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Carbohydrate Composition of Endotoxins from R-Type Isogenic Mutants of Shigella Sonnei Studied by Capillary Electrophoresis and GC-MS†

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Carbohydrate Composition of Endotoxins from R-Type Isogenic Mutants of Shigella Sonnei Studied by Capillary Electrophoresis and GC-MS† CROATICA CHEMICA ACTA CCACAA, ISSN 0011-1643, e-ISSN 1334-417X Croat. Chem. Acta 84 (3) (2011) 393–398. CCA-3487 Original Scientific Article Carbohydrate Composition of Endotoxins from R-type Isogenic Mutants of Shigella sonnei Studied by Capillary Electrophoresis and GC-MS† Annamária Bui,a Anikó Kilár,b,c Ágnes Dörnyei,a,c Viktória Poór,a Krisztina Kovács,b Béla Kocsis,b and Ferenc Kilára,c,* aInstitute of Bioanalysis, Faculty of Medicine, University of Pécs, Szigeti út 12., H-7624 Pécs bDepartment of Medical Microbiology and Immunology, Faculty of Medicine, University of Pécs, Szigeti út 12., H-7624 Pécs, Hungary cDepartment of Analytical and Environmental Chemistry, Faculty of Sciences, University of Pécs, Ifjúság útja 6., H-7624 Pécs, Hungary RECEIVED NOVEMBER 9, 2010; REVISED FEBRUARY 5, 2011; ACCEPTED MAY 13, 2011 Abstract. The carbohydrate composition of the rough-type endotoxins (lipopolysaccharides, LPSs) from Shigella sonnei mutant strains (Shigella sonnei phase II - 4303, 562H, R41 and 4350) was investigated by capillary electrophoresis and GC-MS. The monosaccharides obtained by hydrolysis were determined by capillary electrophoresis combined with laser induced fluorescence detection (CE-LIF) after labeling with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) and by GC-MS as alditol-acetate derivatives. It was ob- tained that the lipopolysaccharides of the isogenic rough mutants are formed in a step-like manner, con- taining no heptose (4350), one D-glycero-D-mannoheptose (562H), or two or three L-glycero-D- mannoheptoses (R41, 4303, respectively) in the deep core region. Besides the heptoses, the longest LPS from the mutant Shigella sonnei 4303 contains hexoses, such as glucoses and galactoses, in a pro- portion of approximately 3:2. This study provides a comprehensive comparison of the variability in the carbohydrate part of the rough endotoxins extracted from Shigella sonnei isogenic mutants. (doi: 10.5562/cca1795) Keywords: lipopolysaccharide structure, Shigella sonnei rough mutant, D-glycero-D-mannoheptose, capillary electrophoresis, CE-LIF, GC-MS INTRODUCTION i.e., L-glycero-D-mannoheptose or D-glycero-D-manno- heptose) and neutral hexoses like D-glucose (D-Glc), The structural composition of the bacterial endotoxins D-galactose (D-Gal) or their derivatives. One (or more) (also known as lipopolysaccharides or LPSs) is of im- Kdo residue(s) is (are) found in the connection part portance due to their pathophysiological impact. The between the core OS and the lipid A. The lipid A usual- endotoxins are cell wall components of the Gram- ly consists of a D-glucosamine (GlcN) disaccharide negative smooth (S) or rough (R) bacteria,1 and consist backbone acylated with β-hydroxylated fatty acids. of two or three main parts: (i) a glycolipid region, called The significance of the endotoxins in living organ- lipid A, (ii) an oligosaccharide (OS) region, called core, isms is due to the antigenicity of the O-antigen as well to which (iii) a polysaccharide region, called O-chain, is as the toxic character of the lipid A moiety4. The inner- attached in the S-type endotoxins.2 Spontaneous or in- most region of an endotoxin, consisting of the lipid A duced mutations of S-type strains may cause loss of and the Kdo residues, is essential for the viability of the carbohydrate constituents (O-chains and part of the Gram-negative bacteria. core) in the endotoxins, resulting in the R-type lipopoly- Shigella sonnei (S. sonnei) phase I3 is an important saccharides.3 There is a high degree in the variability of member of the Enterobacteriaceae family, and causes the polysaccharide chains (O-chains) amongst the dif- dysentery. This S-type strain can spontaneously mutate ferent bacteria, while the core oligosaccharides have a to the non-pathogenic R-type S. sonnei 4303 (phase II),3 more conservative structure. The oligosaccharide core which has the same core region but it lacks the is formed by several sugar units such as 3-deoxy- O-chain3,5,6 due to plasmid-loss. A series of R-type iso- 7 8 D-manno-2-octulosonic acid (Kdo), heptose(s) (Hep, genic mutants, namely S. sonnei R41, 562H and 4350 † Presented at the 10th International Symposium and Summer School on Bioanalysis within the CEEPUS Network CII-HU-0010- 04-0910, Zagreb, Croatia, July 2010. * Author to whom correspondence should be addressed. (E-mail: [email protected]) 394 A. Bui et al., Endotoxins from R-type Mutants of Shigella sonnei have been developed by ethyl-methyl-sulfonate induced Preparation of Monosaccharides from Endotoxins mutagenesis9 from the parent strain, S. sonnei 4303 The endotoxins (50–200 mg) were hydrolyzed with 1 % (being already a mutant). The mutant strains were iso- lated with the help of R-specific phages. Previous stu- acetic acid (“mild hydrolysis”) at 100 °C for 90 min to dies claim that the S. sonnei 4350 strain is a heptose- separate the carbohydrate part from the lipid A. The transferase-less mutant,8 while the S. sonnei 562H strain lipid A part of each endotoxin was removed by centri- lacks the ADP-L-glycero-D-mannoheptose-6-epimerase fugation (10000 × g, 15 min, 4 °C). The supernatants enzyme.7,10 (containing the truncated core parts and other small Kdo Although, the carbohydrate regions of the endo- fragments, as well as minor amount of intact endotoxin toxins from the S-type S. sonnei phase I and the R-type due to incomplete hydrolysis), were freeze-dried, redis- S. sonnei 4303 (phase II) have been studied extensively solved in 1 ml of pyridine-acetate buffer (4 ml pyridine, by different methods,11–15 the structures of the LPS 20 ml acetic acid in 1000 ml deionized water) and frac- extracted from the isogenic deep rough mutants (562H, tionated by gel chromatography on a Sephadex G-50 R41, 4350) have not been fully characterized (the mole- (Sigma-Aldrich, USA) column (2.5 × 80 cm) using the same buffer.19 Fractions (4 ml each) were collected and cular masses of these LPSs were determined by mass 20 spectrometry).16 tested by the phenol-sulfuric acid method for the car- In this paper we describe the usefulness of bohydrate content. Following lyophilization, the frac- capillary electrophoresis combined with laser indu- tion materials were hydrolyzed in 0.5 M sulfuric acid ced fluorescence detection (CE-LIF) and GC-MS for 14 h at 100 °C in order to obtain the monosaccharide methods in the identification of the sugar moiety of components. Barium hydroxide was used after hydroly- endotoxins. These methods were used to provide a sis to remove the sulfate. The hydrolyzed samples, con- detailed profiling of the LPSs of isogenic deep rough taining the monosaccharides, were then lyophilized. mutants of S. sonnei. Thin Layer Chromatography EXPERIMENTAL Samples, containing the monosaccharide mixture from the endotoxins, were run in a Shandon's chromatogra- 21 Materials phy vessel on a Cellulose DC Plastikfolia (Merck, Darmstadt, Germany) thin layer with buthanol/pyri- All monosaccharide standards (D-ribose, D-mannose, dine/water mixture (6:4:3), which is optimal for neu- D-glucose, D-galactose, inositol), as well as APTS tral sugars. An aqueous mixture of D-ribose, D-mannose, (8-aminopyrene-1,3,6-trisulfonic acid), were supplied D-galactose and D-glucose was used as the standard by Sigma-Aldrich (St. Louis, Missouri, USA). Sodium solution. The samples were detected by silver staining as 22 cyanoborohydride (1.0 M NaBH3CN solution in tetra- described by Trevelyan et al. hydrofuran) was obtained from Aldrich (St. Louis, Mis- souri, USA) and citric acid and boric acid were pur- Gas-Chromatography with MS Detection chased from Reanal (Budapest, Hungary). All other reagents were of analytical grade. The monosaccharides that were isolated from the endo- toxins were analyzed in their alditol-acetate forms by Bacterial Strains GC-MS. The derivatization procedure was used accord- ing to Sawardeker et al.23 Briefly, the hydrolyzates S. sonnei phase I bacteria were cultured at 37 °C in a were reduced with sodium-borohydride at room temper- laboratory fermentor in a beef extract peptone broth at ature for 4 h. The excess borohydride was quenched pH = 7.2, until they reached the late logarithmic phase with acetic acid and then the borate was removed with 7 (about 10 h), and then collected by centrifugation. methanol. The samples containing the reduced sugars Spontaneous mutation of the smooth colonies of this were then desiccated. Peracetylation was performed strain leads to the rough or R-type S. sonnei 4303 (phase with acetic anhydride at 100 °C for 12 h. The samples, II) of which further R mutants, named S. sonnei R41, containing the sugar derivatives, were washed with 562H and 4350, were produced by induced mutation water, desiccated and resuspended in chloroform. The 17 using ethyl-methyl-sulfonate as mutagenic agent. The GC-MS system (Agilent Technologies, Waldbronn, mutant bacteria were isolated and classified on the basis Germany) consisted of a gas chromatograph (6890N) 8 of their core-specific phage sensitivity. The bacterial and a mass spectrometer (5975) equipped with a endotoxins were extracted from acetone-dried organ- quadrupole mass analyzer. The separation was per- isms by the phenol-chloroform-petroleum ether me- formed on a DB-225 column (Agilent Technologies, 18 thod and were lyophilized. Waldbronn, Germany, length: 30 m; inner diameter: Croat. Chem. Acta 84 (2011) 393. A. Bui et al., Endotoxins from R-type Mutants of Shigella sonnei 395 0.25 mm; film thickness: 0.15 μm). Helium was used as phy Inc., Moorpark, CA, USA). After the electrophoret- a carrier gas (initial flow, 1.5 ml min–1) and splitless ic run the capillary was washed with 0.1 M NaOH for 4 injection was applied. The GC temperature program was min.
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