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Báo cáo khoa học: Structure of the polysaccharide chain of the lipopolysaccharide from Flexibacter maritimus pptx

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Structure of the polysaccharide chain of the lipopolysaccharide from Flexibacter maritimus Evgeny Vinogradov 1 , Leann L. MacLean 1 , Elizabeth M. Crump 2 , Malcolm B. Perry 1 and William W. Kay 2 1 Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada; 2 Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada Flexibacter maritimus, a Gram-negative bacterium, is a fish pathogen responsible for disease in finfish species and a cause of cutaneous erosion disease in sea-caged salmonids. For the development of serology based diagnostics, protective vac- cines, and a study of pathogenesis, the structural analysis of the lipopolysaccharide (LPS) produced by the bacterium has been undertaken. We now report that an acidic O-specific polysaccharide, obtained by mild acid degradation of the F. maritimus LPS was found to be composed of a disac- charide repeating unit built of 2-acetamido-3-O-acetyl- 4-[(S)-2-hydroxyglutar-5-ylamido]-2,4,6-trideoxy-b-glucose and 5-acetamido-7-[(S)-3-hydroxybutyramido]-8-amino- 3,5,7,8,9-pentadeoxynonulopyranosonic acid (Sug) having the structure: The configuration of the C-2–C-7 fragment of the latter monosaccharide (B) was assigned b-manno; however, the configuration at C-8 could not be established. NMR data indicate that the two monosaccharides have opposite abso- lute configurations. The repeating unit includes a linkage via a (S)-2-hydroxyglutaric acid residue, reported here for the first time as a component of a bacterial polysaccharide. The LPS was also found to contain a minor amount of a disac- charide b-Sug-(2-3)- L -Rha, isolated from the products of the acidic methanolysis of the LPS. Keywords: Flexibacter maritimus; lipopolysaccharide; NMR; polysaccharide. The Cytophaga ) Flavobacterium ) Flexibacter bacteria are a large, somewhat heterogeneous group of filamentous, gliding, Gram-negative bacteria with unusual surface pro- perties [1]. At least seven members of this group are considered to be important fish pathogens. They infect a wide variety of fish species and usually form filamentous biofilms, primarily on the tissues associated with the oral cavity. Among these, Flexibacter maritimus has recently emerged as a cause of widespread severe mortality and economic losses in farmed marine species worldwide [2]. F. maritimus, a long rod-shaped, Gram-negative bacter- ium, has been associated with disease (Flexibacteriosis) in a number of fish species [3–5] and its economic importance has been related to a cause of cutaneous erosion disease particularly in sea-caged salmonids [6,7]. In grouper, F. maritimus causes Ôred boilÕ disease [8] related to its clinical signs of reduced scales and severe hemorrhage on the body surface, resembling boiled skin and causing a high mortality rate. No effective vaccine has been developed against this pathogen. A clearer definition of the relevant immunoreactive macromolecules of these bacterial fish pathogens is funda- mentally important particularly with regard to the mecha- nisms of pathogenesis and the role of infective biofilms. This information is important for the development of appropri- ate immunochemical reagents to facilitate speciation and the design of cost-effective, efficacious vaccines. Lipopolysaccharides (LPS, endotoxins) play a role in the pathogenesis of Gram-negative infections and the structural analysis of their antigenic LPS O-polysaccharide (O-PS) components is important in providing a molecular level understanding of their serological specificities, role in pathogenesis, development of diagnostic agents, and the production of O-PS based conjugate vaccines. As part of a study of bacterial fish infections, this paper records the determination of the unusual structure of the LPS O-PS- antigen of F. maritimus. Experimental procedures Bacterial cell growth and LPS and O-PS production F. maritimus was grown in a 35-L Chemap fermenter (Chemap AG, Volketswil, Switzerland) in 30 L MAT made in an Instant Ocean (Aquarium Systems, Mentor, OH, USA) at 25 °C, stirring at 300 r.p.m., aeration rate of 15 LÆmin )1 , for 42 h. Cells were collected by low speed centrifugation and Correspondence to M. B. Perry, Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada K1A 0R6. Fax: + 1 613 941 1327, Tel.: + 1 613 990 0837. E-mail: Malcolm.Perry@nrc.ca Abbreviations: LPS, lipopolysaccharide; O-PS, O-polysaccharide; S-LPS, smooth LPS; R-LPS, rough LPS; DOC/PAGE, deoxycholate polyacrylamide gel electrophoresis. (Received 6 November 2002, revised 13 February 2003, accepted 26 February 2003) Eur. J. Biochem. 270, 1810–1815 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03543.x the cell pellet (355 g) was washed with 0.9% NaCl and extracted with stirred 50% aqueous phenol (400 mL) for 10 min at 60–70 °C. Following low speed centrifugation of the cooled (4 °C) extract to remove solid material, the clear phenol and water phases were collected separately and dialysed against running tap water until they were free from phenol. The lyophilized retentates were dissolved in 35 mL 10 m M Tris pH 8.0 and treated sequentially with DNase and RNase for 3 h at 37 °C, followed by proteinase K(Sigma) for a further 6 h. The digests were dialysed against distilled water and ultracentrifuged at 105 000 g (4 °C) for 10 h to yield LPS as gel pellets (yields: 910 mg water phase, 290 mg PhOH phase) which were dissolved in distilled water and lyophi- lized. DOC/PAGE (13%) revealed that the water phase extract contained essentially smooth LPS (S-LPS) and the phenol phase rough LPS (R-LPS). Aqueous phase S-LPS (0.4 g) in 2% (v/v) acetic acid (100 mL) was kept at 100 °C for 2 h and following low speed centrifugation to remove precipitated Lipid A (61 mg), the lyophilized centrifugate dissolved in pyridinium acetate buffer (pH 4.5, 5 mL), was fractionated by Sepha- dex G-50 column chromatography (2.5 · 90 cm) using the same buffer as the eluant. The high molecular mass O-PS fraction (K av , 0.02–0.15) of the eluant was lyophilyzed (218 mg). The O-PS had [a] D )60° (c.0.2,water)andwas used in all further analyses. NMR spectroscopy and general methods 1 Hand 13 C NMR spectra were recorded on a Varian Inova 500 spectrometer in D 2 Oat25°C with acetone standard (2.225 p.p.m. for 1 H and 31.5 p.p.m. for 13 C) using standard pulse sequences COSY, TOCSY (mixing time 120 ms), NOESY (mixing time 250 ms), HSQC, gHMBC (optimized for 5 Hz long range coupling constant). GLC, GLC-MS, electrospray MS, monosaccharide and chemical analyses were performed as previously described [9]. Preparation of oligosaccharides 1 and 3 O-PS (60 mg) or LPS (200 mg) were suspended in dry methanol (3 or 6 mL, respectively), cooled in a dry ice/ acetone bath, and acetyl chloride (0.2 or 0.4 mL) was added and the dissolved material were kept at 80 °C for 24 h. Followed by drying in an air stream, the products were separated by HPLC on a C18 column (Phenomenex Aqua, 0.9 · 25 cm) in 2% MeCN in water. Compound 3was eluted ahead of compound 1 (from LPS), the a-andb-anomers of 1 were not separated under these conditions: Yield of 1,23 mg from O-PS, 38 mg from LPS; yield of 3, 3 mg from LPS. The acetate derivative 2 was prepared by treatment of 1 (60 mg) with Ac 2 O/Py (1 : 1 v/v, 6 mL) at 100 °Cfor1h, concentration by drying under air flow, and fractionation on the HPLC system described using a 10–100% MeCN gradient to yield 2 (56 mg). Characterization of ( S )-3-hydroxybutanoic acid and ( S )-2-hydroxyglutaric acid substituents O-PS (15 mg) was hydrolysed with 2 M HCl (0.2 mL, 100 °C, 4 h), and, after concentration in a nitrogen stream, the solution of the residue in water was passed through Dowex (H + )50W· 8–200 ion-exchange resin to remove basic materials and the concentrated eluate was subjected to 1 H NMR identification and analysis according to the directions of the Sigma b-hydroxybutyrate dehydrogenase diagnostic system kit (procedure no. 310 UV) [10]. (S)-2- hydroxyglutaric acid released by methanolysis (M MeOH/ HCl, 100 °C, 16 h) and characterized by GLC of its O-trimethylsilylated (S)-2-butyl ester derivative as described previously [11,12]. Authentic standards of optically active acids were from Sigma. Results and discussion Fermenter grown cells of F. maritimus were extracted by hot aqueous phenol and yielded S-LPS and R-LPS, obtained as precipitated gels after ultracentrifugation of the aqueous and phenol phases respectively. DOC/PAGE analysis showed the S-LPS product to give silver stained ladder bands indicative of the LPS being composed of disaccharide repeating units forming a high molecular mass O-PS of a restricted mass range [13]. The O-PS was isolated from the S-LPS of F. maritimus by mild acid hydrolysis followed by Sephadex G50 gel- filtration chromatography. No monosaccharides were iden- tified by GLC analysis of reduced and acetylated products from the use of conventional acid hydrolysis conditions. NMR analysis of the polysaccharide using 1 H, 13 C, 2D-COSY, TOCSY, NOESY, HSQC, HSQC-TOCSY, and HMBC spectra led to the complete assignment of all 1 Hand 13 C signals and observed correlations, as presented in Table 1. These data revealed the presence of the spin systems of 2,4-diacylamino-2,4,6-trideoxy-b-glucopyranose (A), 3-hydroxybutyrate (Bu), 2-hydroxyglutarate (C), and 5,7,8-triamino-3,5,7,8,9-pentadeoxynonulosonic acid (B). The positions of the amino groups were determined from the chemical shifts of the carbon atoms linked to nitrogen at 46.5–56.3 p.p.m. (Fig. 1). The position of N- and O-acyl groups was determined from three experiments: (a) in the HMBC spectrum, the carboxyl carbon of each acyl group gave a cross peak with the proton at the acylation position; (b) the H-2 (H-4 in case of 2-hydroxyglutaric acid residue) of the acyl residues gave a NOE to the respective protons at the acylation position; (c) NOEs measured in 9 : 1 H 2 O/D 2 O solution showed correlations between the amide protons and H-2 of acyl groups (Fig. 2). Additionally, O-acetylation at O-3 residue A is supported by the low field position of A-3 1 H signal at 5.02 p.p.m. Pyranosidic ring size of residues Aand B follows from the absence of other possibilities for ring size. The b-gluco-configuration of residue A followed from measured vicinal coupling constants (Table 1). The confi- guration of the nonulosonic acid residue B was determined on the basis of interproton coupling constants and NOE data. The orientation of the substituents at C-4 and C-5 of residue B follows from measured coupling constants: a large coupling  13 Hz between H-3ax and H-4 indicated the axial position of H-4. Small coupling constants J 4,5 and J 5,6 < 5 Hz indicated an equatorial orientation of H-5. A large coupling J 6,7 ¼ 10.2 Hz corresponds to a trans-orientation of the H-6 and H-7 protons. A strong NOE observed between NH-7 and H-5 is only possible in the case of a manno-configuration of the C-4–C-7 fragment, as confirmed by molecular modelling INSIGHT II - DISCOVER Ó FEBS 2003 Polysaccharide structure of F. maritimus LPS (Eur. J. Biochem. 270) 1811 (MSI-Accelrys). Futher NOEs observed between NH-5 and H-3ax, and between NH-5 and H-7, were also consistent in molecular modelling of the monosaccharide having a manno-configuration. Data establishing the configuration at C-8 was not obtained. The anomeric configuration of the nonulosonic acid (B) was deduced from the position of the H-3 proton signals. As shown previously for nonulosonic acids with the manno- configuration of the C-4–C-7 fragment, the chemical shift of the H-3eq signal > 2.5 p.p.m. corresponded to an axial orientation of the C-1 carboxyl group, and thus to the b-configuration assigned [14]. The sequence of the component glycose residues were determined from NOE and HMBC data. The H-1 of residue A gave a NOE to H-4 and a HMBC correlation to C-4 of residue B, thus proving the linkage of A-(1–4)-B. HMBC correlation between C-2 of the nonulosonic acid residue B and the H-2 of the 2-hydroxyglutaric acid (residue C) indicated that residue B glycosylates C at the O-2 position. The 13 CNMRchemicalshiftoftheC-1ofresidueA had the same value ( 100 p.p.m) as that previously published for C-1 of b- D -QuiNAc in the b- D -QuiNAc-(1–4)-5-acet- amido-7-formamido-3,5,7,9-tetradeoxy- L -glycero-a- L -manno- nonulosonic acid fragment in the polysaccharide from Pseudoalteromonas distincta [15]. This parameter is sensitive to the combination of the absolute configurations of both monosaccharides, and in the case of the Ps. distincta polysaccharide it reflects different configurations of the monosaccharides. The A-B-fragment of the O-PS from F. maritimus is isosteric to that of Ps. distincta in the vicinity of this glycoside bond and therefore must follow the same rules for glycosylation effects. Thus it can be concluded that residues A and B have different absolute configurations (in the case of nonulosonic acid the absolute configuration is referenced to C-7, the last chiral centre of the Table 1. 1 H and 13 C-NMR Chemical shift and coupling data for the F. maritimus O-PS. OMe at B1 in 1, 3.91/56.0 p.p.m.; in 2, 3.83/54.0; in 3, 3.88/ 55.8. Residue Nucleus 1 J 1,2 (J 3ax,3eq ) 2 (3ax)/NH J 2,3 (J 3ax,4 ) 3 (3eq) J 3,4 (J 3eq,4 ) 4/NH J 4,5 5/NH J 5,6 6 J 6,7 7/NH J 7,8 8/NH J 8,9 9 A,PS 1 H 4.76 3.81/7.62 5.02 3.72/8.06 3.73 1.24 13 C 100.0 55.3 74.6 56.3 71.8 18.1 J, Hz 8.1 9.5 9.5 6.5 A,1 1 H 4.99 3.40 3.86 3.66 3.87 1.21 OMe 3.44 13 C 98.0 56.5 69.5 58.7 69.0 18.7 57.3 A,2 1 H 4.68 4.33/6.32 5.15 3.85/6.99 3.93 1.25 OMe 3.34 13 C 99.1 52.4 73.8 56.1 66.3 18.6 56.0 B,PS 1 H 1.82 2.64 3.98 4.32/8.38 4.01 4.15/8.11 3.59/7.82 1.31 13 C 173.4 103.6 36.0 74.2 46.5 74.2 51.7 50.3 14.5 J, Hz 12.8 12.8 < 3 < 3 < 3 10.2 8.5 6.5 B,1 1 H 1.89 2.74 4.14 3.54 4.08 4.45 3.61 1.41 13 C 170.6 102.5 35.7 64.6 51.8 72.4 51.7 48.9 12.9 J, Hz 13.4 13.4 5.0 3.8 0 10.7 2.7 6.9 B,2 1 H 1.84 2.39 4.84 4.45/6.85 3.78 4.34/6.24 4.37/6.61 1.17 13 C 168.7 100.4 31.5 68.8 45.5 74.0 52.4 47.0 15.6 B,3 1 H 1.83 2.80 4.14 3.54 3.92 4.43 4.03 1.41 13 C 170.9 100.3 35.9 64.7 51.8 72.3 52.0 49.3 13.6 C,PS 1 H 4.12 1.88 2.27 13 C 179.1 75.5 30.0 32.6 177.0 C,1 1 H 4.37 2.02 2.36 OMe 3.83 13 C 176.4 75.2 30.0 32.3 177.0 55.1 C,2 1 H 4.41 1.93; 2.19 2.35 OMe 3.77 13 C 173.8 72.6 29.3 32.3 173.0 53.2 D,3 1 H 4.64 3.63 4.13 3.51 3.72 1.30 OMe 3.38 13 C 102.6 70.3 75.9 72.1 70.4 18.8 56.9 Bu, PS 1 H 2.34; 2.43 4.18 1.22 13 C 175.4 46.3 66.0 23.3 Bu, 1 and 3 1 H 2.46; 2.59 4.25 1.24 13 C 178.9 46.3 67.0 24.3 Bu, 2 1 H 2.38 5.25 1.29 13 C 171.1 44.6 68.9 21.2 N-Ac at A2 1 H 1.93 13 C 175.4 23.4 O-Ac at A3 1 H 2.01 13 C 174.5 21.2 N-Ac at B7 1 H 1.97 13 C 23.2 175.4 1812 E. Vinogradov et al. (Eur. J. Biochem. 270) Ó FEBS 2003 manno-fragment). Since the residue of 2,4-diamino- 2,4,6-trideoxyglucose (bacillosamine) has only been found in the D -configuration [9,16–20], we tentatively present it as D -, and therefore the nonulosonic acid derivative B in the L -configuration. Acidic methanolysis of the O-PS gave a high yield of disaccharide 1 (isolated by reverse-phase HPLC, it contained  20% b-anomer). NMR analysis showed 1 to represent the repeating unit of the O-PS lacking both N-acetyl and O-acetyl substituents. Its structure was confirmed by elec- trospray MS data ([M + 2] 2+ ion at m/z 326.8). Complete acetylation of 1 gave derivative 2, prepared pure in a significant amount ( 50 mg) (the b-anomer was removed at this stage by HPLC) in the unrealized, but expected hope of obtaining crystals for the determination of the configur- ation of Sug by X-ray diffraction. The absolute configuration of (S)-2-hydroxyglutarate was determined by GLC of its O-trimethylsilylated 2-butyl ester derivatives prepared with optically pure 2-(S)-butanol, and the absolute configuration of the (S)-3-hydroxybutyrate substituent was established from the negative reaction of the liberated acid in the enzymic D -3-hydroxybutyric dehydrogenase analytical procedure [10]. The combined experimental data led to the following structure of the O-PS repeat unit: Acidic methanolysis of intact LPS gave, in addition to disaccharide 1, a minor amount of disaccharide 3 in which the nonulosonic acid residue was linked to the O-3 position of a rhamnose unit. This product originated from the LPS core or was present as a single unit lost from the O-PS during mild hydolysis. LPS showed the presence of Rha in a similar amount to those of GlcN, Glc and Man: each of these monosaccharide constituted 0.5–1.5% by weight of the LPS. Rhamnose was not present in the isolated O-PS. Fig. 1. 1 H- 13 CHSQCspectrumofF. maritimus O-PS. The anomeric signal of residue A in the 1 H NMR spectrum resides under the water signal. Ó FEBS 2003 Polysaccharide structure of F. maritimus LPS (Eur. J. Biochem. 270) 1813 Nonulosonic acids have been found in natural sources in three major variants: 5-amino-3,5-dideoxy- D -glycero- D -galacto-nonulosonic acid (neuraminic acid), 3-deoxy- D -glycero- D -galacto-nonulosonic acid (Kdn) and 5,7-diamino-3,5,7,9-tetradeoxy-nonulosonic acids. The latter class was found in four configurations: L -glycero- L -manno (pseudaminic acid), D -glycero- D -galacto (legio- naminic acid), L -glycero- D -galacto (8-epi-legionaminic acid), and D -glycero- D -talo (4-epilegionaminic acid) [12,21]. All of these monosaccharides can have variable N- and O-acyl substituents and other modifications. O-PS from Flexibacter, described herein, contains a new nonulosonic acid derivative, 5-(3-hydroxybutyramido)-7- acetamido-8-amino-3,5,7,8,9-pentadeoxy-b-manno-nonulo- pyranosonic acid, with as yet undetermined configuration at C-8 and tentatively assigned the L -absolute configur- ation. Biosynthesis of sialic acids and possibly 5,7- diamino-3,5,7,9-tetradeoxy-nonulosonic acids proceeds by condensation of hexose derivatives with phosphoenol- pyruvate, so that atoms C1–C6 of the hexose become C4–C9 of the nonulosonic acid. As in the 5,7,8-triamino- 3,5,7,8,9-pentadeoxynonulopyranosonic acid position 8, which would be C-5 in a possible hexose precursor, is occupied by an amino group, its biosynthesis must be different from that of other nonulosonic acids or include an introduction of the amino group following the condensation step. The O-PS of F. maritimus contains a linkage involving a (R)-2-hydroxyglutaric acid residue reported here for the first time as a bacterial polysaccharide component. A similar component, O-glycosylated amide linked (R)-malic acid was recently reported as a component of the O-PS from another fish pathogen Flavobacterium psychrophilum [9]. The structural differences exhibited by the O-PSs from F. maritimus and Fl. psychrophilum [9] are reflected in the observed serological specificities shown by rabbit antisera prepared against synthetic respective O-PS glycoconjugates (unpublished data) and their use as diagnostic agents and Fig. 2. Partial NOESY spectrum of F. maritimus O-PS (H 2 O/D 2 O, 9 : 1) showing correlations from NH-protons. 1814 E. Vinogradov et al. (Eur. J. Biochem. 270) Ó FEBS 2003 possible vaccines. It is also interesting that the prolific production of the O-PS of these species probably contri- butes to the surface properties and consequent biofilming characteristics of these pathogens, especially in the case of F. maritimus. Acknowledgements This work was supported by the Canadian Bacterial Diseases Network. References 1. Bernadet, J.F., Segers, P., Vancanneyt, M., Berthe, F., Kersters, K. & Vandamme, P. (1996) Cutting the Gordian knot: emended classification and description of the genus Flavobacter- ium, emended description of the family Flavobacteriaceae,and proposal of Flavobacterium hydatis nom. nov, (Basonym, Cyto- phaga aquatalis Strohl and Tait (1978). Int. J. Syst. Bacteriol. 46, 128–148. 2. Bernadet, J.F. (1997) Immunization with bacterial antigens: Flavobacterium and Flexibacterium infections. Dev. Biol. Stand. 90, 179–188. 3. Handlinger, J., Soltani, M. & Percival, S. (1997) The pathology of Flexibacter maritimus in aquaculture species in Tasmania, Aus- tralia. J. 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Carbohydr. Res. 275, 147–154. 20. Young N.M., Brisson, J–R., Kelly, J., Watson, D.C., Tessier, L., Lanthier, P.H., Jarrell, H.C., Cadotte, N., St. Michael, F., Aberg, E. & Szymanski, C.M. (2002) Structure of the N-linked glycan present on multiple glycoproteins in the Gram-negative bacterium, Campylobacter jejuni. J. Biol Chem. 277, 42530–42539. 21. Tsvetkov, Y.E., Shashkov, A.S., Knirel, Y.A. & Za ¨ hringer, U. (2001) Synthesis and identification in bacterial lipopolysaccharides of 5,7-diacetamido-3,5,7,9-tetradeoxy- D -glycero- D -galacto-and- D - glycero- D -talo-non-2-ulosonic acids. Carbohydr. Res. 331, 233–237. Ó FEBS 2003 Polysaccharide structure of F. maritimus LPS (Eur. J. Biochem. 270) 1815 . Structure of the polysaccharide chain of the lipopolysaccharide from Flexibacter maritimus Evgeny Vinogradov 1 , Leann. b-Sug-(2-3)- L -Rha, isolated from the products of the acidic methanolysis of the LPS. Keywords: Flexibacter maritimus; lipopolysaccharide; NMR; polysaccharide. 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