Chemical structure and immunobiological activity of Porphyromonas gingivalis lipid A.

In 1933, Boivin et al. extracted an endotoxin from Salmonella typhimurium for the first time, after which a variety of chemical and biological studies on endotoxins have been performed. In 1952, the structural and functional properties of endotoxic lipopolysaccharide (LPS), extracted by a hot phenol and water method devised by Westphal et al., were reported, which led to a number of studies of Gram-negative bacteria in regards to the host defense mechanism. Since 1960, the unique chemical structure and biological activity of Bacteroides species LPS have received a great deal of attention, and there is a long history of such studies. In addition, among oral bacterial strains that have received attention as causative periodontopathic bacteria, many have been classified as Bacteroides species. In particular, a number of researchers have investigated whether LPS of Porphyromonas gingivalis (formerly Bacteroides gingivalis), a black-pigmented oral anaerobic rod, is a virulent factor of the bacterium. The active center of the LPS of these Bacteroides species, the lipid A molecule, is known to be an active participant in endotoxic activation, though its other biological activities are weak, due to its unique chemical structure and action as an antagonist of LPS. On the other hand, many reports have noted that the LPS of those species activate cells in C3H/HeJ mice, which generally do not respond to LPS. We were the first to reveal the chemical structure of P. gingivalis lipid A and, together with other researchers, reported that P. gingivalis LPS and its lipid A have activities toward C3H/HeJ mice. Since that time, because of the popularity of Toll-like receptor (TLR) studies, a great deal of evidence has been reported indicating that P. gingivalis LPS and its lipid A are ligands that act on TLR2. In order to solve such problems as heterogeneity and contamination of the biologically active components of P. gingivalis lipid A, we produced a chemical synthesis counterpart of lipid A and test results indicated it to be a TLR4 agonist. Furthermore, in order to disprove the common belief that P. gingivalis LPS and its lipid A are TLR2 ligands, the TLR2-active component contained in a P. gingivalis LPS fraction was separated and purified, after which we showed its chemical structure to be a lipoprotein consisting of three fatty acid residues, thus answering a longstanding question regarding Bacteroides species LPS. In addition to the field of dentistry, many studies based on the misconception of "TLR2-active LPS/lipid A" still exist in the field of innate immunity. Based on the history of studies of ligands acting on TLR4, Bacteroides species LPS findings were reviewed and are presented here. In particular, we investigated P. gingivalis LPS and its lipid A.

Toll-like receptor 2-mediated dendritic cell activation by a Porphyromonas gingivalis synthetic lipopeptide.

A PG1828 gene-encoded triacylated lipoprotein was previously isolated from a Porphyromonas gingivalis lipopolysaccharide preparation as a Toll-like receptor (TLR) 2 agonist and its lipopeptide derivatives were synthesized based on the chemical structure. In the present study, granulocyte-macrophage colony stimulating factor-differentiated bone marrow-derived dendritic cells (BMDDCs) were stimulated separately with the P. gingivalis synthetic lipopeptide N-palmitoyl-S-[2-pentadecanoyloxy, 3-palmitoyloxy-(2R)-propyl]-l-Cys-Asn-Ser-Gln-Ala-Lys (PGTP2-RL) and its glyceryl stereoisomer (PGTP2-SL). Only PGTP2-RL activated BMDDCs from wild-type mice to secrete tumour necrosis factor-alpha, interleukin (IL)-6, IL-10 and IL-12p40, whilst PGTP2-RL-induced cytokine production was eliminated in TLR2 knockout (-/-) BMDDCs. BMDDCs from wild-type mice but not TLR2-/- mice responded to PGTP2-RL as well as Pam(3)CSK(4) by increasing the expression of maturation markers, including CD80 (B7-1), CD86 (B7-2), CD40, CD275 (B7RP-1/inducible T-cell co-stimulatory ligand) and major histocompatibility complex class II. Taken together, these results indicate that the fatty acid residue at the glycerol position in the P. gingivalis lipopeptide plays a pivotal role in TLR2-mediated dendritic cell activation.

Genetic variation of a fimbrial protein from Porphyromonas gingivalis and its distribution in patients with periodontal diseases.

Pg-II fim from various strains of Porphyromonas gingivalis was classified on the basis of each nucleotide sequence, while the distribution of Pg-II fim types in 141 subgingival plaque samples was analyzed using PCR assays. Pg-II fim was divided into two types as follows: strains OMZ409, HG405, 381, ATCC 33277 and BH18/10 (type 1) and strains OMZ314 and HW24D-1 (type 2). The presence of P. gingivalis was demonstrated in 2.8% of healthy subjects and 56.1% of patients with periodontal diseases, and Pg-II fim was detected in 91.8% of the P. gingivalis-positive subjects. We also analyzed the distribution of the Pg-II fim types among Pg-II fim-positive patients, with the following results: type 1 (38.2%), type 2 (56.4%) and types 1 and 2 (5.4%). These findings strongly suggest that P. gingivalis organisms possessing Pg-II fim type 2 was principally detected in patients with periodontal diseases.

Chemical structure and immunobiological activity of lipid A from Prevotella intermedia ATCC 25611 lipopolysaccharide.

The novel chemical structure and immunobiological activities of Prevotella intermedia ATCC 25611 lipid A were investigated. A lipopolysaccharide (LPS) preparation of P. intermedia was extracted using a phenol-chloroform-petroleum ether method, after which its purified lipid A was prepared by weak acid hydrolysis followed by chromatographic separations. The lipid A structure was determined by mass spectrometry and nuclear magnetic resonance to be a diglucosamine backbone with a phosphate at the 4-position of the non-reducing side sugar, as well as five fatty acids containing branched long chains. It was similar to that of Bacteroides fragilis and Porphyromonas gingivalis, except for the phosphorylation site. P. intermedia lipid A induced weaker cytokine production and NF-kappaB activation in murine cells via Toll-like receptor (TLR) 4 as compared to Escherichia coli synthetic lipid A (compound 506). Our results indicate that P. intermedia lipid A activates cells through a TLR4-dependent pathway similar to E. coli-type lipid A, even though these have structural differences.

Binding of Porphyromonas gingivalis fimbriae to Treponema denticola dentilisin.

Treponema denticola has been reported to coaggregate with Porphyromonas gingivalis and localize closely together in matured subgingival plaque. In this study of the interaction of T. denticola with P. gingivalis, the P. gingivalis fimbria-binding protein of T. denticola was identified by two-dimensional electrophoresis followed by a ligand overlay assay with P. gingivalis fimbriae, and was determined to be dentilisin, a chymotrypsin-like proteinase of T. denticola. The binding was further demonstrated with a ligand overlay assay using an isolated GST fusion dentilisin construct. Our results suggest that P. gingivalis fimbriae and T. denticola dentilisin are implicated in the coaggregation of these bacteria.

Possible requirement of intercellular adhesion molecule-1 for invasion of gingival epithelial cells by Treponema medium.

Oral treponemes are members of the spirochete family of bacteria associated with periodontal diseases. In the present study, we demonstrate that intercellular adhesion molecule-1 (ICAM-1) on human gingival epithelial cells (HGEC) contributed to the invasion of Treponema medium, a medium-sized oral Treponema, into those cells. The quantity of T. medium in HGEC was found to peak at 2 h after inoculation and then decreased gradually. Immunofluorescence microscopy findings showed that the bacteria were colocalized with ICAM-1 on HGEC. Furthermore, knockdown of ICAM-1 in HGEC resulted in inhibition of T. medium invasion by RNA interference, whereas that of Toll-like receptor 2 did not. These results suggest that ICAM-1 may be required for the invasion of T. medium into HGEC, and they indicate that the molecule plays a principal role in the primary stages of the development and progression of chronic periodontitis.

Requirement for intercellular adhesion molecule 1 and caveolae in invasion of human oral epithelial cells by Porphyromonas gingivalis.

Porphyromonas gingivalis, a periodontopathic bacterium, is known to invade oral epithelial cells in periodontal lesions, although the mechanism is unclear. In the present study, goat polyclonal anti-intercellular adhesion molecule 1 (anti-ICAM-1) antibody inhibited the invasion of P. gingivalis into KB cells (human oral epithelial cells). Further, the P. gingivalis fimbria, a pathogenic adhesion molecule, bound to recombinant human ICAM-1, as shown by enzyme-linked immunosorbent assay. P. gingivalis was also found to colocalize with ICAM-1 on KB cells, as seen with an immunofluorescence microscope, and the knockdown of ICAM-1 in KB cells resulted in the inhibition of P. gingivalis invasion by RNA interference. In addition, methyl-beta-cyclodextrin, a cholesterol-binding agent, inhibited the colocalization of P. gingivalis with ICAM-1 and invasion by the microorganism. The colocalization of caveolin-1, a caveolar marker protein, on KB cells with P. gingivalis was also shown, and the knockdown of caveolin-1 in KB cells caused a reduced level of P. gingivalis invasion. These results suggest that ICAM-1 and caveolae are required for the invasion of P. gingivalis into human oral epithelial cells, and these molecules appear to be associated with the primary stages of the development and progression of chronic periodontitis.

Lipopolysaccharide preparation extracted from Porphyromonas gingivalis lipoprotein-deficient mutant shows a marked decrease in toll-like receptor 2-mediated signaling.

We recently demonstrated that a new PG1828-encoded lipoprotein (PG1828LP) was able to be separated from a Porphyromonas gingivalis lipopolysaccharide (LPS) preparation, and we found that it exhibited strong cell activation, similar to that of Escherichia coli LPS, through a Toll-like receptor 2 (TLR2)-dependent pathway. In order to determine the virulence of PG1828LP toward cell activation, we generated a PG1828-deficient mutant of P. gingivalis strain 381 by allelic exchange mutagenesis using an ermF-ermAM antibiotic resistance cassette. A highly purified preparation of LPS from a PG1828-deficient mutant (DeltaPG1828-LPS) showed nearly the same ladder-like patterns in silver-stained gels as a preparation of LPS from a wild-type strain (WT-LPS), as well as Limulus amoebocyte lysate activities that were similar to those of the WT-LPS preparation. However, the ability of the DeltaPG1828-LPS preparation to activate NF-kappaB in TLR2-expressing cells was markedly attenuated. Cytokine production by human gingival fibroblasts was also decreased in response to the DeltaPG1828-LPS preparation in comparison with the WT-LPS preparation, and the activity was comparable to the stimulation of highly purified lipid A of P. gingivalis by TLR4. Further, lethal toxicity was rarely observed following intraperitoneal injection of the PG1828-deficient mutant into mice compared to that with the wild-type strain, while the DeltaPG1828-LPS preparation showed no lethal toxicity. Taken together, these results clearly indicate that PG1828LP plays an essential role in inflammatory responses and may be a major virulence factor of P. gingivalis.

Antimicrobial activity of perilla seed polyphenols against oral pathogenic bacteria.

A perilla seed (Perilla frutescens Britton var. japonica Hara) extract was examined for its antimicrobial activity against oral cariogenic streptococci and periodontopathic Porphyromonas gingivalis. Luteolin, one of the components of perilla seed, showed the strongest antimicrobial effect among the phenolic compounds. According to our results, perilla seed may be the source of an antimicrobial agent that could prevent dental caries and periodontal diseases.

Oral treponemes and their outer membrane extracts activate human gingival epithelial cells through toll-like receptor 2.

Oral treponemes are considered to be important in the development and progression of periodontal diseases. We investigated the mechanisms of recognition and activation of human gingival epithelial cells (HGEC) with the oral treponemes Treponema denticola, Treponema vincentii, and Treponema medium and their outer membrane extracts (OMEs). T. vincentii and T. medium but not T. denticola produced interleukin 8 (IL-8) in an HGEC culture. Further, all three treponemes induced IL-8 mRNA expression and NF-kappaB activation in HGEC. Among them, T. denticola especially exhibited trypsin- and chymotrypsin-like protease activities, and the addition of chymostatin, a chymotrypsin protease inhibitor, resulted in detectable IL-8 production by HGEC cultured with T. denticola. Additionally, IL-8 mRNA expression in HGEC cultured with the three treponemes and their OMEs was definitely inhibited by the mouse anti-human Toll-like receptor 2 (TLR2) monoclonal antibody TL2.1. These findings suggest that oral treponemes and their OMEs activate HGEC through TLR2.

Separation and structural analysis of lipoprotein in a lipopolysaccharide preparation from Porphyromonas gingivalis.

Lipopolysaccharide (LPS) preparations from the periodontopathic bacterium Porphyromonas gingivalis (Pg-LPS) are thought to require Toll-like receptor (TLR)2 rather than TLR4, a receptor of Escherichia coli LPS (Ec-LPS), for activation of immune cells. However, we previously reported that P. gingivalis lipid A, an immunostimulatory principal component of LPS, and its synthetic counterpart activate cells through a TLR4-dependent pathway but not via TLR2. In the present study, a lipoprotein from Pg-LPS (Pg-LP) was shown to be a principal component for TLR2-mediated cell activation. Pg-LP was separated by hydrophobic interaction chromatography followed by preparative electrophoresis and identified by internal peptide sequencing as PG1828, a putative lipoprotein encoded in the P. gingivalis genome. The N-terminal structure was characterized as a triacylated lipopeptide using mass spectrometry. Pg-LP, as well as Ec-LPS, was potent in inducing IL-8 production in human gingival fibroblasts. From our results, we propose that Pg-LP is a powerful inflammatory factor of P. gingivalis.

Porphyromonas gingivalis lipopolysaccharides induce maturation of dendritic cells with CD14+CD16+ phenotype.

Primary immune responses are initiated by dendritic cells (DC) that inform naive T helper cells about invading pathogens. DC undergo sequential events leading to irreversible maturation upon bacterial stimulation. To investigate the responses of DC during periodontal infection, we studied the effects of LPS from Porphyromonas gingivalis on DC. DC generated from human peripheral monocytes by culture with IL-4 and GM-CSF were incubated with P. gingivalis LPS (Pg LPS) or Escherichia coli LPS (Ec LPS). Flow cytometry and real-time quantitative RT-PCR analysis revealed that Pg LPS, but not Ec LPS, preferentially up-regulated CD14 and CD16 expression at protein and mRNA levels. Furthermore, Pg LPS preferentially induced the secretion of soluble CD14. CD1a, HLA-DR and CD54 were highly expressed on DC stimulated with both kinds of LPS; however, CD40, CD80, CD83 and CD86 expression on Pg LPS-stimulated DC was lower than on Ec LPS-stimulated DC. With regard to IL-6, IL-8, IL-10, IL-12 and RANTES production from DC and allogeneic T cell proliferation, Pg LPS was a weaker stimulator than Ec LPS. These results suggested that Pg LPS triggers maturation of DC with unique characteristics, which exhibited weak immunostimulatory activity and may contribute to induction of chronic inflammation at the site of periodontal infection.

Bacterial fimbriae activate human peripheral blood monocytes utilizing TLR2, CD14 and CD11a/CD18 as cellular receptors.

Bacterial fimbriae are associated with a specific adherence factor, adhesin, in their microbial etiology. Porphyromonas gingivalis, as an anaerobic Gram-negative periodontopathogenic organism, is known to possess fimbriae on its cell surfaces. In this study, we demonstrated that P. gingivalis fimbriae and an active synthetic peptide composed of residues 69 - 73 of thefimbrial subunit protein, ALTTE, induced IL-6 mRNA expression and cytokine production, p38 mitogen-activated protein (MAP) kinase phosphorylation, and NF-kappaB activation in human peripheral blood monocytes. P. gingivalis fimbriae and ALTTE also induced IL-6 production via human Toll-like receptor (TLR) 2, CD14, and CD11a/CD18 (LFA-1) molecules on human monocytes. These results suggest that P. gingivalis fimbriae and these degraded peptides may play an important role in the inflamed gingival and periodontal tissues seen in the development and progression of periodontal diseases.

Detection and quantification of oral treponemes in subgingival plaque by real-time PCR.

Oral treponemes have been associated with periodontal diseases. We developed a highly sensitive and specific method to detect and quantify cultivable oral treponemes (Treponema denticola, Treponema vincentii, and Treponema medium) in 50 subgingival plaque samples from 13 healthy subjects as well as 37 patients with periodontal diseases using real-time PCR assays with specific primers and a TaqMan probe for each 16S rRNA sequence. The specificity for each assay was examined by using DNA specimens from various treponemal and other bacterial species. The TaqMan real-time PCR was able to detect from 10(3) to 10(8) cells of the oral treponemes, with correlation coefficients as follows: T. denticola, 0.984; T. vincentii, 0.991; and T. medium, 0.984. The frequencies of occurrence of these three oral treponemes in subgingival plaque samples were as follows: T. denticola, 68.0%; T. vincentii, 36.0%; and T. medium, 48.0%. In addition, the number of T. denticola, T. vincentii, and T. medium cells in plaque samples detected by real-time PCR ranged from 3 to 15,184, 1 to 447, and 1 to 7,301 cells/pg of plaque DNA, respectively. Increased numbers of T. denticola cells were detected in plaque samples from deep periodontal pockets, and T. medium was also detected in deep pockets. On the other hand, T. vincentii was mainly found in shallow pockets. These results suggest that various oral treponemes are associated with the formation of each stage of periodontal disease.

Cell activation by Porphyromonas gingivalis lipid A molecule through Toll-like receptor 4- and myeloid differentiation factor 88-dependent signaling pathway.

Porphyromonas gingivalis lipopolysaccharide (LPS) and its bioactive center, lipid A, are known to exhibit very low endotoxic activities and activate LPS-hyporesponsive C3H/HeJ mice that have a point mutation in the cytoplasmic portion of Toll-like receptor (TLR) 4, in contrast to classical enterobacterial LPS and their lipid A. In the present study, we attempted to determine which TLR mediates the response to lipid A from P. gingivalis strain 381. P. gingivalis LPS and its natural lipid A fraction induced NF-kappa B activation primarily in Ba/F3 cells expressing mouse TLR 2 (Ba/mTLR2), rather than in those expressing mouse TLR4 and its accessory protein MD2 (Ba/mTLR4/mMD2). Further purification of the natural lipid A fraction resulted in a significant decrease of NF-kappa B activation in Ba/mTLR2, although not in Ba/mTLR4/mMD2. The synthetic counterpart of P. gingivalis strain 381-lipid A (compound PG-381) also elicited NF-kappa B activation in Ba/mTLR4/mMD2, but not Ba/mTLR2. Furthermore, P. gingivalis purified natural lipid A and compound PG-381 lacked the ability to activate gingival fibroblasts from C3H/HeJ, TLR4 knockout (KO) and myeloid differentiation factor 88 (MyD88) KO mice. These findings demonstrate that the P. gingivalis lipid A molecule induces cell activation via a TLR4/MD2-MyD88-dependent pathway, and suggest the possibility that unknown bacterial components in P. gingivalis LPS and its lipid A may induce cell activation via TLR2.

Relationship between structures and biological activities of mycoplasmal diacylated lipopeptides and their recognition by toll-like receptors 2 and 6.

The lipopeptide FSL-1 [S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe, Pam(2)CGDPKHPKSF] synthesized on the basis of the N-terminal structure of a Mycoplasma salivarium lipoprotein capable of activating normal human gingival fibroblasts to induce the cell surface expression of ICAM-1 revealed an activity to induce production of monocyte chemoattractant protein 1, interleukin-6 (IL-6), and IL-8. FSL-1 also activated macrophages to produce tumor necrosis factor alpha as the Mycoplasma fermentans-derived lipopeptide MALP-2 (Pam(2)CGNNDESNISFKEK), a potent macrophage-activating lipopeptide, did. The level of the activity of FSL-1 was higher than that of MALP-2. This result suggests that the difference in the amino acid sequence of the peptide portion affects the activity because the framework structure other than the amino acid sequence of the former is the same as that of the latter. To determine minimal structural requirements for the activity of FSL-1, the diacylglyceryl Cys and the peptide portions were examined for this activity. Both portions did not reveal the activity. A single amino acid substitution from Phe to Arg and a fatty acid substitution from palmitic acid to stearic acid drastically reduced the activity. Similar results were obtained in measuring the NF-kappaB reporter activity of FSL-1 to human embryonic kidney 293 cells transfected with Toll-like receptor 2 and 6, together with a NF-kappaB-dependent luciferase reporter plasmid. These results suggest that both the diacylglyceryl and the peptide portions of FSL-1 are indispensable for the expression of biological activities and for the recognition by Toll-like receptors 2 and 6 and that the recognition of FSL-1 by Toll-like receptors 2 and 6 appears to be hydrophobic.