The importance of extracellular antigens in Pseudomonas cepacia infections (original) (raw)
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Journal of Medical Microbiology, 1990
Six clinical isolates of Pseudomonas cepacia (representing the five serotypes of the organism) were examined for the presence of 3-deoxy-~-manno-2-octu~osonic acid (KDO) in their lipopolysaccharide (LPS). Purified LPS was examined for the presence of KDO by the thiobarbituric acid (TBA) assay and by gas chromatography. All strains possessed KDO. One strain possessed KDO that was detectable by the TBA assay after mild acid hydrolysis with 0.04 M H2S04 at 100°C for 20 min. The other strains also possessed KDO but it was only demonstrable by the TBA assay after strong acid hydrolysis (4~ HCl for 60 min at 100OC). All six purified LPS preparations were shown to possess KDO by two separate gas chromatography procedures. LPS isolated from the six strains of P. cepacia was toxic for mice.
Characterization of Pseudomonas cepacia isolates from patients with cystic fibrosis
Journal of Clinical Microbiology, 1984
Pseudomonas cepacia infections which follow a fulminant course and which include septicemia are being reported with increasing frequency from cystic fibrosis patients. Forty-eight P. cepacia isolates from cystic fibrosis patients were screened for production of potential virulence factors. A majority of strains tested produced protease and lipase. Eleven strains harbored plasmids of approximate molecular weights in the range 50 X 10(6) to 100 X 10(6). Twenty-two strains produced a smooth lipopolysaccharide. Studies are presently under way to determine the role of these potential virulence factors in the pathogenesis of P. cepacia disease.
Pathogenic factors of Pseudomonas cepacia isolates from patients with cystic fibrosis
Journal of Medical Microbiology, 1990
One hundred and nineteen isolates of Pseudomonas cepacia, 98 of which were from cystic fibrosis (CF) patients and 21 from environmental and other human sources, were examined for biochemical and exo-enzymatic properties that may contribute to the pathogenicity of this bacterium. The following characteristics were demonstrated significantly more frequently in isolates from CF patients than in control isolates : production of catalase, ornithine decarboxylase, valine aminopeptidase, C14 lipase, alginase and trypsin; reduction of nitrate to nitrite; hydrolysis of urea and xanthine ; complete haemolysis on bovine red blood cells; cold-sensitive haemolysis on human red blood cells; greening of horse and rabbit red blood cells. The role of these factors in the pulmonary disease associated with cystic fibrosis is not clear. However, several factors which have been reported previously as being associated with pathogenic processes with other bacteria have now been described in P. cepacia. Additional factors not previously reported as "pathogenicity factors" are also described.
Journal of Medical Microbiology, 1992
Monoclonal antibodies (MAbs) to the core antigen region of lipopolysaccharide (LPS) of Pseudomonas aeruginosa were produced from mice immunised with whole cells of heat-killed rough mutants of Pseudomonas aeruginosa expressing partial or complete core LPS. MAbs were screened in an enzyme-linked immunosorbent assay (ELISA) against three different antigen cocktails : S-form LPS from three P . aeruginosa strains, R-form LPS from six P . aeruginosa strains and, as a negative control, R-form LPS from Salmonefla typhimurium and Escherichia coli. Selected MAbs were subsequently screened against a range of extracted LPS and whole cells from both reference strains and clinical isolates of P . aeruginosa. The antibodies were also screened in ELISA against whole-cell antigens from other Pseudomonas spp. as well as strains of Haemophilus influenzae, Neisseria subjlava and Staphylococcus aureus. Five MAbs reacting with the core component of P. aeruginosa LPS were finally selected. Two of these, MAbs 360.7 and 304.1.4, were particularly reactive in immunoblots against unsubstituted core LPS, including that from 0-antigenic serotypes of P. aeruginosa. The MAbs also reacted with some of the other Pseudomonas spp., but not with P. cepacia or Xanthomonas (Pseudomonas) maltophilia. Cross-reactivity with whole cells from other bacterial species was minimal or not observed. Reactivity of MAbs with some Staph. aureus strains was observed, and binding to the protein A component was implicated. The reactivity of the M Abs was investigated further by flow cytometry and immunogold electronmicroscopy. The suitability of the MAbs for an immunological assay for detection of P. aeruginosa in respiratory secretions from C F patients is discussed.
Journal of Clinical Microbiology, 1989
The lipopolysaccharide (LPS) of Pseudomonas aeruginosa PAO1 contains two species of O polysaccharide termed A and B bands. The high-molecular-weight B-band LPS determines the O specificity of the bacterium, while the antigenically distinct A-band LPS consists of only shorter-chain polysaccharides. Seven hybridomas secreting A-band-specific monoclonal antibodies were produced and used to study the LPS of standard and clinical strains. Although A-band antibodies did not agglutinate any of the serotype strains presently in the International Antigenic Typing Scheme, Western immunoblots revealed that 11 of the 17 serotype strains possessed A-band LPS. In a group of 250 clinical isolates from patients with cystic fibrosis, 170 (68%) had A-band LPS on the basis of agglutination tests, but in silver-stained gels all were shown to be deficient in O-antigen-containing B band. Investigation of serial isolates from a single patient revealed a pattern of antigenic variation. During the course of...
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), [ .
Journal of Immunological Methods, 1989
A method for identification of the components of immune complexes would increase our understanding of the pathogenesis of chronic diseases such as Pseudomonas aeruginosa lung infection in cystic fibrosis. Capillary tube, gel diffusion and turbidimetric methods of determining immune complex formation were investigated with antigens from P. aeruginosa and the homologous rabbit antisera. Visible complexes were formed in the first two methods with flagella antigens. Purified lipopolysaccharide from P. aeruginosa would not form visible precipitates and a rapid and economical turbidimetric method was developed with 96-well microtiter plates. Larger quantities of immune complexes were formed in vitro with antigen/antibody ratios determined by the above methods. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting (Western blotting) were evaluated and found to be useful in determining the antigen and antibody components of these immune complexes.
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), [ .