The extraction and analysis of lipopolysaccharides from <i>Pseudomonas aeruginosa</i> strain PAO, and three rough mutants

Kropinski, Andrew M.; Chan, L.; Milazzo, F. H.

doi:10.1139/m79-060

Cited by 60 publications

(60 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These populations represent sets of molecules with 0 antigens of different lengths which are made in large amounts. Other investigators have also demonstrated, using gel permeation chromatography in combination with other methods, that the LPS from members of the family Enterobacteriaceae and other gram-negative bacteria could be resolved into at least two main populations of LPS, differing in the length of their O-polysaccharide chain (9,26,30,32,34). In the results presented here, we found that the LPS from strains 503 and 1715 ( Fig.…”

Section: Discussionsupporting

confidence: 78%

“…Structural microheterogeneity in several regions of LPS molecules from members of the family Enterobacteriaceae (2,14,22,37,38,48,51) and Pseudomonas aeruginosa strains (33,57) has been demonstrated. Of the several methods used to separate the subclasses of LPS from individual strains, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (20,24,45,48) and gel filtration (9,26,30,32,34,48) are the best. Either of these two methods by themselves, however, may be insufficient to completely characterize the high-and low-molecular-weight fractions of LPS.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Heterogeneity of lipopolysaccharides from Pseudomonas aeruginosa: analysis of lipopolysaccharide chain length

et al. 1988

View full text Add to dashboard Cite

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.Lipopolysaccharide (LPS), a major component of the outer membranes of gram-negative bacteria, is important in the structure (33, 37) and function (33,36) of this membrane. Structural microheterogeneity in several regions of LPS molecules from members of the family Enterobacteriaceae (2,14,22,37,38,48,51) and Pseudomonas aeruginosa strains (33, 57) has been demonstrated. Of the several methods used to separate the subclasses of LPS from individual strains, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (20,24,45,48) and gel filtration (9, 26, 30, 32, 34, 48) are the best. Either of these two methods by themselves, however, may be insufficient to completely characterize the high-and low-molecular-weight fractions of LPS. Peterson and McGroarty (48) demonstrated that the SDS-PAGE of the column fractions of samples from Salmonella typhimurium, Salmonella minnesota, and Escherichia coli was instrumental in characterizing the various-sized fractions. Analysis of the isolated fractions allowed for the estimation of the average number of 0-antigen repeat units per LPS from each of the si...

show abstract

Section: Discussionsupporting

confidence: 78%

mentioning

confidence: 99%

Heterogeneity of lipopolysaccharides from Pseudomonas aeruginosa: analysis of lipopolysaccharide chain length

et al. 1988

View full text Add to dashboard Cite

show abstract

“…Chemical structures of the O-antigen polysaccharides from 17 (International Antigenic Typing Scheme [IATS]) standard serotypes and of lipid A from P. aeruginosa LPS have been reported previously (22,24). Until recently (34), only partial or tentative structures of the core regions of a few strains of P. aeruginosa had been reported (18,27,38). The complete and detailed structure is still unknown.…”

mentioning

confidence: 99%

Structural elucidation of the lipopolysaccharide core region of the O-chain-deficient mutant strain A28 from Pseudomonas aeruginosa serotype 06 (International Antigenic Typing Scheme)

et al. 1995

View full text Add to dashboard Cite

The lipopolysaccharide (LPS) of the Pseudomonas aeruginosa serotype 06 rough-type mutant A28 was isolated by a modified phenol-chloroform-petroleum ether extraction method. Deoxycholate-polyacrylamide gel electrophoresis indicated a single band with mobility similar to that of the complete core region of the wild-type parent serotype 06 (International Antigenic Typing Scheme) strain. Compositional analysis of the LPS indicated that the core oligosaccharide was composed of D-glucose (three units), L-rhamnose (one unit), 2-amino-2-deoxy-D-galactose (one unit), L-glycero-D-manno-heptose (two units), 3-deoxy-D-manno-octulosonic acid (two units), L-alanine (one unit), and phosphate (two units). Under the mild conditions of hydrolysis with methanolic hydrogen chloride, a 7-O-carbamoyl substituent was observed on the second heptose residue. The glycan structure of the LPS was determined by employing one-and two-dimensional nuclear magnetic resonance spectroscopy and mass spectrometry-based methods with a backbone oligosaccharide that was obtained from the LPS by deacylation, dephosphorylation, and reduction of the terminal glucosamine. On the basis of the results of the present study and our earlier work with the P. aeruginosa 06-derived core-defective mutant R5 (H. Masoud, E. Altman, J. C. Richards, and J. S. Lam, Biochemistry, 33:10568-10578, 1994), a structural model for the complete core oligosaccharide is proposed.Pseudomonas aeruginosa is an opportunistic pathogen that can cause fatal infections in immunocompromised or debilitated individuals, including those with severe burn wounds, cystic fibrosis, and cancer (1, 36). Lipopolysaccharide (LPS), an integral component of the outer membrane, is the most immunoreactive surface antigen of gram-negative bacteria. It is responsible for determining the O-antigen groups of bacteria and is implicated as a virulence factor involved in the pathogenesis of bacterial infections (13,14,23,33). LPS of P. aeruginosa possesses a molecular architecture similar to that of members of the family Enterobacteriaceae, being composed of a hydrophilic polysaccharide region that is linked to hydrophobic lipid A via a core oligosaccharide (41). Twenty major serotypes of P. aeruginosa have been described on the basis of the structural diversity of their O antigens (30,31). In order to understand the biochemistry, immunochemistry, and biosynthesis of LPS as well as to provide for future purposes a possible basis for developing specific reagents for intervention in P. aeruginosa infections, the complete and detailed structure of the LPS should be defined. Chemical structures of the O-antigen polysaccharides from 17 (International Antigenic Typing Scheme [IATS]) standard serotypes and of lipid A from P. aeruginosa LPS have been reported previously (22,24). Until recently (34), only partial or tentative structures of the core regions of a few strains of P. aeruginosa had been reported (18,27,38). The complete and detailed structure is still unknown.The core region of P. aeruginosa consists of an...

show abstract

“…Strain AK1401 contained core plus one 0 side chain (1), and strain AK44 contained complete core but was 0 antigen deficient (17). All P. aeruginosa strains were maintained on tryptic soy agar (Difco Laboratories, Detroit, Mich.).…”

mentioning

confidence: 99%

“…core-plus-one-0-antigen band (the second fastest migrating band) of a mutant strain, AK1401, which contains complete core plus one 0 repeat (1), while no binding was observed with the LMW band of AK44, a rough mutant with complete core and noO repeat units (17) (Fig. 6b).…”

mentioning

confidence: 99%

Monoclonal antibodies as probes to examine serotype-specific and cross-reactive epitopes of lipopolysaccharides from serotypes O2, O5, and O16 of Pseudomonas aeruginosa

et al. 1992

View full text Add to dashboard Cite

Liu and Wang (27), another 3 new serotypes were added to the scheme. Until recently, serotyping of P. aeruginosa was largely done by using rabbit antisera raised against each of the serotypes. The use of these typing sera has certain disadvantages, including variability from batch to batch and a high incidence of cross-reactivity. Even after extensive cross absorptions, cross-reactions among closely related serotypes could be observed (10,32 aeruginosa, it became apparent that these cross-reactions could be attributed to the similarities in the chemical structure of the 0-antigen repeats common to all strains of the same group. Among 02, 05, and 016 serotypes, the 0 antigens share a trisaccharide repeating unit which consists of two uronic acid derivatives and one N-acetyl fucosamine residue (Fig. 1). These three serotypes were designated by Lanyi and Bergan (25) the 3-acetamidino-2-acetamido-2,3-dideoxy-D-mannuronic acid residue. Serotype differences among strains in this group were believed to be caused by configurational variations in the diacetamidohexuronic acid and the glycosidic bond of N-acetyl fucosamine (15). In order to improve the efficiency of serotyping P. aeruginosa, monoclonal antibodies (MAbs) would be a logical alternative to the polyclonal antisera. Gaston et al. (7)

show abstract

The extraction and analysis of lipopolysaccharides from Pseudomonas aeruginosa strain PAO, and three rough mutants

Cited by 60 publications

References 0 publications

Heterogeneity of lipopolysaccharides from Pseudomonas aeruginosa: analysis of lipopolysaccharide chain length

Heterogeneity of lipopolysaccharides from Pseudomonas aeruginosa: analysis of lipopolysaccharide chain length

Structural elucidation of the lipopolysaccharide core region of the O-chain-deficient mutant strain A28 from Pseudomonas aeruginosa serotype 06 (International Antigenic Typing Scheme)

Monoclonal antibodies as probes to examine serotype-specific and cross-reactive epitopes of lipopolysaccharides from serotypes O2, O5, and O16 of Pseudomonas aeruginosa

Contact Info

Product

Resources

About