O-specific Polysaccharide Chain Research Articles

Structures of the O‐specific Polysaccharide Chains of Pectinatus cerevisiiphilus and Pectinatus frisingensis Lipopolysaccharides

Mild acid hydrolysis of the smooth-type lipopolysaccharide (LPS) of Pectinatus frisingensis afforded no polysaccharide but monomeric 6-deoxy-L-altrose (L-6dAlt) which was identified by anion-exchange chromatography in borate buffer, GLC/MS, 1H-NMR spectroscopy, and optical rotation. LPS was degraded with alkali under reductive conditions to give a completely O-deacylated polysaccharide, which was studied by methylation analysis, 1H-NMR and 13C-NMR spectroscopy, including sequential, selective spin-decoupling, two-dimensional correlation spectroscopy (COSY), COSY with relayed coherence transfer, two-dimensional heteronuclear 13C, 1H-COSY, one-dimensional NOE and two-dimensional rotating-frame NOE spectroscopy. It was found that the O-specific polysaccharide chain of P. frisingensis LPS is a homopolymer of 6-deoxy-L-altrofuranose built up of tetrasaccharide-repeating units having the following structure: [sequence: see text] Similarly, mild acid degradation of smooth-type LPS of Pectinatus cerevisiiphilus resulted in depolymerisation of the polysaccharide chain to give a disaccharide consisting of D-glucose and D-fucose. Study of the disaccharide by methylation analysis and alkali-degraded LPS by one-dimensional and two-dimensional 1H-NMR and 13C-NMR spectroscopy showed that the O-specific polysaccharide of P. cerevisiiphilus has the following structure: -->2)-beta-D-Fucf-(1-->2)-alpha-D-Glcp-(1-->.

The structure of the O-specific polysaccharide chain of Proteus penneri strain 42 lipopolysaccharide

The structure of the O-specific polysaccharide chain of Proteus penneri strain 42 lipopolysaccharide

Structural studies of O-specific polysaccharide chains of the lipopolysaccharide from Yersinia enterocolitica serovar O:10

Lipopolysaccharide (LPS) was isolated from Yersinia enterocolitica serovars O:10 and O:10 KL and the structural pattern of O-specific sugar chains elucidated. The rhamnan and l-xylulose ( l- threo-pent-2-ulose) as constituents of the O-specific polysaccharide were obtained by autohydrolysis of the LPS. The rhamnan was shown to be a linear, α-(1 → 3)-linked polysaccharide in the d configuration. l-Xylulose was purified using paper chromatography on a preparative scale and its structure was confirmed by 13C NMR spectroscopy. Using sugar and methylation analysis and 13C NMR spectroscopy of the LPS and the rhamnan, the structural features of the disaccharide repeating unit of the Y. enterocolitica O:10 O-specific polysaccharide were elucidated as: →3)-α- d ▪p-(1→

Tracking the Response of Burkholderia cepacia G4 5223-PR1 in Aquifer Microcosms

The introduction of bacteria into the environment for bioremediation purposes (bioaugmentation) requires analysis and monitoring of microbial population dynamics to define persistence and activity from both efficacy and risk assessment perspectives. Burkholderia cepacia G4 5223-PR1 is a Tn5 insertion mutant which constitutively expresses a toluene ortho-monooxygenase that degrades trichloroethylene (TCE). This ability of G4 5223-PR1 to degrade TCE without aromatic induction may be useful for bioremediation of TCE-containing aquifers and groundwater. Thus, a simulated aquifer sediment system and groundwater microcosms were used to monitor the survival of G4 5223-PR1. The fate of G4 5223-PR1 in sediment was monitored by indirect immunofluorescence microscopy, a colony blot assay, and growth on selective medium. G4 5223-PR1 was detected immunologically by using a highly specific monoclonal antibody which reacted against the O-specific polysaccharide chain of the lipopolysaccharides of this organism. G4 5223-PR1 survived well in sterilized groundwater, although in nonsterile groundwater microcosms rapid decreases in the G4 5223-PR1 cell population were observed. Ten days after inoculation no G4 5223-PR1 cells could be detected by selective plating or immunofluorescence. G4 5223-PR1 survival was greater in a nonsterile aquifer sediment microcosm, although after 22 days of elution the number of G4 5223-PR1 cells was low. Our results demonstrate the utility of monoclonal antibody tracking methods and the importance of biotic interactions in determining the persistence of introduced microorganisms.

The effect of different Escherichia coli endotoxins on red blood cell deformability

Lipopolysaccharide (LPS) of grant-negative bacteria consists of O-specific polysaccharide chain, core oligosaccharide chain and lipid A. Studies on the effect of various endotoxins on red blood cell (RBC) deformability gave conflicting results. To evaluate whether the effect of endotoxin on RBC deformability depends on the presence and exposure of lipid A, we studied the effect of five E.coli LPS components on RBC deformation by means of a shear stress diffractometer: 1) complete E.coli OIll:B4 LPS; 2) delipidated E.coli 0111 B4 LPS; 3) R-mutant E.coli F-583 LPS lacking the O-specific polysaccharide chain; 4) E.coli F-583 lipid A, and 5) electrodialysed E.coli F- 515 lipid A. Electrodialysis results in highly dispersed molecules whereas unelectrodialysed LPS tends to form aggregates. At a shear stress of 6 Pa RBC deformation was not changed by complete and delipidated LPS, but RBC deformation was impaired by Rd-mutant LPS (-12 %) and lipid A F-583 (-10 %) after 30 min incubation. Electrodialysed lipid A F-515 showed the strongest effect and decreased RBC deformation (-24 %) after 30 min incubation. These results indicate that the effect of LPS on RBC deformation depends on the exposure of lipid A for binding to RBC membranes. The polysaccharide chain of LPS weakens the interaction of lipid A and RBC membranes. Furthermore, aggregates of lipid A impair RBC deformation less than highly dispersed lipid A.

Sudies of O-specific polysaccharide chains of Pseudomonassolanacearum lipopolysaccharides consisting of structually different repeating units.

The structure of the O-antigenic ploysaccharide chains of lipopolysaccharides of a number of Pseudomonas solanacearum strains were elucidated mainly with the help of methylation analysis and 13 CNMR spectroscopy, including a computer-assisted 13 CNMR-based analysis. Six structually distinct but related polysaccharides were identified. They have a backbone which is built up of three l-rhammoyranosyl resides and one 2-acetamido-2-deoxy- d-glucopyranosyl residue, and is unsubtituted or substituted with a residue of l-xylopyranose or l-rhamnopyranose as a monosaccharide side chain. The lipopolysaccharides of most of the strains contain polysaccharide chains consisting of at least two structually different types of repeating units. Three of the polysaccharides are common to more than one strain.

Structural Study of O-Specific Polysaccharides ofProteus

Abstract Proteus bacteria are important human opportunistic pathogens which frequently cause urinary tract infections. According to Bergey's Manual of Systematic Bacteriology,1 this genus includes three species: P. mirabilis, P. vulgaris, and P. myxofaciens. A novel species of P. penneri has been recently proposed2,3 for strains formerly called P. vulgaris biogroup I. Proteus is an antigenically heterogeneous group of bacteria, and this is mainly associated with diverse composition and structures of O-specific polysaccharide chains of outer-membrane lipopolysaccharides (O-antigens). The Kauffman-Perch serological classification4 of P. mirabilis and P. vulgaris includes 49 O-serogroups. However, a number of S-strains remain unclassified,5 including strains of P. penneri.

The structure of the sialic acid-containing Escherichia coli O104 O-specific polysaccharide and its linkage to the core region in lipopolysaccharide

Mild acid hydrolysis of Escherichia coli O104 lipopolysaccharide released an O-specific polysaccharide, a tetrasaccharide repeating unit, the corresponding dimer, and a disaccharide fragment of the repeating unit. Complete and incomplete cores, and oligosaccharides comprising fragments of the repeating unit and the core region, were also obtained. On the basis of sugar and methylation analysis, FAB-mass spectrometry and NMR spectroscopy of the hydrolysis products, the repeating unit of the O-specific polysaccharide was shown to be the tetrasaccharide: → 4)-α-D-Gal p-(1 → 4)-α-Neu p5,7,9,Ac 3-(2 → 3)-β- d-Gal p-(1 → 3)-β- d-Gal pNAc (1 →. The linkage between the O-specific polysaccharide chain and the core region, which appeared to be of the R2 type, was established. These results indicate that N-acetylneuraminic acid, located in the O-specific polysaccharide, is an inherent lipopolysaccharide component.

Structure of the O-specific polysaccharide chain of Klebsiella pneumoniae O1K2 (NCTC 5055) lipopolysaccharide. A complementary elucidation

Structure of the O-specific polysaccharide chain of Klebsiella pneumoniae O1K2 (NCTC 5055) lipopolysaccharide. A complementary elucidation

Pathogenicity and molecular genetics of O-specific side-chain lipopolysaccharides of Escherichia coli.

Lipopolysaccharide (LPS), a glycolipid molecule found on the outer leaflet of outer membranes of gram-negative bacteria, consists of three moieties: lipid A, core oligosaccharide, and the O-specific polysaccharide chain. The O-specific side chain, which extends to the extracellular milieu, plays an important role in pathogenicity, especially during the initial stages of infection, because of its ability to interact with serum complement. In recent years, several laboratories have used recombinant DNA tools to determine, at the molecular level, the organization, expression, and regulation of genes involved in LPS biosynthesis in Salmonella and Escherichia coli. An increased understanding of the molecular aspects of the O-specific side-chain genes will shed light on the intimate details related with the formation of the O-specific side chain, its assembly onto the lipid A--core, and the translocation and insertion of the complete LPS molecule into the outer membrane. It will also contribute to the understanding of the evolution of these genes and the correlation of chemical diversity of O-specific side chains with the genetic diversity of O-specific side-chain genes. In addition, since the O-specific side chains are involved in the pathogenicity of medically important gram-negative bacteria, a basic understanding of the regulation and expression of O-specific side chain LPS genes will contribute to the field of molecular pathogenesis. This article provides an overview of the role of O-specific side chains in septicemic infections and also discusses the current status of molecular genetic studies on O-specific side-chain genes from E. coli.

The structure of the O-specific polysaccharide chain of the lipopolysaccharide of Salmonella arizonae O61

The O-specific polysaccharide was obtained by mild degradation of the Salmonella arizonae O61 lipopolysaccharide with acid. It contained 2-acetamido-2-deoxy- d-glucose, 2-acetamidino-2,6-dideoxy- l-galactose (FucAm), and 7-acetamido-3,5,7,9-tetradeoxy-5-[( R)-3-hydroxybutyramido]- d- glycero- l- galacto-nonulosonic acid (Sug). On the basis of partial acid hydrolysis with 0.1 M HCl, solvolysis with anhydrous HF in methanol, and 1H- and 13C-NMR analysis (including 1H/ 13C inversely correlated spectroscopy for localisation of N-acyl substituents), it was concluded that the O-specific polysaccharide had the following structure. →3)-α- l-FucAm-(1 → 3)-α- d-GlcNAc-(1 → 8)-β-Sug-(2 → The O-antigen of S. arizonae O61 is structurally related to that of Pseudomonas aeruginosa O12, thus explaining the known serological cross-reactivity between these micro-organisms.

Chemical and serological study of lipopolysaccharide isolated from Shigella flexneri 88-893 possessing a new type-antigen

The O-specific polysaccharide chain which represents a new type-antigen in lipopolysaccharide (LPS) of Shigella flexneri 88-893 was investigated. The O-polysaccharide chain was found to be composed of repeating units comprising rhamnose, N-acetylglucosamine and glucose (3:1:2). In the passive hemolysis test, group-6 antiserum of S. flexneri exhibited a high hemolytic titer (50% hemolysis titer: 7,900) against sheep red blood cells (SRBC) sensitized with intact 893 LPS, but virtually no hemolytic activity against SRBC sensitized with alkali-treated 893 LPS. None of the type-specific antisera (I-VI), showed any significant hemolytic titer against SRBC sensitized with either intact or alkali-treated 893 LPS. Thus, 893 LPS contained both the group-6 antigen and a new type-antigen which is distinct from any known type-antigen of S. flexneri.

Structure of the O-specific, sialic acid containing polysaccharide chain and its linkage to the core region in lipopolysaccharide from Hafnia alvei strain 2 as elucidated by chemical methods, gas-liquid chromatography/mass spectrometry, and proton NMR spectroscopy

Mild acid hydrolysis of Hafnia alvei strain 2 lipopolysaccharide released no O-specific polysaccharide but instead gave a monomeric octasaccharide repeating unit with N-acetylneuraminic acid as the reducing terminus. In addition, a dimer of the octasaccharide repeating unit, and also a decasaccharide composed of a fragment of the O-specific polysaccharide chain and the core region, were obtained in minute amounts. On the basis of the sugar and methylation analyses, periodate oxidation, and 1H NMR spectroscopy of the lipopolysaccharide hydrolytic products, the biological repeating unit of the O-specific polysaccharide was shown to be a branched octasaccharide: (Formula; see text) The linkage between the O-specific polysaccharide chain and core region has also been determined and has yield strong evidence that N-acetylneuraminic acid is an inherent lipopolysaccharide component. The lipopolysaccharide of H. alvei strain 2 is the first lipopolysaccharide reported to contain 4-substituted neuraminic acid in its O-specific polysaccharide region.

Localization of the terminal steps of O-antigen synthesis in Salmonella typhimurium

Previous immunoelectron microscopic studies have shown that both the final intermediate in O-antigen synthesis, undecaprenol-linked O polymer, and newly synthesized O-antigenic lipopolysaccharide are localized to the periplasmic face of the inner membrane (C. A. Mulford and M. J. Osborn, Proc. Natl. Acad. Sci. USA 80:1159-1163, 1983). In vivo pulse-chase experiments now provide further evidence that attachment of O antigen to core lipopolysaccharide, as well as polymerization of O-specific polysaccharide chains, takes place at the periplasmic face of the membrane. Mutants doubly conditional in lipopolysaccharide synthesis [kdsA(Ts) pmi] were constructed in which synthesis of core lipopolysaccharide and O antigen are temperature sensitive and mannose dependent, respectively. Periplasmic orientation of O antigen:core lipopolysaccharide ligase was established by experiments showing rapid chase of undecaprenol-linked O polymer, previously accumulated at 42 degrees C in the absence of core synthesis, into lipopolysaccharide following resumption of core formation at 30 degrees C. In addition, chase of the monomeric O-specific tetrasaccharide unit into lipopolysaccharide was found in similar experiments in an O-polymerase-negative [rfc kdsA(Ts) pmi] mutant, suggesting that polymerization of O chains also occurs at the external face of the inner membrane.

Membrane receptors on rat hepatocytes for the inner core region of bacterial lipopolysaccharides.

Bacterial lipopolysaccharides (LPS) are potent endotoxins that are thought to be involved in the pathogenesis of Gram-negative septicemia. The liver is known to be the primary organ responsible for the clearance of LPS from the systemic circulation in mammals. In this work, 125I-labeled LPS have been used in a filtration assay for the specific binding of LPS to intact rat hepatocytes. Eight S-form (smooth) LPS with complete O-specific polysaccharide chains isolated from different O-serotypes of Salmonella and Escherichia coli as well as nine R-form (rough) LPS isolated from Salmonella mutants deficient in synthesis of their core oligosaccharides were used in this study. All 125I-labeled S-form LPS and R-form LPS, except Re, show specific binding to isolated hepatocytes. The binding is saturable, is inhibited with excess unlabeled homologous or heterologous LPS but not lipid A, and is trypsin sensitive. L-Glycero-D-mannoheptose (heptose), a constituent of the inner core region of almost all LPS, is a potent inhibitor of the specific binding of 125I-labeled Rb2 LPS, whereas other monosaccharides, including 3-deoxy-D-manno-2-octulosonic acid (KDO), have weak or negligible inhibitor activity. These results strongly suggest the presence of a lectin-like receptor for the LPS inner core region (heptose-KDO region) on the plasma membrane of rat hepatocytes.

Retention of bacterial lipopolysaccharide at the site of subcutaneous injection.

The tissue distribution of Klebsiella pneumoniae O3 lipopolysaccharide (KO3 LPS) was studied in mice injected subcutaneously (s.c.) or intraperitoneally (i.p.) with 125I-labeled KO3 LPS. Marked retention of KO3 LPS radioactivity could be found at the site of s.c. injection for several weeks. On the other hand, about 85% of the radioactivity rapidly disappeared from the peritoneal cavity within 6 h after i.p. injection. The long-term presence of KO3 LPS at the injection site was also supported by experiments with 51Cr-labeled KO3 LPS and immunoblotting and immunofluorescence staining methods. The R-form LPS lacking the O-specific polysaccharide chain of KO3 LPS and the lipid A fraction of KO3 LPS seemed to remain at the site in larger amounts and for longer times than KO3 LPS. There were no marked differences in the retention pattern at the injection site among KO3 LPS, Escherichia coli LPS, Salmonella typhosa LPS, and Salmonella enteritidis LPS. However, much less radioactivity accumulated in the livers and spleens of mice injected with either KO3 LPS or S. typhosa LPS compared with the other LPS preparations. It was suggested that retention of LPS at the site of s.c. injection may play an important role in the development of various biological actions of s.c. injected LPS.

Staining of O‐Specific Polysaccharide Chains of Lipopolysaccharides with Ruthenium Red

S-form lipopolysaccharides (LPS) from Klebsiella strain LEN-1 (O3: K1-) and from Salmonella minnesota strain 1114 were positively stained with ruthenium red, whereas R-form LPS from Klebsiella strain LEN-111 (O3-: K1-) and Ra, Rb1, RcP+, Rd1P-, and Re LPS from the respective mutant strains of S. minnesota were not or only faintly stained by such treatment. From these results it was concluded that ruthenium red stains the O-specific polysaccharide chains of LPS. The appearance of stained preparations of S-form LPS suggested that the material responsible for this positive staining corresponded to the surface projections which were seen by the negative staining technique as attached to the ribbon-like structures and spherules of the LPS.

Somatic antigens of Pseudomonas aeruginosa

Lipopolysaccharides from Pseudomonas aeruginosa O1 (Lányi classification), O3 (Habs classification), O13 and O14 (Wokatsch classification), and strain NCTC 8505, which is also related to serogroup O3 (Habs), have structurally similar O-specific polysaccharide chains built up of tetrasaccharide repeating units involving L-rhamnose (Rha), 2-acetamido-2-deoxy-D-glucose (GlcNAc), 2-acetamido-2-deoxy-L-galacturonic acid (GalNAcA), and a di-N-acyl derivative of bacillosamine (BacN): 2,4-diacetamido-2,4,6-trideoxy-D-glucose or 2-acetamido-2,4,6-trideoxy-4-[(S)-3-hydroxybutyramido]-D-glucose. The latter derivative was obtained free by solvolysis with hydrogen fluoride of carboxyl-reduced Habs O3 polysaccharide, and was identified by 1H-nuclear magnetic resonance spectroscopy and by mass spectrometry of the corresponding methylated alditol. Habs O3, Lányi O1, and Wokatsch O14 polysaccharides contained O-acetyl groups. Solvolysis with hydrogen fluoride of the native Habs O3 polysaccharide resulted in selective cleavage of the glycosidic linkages of 6-deoxy sugars to give the trisaccharide fragment involving all three N-acylated amino sugars. Similar solvolysis of NCTC 8505 polysaccharide afforded a mixture of disaccharide and trisaccharide with N,N'-diacetylbacillosamine at the reducing end. Smith degradation of Habs O3 polysaccharide resulted in selective oxidation of rhamnose to give a glycoside of a trisaccharide with glyceraldehyde as the aglycone. Smith degradation of NCTC 8505 polysaccharide was complicated by the formation of the glycoside of a trisaccharide with an aglycone of unknown structure. A trisaccharide with rhamnose at the reducing end was also isolated after Smith degradation of the latter polysaccharide. Analysis of the composition and structure of all oligosaccharides obtained, and detailed examination of the 13C-nuclear magnetic resonance spectra of these oligosaccharides, and of both intact and modified polysaccharides, revealed the following structures of the repeating units. The structure for the NCTC 8505 polysaccharide differs from that proposed previously [Tahara, Y. and Wilkinson, S.G. (1983) Eur. J. Biochem. 134, 299-304] in the configurations assigned to the glycosidic linkages of rhamnose and bacillosamine. The results obtained show the P. aeruginosa strains studied to represent three different O-serotypes in a single O-serogroup (Formula: see text).

Somatic antigens of Pseudomonas aeruginosa. The structure of the O-specific polysaccharide chains of lipopolysaccharides of P. aeruginosa serogroup O4 (Lanyi) and related serotype O6 (Habs) and immunotype 1 (Fisher)

Acidic O-specific polysaccharides were isolated on mild acidic degradation of lipopolysaccharides of Pseudomonas aeruginosa serotypes O4a,b, O4a,c, O4a,d (Lányi classification) and serologically related to them serotype O6 (Habs classification) and immunotype 1 (Fisher classification). The polysaccharides had identical monosaccharide composition and were built up of L-rhamnose, 2-acetamido-2,6-dideoxy-D-glucose,2-formamido-2-deoxy-D-galacturonic acid and 2-acetamido-2-deoxy-D-galactouronamide residues. The latter two derivatives of D-galactosaminuronic acid were found in nature for the first time. All the polysaccharides, but Lányi serotype O4a,c, contained O-acetyl groups. The polysaccharides were readily de-O-acetylated with aqueous triethylamine and de-N-formylated with dilute hydrochloric acid. De-N-formylated polysaccharide of serotype O4a,c was selectively cleaved with nitrous acid upon 2-amino-2-deoxygalacturonic acid residues to form a tetrasaccharide with a 2,5-anhydrotaluronic acid residue on the reducing end. The tetrasaccharide represented a modified repeating unit of the polysaccharide. Solvolysis of all intact polysaccharides with hydrogen fluoride selectively split the glycosidic linkages of 6-deoxy sugars to give the same trisaccharide, including both derivatives of galactosaminuronic acid and having 2-acetamido-2,6-dideoxyglucose on the reducing end. Structural investigation of the oligosaccharides obtained together with methylation analysis and 13C nuclear magnetic resonance data revealed the following structures of the O-specific polysaccharides: (Formula: see text) An independent confirmation of the structures of the repeating units was obtained as the result of full interpretation of the 13C nuclear magnetic resonance spectra of the intact and modified polymers. Spectral data analysis revealed a number of regularities in the effects of glycosidation connecting their values with the anomeric and absolute configuration of pyranose residues. The data on the structures of the O-specific polysaccharides indicated that each of the five P. aeruginosa strains under study should be considered as an individual O-serotype within one O-serogroup.

Inhibition by succinyl concanavalin A of strong adjuvant activity of lipopolysaccharides which possess mannans as the O-specific polysaccharide chains

It has been reported that lipopolysaccharides (LPS) from Klebsiella O3 and O5 and Escherichia coli O8 and O9 exhibit extraordinarily strong adjuvant activity in augmenting antibody responses against protein antigens in mice as compared with other kinds of LPS. These four kinds of LPS all possess homopolysaccharides consisting of mannose (mannans) as the O-specific side chains. When these kinds of LPS were mixed in vitro with succinyl concanavalin A (Con A) which is known to bind specifically to α-mannoside and α-glucoside, their strong adjuvant activity was inhibited. Degree of the inhibition of the adjuvant activity of Klebsiella O3 LPS by succinyl Con A was dependent upon the dose of succinyl Con A. However, phytohemagglutinin, which is known to bind specifically to N-acetyl- d-galactosamine, did not inhibit the adjuvant activity of Klebsiella O3 LPS and O5 LPS. When Klebsiella O3 LPS was mixed with succinyl Con A in the presence of excess amounts of α-methyl mannoside or the polysaccharide fraction isolated from Klebsiella O3 LPS, the inhibitory effect of succinyl Con A on the adjuvant activity of Klebsiella O3 LPS was blocked. By contrast, the activity of Klebsiella O3 LPS as a polyclonal B-cell activator was not affected by treatment with succinyl Con A. From these results it is concluded that the mannans, as the O-specific polysaccharide chains of the LPS, significantly contribute to expression of their strong adjuvant activity.