Biochimica et Biophysica Acta 1594 (2002) 199^205 www.bba-direct.com Structure of the major glycolipid from Rothia dentocariosa Mariola Pas̈ciak a , Irena Ekiel b , Anna Grzegorzewicz a , Halina Mordarska a , Andrzej Gamian a; * a Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, PL-53-114 WrocIaw, Poland b Biotechnology Research Institute, NRC, Montreal, QC, Canada H4P 2R2 Received 8 June 2001; received in revised form 10 October 2001; accepted 12 October 2001 Abstract Structural studies of the major glycolipid isolated from Rothia dentocariosa were carried out by specific chemical degradation and nuclear magnetic resonance spectroscopy. The glycolipid was found to be a dimannosylacylmonoglyceride in which the carbohydrate part was the glycerol-linked dimannoside K-D-Manp-(1C3)-K-D-Manp-(1C3)-sn-Gro, and the internal mannose was esterified at C-6 by fatty acid residue. The other fatty acyl chain substituted the primary methylene position of glycerol. The occurrence of this glycolipid is limited to the related microorganisms. The structural characteristics can facilitate the differentiation of some genera. ß 2002 Elsevier Science B.V. All rights reserved. Keywords: Opportunistic agent; Glycolipid; Structure; Nuclear magnetic resonance; Rothia dentocariosa 1. Introduction Rothia dentocariosa, which has been known as a natural inhabitant of human oral cavity, has been more and more frequently isolated from supragingival dental plaque or identi¢ed as one of the microbial strains associated with dental caries and periodontal disease [1]. The role of these bacteria, however, remains speculative. Recently, R. dentocariosa was shown to be a typical opportunistic pathogen involved in di¡erent in£ammatory processes, mainly infective endocarditis [2^6], but also septicemia [7] Abbreviations: T, type strain; HMQC, 1 H-detected heteronuclear multiple quantum correlation spectroscopy; HMBC, 1 H-detected heteronuclear multiple bond correlation spectroscopy; TMS, tetramethylsilane * Corresponding author. Fax: +48-71-3732587. E-mail address: gamian@immuno.iitd.pan.wroc.pl (A. Gamian). and pneumonia [8,9], or in other diseases in immunocompromised patients [10^12]. R. dentocariosa was also found to be dominant in the sputum cultures of male subjects su¡ering from AIDS [13]. R. dentocariosa a¡orded some taxonomic di¤culties because its position has not yet been ¢rmly established. The aerobic or facultative anaerobic, pleomorphic (coccoid- to rod-shaped or branched ¢laments) Gram-positive microorganisms, previously classi¢ed as Actinomyces dentocariosus, Nocardia dentocariosa and Nocardia salivae [14], could be properly distinguished from two genera mentioned and other related taxa by chemotaxonomic features. They included mainly the presence of an L-Lys-LAla3 type cell wall [15], a fatty acid pro¢le composed of iso-, anteiso- and normal fatty acids [16], a major glycolipid characterized previously as the dimannosyl diglyceride [17,18], and a lipomannan identi¢ed recently [19]. In the present study, we examined the structure of 0167-4838 / 02 / $ ^ see front matter ß 2002 Elsevier Science B.V. All rights reserved. PII : S 0 1 6 7 - 4 8 3 8 ( 0 1 ) 0 0 3 0 1 - 6 200 M. Pas̈ciak et al. / Biochimica et Biophysica Acta 1594 (2002) 199^205 the major glycolipid from R. dentocariosa. It was found to di¡er from that of diglycosyl diglycerides, known as the widespread glycolipids among bacteria. This glycolipid may be a valuable supplement for the taxonomy, classi¢cation and identi¢cation of these opportunistic microorganisms. 2. Materials and methods 2.1. Bacterial strains and cultivation R. dentocariosa LL-Pba2 obtained from the Lechevalier's Collection of Waksman Institute of Microbiology (USA) was used throughout this study. Use was also made of other strains, namely Arthrobacter globiformis ATCC 8010T (PCM 2134), Micrococcus luteus ATCC 4698T (PCM 525), R. dentocariosa ATCC 17931T (PCM 2249), Saccharopolyspora hirsuta ATCC 27875T and Stomatococcus mucilaginosus PCM 2403. The microorganisms were grown in a yeast extract^glucose medium in submerged cultures at 37³C for 48 h [20]. The cultures were checked for purity, heat-killed and harvested by centrifugation and then washed with phosphate-bu¡ered saline. 2.2. Lipid extraction and thin-layer chromatography (TLC) The wet cell mass (10 g) of the R. dentocariosa strain LL-Pba2 was extracted twice by shaking with a chloroform^methanol mixture (2:1, v/v, 150 ml) at 30³C for 12 h [21]. The crude lipid extract was partially puri¢ed to remove non-lipid material, using a modi¢ed Bligh and Dyer procedure [22]. The samples were monitored for glycolipids by TLC, using Silica Gel H plates (Merck), the CHCl3 ^methanol^water (65:25:4, v/v) solvent system and orcinol as visualizing spray reagent. The plates were also stained with vanillin, ninhydrin and a reagent speci¢c to phosphorus [23]. For analytical comparative examination of the crude lipids of all the strains under study, smallscale extractions from the biomass (200 mg) were performed, without partial puri¢cation [23]. 2.3. Puri¢cation of the major glycolipid [21] The crude extract from R. dentocariosa (LL-Pba2), free from water-soluble non-lipid contaminants, was separated on a column of activated silica gel (HIFlosil 60^200 mesh), using successively chloroform, acetone and methanol as eluents. The acetone fractions which contained the major glycolipid were combined for further puri¢cation by preparative TLC, performed on silica gel PF 254 plates (Merck) with chloroform^methanol^water as the solvent system. The procedure was repeated (up to three times) until a single spot of the glycolipid was obtained in two-dimensional (2D) TLC, using the solvent system as previously [21]. 2.4. Analytical procedures Total neutral sugar determinations, as well as fatty acid, sugar and methylation analyses, were carried out as previously [21,24]. Absolute con¢guration of monosaccharide was established with an enzymatic procedure. The glycolipid sample (0.4 mg) was hydrolyzed (1 M HCl, 4 h, 100³C) and the released sugar residues were treated with hexokinase in a 0.2 M ammonium acetate bu¡er pH 8.0, in the presence of ATP. Phosphorylation of mannose was monitored by high voltage electrophoresis. Determination of the absolute con¢guration of glycerol was performed according to [25] with prior deacylation of glycolipid with 12.5% NH4 OH for 25 h at room temperature. Gas^liquid chromatography/mass spectrometry analyses of the derivatives of sugars and fatty acids were performed with a Hewlett-Packard 5971A system, using an HP-1 glass capillary column (0.2 mmU12 m) and a temperature program of 150^ 270³C, 8³C/min. 2.5. Nuclear magnetic resonance (NMR) spectroscopy NMR spectra were measured in CDCl3 , in a 6:4 CDCl3 :CD3 OD mixture, or in [CD3 ]2 SO (DMSO) at room temperature, using tetramethylsilane as a standard. Signal assignments in the 1 H- and 13 C-NMR spectra of glycolipids were performed using 2D techniques, namely homonuclear shift correlation spectroscopy (COSY), relayed COSY, phase-sensitive COSY and proton-detected carbon-proton shift correlated (1 H-detected heteronuclear multiple (singular) quantum correlation spectroscopy (HMQC and M. Pas̈ciak et al. / Biochimica et Biophysica Acta 1594 (2002) 199^205 201 HSQC)) experiments. Typically, 200^400 free induction decays, each of 2 K size, were acquired for 2D NMR experiments. Additional con¢rmation of the assignments in the crowded regions of 13 C NMR spectra was obtained from distortionless enhancement by polarization transfer (DEPT) spectra. Proton-detected NMR experiments were performed using Bruker AM-360, AM500 and AMX500 spectrometers, and 13 C NMR spectra were obtained with Bruker AM-360 and MSL-300 spectrometers. 3. Results The thin-layer chromatogram of crude lipid extracts from the representatives of ¢ve related species revealed that all of them contained a major glycolipid of the same Rf = 0.6 mobility (Fig. 1). Each of them was characterized by a positive reaction with Fig. 1. TLC of major glycolipids (G) from cells of representatives of R. dentocariosa and some related taxa: (1) A. globiformis (PCM 2134), (2) S. mucilaginosus (PCM 2403), (3) M. luteus (ATCC 4698), (4 and 5) R. dentocariosa (Pba2 and ATCC 17931), (6) S. hirsuta (ATCC 27875). Solvent system: chloroform^methanol^water (65:25:4, v/v); detection by orcinol reagent, followed by heating at 120³C. Fig. 2. Double quantum ¢ltered-COSY spectrum of R. dentocariosa Pb2a glycolipid dissolved in [CD3 ]2 SO (DMSO); a, b, g refer to terminal mannose, internal mannose and glycerol residues, respectively. vanillin and orcinol, which had been used as reagents speci¢c to lipid and sugar compounds, respectively. They were negative in staining for amino groups and phosphorus. Fractionation by column chromatography of the crude lipid extract from the R. dentocariosa strain, yielded neutral lipids in the chloroform fraction, glycolipids in the acetone fraction and, ¢nally, phospholipids in the methanol eluate. The major glycolipid found in the acetone fraction was puri¢ed with preparative TLC. Sugar analysis of the glycolipid revealed the presence of glycerol and two residues of mannose. Analysis of fatty acids disclosed the presence of anteiso-C17:0 (45.0%), nC18:0 (24.0%), nC16:0 (9.5%) with smaller amounts of iso-C16:0 (7.0%) and nC19:0 (4.0%). Mannose was found to have the D-con¢guration because of complete phosphorylation with hexokinase, which speci¢cally phosphorylates the D-isomers. Methylation analysis, involving methylation by the Hakomori method, revealed the presence of terminal and 3-substituted residues of mannose. The 1 H NMR spectrum of the glycolipid from R. dentocariosa is shown in Fig. 2. Assignments of proton signals were made using 2D-COSY and relayed 202 M. Pas̈ciak et al. / Biochimica et Biophysica Acta 1594 (2002) 199^205 Fig. 3. HMQC/HMBC spectrum (D) and long-range correlation experiment (A,B,C) for glycolipid isolated from R. dentocariosa strain Pb2a. COSY. 13 C NMR spectra were assigned using HMQC (Fig. 3D) and HSQC experiments. An additional DEPT experiment con¢rmed the assignments of the methylene groups of glycerol and hexoses, as well as the methine carbons of branched fatty acyl chains. The 1 H NMR spectrum of the glycolipid (Table 1) displayed two signals of equal intensity attributed to the anomeric protons at 5.09 and 4.77 ppm with coupling constants of J1:2 1.8 Hz typical of two K-mannose residues [26]. The 13 C NMR spectrum of the glycolipid had two anomeric carbon signals at 102.46 and 101.28 ppm, as well as 13 well-resolved resonances between 60 and 80 ppm for the remaining part of two mannose rings and for glycerol. In the high-¢eld part of the spectrum (18^40 ppm), signals typical of fatty acyl chains were observed, including those characteristic of branched iso and anteiso chains (Table 2). 13 C NMR chemical shifts for C-5 and C-6 (Table 2) are indicative of a substitution in position 6 in the internal, but not in the terminal hexose. Glycerol has a signal of its CH group at 4.0 ppm, shifted up-¢eld by 1 ppm as compared to the signal of typical glycolipids, where the CH group is substituted [27,28], which is in agreement with the presence of a free hydroxyl group at C-2. The 1 H NMR COSY spectrum performed in DMSO as a solvent shows that the CH2 groups of glycerol have no cross-peaks with free OH groups, which means that both CH2 groups of glycerol are substituted. Instead, the methine group of glycerol has a crosspeak with an OH group (Fig. 2). The 13 C NMR chemical shifts for glycerol additionally support the presence of a free hydroxyl group in position 2. The long-range correlation experiment revealed that one CH2 group of glycerol had a cross-peak with C-1 of the internal mannose (Fig. 3C), whereas the other CH2 group (g1) was correlated with the CNO group of the fatty acid (Fig. 3A). The signals of the CNO group of the fatty acid at 174.4 ppm correlate with the protons of the CH2 group of glycerol, whereas the second carbonyl at 174.6 ppm corresponds with position 6 of mannose b (Fig. 3). The 1 H NMR and 13 C NMR spectra of the glycolipid from R. dentocariosa were identical to the spectra of the glycolipids from Saccharopolyspora erythraea [21] (Tables 1 and 2). However, some di¡erences in intensities were observed in the up-¢eld regions, containing signals of fatty acyl chains, in both 1 H and 13 C NMR spectra. The glycerol substituted by sugar moiety at C-3 and the fatty acid residue and C-1, respectively, has chirality introduced to C-2 position. Thus the absolute con¢guration of the glycerol group could be determined to be L, after saponi¢cation of glycolipid, using the method described by Rundloªf and Widmalm [25]. It is consistent with sn-con¢guration when carbons are stereospeci¢cally numbered. 4. Discussion The components detected in the major glycolipid from R. dentocariosa were mannose, glycerol and fatty acids in a molar ratio corresponding to that of dimannosyl diglyceride [17,18]. The investigations presented in this paper revealed further structural details of this glycolipid. Its hydrophilic moiety consisted of one terminal K-D-mannopyranose and one 3substituted K-D-mannopyranose residue. Only one position of glycerol was esteri¢ed by fatty acid, whereas the other fatty acid chain was attached to the internal sugar molecule. Surprising was the ¢nding that the same structural type of glycolipid was 203 M. Pas̈ciak et al. / Biochimica et Biophysica Acta 1594 (2002) 199^205 Table 1 1 H NMR data for glycolipids from R. dentocariosa Pb2a and S. erythraea K600 Residue K-D-Manp-(1C C3)-K-D-Manp-(1C Gro Proton Pb2a (in CDCl3 :CD3 OD) K600 (in DMSO) 1 2 3 4 5 6 6P 2-OH 3-OH 4-OH 6-OH 1 2 3 4 5 6 6P 2-OH 4-OH 1 1P 2 3 3P 2-OH 5.09 3.98 3.80 3.59 3.77 3.88 3.72 4.83 3.73 3.57 3.40 3.57 3.62 3.45 4.62 4.49 4.65 4.46 4.59 3.85 3.55 3.53 3.55 4.29 4.03 4.76 5.16 3.98 3.94 3.82 3.52 3.29 5.09 (1.8) (1.8; 3.4) (3.4; 9.5) (9.3; 9.5) (1.8; 311.4) (6.1; 311.2) 4.77 4.07 3.85 3.75 3.76 4.40 4.31 (1.8) (1.8; 3.4) (3.3; 9.3) (1.2; 311.7) (6.1; 311.7) 4.15 4.10 4.00 3.44 3.78 (4.8; 11.4) (5.8; 11.2) (5.8) (6.7; 310.4) (4.4) (1.7) (4.6; 3.4) (5.2; 9.6) (4.6) (6.0) (5.2) (5.6 av.) (1.5) (311.6; (311.6; (5.3) (5.7) (311.1; (311.1; 1.8) 6.8) 5.4) 4.7) (5.3) Chemical shifts were referenced relative to internal TMS. Coupling constants (in Hz) are in parentheses. present in the Sacharopolyspora strains [20,21] despite its absence in the other members of the Pseudonocardiaceae family [29]. So, this glycolipid is of practical value when di¡erentiating R. dentocariosa from other taxa of Actinomycetaceae. It should be noted that the representatives of three other phylogenetically related taxa, Arthrobacter, Micrococcus and Stomatococcus, possess major glycolipids with the same TLC mobility. Analysis of two representatives, Arthrobacter and Micrococcus, which was carried out in the past, led to the identi¢cation of dimannosyl diglycerides in their cells [30]. R. dentocariosa was found to be phylogenetically related to some genera of the Micrococcaceae family [31]. It is worth mentioning that R. dentocariosa produces lipomannan comparable to that produced by the micrococcal strains [19]. It was suggested that the presence of mannosyl glycerides and lipomannan compounds in the bacterial cells might re£ect a biosynthetic rela- tionship between the two lipid components of the cell envelope [32]. That could be of help in explaining their occurrence in some systematically diverse microorganisms like Rothia and Saccharopolyspora, having type VI cell walls and type IV cell walls, respectively. The usefulness of the major envelope glycolipids lies in the fact that they can facilitate the phenotypic di¡erentiation of related genera. Further investigations are needed because of the heterogeneity within R. dentocariosa taxon reported lately [33]. Acknowledgements This study was supported by the Committee for Scienti¢c Research (KBN), Grant No. 4 PO5A 038 09 and 4 PO5A 073 19. 204 M. Pas̈ciak et al. / Biochimica et Biophysica Acta 1594 (2002) 199^205 Table 2 13 C NMR data for glycolipids from R. dentocariosa Pb2a, S. erythraea K600 and S. hirsuta K52 strains Chemical shift in ppm Residue K-D-Manp-(1C C3)-K-D-Manp-(1C Gro Fatty acids Carbon Pb2a (292 K) K600 (292 K) K600 (298 K) K52 (298 K) 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 C(2) C(3) CH(i) CH3 (i) CH(ai) CH2 -CH(ai) CH2 -CH(i) CH3 (ai) CH3 (ai) CH-CH2 (ai) 102.46 70.87 71.56 67.96 73.71 62.14 101.28 69.99 79.09 66.72 71.31 64.25 65.46 68.53 69.34 34.44 25.22 28.29 22.80 34.75 36.98 39.40 19.37 11.53 102.46 70.92 71.53 68.03 73.75 62.08 101.14 70.07 79.31 66.63 71.41 64.07 65.35 68.50 69.23 34.49 25.13 28.22 22.94 34.81 37.06 39.53 19.45 11.48 29.86 102.50 70.92 71.62 68.17 73.75 62.19 101.25 70.09 79.17 66.63 71.35 64.18 65.47 68.56 69.29 34.57 25.24 28.30 22.82 34.82 37.10 39.52 19.53 11.61 29.87 102.38 70.80 70.56 68.06 73.66 62.14 101.24 69.93 79.10 66.63 71.31 64.11 65.39 68.50 69.32 34.47 25.12 28.13 22.87 34.74 36.98 39.40 19.38 11.46 29.89 References [1] D. 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