SummaryWe recently separated a PG1828-encoded triacylated lipoprotein (Pg-LP), composed of two palmitoyl and one pentadecanoyl groups at the N-terminal of glycerocysteine from Porphyromonas gingivalis, a periodontopathic bacteria, and found that Pg-LP exhibited definite biological activities through Tolllike receptor (TLR) 2. In the present study, we synthesized 12 different Pg-LP N-terminal peptide moieties (PGTP) using four combinations of glyceryl (R and S) and cysteinyl (l and d) stereoisomers, and three different acyl group regioisomers, N-pentadecanoyl derivative (PGTP1), S-glycero 2-pentadecanoyl derivative (PGTP2) and S-glycero 3-pentadecanoyl derivative (PGTP3). All the PGTP compounds (RL, SL, SD, RD) tested showed TLR2-dependent cell activation. The activating capacities of the PGTP-R compounds were more potent than those of the PGTP-S compounds, whereas there were no differences between the PGTP-L and -D compounds. Furthermore, the production of interleukin (IL)-6 following stimulation with the PGTP1-RL, PGTP2-RL and PGTP3-RL compounds was impaired in peritoneal macrophages from TLR2 knock-out (KO), but not those from TLR1 KO or TLR6 KO mice. These results suggest that P. gingivalis triacylated lipopeptides are capable of activating host cells in a TLR2-dependent and TLR1-/ TLR6-independent manner, and the fatty acid residue at the glycerol position in the PGTP molecule plays an important role in recognition by TLR2.

Oral treponemes are well-known as causative agents of periodontal diseases; however, the details have not been fully clarified. Here, we examined the effects of Treponema medium glycoconjugate on the activation of human gingival fibroblasts using phenol-water extracts from Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum subsp. nucleatum, and Actinobacillus actinomycetemcomitans. The phenol-water extracts activated human gingival fibroblasts to mediate IL-8 production, as well as IL-8 mRNA expression, phosphorylation of p38 mitogen-activated protein kinase, and expression of intercellular adhesion molecule-1. T. medium glycoconjugate exhibited no activation of human gingival fibroblasts, while phenol-water extract-induced activation of human gingival fibroblasts was clearly inhibited by T. medium glycoconjugate. Furthermore, binding of biotinylated phenol-water extracts to CD14 in the presence of LPS-binding protein was blocked with T. medium glycoconjugate. These results suggest that T. medium glycoconjugate has an inhibitory effect on host cell activation by periodontopathic bacteria caused by binding to CD14- and LPS-binding protein.

Abstract:The motility and chemotaxis of human oral spirochetes Treponemadenticola ATCC 35404, T. medium ATCC 700293, and T. vincentii ATCC 35580 were examined by a capillary assay method. Of five sera three human oral treponemes were dominantly chemoattractant to the rabbit serum. The checkerboard analysis of chemotaxis toward rabbit serum clearly showed that the motile T. denticola cells swam toward the culture media containing higher concentrations of the rabbit serum. T. denticola chemotaxis to the rabbit serum was clearly reduced by heating serum, and rabbit albumin contributed by 60 to 70% to its chemotaxis to the rabbit serum. Western blotting analysis demonstrated that these treponemes possessed rabbit albumin-binding polypeptides with approximate molecular sizes of 65 kDa and 70 kDa. Immunoelectron microscopy demonstrated that a 65 kDa rabbit albumin-binding polypeptide was located on the outer envelopes, suggesting that the rabbit albumin-binding polypeptide is responsible for chemotaxis toward rabbit serum.Key words: Chemotaxis, Oral spirochetes, Serum albumin, Albumin-binding polypeptides Many flagellated microorganisms move either towards or away from chemical stimulants (1,5,27) and possess specific receptors for reacting to the attractants or repellents (2). It has been suggested that flagella may playa role in spirochetal motility (4, 9, 15), because flagelladeficient mutants have been seen to lose their motility (6). In the human oral cavity, anaerobic treponemes are frequently detected in subgingival plaque samples from both healthy and diseased sites (12,23,29). Specific nutrients in sera, such as albumin or fatty acids, seem to be essential requisites for in vitro cultivation of their individual species (28). Chemotaxis toward serum in the crevice fluid would benefit these nutritionally strict treponemes in those limited ecological environments where many kinds of oral microorganisms compete with neighboring organisms for growth nutrients. In this study, we examined the chemotactic characterizations of human oral spirochetes for sera and predominant serum albumin. Materials and MethodsBacterial strains, culture, and growth conditions. Human oral treponemes, T. denticola ATCC 35404, T. medium ATCC 700293, and T. vincentii ATCC 35580, *Addresscorrespondence to Dr. Tomohiko Ogawa, Department of Oral Microbiology, Asahi University School of Dentistry, Hozumi, Hozumi-cho, Motosu-gun, Gifu 501-0296, Japan. Fax: +81-58-329-1422. E-mail: tomo527@dent.asahi-u.ac.jp 571 were used in this study. These spirochetes were all anaerobically grown in trypticase-yeast extract-gelatinevolatile fatty acid-rabbit serum (TYGVS) medium, in an anaerobic chamber (Nz:Hz:COz, 80%: 10%: 10%) at 37 C (16). The treponemes of each 4-day culture were gently washed twice with 10% rabbit serum-free TYGVS medium (TYGV), suspended gently again in the TYGV medium, and counted by a Petroff-Hausser bacteria counter (C.A. Hausser & Son, Pa., U.S.A.). Bacterial cells were suspended in the TYGV (negative control), serum-, or albumin-c...

A synthetic lipid A of Porphyromonas gingivalis strain 381 (compound PG-381), which is similar to its natural lipid A, demonstrated no or very low endotoxic activities as compared to Escherichia coli-type synthetic lipid A (compound 506). On the other hand, compound PG-381 had stronger hemagglutinating activities on rabbit erythrocytes than compound 506. Compound PG-381 also induced mitogenic responses in spleen cells from lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ mice, as well as LPS-responsive C3H/HeN mice. The addition of polymyxin B resulted in the inhibition of mitogenic activities, however, compound 506 did not show these capacities. Additionally, compound PG-381 showed a lower level of activity in inducing cytokine production in peritoneal macrophages and gingival fibroblasts from C3H/HeN mice, but not C3H/HeJ mice, in comparison to compound 506. Thus, this study demonstrates that the chemical synthesis of lipid A, mimicking the natural lipid A portion of LPS from P. gingivalis, confirms its low endotoxic potency and immunobiological activity.