3-Deoxy-D-manno-2-octulosonic acid in the lipopolysaccharide of various strains of Pseudomonas cepacia (original) (raw)
Related papers
The importance of extracellular antigens in Pseudomonas cepacia infections
Journal of medical …, 1988
A clinical isolate of Pseudomonas cepacia from a cystic fibrosis patient was examined for its ability to produce extracellular toxic material. The organism was grown to stationary phase in a defined medium and toxic material was isolated by ultrafiltration, ion-exchange chromatography on DEAE-Sephacel and gel-filtration chromatography on Sepharose 4B. It consisted of a surface carbohydrate antigen, lipopolysaccharide and protein, and had an LD50 (when injected intraperitoneally into mice) of 395k2Opg. The toxicity appeared to be associated with the lipopolysaccharide portion of the complex, because boiling for 15 min and exposure to proteolytic enzymes had no effect on toxicity. However, saponification destroyed the toxicity of the compound. Studies employing radial immunodiffusion with the sera of mice infected with this organism demonstrated production of the complex in uiuo at levels approaching those sufficient to produce death. When sublethal amounts of this complex were placed in the lungs of specific-pathogen-free rats, the lung pathology observed after 12, 24, 36 and 48 h was extensive. However, antibody generated in rabbits against this material could protect mice against the complex, as well as against challenge by the homologous organism. These data indicate that extracellular toxic material produced by P. cepacia may be responsible for the lethality and lung tissue destruction normally associated with an active pneumonia caused by this organism.
European journal of biochemistry, 1986
EJB 86 1041 related immunotype 6 (Fisher classification [l 11) lipopolysacremoved by centrifugation and the supernatant was subjected charides. Two new diN -acyl derivatives of 5,7-diaminoto gel filtration on Sephadex G-50. The fraction B (Fig.4) 3,5,7,9-tetradeoxy-~-glycero-~-manno-nonulosonic acid, which corresponding to small oligosaccharides was rechromawas called by us pseudaminic acid [8], were identified as the tographed in each case on Sephadex G-15 to give trisacconstituents of these polysaccharides. For immunotype 6 0charides I, 2 and 3 with the yields of 25-35%; [KID-63.3", specific polysaccharide see preliminary communication [12].-69.8" and-65.5" (c 1) respectively. MATERIALS AND METHODS Miscellaneous methods 'H and 13C nuclear magnetic resonance spectra were recorded with a WM-250 (Bruker) spectrometer in DzO at 60°C for lipopolysaccharides and at 30 "C for oligosaccharides and monosaccharides with acetone (6, 2.23 ppm) or methanol (6, 50.15 ppm) as internal standards. Optical rotations were measured with a Perkin-Elmer polarimeter (model 141) in water at 20°C. Solutions were freeze-dried or evaporated in vacuo at 40°C. Serological tests were performed as described earlier 1131. Chromatography and electrophoresis Ascending paper chromatography was carried out on FN-11 paper with n-butanollpyridinelwater (61413, v/v, system A) and ethyl acetatelpyridinelacetic acidlwater (5151113, v/v, system B). Paper electrophoresis was performed in 0.025 M pyridine acetate buffer, pH 4.5, at 28 V/cm. Substances were detected on paper using alkaline silver nitrate or benzidine with potassium iodide after treatment with chlorine [14]. Gel filtration was performed on columns of Sephadex G-50 (70 x 3.7 cm) and Sephadex G-15 (75 x 1.5 cm) in pyridine acetate buffer, and of Fractogel TSK 40 (90 x 2 cm) in water. Ion-exchange chromatography was carried out on a column (20 x 1 cm) of DEAE-Trisacryl M. Elution profiles were recorded by using a Technicon sugar analyzer. Monosaccharides were determined with a Technicon sugar analyzer and a BC-200 amino acid analyzer as described earlier [15]. Gas-Liquid chromatography was carried out on a Pye-Unicam 104 instrument (model 64) using a column (150 x 0.1 cm) packed with 5% OV-1 on Diatomite CQ (100-200 mesh) at 190-270°C; carrier gas nitrogen, flow rate 30 ml/min. Combined gas-liquid chromatography/mass spectrometry was performed on a Varian MAT Gnom 111 instrument using the same phase. Bacterial cultures and isolation of lipopolysaccharides Bacterial cultures of P. aeruginosa OSa,b,c, OSa,b,d, 05a,d and immunotype 6 (strains 170011, 170012, 170013 and 170046 respectively) were kindly provided by Dr Lhnyi (Institute of Hygiene, Budapest). Cultures were grown as described previously [ 131. Acetone-dried cells (30 g each strain) were extracted with 45% aqueous phenol [16], nucleic acids precipitated with Cetavlon and lipopolysaccharides recovered from the aqueous solution by the addition of ethanol (10 volumes) followed by dialysis and freeze-drying [16]. The yield of the lipopolysaccharides was 10-3 5% of the dry cell weight. Mild acid degradation Lipopolysaccharides (1 g of each) were heated with 1% acetic acid (100 ml, lOO"C, 1.5 h), the lipid precipitate was Solvolysis with hydrogen fluoride Trisaccharide 1 (50 mg) was treated with hydrogen fluoride (10 ml, 20"C, 3 h) freshly distilled over cobalt trifluoride [17]; hydrogen fluoride was removed in vacuo by absorption with solid sodium hydroxide. The residue was dissolved in water (10 ml), evaporated, again dissolved in water and passed through a column (3 x 1 cm) with Amberlite CG 120 resin (H+-form). The column was washed with water, the eluate was evaporated and D-xylose (8 mg), [ .
European journal of biochemistry, 1987
EJB 86 1041 related immunotype 6 (Fisher classification [l 11) lipopolysacremoved by centrifugation and the supernatant was subjected charides. Two new diN -acyl derivatives of 5,7-diaminoto gel filtration on Sephadex G-50. The fraction B (Fig.4) 3,5,7,9-tetradeoxy-~-glycero-~-manno-nonulosonic acid, which corresponding to small oligosaccharides was rechromawas called by us pseudaminic acid [8], were identified as the tographed in each case on Sephadex G-15 to give trisacconstituents of these polysaccharides. For immunotype 6 0charides I, 2 and 3 with the yields of 25-35%; [KID-63.3", specific polysaccharide see preliminary communication [12].-69.8" and-65.5" (c 1) respectively. MATERIALS AND METHODS Miscellaneous methods 'H and 13C nuclear magnetic resonance spectra were recorded with a WM-250 (Bruker) spectrometer in DzO at 60°C for lipopolysaccharides and at 30 "C for oligosaccharides and monosaccharides with acetone (6, 2.23 ppm) or methanol (6, 50.15 ppm) as internal standards. Optical rotations were measured with a Perkin-Elmer polarimeter (model 141) in water at 20°C. Solutions were freeze-dried or evaporated in vacuo at 40°C. Serological tests were performed as described earlier 1131. Chromatography and electrophoresis Ascending paper chromatography was carried out on FN-11 paper with n-butanollpyridinelwater (61413, v/v, system A) and ethyl acetatelpyridinelacetic acidlwater (5151113, v/v, system B). Paper electrophoresis was performed in 0.025 M pyridine acetate buffer, pH 4.5, at 28 V/cm. Substances were detected on paper using alkaline silver nitrate or benzidine with potassium iodide after treatment with chlorine [14]. Gel filtration was performed on columns of Sephadex G-50 (70 x 3.7 cm) and Sephadex G-15 (75 x 1.5 cm) in pyridine acetate buffer, and of Fractogel TSK 40 (90 x 2 cm) in water. Ion-exchange chromatography was carried out on a column (20 x 1 cm) of DEAE-Trisacryl M. Elution profiles were recorded by using a Technicon sugar analyzer. Monosaccharides were determined with a Technicon sugar analyzer and a BC-200 amino acid analyzer as described earlier [15]. Gas-Liquid chromatography was carried out on a Pye-Unicam 104 instrument (model 64) using a column (150 x 0.1 cm) packed with 5% OV-1 on Diatomite CQ (100-200 mesh) at 190-270°C; carrier gas nitrogen, flow rate 30 ml/min. Combined gas-liquid chromatography/mass spectrometry was performed on a Varian MAT Gnom 111 instrument using the same phase. Bacterial cultures and isolation of lipopolysaccharides Bacterial cultures of P. aeruginosa OSa,b,c, OSa,b,d, 05a,d and immunotype 6 (strains 170011, 170012, 170013 and 170046 respectively) were kindly provided by Dr Lhnyi (Institute of Hygiene, Budapest). Cultures were grown as described previously [ 131. Acetone-dried cells (30 g each strain) were extracted with 45% aqueous phenol [16], nucleic acids precipitated with Cetavlon and lipopolysaccharides recovered from the aqueous solution by the addition of ethanol (10 volumes) followed by dialysis and freeze-drying [16]. The yield of the lipopolysaccharides was 10-3 5% of the dry cell weight. Mild acid degradation Lipopolysaccharides (1 g of each) were heated with 1% acetic acid (100 ml, lOO"C, 1.5 h), the lipid precipitate was Solvolysis with hydrogen fluoride Trisaccharide 1 (50 mg) was treated with hydrogen fluoride (10 ml, 20"C, 3 h) freshly distilled over cobalt trifluoride [17]; hydrogen fluoride was removed in vacuo by absorption with solid sodium hydroxide. The residue was dissolved in water (10 ml), evaporated, again dissolved in water and passed through a column (3 x 1 cm) with Amberlite CG 120 resin (H+-form). The column was washed with water, the eluate was evaporated and D-xylose (8 mg), [ .
European journal of biochemistry, 1987
EJB 86 1041 related immunotype 6 (Fisher classification [l 11) lipopolysacremoved by centrifugation and the supernatant was subjected charides. Two new diN -acyl derivatives of 5,7-diaminoto gel filtration on Sephadex G-50. The fraction B (Fig.4) 3,5,7,9-tetradeoxy-~-glycero-~-manno-nonulosonic acid, which corresponding to small oligosaccharides was rechromawas called by us pseudaminic acid [8], were identified as the tographed in each case on Sephadex G-15 to give trisacconstituents of these polysaccharides. For immunotype 6 0charides I, 2 and 3 with the yields of 25-35%; [KID-63.3", specific polysaccharide see preliminary communication [12].-69.8" and-65.5" (c 1) respectively. MATERIALS AND METHODS Miscellaneous methods 'H and 13C nuclear magnetic resonance spectra were recorded with a WM-250 (Bruker) spectrometer in DzO at 60°C for lipopolysaccharides and at 30 "C for oligosaccharides and monosaccharides with acetone (6, 2.23 ppm) or methanol (6, 50.15 ppm) as internal standards. Optical rotations were measured with a Perkin-Elmer polarimeter (model 141) in water at 20°C. Solutions were freeze-dried or evaporated in vacuo at 40°C. Serological tests were performed as described earlier 1131. Chromatography and electrophoresis Ascending paper chromatography was carried out on FN-11 paper with n-butanollpyridinelwater (61413, v/v, system A) and ethyl acetatelpyridinelacetic acidlwater (5151113, v/v, system B). Paper electrophoresis was performed in 0.025 M pyridine acetate buffer, pH 4.5, at 28 V/cm. Substances were detected on paper using alkaline silver nitrate or benzidine with potassium iodide after treatment with chlorine [14]. Gel filtration was performed on columns of Sephadex G-50 (70 x 3.7 cm) and Sephadex G-15 (75 x 1.5 cm) in pyridine acetate buffer, and of Fractogel TSK 40 (90 x 2 cm) in water. Ion-exchange chromatography was carried out on a column (20 x 1 cm) of DEAE-Trisacryl M. Elution profiles were recorded by using a Technicon sugar analyzer. Monosaccharides were determined with a Technicon sugar analyzer and a BC-200 amino acid analyzer as described earlier [15]. Gas-Liquid chromatography was carried out on a Pye-Unicam 104 instrument (model 64) using a column (150 x 0.1 cm) packed with 5% OV-1 on Diatomite CQ (100-200 mesh) at 190-270°C; carrier gas nitrogen, flow rate 30 ml/min. Combined gas-liquid chromatography/mass spectrometry was performed on a Varian MAT Gnom 111 instrument using the same phase. Bacterial cultures and isolation of lipopolysaccharides Bacterial cultures of P. aeruginosa OSa,b,c, OSa,b,d, 05a,d and immunotype 6 (strains 170011, 170012, 170013 and 170046 respectively) were kindly provided by Dr Lhnyi (Institute of Hygiene, Budapest). Cultures were grown as described previously [ 131. Acetone-dried cells (30 g each strain) were extracted with 45% aqueous phenol [16], nucleic acids precipitated with Cetavlon and lipopolysaccharides recovered from the aqueous solution by the addition of ethanol (10 volumes) followed by dialysis and freeze-drying [16]. The yield of the lipopolysaccharides was 10-3 5% of the dry cell weight. Mild acid degradation Lipopolysaccharides (1 g of each) were heated with 1% acetic acid (100 ml, lOO"C, 1.5 h), the lipid precipitate was Solvolysis with hydrogen fluoride Trisaccharide 1 (50 mg) was treated with hydrogen fluoride (10 ml, 20"C, 3 h) freshly distilled over cobalt trifluoride [17]; hydrogen fluoride was removed in vacuo by absorption with solid sodium hydroxide. The residue was dissolved in water (10 ml), evaporated, again dissolved in water and passed through a column (3 x 1 cm) with Amberlite CG 120 resin (H+-form). The column was washed with water, the eluate was evaporated and D-xylose (8 mg), [ .
1988
Lipopolysaccharide (LPS) from smooth strains of Pseudomonas aeruginosa 503, PAZ1, PA01715, PA01716, and Z61 was fractionated by gel filtration chromatography. LPS samples from the first four strains, all PAO1 derivatives, separated into three major size populations, whereas LPS from strain Z61, a Pac K799/WT mutant strain, separated into two size populations. When column fractions were applied to sodium dodecyl sulfate-polyacrylamide gels in their order of elution, molecules of decreasing size were resolved, and the ladder of molecules with different-length 0 antigens formed a diagonal across the gel. The LPS from the PAO1 derivatives contained two distinct sets of bands, distinguished on the gels as two sets of diagonals. The set of bands with the faster mobility, the B bands, was found in column fractions comprising the three major amino sugar-containing peaks. In the sample from strain 503, a fourth minor peak which contained B bands was resolved. The slower-moving set of bands, the A bands, were recovered in a minor peak. LPS from strain Z61 contained only one set of bands, with the higher-molecular-weight molecules eluting from the column in a volume similar to that of the B bands of the PAO1 strains. Analysis of the fractions of LPS from all strains indicated that less than 8% of the LPS molecules had a long, attached 0 antigen. Analysis of the peak that contained mainly A bands indicated a lack of reactive amino sugar and phosphate, although heptose and 2-keto-3-deoxyoctulosonic acid were detected. Reaction of isolated fractions with monoclonal antibody specific for the PA01 0-antigen side chain indicated that only the B bands from the PAO1 strains were antigenically reactive. The bands from strain Z61 showed no reactivity. The data suggest that the A and B bands from the PAO1 strains are antigenically distinct. We propose that PAO1 strains synthesize two types of molecules that are antigenically different.
European journal of biochemistry, 1987
EJB 86 1041 related immunotype 6 (Fisher classification [l 11) lipopolysacremoved by centrifugation and the supernatant was subjected charides. Two new diN -acyl derivatives of 5,7-diaminoto gel filtration on Sephadex G-50. The fraction B (Fig.4) 3,5,7,9-tetradeoxy-~-glycero-~-manno-nonulosonic acid, which corresponding to small oligosaccharides was rechromawas called by us pseudaminic acid [8], were identified as the tographed in each case on Sephadex G-15 to give trisacconstituents of these polysaccharides. For immunotype 6 0charides I, 2 and 3 with the yields of 25-35%; [KID-63.3", specific polysaccharide see preliminary communication [12].-69.8" and-65.5" (c 1) respectively. MATERIALS AND METHODS Miscellaneous methods 'H and 13C nuclear magnetic resonance spectra were recorded with a WM-250 (Bruker) spectrometer in DzO at 60°C for lipopolysaccharides and at 30 "C for oligosaccharides and monosaccharides with acetone (6, 2.23 ppm) or methanol (6, 50.15 ppm) as internal standards. Optical rotations were measured with a Perkin-Elmer polarimeter (model 141) in water at 20°C. Solutions were freeze-dried or evaporated in vacuo at 40°C. Serological tests were performed as described earlier 1131. Chromatography and electrophoresis Ascending paper chromatography was carried out on FN-11 paper with n-butanollpyridinelwater (61413, v/v, system A) and ethyl acetatelpyridinelacetic acidlwater (5151113, v/v, system B). Paper electrophoresis was performed in 0.025 M pyridine acetate buffer, pH 4.5, at 28 V/cm. Substances were detected on paper using alkaline silver nitrate or benzidine with potassium iodide after treatment with chlorine [14]. Gel filtration was performed on columns of Sephadex G-50 (70 x 3.7 cm) and Sephadex G-15 (75 x 1.5 cm) in pyridine acetate buffer, and of Fractogel TSK 40 (90 x 2 cm) in water. Ion-exchange chromatography was carried out on a column (20 x 1 cm) of DEAE-Trisacryl M. Elution profiles were recorded by using a Technicon sugar analyzer. Monosaccharides were determined with a Technicon sugar analyzer and a BC-200 amino acid analyzer as described earlier [15]. Gas-Liquid chromatography was carried out on a Pye-Unicam 104 instrument (model 64) using a column (150 x 0.1 cm) packed with 5% OV-1 on Diatomite CQ (100-200 mesh) at 190-270°C; carrier gas nitrogen, flow rate 30 ml/min. Combined gas-liquid chromatography/mass spectrometry was performed on a Varian MAT Gnom 111 instrument using the same phase. Bacterial cultures and isolation of lipopolysaccharides Bacterial cultures of P. aeruginosa OSa,b,c, OSa,b,d, 05a,d and immunotype 6 (strains 170011, 170012, 170013 and 170046 respectively) were kindly provided by Dr Lhnyi (Institute of Hygiene, Budapest). Cultures were grown as described previously [ 131. Acetone-dried cells (30 g each strain) were extracted with 45% aqueous phenol [16], nucleic acids precipitated with Cetavlon and lipopolysaccharides recovered from the aqueous solution by the addition of ethanol (10 volumes) followed by dialysis and freeze-drying [16]. The yield of the lipopolysaccharides was 10-3 5% of the dry cell weight. Mild acid degradation Lipopolysaccharides (1 g of each) were heated with 1% acetic acid (100 ml, lOO"C, 1.5 h), the lipid precipitate was Solvolysis with hydrogen fluoride Trisaccharide 1 (50 mg) was treated with hydrogen fluoride (10 ml, 20"C, 3 h) freshly distilled over cobalt trifluoride [17]; hydrogen fluoride was removed in vacuo by absorption with solid sodium hydroxide. The residue was dissolved in water (10 ml), evaporated, again dissolved in water and passed through a column (3 x 1 cm) with Amberlite CG 120 resin (H+-form). The column was washed with water, the eluate was evaporated and D-xylose (8 mg), [ .
Carbohydrate Research, 2008
The lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans strains Y4 and N27 was isolated by the phenol-water procedure. Morphologically, the molecule consisted of ribbon and branched filaments which comprised 3% of the cellular dry weight. Chemical analysis of the isolated and purified LPSs of both strains showed them to consist of carbohydrate, lipid, 2-keto-3-deoxyoctonate, heptose, hexosamine, and phosphate. The major fatty acids of the lipid A moiety were saturated C14 and f-OH C14 compounds. Rhamnose, fucose, galactose, glucose, heptose, glucosamine, and galactosamine comprised the monosaccharide portion of the LPS. Biological activity studies revealed both LPS molecules to be active in the Schwartzman reaction and in in vitro 45Ca bone resorption, as well as in macrophage activation and lethality and in platelet aggregation.
European journal of biochemistry, 1986
EJB 86 1041 related immunotype 6 (Fisher classification [l 11) lipopolysacremoved by centrifugation and the supernatant was subjected charides. Two new diN -acyl derivatives of 5,7-diaminoto gel filtration on Sephadex G-50. The fraction B (Fig.4) 3,5,7,9-tetradeoxy-~-glycero-~-manno-nonulosonic acid, which corresponding to small oligosaccharides was rechromawas called by us pseudaminic acid [8], were identified as the tographed in each case on Sephadex G-15 to give trisacconstituents of these polysaccharides. For immunotype 6 0charides I, 2 and 3 with the yields of 25-35%; [KID-63.3", specific polysaccharide see preliminary communication [12].-69.8" and-65.5" (c 1) respectively. MATERIALS AND METHODS Miscellaneous methods 'H and 13C nuclear magnetic resonance spectra were recorded with a WM-250 (Bruker) spectrometer in DzO at 60°C for lipopolysaccharides and at 30 "C for oligosaccharides and monosaccharides with acetone (6, 2.23 ppm) or methanol (6, 50.15 ppm) as internal standards. Optical rotations were measured with a Perkin-Elmer polarimeter (model 141) in water at 20°C. Solutions were freeze-dried or evaporated in vacuo at 40°C. Serological tests were performed as described earlier 1131. Chromatography and electrophoresis Ascending paper chromatography was carried out on FN-11 paper with n-butanollpyridinelwater (61413, v/v, system A) and ethyl acetatelpyridinelacetic acidlwater (5151113, v/v, system B). Paper electrophoresis was performed in 0.025 M pyridine acetate buffer, pH 4.5, at 28 V/cm. Substances were detected on paper using alkaline silver nitrate or benzidine with potassium iodide after treatment with chlorine [14]. Gel filtration was performed on columns of Sephadex G-50 (70 x 3.7 cm) and Sephadex G-15 (75 x 1.5 cm) in pyridine acetate buffer, and of Fractogel TSK 40 (90 x 2 cm) in water. Ion-exchange chromatography was carried out on a column (20 x 1 cm) of DEAE-Trisacryl M. Elution profiles were recorded by using a Technicon sugar analyzer. Monosaccharides were determined with a Technicon sugar analyzer and a BC-200 amino acid analyzer as described earlier [15]. Gas-Liquid chromatography was carried out on a Pye-Unicam 104 instrument (model 64) using a column (150 x 0.1 cm) packed with 5% OV-1 on Diatomite CQ (100-200 mesh) at 190-270°C; carrier gas nitrogen, flow rate 30 ml/min. Combined gas-liquid chromatography/mass spectrometry was performed on a Varian MAT Gnom 111 instrument using the same phase. Bacterial cultures and isolation of lipopolysaccharides Bacterial cultures of P. aeruginosa OSa,b,c, OSa,b,d, 05a,d and immunotype 6 (strains 170011, 170012, 170013 and 170046 respectively) were kindly provided by Dr Lhnyi (Institute of Hygiene, Budapest). Cultures were grown as described previously [ 131. Acetone-dried cells (30 g each strain) were extracted with 45% aqueous phenol [16], nucleic acids precipitated with Cetavlon and lipopolysaccharides recovered from the aqueous solution by the addition of ethanol (10 volumes) followed by dialysis and freeze-drying [16]. The yield of the lipopolysaccharides was 10-3 5% of the dry cell weight. Mild acid degradation Lipopolysaccharides (1 g of each) were heated with 1% acetic acid (100 ml, lOO"C, 1.5 h), the lipid precipitate was Solvolysis with hydrogen fluoride Trisaccharide 1 (50 mg) was treated with hydrogen fluoride (10 ml, 20"C, 3 h) freshly distilled over cobalt trifluoride [17]; hydrogen fluoride was removed in vacuo by absorption with solid sodium hydroxide. The residue was dissolved in water (10 ml), evaporated, again dissolved in water and passed through a column (3 x 1 cm) with Amberlite CG 120 resin (H+-form). The column was washed with water, the eluate was evaporated and D-xylose (8 mg), [ .