Factor
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ABSTRACT: Periodontal (gum) disease is one of the main global oral health burdens and severe periodontal disease (periodontitis) is a leading cause of tooth loss in adults globally. It also increases the risk of cardiovascular disease and diabetes mellitus. Porphyromonas gingivalis lipopolysaccharide (LPS) is a key virulent attribute that significantly contributes to periodontal pathogenesis. Baicalin is a flavonoid from Scutellaria radix, an herb commonly used in traditional Chinese medicine for treating inflammatory diseases. The present study examined the modulatory effect of baicalin on P. gingivalis LPS-induced expression of IL-6 and IL-8 in human oral keratinocytes (HOKs). Cells were pre-treated with baicalin (0-80 µM) for 24 h, and subsequently treated with P. gingivalis LPS at 10 µg/ml with or without baicalin for 3 h. IL-6 and IL-8 transcripts and proteins were detected by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The expression of nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) proteins was analyzed by western blot. A panel of genes related to toll-like receptor (TLR) signaling was examined by PCR array. We found that baicalin significantly downregulated P. gingivalis LPS-stimulated expression of IL-6 and IL-8, and inhibited P. gingivalis LPS-activated NF-κB, p38 MAPK and JNK. Furthermore, baicalin markedly downregulated P. gingivalis LPS-induced expression of genes associated with TLR signaling. In conclusion, the present study shows that baicalin may significantly downregulate P. gingivalis LPS-upregulated expression of IL-6 and IL-8 in HOKs via negative regulation of TLR signaling.PLoS ONE 01/2012; 7(12):e51008. · 3.73 Impact Factor
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ABSTRACT: BACKGROUND: Porphyromonas gingivalis is a major pathogen of periodontal disease that affects a majority of adults worldwide. Increasing evidence shows that periodontal disease is linked to various systemic diseases like diabetes and cardiovascular disease, by contributing to increased systemic levels of inflammation. Lipopolysaccharides (LPS), as a key virulent attribute of P. gingivalis, possesses significant amount of lipid A heterogeneity containing tetra- (LPS1435/1449) and penta-acylated (LPS1690) structures. Hitherto, the exact molecular mechanism of P. gingivalis LPS involved in periodontal pathogenesis remains unclear, due to limited understanding of the specific receptors and signaling pathways involved in LPS-host cell interactions. METHODOLOGYPRINCIPAL FINDINGS: This study systematically investigated the effects of P. gingivalis LPS1435/1449 and LPS1690 on the expression of TLR2 and TLR4 signal transduction and the activation of pro-inflammatory cytokines IL-6 and IL-8 in human gingival fibroblasts (HGFs). We found that LPS1435/1449 and LPS1690 differentially modulated TLR2 and TLR4 expression. NF-κB pathway was significantly activated by LPS1690 but not by LPS1435/1449. In addition, LPS1690 induced significant expression of NF-κB and p38 MPAK pathways-related genes, such as NFKBIA, NFKB1, IKBKB, MAP2K4 and MAPK8. Notably, the pro-inflammatory genes including GM-CSF, CXCL10, G-CSF, IL-6, IL-8 and CCL2 were significantly upregulated by LPS1690 while down-regulated by LPS1435/1449. Blocking assays confirmed that TLR4-mediated NF-κB signaling was vital in LPS1690-induced expression of IL-6 and IL-8 in HGFs. CONCLUSIONSSIGNIFICANCE: The present study suggests that the tetra- and penta-acylated lipid A structures of P. gingivalis LPS differentially activate TLR4-mediated NF-κB signaling pathway, and significantly modulate the expression of IL-6 and IL-8 in HGFs. The ability to alter the lipid A structure of LPS could be one of the strategies carried-out by P. gingivalis to evade innate host defense in gingival tissues, thereby contributing to periodontal pathogenesis.PLoS ONE 01/2013; 8(3):e58496. · 3.73 Impact Factor