Báo cáo Y học: Structural determination of lipid A of the lipopolysaccharide from Pseudomonas reactans A pathogen of cultivated mushrooms

Structural Determination Of Lipid A Of The Lipopolysaccharide From Pseudomonas Reactans

Tác giả: Alba Silipo, Rosa Lanzetta, Domenico Garozzo, Pietro Lo Cantore, Nicola Sante Iacobellis, Antonio Molinaro, Michelangelo Parrilli, and Antonio Evidente

Lĩnh vực: Hóa sinh, Vi sinh vật học

Nội dung tài liệu: Nghiên cứu này tập trung vào việc xác định cấu trúc hóa học của lipid A, một thành phần quan trọng của lipopolysaccharide (LPS) từ vi khuẩn Pseudomonas reactans, một tác nhân gây bệnh trên nấm ăn. Bằng cách sử dụng các phương pháp phân tích thành phần và kỹ thuật phổ (MALDI-TOF và NMR hai chiều), các nhà khoa học đã làm sáng tỏ cấu trúc của lipid A, bao gồm chuỗi đường disaccharide D-glucosamine liên kết β-(1′→6) và gốc phosphate. Nghiên cứu cũng chỉ ra sự không đồng nhất đáng kể về thành phần axit béo và phosphate trong các phân tử lipid A, với các dạng chính là hexacyl hóa và pentacyl hóa. Đặc biệt, một số đặc điểm cấu trúc độc đáo đã được phát hiện, bao gồm sự thiếu vắng axit béo chính ở vị trí C-3′ trong dạng pentacyl lipid A và sự thay thế phosphate không theo tỷ lệ ở vị trí C-4′ của glucose. Những phát hiện này có ý nghĩa quan trọng trong việc hiểu rõ hơn về cơ chế gây bệnh và tiềm năng ứng dụng của lipid A.

Mục lục chi tiết:

• Structural determination of lipid A of the lipopolysaccharide from Pseudomonas reactans
• A pathogen of cultivated mushrooms
• A. Silipo, R. Lanzetta, D. Garozzo, P. Lo Cantore, N. S. Iacobellis, A. Molinaro, M. Parrilli, and A. Evidente
• The chemical structure of lipid A from the lipopolysaccharide of the mushroom-associated bacterium Pseudomonas reactans, a pathogen of cultivated mushroom, was elucidated by compositional analysis and spectroscopic methods (MALDI-TOF and two-dimensional NMR). The sugar backbone was composed of the β-(1′ → 6)-linked D-glucosamine disaccharide 1-phosphate. The lipid A fraction showed remarkable heterogeneity with respect to the fatty acid and phosphate composition. The major species are hexacylated and pentacylated lipid A, bearing the (R)-3-hydroxydodecanoic acid [C12:0 (3OH)] in amide linkage and a (R)-3-hydroxydecanoic [C10:0 (3OH)] in ester linkage while the secondary fatty acids are present as C12:0 and/or C12:0 (2-OH). A nonstoichiometric phosphate substitution at position C-4′ of the distal 2-deoxy-2-amino-glucose was detected. Interestingly, the pentacyl lipid A is lacking a primary fatty acid, namely the C10:0 (3-OH) at position C-3′. The potential biological meaning of this peculiar lipid A is also discussed.
• Keywords: cultivated mushrooms; lipid A; MALDI-TOF; NMR; Pseudomonas reactans.
• Lipopolysaccharides (LPS) of Gram-negative bacteria are composed of three genetically and structurally distinct regions: the O-specific polysaccharide (O-chain, O-antigen), the core oligosaccharide and a lipophilic portion, termed lipid A, which anchors the molecule to the bacterial outer membrane.
• In animal pathogenic bacteria, lipid A is the endotoxic portion of LPS and its conservative structure usually consists of a glucosamine (GlcN) disaccharide backbone which is phosphorylated at positions 1 and 4′ and is acylated at the positions 2, 3, 2′ and 3′ of the GlcN I (proximal) and GlcN II (distal) residue with 3-hydroxy fatty acids [1].
• To date, very little is known about the structure and functions of lipid A in nonanimal associated bacteria [2] but they should be important in understanding of mechanisms of infection. Moreover, the study of lipid A structures from nontoxic Gram-negative bacteria is extremely important in order to identify lipid A analogues which can antagonize the biological activation of competent mammalian host-cells by lipid A. This was the case of the lipid A of Rhodobacter capsulatus and its synthesized analogue labelled as E5531 [3].
• The LPS fraction of the bacterium Pseudomonas reactans was analysed within this context and also with the purpose of a polyphenetic characterization of this still unclassified bacteria entity.
• Ps. reactans is considered to be a saprophytic mushroom-associated bacterium [4]; however, recent studies have shown that the bacterium is responsible for alteration of Pleurotus and Agaricus spp. cultivated mushrooms. In particular, it appears that brown and yellow blotch diseases of A. bisporus and P. ostreatus are complex diseases caused by both Ps. tolaasii and Ps. reactans [5,6]. The latter bacterium is also the causal agent of yellowing of P. eryngii [7].
• MATERIALS AND METHODS
• Growth of bacteria, isolation of LPS and lipid A and de-O-acylation of lipid A
• Strain NCPPB1311 of Ps. reactans, was maintained lyophilized at 4 °C and routinely grown on KB agar slants at 25 °C. Bacterial cells for LPS extraction were obtained by growing the above strain in 500-mL conical flasks filled with 200 mL liquid KB on a rotary shaker at 150 r.p.m. at 25 °C for 48 h. Cultures were centrifuged (12 000 g, 15 min), the pellet washed twice with 0.8% NaCl and the cells were freeze-dried. The dried cells (9 g) from 4.8 L culture filtrates of Ps. reactans were suspended in 390 mL ultrapure water and extracted with hot phenol according to the conventional procedure [8] (yield of LPS: 300 mg, 3% of bacterial dry mass).
• The LPS content of both phases was checked by SDS/PAGE [9], Kdo (3-deoxy-D-manno-oct-2-ulosonic acid) and 3-hydroxy fatty acid content [10]. To obtain lipid A, the LPS (100 mg) was hydrolysed with aqueous 1% AcOH for 2 h at 100 °C and ultracentrifuged (110 000 g, 4 °C, 1 h). The precipitate thus obtained was washed twice with water and lyophilized (lipid A, yield: 7 mg, 7% of LPS). Alternatively, LPS (200 mg) was hydrolysed with acetate buffer (25 mL) at pH 4.4, containing 0.1% SDS at 100 °C for 2 h. Then the solution was lyophilized, extracted once with 2 M HCl/ethanol and twice with ethanol, dried, re-dissolved in water and ultracentrifuged (110 000 g, 4 °C, 1 h). The sediment was washed four times with water and lyophilized (lipid A, yield: 12 mg, 6% of LPS).
• An aliquot of lipid A (10 mg) was de-O-acylated with anhydrous hydrazine in tetrahydrofurane at 37 °C for 90 min, cooled, poured into ice-cold acetone (30 mL) and centrifuged (5000g, 15 min). The precipitate was washed twice with ice-cold acetone, dried, then dissolved in water and lyophilized.
• Mild de-O-acylation with ammonium hydroxide was achieved by treatment of the lipid A fraction (1 mg) with 12% aqueous NH3 (200 µL) at 20 °C 18 h.
• MALDI-TOF analysis
• MALDI-TOF analyses were conducted using a Perseptive (Framingham, MA, USA) Voyager STR instrument equipped with delayed extraction technology and with a reflectron. Ions formed by a pulsed UV laser beam (nitrogen laser, λ = 337 nm) were accelerated through 20 kV. Mass spectra reported are the result of 128 laser shots, and mass accuracy < 10 p.p.m. in reflectron mode. Insulin and myoglobin were used for external calibration. The dried samples was dissolved in CHCl3/CH3OH (50/50, v/v) at a concentration of 25 pmol·mL⁻¹. The matrix solution was prepared by dissolving 2,5-dihydroxybenzoic acid in CH3OH at a concentration of 30 mg·mL⁻¹ or trihydroxyacetophenone in CH3OH/0.1% trifluoroacetic acid/CH3CN (7 : 2 : 1, v/v) at a concentration of 75 mg·mL⁻¹. A sample/matrix solution mixture (1 : 10, v/v) was deposited (1 mL) onto a stainless steel gold-plated 100-sample MALDI probe tip, and dried at 20 °C.
• NMR spectroscopy
• The ¹H-, ¹³C- and ³¹P-NMR spectra were obtained at 333 K in DMSO-d₆ at 400, 100 and 162 MHz, respectively, with a Bruker DRX 400 spectrometer equipped with a reverse probe. ¹³C and ¹H chemical shifts are expressed in δ relative to dimethyl sulfoxide (δн 2.49, δc 39.7). Two-dimensional spectra (DQF-COSY, TOCSY, ROESY, HSQC and HMBC) were measured using standard Bruker software. All homonuclear experiments were performed acquiring 4096 data points in the F2 dimension with 512 experiments in F1. The data matrix was zero-filled in the F1 dimension to give a matrix of 4096 x 2048 points and was resolution enhanced in both dimensions by a shifted sine-bell function before Fourier transformation. The TOCSY experiment was performed with a mixing time of 80 ms, while a mixing time of 300 ms was used in the ROESY experiment. The heteronuclear experiments were performed using pulse field gradient programs as gHSQC and gHMBC.
• Gas chromatography
• GC was performed on a Hewlett-Packard 5890 instrument, SPB-5 capillary column (0.25 mm × 30 m, Supelco), for methylation analysis of sugars the temperature program was: 150 °C for 5 min, then 5 °C·min⁻¹ to 300 °C and for monosaccharide absolute configuration analysis: 150 °C for 8 min, then 2 °C·min⁻¹ to 200 °C for 0 min, then 6 °C-min⁻¹ to 260 °C for 5 min. For fatty acids analysis the temperature program was 150 °C for 3 min, then 10 °C-min⁻¹ to 280 °C over 20 min.
• Phosphate and monosaccharide analysis
• Phosphate content was determined according to Kaca et al. [11]. The monosaccharides were identified as acetylated O-methyl glycosides derivatives: briefly, samples were methanolysed with 1 M HCl/MeOH at 80 °C for 20 h, dried under reduced pressure and extracted with methanol/hexane. The methanolic phase, containing the O-methyl glycosides, was acetylated with acetic anhydride in pyridine at 80 °C for 30 min. After work-up, the product was analysed by GLC-MS. The absolute configuration of monosaccharides was determined by GLC of their acetylated glycosides according to Leontein and Lönngren [12].
• Methylation analysis was carried out on de-phosphorylated and reduced product: briefly, the sample (1 mg) was kept at 4 °C, 48 h, in HF 48% (200 μL) and then evaporated under a stream of nitrogen. It was dissolved in water and one drop of pyridine and reduced 18 h with NaBH₄. After work-up, methylation was performed with methyl iodide as described by Ciucanu and Kerek [13]. The hydrolysis of the methylated sugar backbone was performed with 4 M trifluoroacetic acid (100 °C, 4 h) and the partially methylated product, after reduction with NaBH₄, was converted into alditol acetates with acetic anhydride in pyridine at 80 °C for 30 min and analysed by GLC-MS as described above.
• Fatty acids analysis
• Total fatty acid and O-linked fatty acid content was determined as described by Wollenweber and Rietschel [10]. Briefly, two successive hydrolyses were performed: first, in 4 M HCl at 100 °C for 4 h and then 5 M NaOH at 100 °C for 30 min Then the pH was adjusted to slight acidity, fatty acids were extracted with chloroform and esterified with diazomethane. Finally, they were analysed by GLC-MS in the above conditions. Alternatively, fatty acids were obtained after methanolysis of the lipid A and extraction of the sample with n-hexane followed by GLC-MS analysis.
• The ester bound fatty acids were released by mild hydrolysis of lipid A with (0.5 M) NaOH/methanol (1 : 1) at 85 °C for 120 min, then the pH was adjusted to slight acidity and the product extracted in chloroform. After methylation with diazomethane it was analysed by GLC-MS.
• The absolute configuration of 2-hydroxy and 3-hydroxy fatty acids was determined by GLC according to Bryn and Rietschel [14,15].
• RESULTS
• Isolation and characterization of lipid A from Ps. reactans
• The extraction of dried bacterial cells using phenol/water method yielded LPS in the phenol phase. The LPS was obtained after extensive dialysis and centrifugation. The compositional analysis revealed the presence of Kdo and hydroxy fatty acids, typical components of LPS. SDS/PAGE revealed a ladder-like pattern typical of an S-form LPS. The LPS was hydrolysed with AcOH or AcONa to obtain the lipid A moiety. Both conditions gave the same lipid A composition as judged by MALDI-TOF spectro-metry and compositional analysis. Compositional analysis further revealed the presence of a phosphate and GlcN. Methylation analysis of the de-phosphorylated and reduced sample showed the presence of 6-substituted GlcNol and terminal GlcN. The absolute configuration of the GlcN was demonstrated to be D. Fatty acid analysis revealed the presence of (R)-3-hydroxydodecanoic [C12:0 (3-OH)] exclusively as amides and (R)-3-hydroxydecanoic [C10:0 (3-OH)] (S)-2-hydroxydecanoic [C12:0 (2-OH)] and dodec-anoic acid (C12:0) linked in ester linkage (molar ratio: GlcN, 2; phosphate, 1.6; fatty acids, 5.2).
• Analysis of de-O-acylated and de-phosphorylated lipid A
• The amide-linked fatty acids were identified using an aliquot of the de-O-acylated lipid A with anhydrous hydrazine in tetrahydrofurane. The resulting negative ion MALDI-TOF mass spectrum (Fig. 1a) showed a peak at m/z 894.9 in agreement with the presence of two C12:0 (3-OH) fatty acids at the 2 and 2′ positions of both GlcN residues and a peak at m/z 815.1 lacking one phosphate (Am/z 80). The positive ion MALDI-TOF mass spectrum (Fig. 1b) contained two oxonium ions produced by cleavage of the glycosidic linkage. One at m/z 440.3 was attributable to the GlcN II unit bearing a C12:0 (3-OH) and a phosphate group, the latter missing in the other ion occurring at m/z 360.4. Accordingly, a nonstoichiometric phosphate substitution was present on the GlcN II residue. Since the product revealed a good solubility in dimethyl sulfoxide at 333 K and the ¹H-NMR spectrum of the product was of good quality (Fig. 2A), a full two-dimensional NMR analysis was performed (COSY, TOCSY, ROESY, HSQC). The NMR data (Table 1) were in agreement with the results obtained by MS. Thus two H anomeric signals at 5.274 and 4.760 with carbon correlation signals at 92.1 and 100.2 p.p.m., respectively, were present. The chemical shifts, the JH1,H2 and the JC.H were diagnostic of two GlcN residues in a and β anomeric configurations (¹JC.H = 173 and 165 Hz for a and β, respectively). In the ROESY spectrum, besides the expected intra-residue correlations typical of the β anomeric configuration, the anomeric proton of GlcN II showed inter-residue cross peaks with the two protons H-6a and H-6b and the H-4 of GlcN I. These data, together with the downfield shift of the C-6 of GlcN I, proved the β (1 → 6) linkage between the two sugars. Methylation analysis confirmed the results obtained by NMR. The phosphate substitution was inferred by a ¹H, ³¹P HMQC spectrum which indicated the anomeric a-substitution of the GlcN I and the 4′ substitution of the GlcN II (Fig. 2B). It is interesting to note that the cross peak relative to the C-4′ position was not as intense as the other one, suggesting a nonstoichiometric substitution by the phosphate at C-4′. Therefore, the de-O-acylated lipid A was demonstrated to be built up of two D-GlcN, two units of fatty acids C12:0 (3-OH) N-linked to both GlcN and phosphate residues at position C-1 and nonstoichiometric at C4.
• A different aliquot of the lipid A was de