In Vitro Anti-Adhesive Activity of an Acidic Polysaccharide from Panax ginseng on Porphyromonas gingivalis Binding to Erythrocytes

• Abstract
• Material and Methods
• Acknowledgements
• References

Abstract

A polysaccharide with high uronic acid content from the roots of Panax ginseng was found to inhibit the ability of Porphyromonas gingivalis to agglutinate erythrocytes. This polysaccharide showed a strong inhibitory activity (minimum inhibitory concentration 0.25 mg/mL), but treatment with pectinase resulted in non-inhibitory hydrolyzed products. In contrast, the inhibition by the acidic polysaccharide from the leaves of Artemisia capillaris was negligible. The carbohydrate composition of the two polysaccharides indicated that the anti-adhesive activity may be correlated with glucuronic acid content, one of the components of glycosaminoglycans. Low molecular weight heparin and sucrose octasulfate revealed stronger inhibitory effects on bacterial binding, than the acidic polysaccharide from P. ginseng.

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium frequently isolated from periodontal pockets of patients with advanced adult periodontitis, and is recognized as a major etiological pathogen in the disease [1]. The bacterium is asaccharolytic, highly proteolytic, and totally dependent on amino acids and peptides for its growth in the gingiva. It utilizes heme and hemoglobin efficiently for its growth, and hemagglutination serves as the first step in heme acquisition [2]. Cysteine proteases, arginine- and lysine-specific gingipains, and hemagglutinin HagA are important for the bacterial hemagglutination and the binding of hemoglobin [3], [4]. P. gingivalis has also been shown to bind to collagen, fibronectin, laminin, vitronectin and cytokeratin [5], [6], [7], and to produce several adherence factors that contribute to bacterial virulence [8]. It was recently reported that glycosaminoglycan-binding proteins, N-acetylneuraminic acid and glucuronic acid were involved in bacterial binding to host cells [9], [10]. Nevertheless, the ability of the bacteria to bind to host cell carbohydrates has been poorly studied. Previously, an acidic polysaccharide from either the roots of Panax ginseng C.A. Meyer (Araliaceae) or the leaves of Artemisia capillaris (Asteracae) showed a remarkable inhibitory effect on Helicobacter pylori adherence to host cells [11], [12], [13]. In the present study, we examined the inhibitory effects of these acidic polysaccharides on the hemagglutinating (HA) activity of P. gingivalis.

The acidic polysaccharide preparation was obtained as described previously [11], [13], which resulted in a single polysaccharide peak (fraction F2) showing no trace of protein. Gas chromatographic analysis of the purified polysaccharides from P. ginseng and A. capillaris revealed that arabinose, galactose, and glucose were substantial components (more than 50 % in relative mol %) of the total carbohydrate, with a large amount of uronic acids (more than 20 %) [11]. The polysaccharide from P. ginseng may be related to highly branched glycans composed of arabinose, galactose, rhamnose and galacturonic acid with a β-1,3-linked galactan reported previously [14]. Interestingly, glucuronic acid was abundant in P. ginseng (18 %), whereas 22 % galacturonic acid (about 60 % as free form) was found in A. capillaris but no glucuronic acid [13]. The acidic polysaccharide F2 from P. ginseng showed significant anti-adhesive activity upon P. gingivalis-mediated hemagglutination, confirmed by microscopic inspection (Fig. [1] A). Its anti-adhesive effect was reflected by the minimum inhibitory concentration of ca. 0.25 mg/mL (∼17 μM) and by the concentration-dependence. In contrast, the inhibitory activity of the corresponding polysaccharide from A. capillaris was not detectable even at 2.5 mg/mL. Both acidic polysaccharides were previously found to effectively inhibit the hemagglutination induced by H. pylori and the adhesion to gastric adenocarcinoma epithelial cells [11], [13]. Complete enzymatic digestion of F2 (2.5 mg/mL) with pectinase [type IV: poly-(1,4-α-D-galacturonide) glycanohydrolase] produced a non-inhibitory fraction (Fig. [1] B). However, limited digestion with pectinase for 30 min resulted in an oligosaccharide fraction of about 1 kDa with high uronic acid content, which showed an activity similar to that of F2 (data not shown). The inhibition by F2 (1.0 - 2.5 mg/mL) was not observed in Escherichia coli-mediated hemagglutination, clearly indicating that the inhibitory activity of F2 is P. gingivalis-specific (Fig. [1] C). It is very likely that the specific activity of F2 to P. gingivalis may be related to one of its components, i. e., glucuronic acid, which is absent in A. capillaris. Recent reports revealed that glycosaminoglycan is involved in P. gingivalis-mediated cell adhesion and at least 25 mM (∼5 mg/mL) glucuronic acid is necessary to significantly inhibit P. gingivalis adherence to host cells [9], [10].

In addition to the carboxy group, we thus tested low molecular weight heparin (LMWH) and sucrose octasulfate (SOS) to determine the role of the negatively charged group in the inhibition of P. gingivalis HA activity, with aspartic acid and glutaric acid (4 - 40 μM) as a non-carbohydrate negative control. Indeed, LMWH and SOS concentration-dependently inhibited erythrocyte binding. LMWH has greater anti-adhesive activity than glucuronic acid: its minimum inhibitory concentration was ca. 0.02 mg/mL (∼7 μM) (Table [1]). The inhibition exhibited by SOS was also remarkable with a minimum concentration of ca. 0.01 mg/mL (∼9 μM), but no significant effects were observed from aspartic acid or glutaric acid.

Therefore, our results strongly suggest that F2 from P. ginseng can potently inhibit P. gingivalis binding to host cells. The anti-HA activities of glucuronic acid, LMWH and SOS upon P. gingivalis suggest that negatively charged groups of specific carbohydrates play an adhesive role in host-bacterial interaction. Natural receptor analogues in secretions such as breast milk and saliva are known to inhibit bacterial adhesion, and act as clearance factors. The polysaccharide may be a useful dietary component for the prevention of periodontal diseases and may be utilized to create carbohydrate-based anti-adhesive drugs, based on the design of synthetic glycomimetics.

Material and Methods

The extracts from the roots of P. ginseng and the leaves of A. capillaris were prepared as previously described [13]. The voucher specimens (MP-03 - 01 and GP-03 - 02 for mugwort leaf and ginseng root extracts, respectively) were deposited at the Laboratory of Structural Biology, School of Life Sciences & Biotechnology, Korea University in Seoul. P. gingivalis (ATCC 33 277) cells were obtained from the Korean Culture Center of Microorganisms (Seoul, Korea). E. coli strains, BL21(DE3) and BL21(DE3)pLysS, were purchased from Novagen (Madison, U.S.A.). Human erythrocyte cells were obtained from a local hospital. Pectinase [EC 3.2.1.15, poly-(1,4-α-D-galactuonide)glycanohydrolase], trypsin (EC 3.4.21.4), D-glucuronic acid, D-galacturonic acid, bovine serum albumin and LMWH were from Sigma (St. Louis, U.S.A.). Sucrose octasulfate (SOS) was purchased from Toronto Research Chemicals (North York, Canada). P. gingivalis was grown in ATCC medium 1490 modified chopped meat medium, supplemented with trypticase (30 g/L), yeast extract (5 g/L), 0.025 % resazurin, cysteine (0.5 g/L), hemin (10 mL/L), and vitamin K1 (0.2 mL/L) at 37 °C under an 80 % N₂, 10 % H₂ and 10 % CO₂ atmosphere for 3 days. E. coli strains were cultured aerobically on Luria-Bertani (LB) broth at 37 °C. Bacterial cells were harvested by centrifugation (6,000 g for 30 min) and kept at -170 °C until required. 2 % trypsinized erythrocytes were obtained as previously described [12]. Total carbohydrate, uronic acid and protein contents were determined using the phenol-sulfuric acid, carbazole [15], and Bradford [16] methods, respectively. The carbohydrate compositions of polysaccharide fractions were analyzed as alditol acetates [15] by gas chromatography (GC). The polysaccharide fraction F2 was obtained, as described previously [12]. Briefly, the extract (20 g) was dissolved in hot distilled water (200 mL). 2 % CPC was used to precipitate polysaccharides, which were dissolved in 10 % sodium chloride (400 mL). 70 % ethanol precipitates were dialyzed against 20 mM Tris-HCl (pH 8.0), which was applied to a DEAE-Sepharose CL-6B column (2.5 × 7 cm), eluted using a linear gradient (0 - 1 M) of NaCl. The fractions with inhibitory activity were dialyzed and lyophilized. Superdex 200 gel filtration FPLC produced a major polysaccharide fraction (fraction F2). For pectinase treatment, F2 (1 mg/mL) in 20 mM sodium acetate (pH 5.0) was treated with pectinase (final activity 5 units/mg polysaccharide) at RT for 24 h. The hydrolysate was fractionated using Superdex peptide gel filtration FPLC eluted with 20 mM Tris-HCl (pH 8.0). The HA activity was evaluated visually and confirmed by microscopic inspection [11]. Erythrocytes in bacterial suspension in the absence of anti-adhesive agents were used as a positive control and those without bacterial suspension as a negative control. The inhibitory effects of LMWH (final concentrations of 0.01 - 1.0 mg/mL), SOS (at 0.01 - 1.0 mg/mL), glucuronic acid, or galacturonic acid were determined by microscopic inspection. Aspartic and glutaric acids were tested as a non-specific reaction.

Acknowledgements

We thank Mr. K. M. Yeo at S&D Co. for supplying us with samples.

References

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  Thieme ConnectPubMedSearch in Google Scholar
• 12 Woo J S, Ha B H, Kim T G, Lim Y, Kim K H. Development of an enzyme-linked glycosorbent method to monitor the inhibition of sialic acid-dependent Helicobacter pylori adhesion.  Biotechnol Lett. 2001;  23 507-11
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• 13 Lee J H, Park E K, Uhm C S, Chung M S, Kim K H. Inhibition of Helicobacter pylori adhesion to human gastric adenocarcinoma epithelial cells by acidic polysaccharides from Artemisia capillaris and Panax ginseng .  Planta Med. 2004;  70 1-4
  Thieme ConnectPubMedSearch in Google Scholar
• 14 Tomoda M, Hirabayashi K, Shimizu N, Gonda R, Ohara N. The core structure of ginsenan PA, a phagocytosis-activating polysaccharide from the root of Panax ginseng .  Biol Pharm Bull. 1994;  17 1287-91
  PubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar

Prof. Kyung Hyun Kim

Department of Biotechnology

School of Life Sciences & Biotechnology

Korea University

Seoul 136-701

Korea

Phone: +82-2-3290-3444

Fax: +82-2-927-9028

Email: khkim@korea.ac.kr

References

• 1 Haffajee A D, Socransky S S. Microbial etiological agents of destructive periodontal diseases.  Periodontology. 2000;  5 78-111
  PubMedSearch in Google Scholar
• 2 Nakayama K, Ratnayake D B, Tsukuba T, Kadowaki T, Yamamoto K, Fujimura S. Hemoglobin receptor protein is intragenially encoded by the cysteine proteinase-encoding genes and the hemagglutinin-encoding gene of Porphyromonas gingivalis .  Mol Microbiol. 1998;  27 51-61
  CrossrefPubMedSearch in Google Scholar
• 3 Potempa J, Pike R, Travis J. The multiple forms of trypsin-like activity present in various strains of Porphyromonas gingivalis are due to the presence of either Arg-gingipain or Lys-gingipain. Infect Immun 1995; 63 : 1176 - 82. 
• 4 Shi Y, Ratnayake D B, Okamoto K, Abe N, Yamamoto K, Nakayama K. Genetic analyses of proteolysis, hemoglobin binding, and hemagglutination of Porphyromonas gingivalis .  J Biol Chem. 1999;  274 17 955-60
  PubMedSearch in Google Scholar
• 5 Kontani M, Ono H, Shibata H, Okamura Y, Tanaka T, Fujiwara T, Kimura S, Hamada S. Cysteine protease of Porphyromonas gingivalis 381 enhances binding of fimbriae to cultured human fibroblasts and matrix proteins.  Infect Immun. 1996;  64 756-62
  PubMedSearch in Google Scholar
• 6 Nakamura T, Amano A, Nakagawa I, Hamada S. Specific interactions between Porphyromonas gingivalis fimbrae and human extracellular matrix proteins.  FEMS Microbiol Lett. 1999;  175 267-72
  CrossrefPubMedSearch in Google Scholar
• 7 Sojar H T, Sharma A, Genco R J. Porphyromonas gingivalis fimbrae bind to cytokeratin of epithelial cells.  Infect Immun. 2002;  70 96-101
  CrossrefPubMedSearch in Google Scholar
• 8 Lamont R J, Jenkinson H F. Subgingival colonization by Porphyromonas gingivalis .  Oral Microbiol Immunol. 2000;  15 341-9
  CrossrefPubMedSearch in Google Scholar
• 9 Wadstrom T, Ljungh A. Glycosaminoglycan-binding microbial proteins in tissue adhesion and invasion: key events in microbial pathogenicity.  J Med Microbiol. 1999;  48 223-33
  PubMedSearch in Google Scholar
• 10 Agnani G, Tricot-Doleux S, Houalet S, Bonnaure-Mallet M. Epithelial cell surface sites involved in the polyvalent adherence of Porphyromonas gingivalis: a convincing role for neuraminic acid and glucuronic acid.  Infect Immun. 2003;  71 991-6
  CrossrefPubMedSearch in Google Scholar
• 11 Belogortseva N I, Yoon J Y, Kim K H. Inhibition of Helicobacter pylori hemagglutination by polysaccharide fractions from Roots of Panax ginseng .  Planta Med. 2000;  66 217-20
  Thieme ConnectPubMedSearch in Google Scholar
• 12 Woo J S, Ha B H, Kim T G, Lim Y, Kim K H. Development of an enzyme-linked glycosorbent method to monitor the inhibition of sialic acid-dependent Helicobacter pylori adhesion.  Biotechnol Lett. 2001;  23 507-11
  CrossrefPubMedSearch in Google Scholar
• 13 Lee J H, Park E K, Uhm C S, Chung M S, Kim K H. Inhibition of Helicobacter pylori adhesion to human gastric adenocarcinoma epithelial cells by acidic polysaccharides from Artemisia capillaris and Panax ginseng .  Planta Med. 2004;  70 1-4
  Thieme ConnectPubMedSearch in Google Scholar
• 14 Tomoda M, Hirabayashi K, Shimizu N, Gonda R, Ohara N. The core structure of ginsenan PA, a phagocytosis-activating polysaccharide from the root of Panax ginseng .  Biol Pharm Bull. 1994;  17 1287-91
  PubMedSearch in Google Scholar
• 15 Chaplin M F. Monosaccharides, 1. Carbohydrate analysis. In: Chaplin MF, Kennedy JF, editors Oxford; IRL Press 1986: pp 1-36
  Search in Google Scholar
• 16 Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.  Anal Chem. 1976;  72 248-54
  CrossrefPubMedSearch in Google Scholar

Prof. Kyung Hyun Kim

Department of Biotechnology

School of Life Sciences & Biotechnology

Korea University

Seoul 136-701

Korea

Phone: +82-2-3290-3444

Fax: +82-2-927-9028

Email: khkim@korea.ac.kr