A comparative study of lipopolysaccharides from two Coxiella burnetii strains considered to be associated with acute and chronic Q fever

Coxiella burnetii strains Nine Mile and Priscilla, considered to be associated with acute and chronic forms of Q fever, were investigated for variation in composition of their lipopolysaccharides. Though SDS-PAGE profiles of the lipopolysaccharides were distinct, chemical analyses showed only small differences in their overall composition. Further studies on lipid A-deprived O-polysaccharide fractions of both lipopolysaccharides, obtained by steric-exclusion chromatography, revealed noticeable differences in distribution and chemical composition of the O-polysaccharide chains. It is likely that C. burnetii strains are capable of synthesizing chemically distinct subclasses of O-specific polysaccharide molecules differing in their antigenic reactivities. The results provide suggestive evidence that virulence of C. burnetii may be modulated through lipopolysaccharide composition and structure.     

Comparative chemical and biological characterization of the lipopolysaccharides of gastric and enterohepatic helicobacters

BACKGROUND: The lipopolysaccharide of Helicobacter pylori plays an important role in colonization and pathogenicity. The present study sought to compare structural and biological features of lipopolysaccharides from gastric and enterohepatic Helicobacter spp. not previously characterized. MATERIALS AND METHODS: Purified lipopolysaccharides from four gastric Helicobacter spp. (H. pylori, Helicobacter felis, Helicobacter bizzozeronii and Helicobacter mustelae) and four enterohepatic Helicobacter spp. (Helicobacter hepaticus, Helicobacter bilis, 'Helicobacter sp. flexispira' and Helicobacter pullorum) were structurally characterized using electrophoretic, serological and chemical methods. RESULTS: Structural insights into all three moieties of the lipopolysaccharides, i.e. lipid A, core and O-polysaccharide chains, were gained. All species expressed lipopolysaccharides bearing an O-polysaccharide chain, but H. mustelae and H. hepaticus produced truncated semirough lipopolysaccharides. However, in contrast to lipopolysaccharides of H. pylori and H. mustelae, no blood group mimicry was detected in the other Helicobacter spp. examined. Intra-species, but not interspecies, fatty acid profiles of lipopolysaccharides were identical within the genus. Although shared lipopolysaccharide-core epitopes with H. pylori occurred, differing structural characteristics were noted in this lipopolysaccharide region of some Helicobacter spp. The lipopolysaccharides of the gastric helicobacters, H. bizzozeronii and H. mustelae, had relative Limulus amoebocyte lysate activities which clustered around that of H. pylori lipopolysaccharide, whereas H. bilis, 'Helicobacter sp. flexispira' and H. hepaticus formed a cluster with approximately 1000-10,000-fold lower activities. H. pullorum lipopolysaccharide had the highest relative Limulus amoebocyte lysate activity of all the helicobacter lipopolysaccharides (10-fold higher than that of H. pylori lipopolysaccharide), and all the lipopolysaccharides of enterohepatic Helicobacter spp. were capable of inducing nuclear factor-Kappa B(NF-kappaB) activation. CONCLUSIONS: The collective results demonstrate the structural heterogeneity and pathogenic potential of lipopolysaccharides of the Helicobacter genus as a group and these differences in lipopolysaccharides may be indicative of adaptation of the bacteria to different ecological niches.     

Complex O-acetylation in Legionella pneumophila serogroup 1 lipopolysaccharide. Evidence for two genes involved in 8-O-acetylation of legionaminic acid.

A putative gene encoding an O-acetyl transferase, lag-1, is involved in biosynthesis of the O-polysaccharide (polylegionaminic acid) in some Legionella pneumophila serogroup 1 strains. To study the effect of the presence and absence of the gene on the O-polysaccharide O-acetylation, lag-1 from strain Philadelphia 1 was expressed in trans in the naturally lag-1-negative OLDA strain RC1, and immunoblot analysis revealed that the lag-1-encoded O-acetyl transferase is active. O-Polysaccharides of different size were prepared from the lipopolysaccharides of wild-type and transformant strains by mild acid degradation followed by gel-permeation chromatography. Using NMR spectroscopy and MALDI-TOF mass spectrometry, it was found that O-acetylation of the first three legionaminic acid residues next to the core occurs in the short-chain O-polysaccharide (<10 sugars) from both strains. Hence, there is another O-acetyl transferase encoded by a gene different from lag-1. In the longer-chain O-polysaccharide, a legionaminic acid residue proximal to the core is N-methylated and could be further 8-O-acetylated in the lag-1-dependent manner. Only strains expressing a functional lag-1 gene were recognized in Western blot analysis by monoclonal antibody 3/1 requiring 8-O-acetylated polylegionaminic acid for binding. The highly O-acetylated outer core region of the lipopolysaccharide is involved in the epitope of another serogroup 1-specific monoclonal antibody termed LPS-1. The O-acetylation pattern of the L. pneumophila serogroup 1 core oligosaccharide was revised using MALDI-TOF mass spectrometry. lag-1-independent O-acetylation of the core and short-chain O-polysaccharide was found to be a common feature of L. pneumophila serogroup 1 strains. The biological importance of conserved lag-1-independent and variable lag-1-dependent O-acetylation is discussed.     

Elucidation of the structure of the lipopolysaccharide core and the linkage between the core and the O-antigen in Pseudomonas aeruginosa immunotype 5 using strong alkaline degradation of the lipopolysaccharide

The products of the strong alkaline degradation of the lipopolysaccharide (LPS) of Pseudomonas aeruginosa immunotype 5 were separated by anion-exchange HPLC and studied by electrospray ionization mass spectrometry and NMR spectroscopy.It was found that two major products have the same inner core region and lipid A carbohydrate backbone but different outer core regions.The difference is in the position of a rhamnose residue,which is substituted with either an additional glucose residue or a disaccharide remainder of the degraded O-polysaccharide.The site and the configuration of the linkage between the O-polysaccharide and the core were determined and,together with published data,the structure of the so-called biological repeating unit of the O-antigen was defined.The glycosidic linkage of the 2-acetamido-2,6-dideoxy D-glucose (N-acetyl-D-quinovosamine) residue is when it links the O-polysaccharide to the core and when it connects the interior repeating units of the O-polysaccharide to each other.     

The structures of core regions from enterobacterial lipopolysaccharides - an update

To the major virulence factors of Gram-negative bacteria belong the lipopolysaccharides (endotoxins), which are very well characterized for their immunological, pharmacological and pathophysiological effects displayed in eucaryotic cells and organisms. In general, these amphiphilic lipopolysaccharides comprise three regions, which can be differentiated by their structures, function, genetics and biosynthesis: lipid A, the core region and a polysaccharide portion, which may be the O-specific polysaccharide, Enterobacterial Common Antigen (ECA) or a capsular polysaccharide. In the past, much emphasis has been laid on the elucidation of the structure-function relation. The lipid A was proven to represent the toxic principle of endotoxic active lipopolysaccharides, however, its toxicity depends not only on its structure but also on that of the core region, which is covalently linked to lipid A. Thus, and since the core region possesses immunogenic properties, complete structural analyses of lipopolysaccharides core regions and of structure-function relation are highly important for a better understanding of lipopolysaccharides action. To date, quite a number of core structures from lipopolysaccharides of various Gram-negative bacteria have been published and summarized in several overviews. This short review adds to this knowledge those structures of enterobacterial lipopolysaccharides that were published between January 2002 and October 2006.     

Elucidation of the full O-polysaccharide structure and identification of the core type of the lipopolysaccharide of Providencia alcalifaciens O9

Opportunistic human pathogens of the genus Providencia from the family Enterobacteriaceae are serotyped by their O-antigens, which represent the O-polysaccharide chains of the lipopolysaccharides (LPSs) on the cell surface. In this work, the O-polysaccharide of Providencia alcalifaciens O9 was obtained by mild acid degradation of a long-chain S-form LPS. The structure of the hexasaccharide repeat (O-unit) of the O-polysaccharide containing one d-Gal, two d-Glc, and three d-GalNAc residues was established by sugar and methylation analyses along with one- and two-dimensional ¹H and ¹³C NMR spectroscopy. Another degradation product was derived from a short-chain SR-form LPS and found to consist of a core oligosaccharide bearing one O-unit. Its studies by NMR spectroscopy and electrospray ionization mass spectrometry enabled identification of one of the GalNAc residues as the first monosaccharide of the O-unit, whose glycosidic linkage links the O-units to each other and the first O-unit to the core. The core is distinguished by the occurrence of two glycoforms differing in the nature of a lateral monosaccharide, which is either d-Glc or d-GlcNAc. Although composed of common monosaccharides, the O-polysaccharide of P. alcalifaciens O9 has a unique structure among bacterial polysaccharides, whereas the oligosaccharide region belongs to one of several core types recognized in the LPSs of Providencia.     

Structure of the O-polysaccharide and serological studies of the lipopolysaccharide of Proteus mirabilis 2002

The structure of the O-polysaccharide of the lipopolysaccharide of Proteus mirabilis 2002 was elucidated by chemical methods and 1H and 13C NMR spectroscopy. It was found that the polysaccharide consists of branched pentasaccharide repeating units having the following structure: [structure in text]. The O-polysaccharide of P. mirabilis 2002 has a common tetrasaccharide fragment with that of P. mirabilis 52/57 from serogroup O29, and the lipopolysaccharides of the two strains are serologically related. Therefore, based on the structural and serological data, we propose to classify P. mirabilis 2002 into the Proteus O29 serogroup as a subgroup O29a,29b.     

Molecular basis for structural diversity in the core regions of the lipopolysaccharides of Escherichia coli and Salmonella enterica

Bacterial lipopolysaccharides (LPS) are unique and complex glycolipids that provide characteristic components of the outer membranes of Gram-negative bacteria. In LPS of the Enterobacteriaceae, the core oligosaccharide links a highly conserved lipid A to the antigenic O-polysaccharide. Structural diversity in the core oligosaccharide is limited by the constraints imposed by its essential role in outer membrane stability and provides a contrast to the hypervariable O-antigen. The genetics of core oligosaccharide biosynthesis in Salmonella and Escherichia coli K-12 have served as prototypes for studies on the LPS and lipo-oligosaccharides from a growing range of bacteria. However, despite the wealth of knowledge, there remains a number of unanswered questions, and direct experimental data are not yet available to define the precise mechanism of action of many gene products. Here we present a comparative analysis of the recently completed sequences of the major core oligosaccharide biosynthesis gene clusters from the five known core types in E. coli and the Ra core type of Salmonella enterica serovar Typhimurium and discuss advances in the understanding of the related biosynthetic pathways. Differences in these clusters reflect important structural variations in the outer core oligosaccharides and provide a basis for ascribing functions to the genes in these model clusters, whereas highly conserved regions within these clusters suggest a critical and unalterable function for the inner region of the core.     

Chemical characterization of lipopolysaccharides from Proteus strains used in Weil-Felix test

The lipopolysaccharides (LPS) extracted from Proteus strains OX2, OX19, and OXK used as antigens in the Weil-Felix test, were characterized by chemical analysis and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). To separate the O-polysaccharide, core-oligosaccharide, and lipid A moieties, each LPS was treated with 2% acetic acid, centrifuged, and applied to Sephadex G-50 column. The core-oligosaccharides contained L-glycero-D-mannoheptose, D-glycero-D-mannoheptose, glucose (Glc), galactose, 3-deoxy-D-mannooctulosonic acid, uronic acid, phosphate, glucosamine (GlcN), and galactosamine (GalN). The lipid A preparations contained GlcN, GlcN-phosphate, and three fatty acids (myristic, plamitic, and beta-hydroxymyristic acids). However, the O-polysaccharides of OX2- and OXK-LPS had different chemical compositions which consisted of Glc, GlcN, and quinovosamine, and Glc, uronic acid, and GalN, respectively, while OX19-LPS seemed to lack O-polysaccharide.     

Structure of the biological repeating unit of the O-antigen of Pseudomonas aeruginosa immunotype 4 containing both 2-acetamido-2,6-dideoxy-D-glucose and 2-acetamido-2,6-dideoxy-D-galactose

A phosphorylated core-lipid A backbone oligosaccharide that carries a disaccharide remainder of the first O-antigen repeating unit was derived by strong alkaline degradation following mild hydrazinolysis of the lipopolysaccharide of Pseudomonas aeruginosa immunotype 4 (serogroup O-1). The structure of the oligosaccharide was determined using ESI MS and NMR spectroscopy and it was demonstrated that 2-acetamido-2,6-dideoxy-D-glucose is the first monosaccharide of the O-polysaccharide that is linked to the LPS core. These data define the structure of the biological repeating unit of the O-antigen.     

Structural studies on the core and the O-polysaccharide repeating unit of Pseudomonas aeruginosa immunotype 1 lipopolysaccharide.

The structure of the lipopolysaccharide (LPS) of Pseudomonas aeruginosa immunotype 1 was studied after mild acid and strong alkaline degradations by MS and NMR spectroscopy. Three types of LPS molecules were found, including those with an unsubstituted glycoform 1 core (A) or an isomeric glycoform 2 core substituted with one O-polysaccharide repeating unit (B) or with a long-chain O-polysaccharide. Therefore, of two core glycoforms, only glycoform 2 accepts the O-polysaccharide. In the structures A and B, Kdo, Hep, Hep7Cm, GalNAcAN3Ac, GalNFoAN, QuiNAc, GalNAla represent 3-deoxy-d-manno-octulosonic acid, l-glycero-d-manno-heptose, 7-O-carbamoyl-l-glycero-d-manno-heptose, 2-acetamido-3-O-acetyl-2-deoxygalacturonamide, 2-formamido-2-deoxygalacturonamide, 2-acetamido-2,6-dideoxyglucose and 2-(l-alanylamino)-2-deoxygalactose, respectively; all sugars are in the pyranose form and have the d configuration unless otherwise stated. One or more phosphorylation sites may be occupied by diphosphate groups. In a minority of the LPS molecules, an O-acetyl group is present in the outer core region at unknown position. The site and the configuration of the linkage between the O-polysaccharide and the core and the structure of the O-polysaccharide repeating unit were defined in P. aeruginosa immunotype 1. The QuiNAc residue linked to the Rha residue of the core was found to have the beta configuration, whereas in the interior repeating units of the O-polysaccharide this residue is in the alpha-configuration. The data obtained are in accordance with the initiation of biosynthesis of the O-polysaccharide of P. aeruginosa O6, which is closely related to immunotype 1, by transfer of d-QuiNAc-1-P to undecaprenyl phosphate followed by synthesis of the repeating O-antigen tetrasaccharide.     

Full Structure of the Lipopolysaccharide of Pseudomonas aeruginosa Immunotype 5

The lipopolysaccharide (LPS) of the opportunistic human pathogen Pseudomonas aeruginosa immunotype 5 was delipidated by mild acid hydrolysis, and the products were separated by high-performance anion-exchange chromatography and analyzed by ESI MS and NMR spectroscopy. LPS species of three types were found, including those with an unsubstituted core and the core substituted with one O-polysaccharide repeating unit or with an O-polysaccharide of a variable number of repeating units. The core region is highly phosphorylated, the major species containing two monophosphate groups and one ethanolamine diphosphate group. Based on these and published data on the O-polysaccharide structure, the full structure of the LPS of P. aeruginosa immunotype 5 was established.     

Structure of the neutral O-polysaccharide and biological activities of the lipopolysaccharide of Proteus mirabilis O20

Mild acid degradation of the lipopolysaccharide (LPS) of Proteus mirabilis O20 resulted in depolymerisation of the O-polysaccharide to give a repeating-unit pentasaccharide. A polysaccharide was obtained by O-deacylation of the LPS followed by nitrous acid deamination. The derived pentasaccharide and polysaccharide were studied by NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C HMQC and HMQC-TOSCY experiments, along with chemical methods, and the following structure of the repeating unit of the O-polysaccharide was established: [Carbohydrate structure: see text]. As opposite to most other P. mirabilis O-polysaccharides studied, that of P. mirabilis O20 is neutral. A week serological cross-reactivity was observed between anti-P. mirabilis O20 serum and LPS of a number of Proteus serogroups with known O-polysaccharide structure. The ability of LPS of P. mirabilis O20 to activate the serine protease cascade was tested in Limulus amoebocyte lysate and in human blood plasma and compared with that of P. mirabilis O14a,14c having an acidic O-polysaccharide. The LPS of P. mirabilis O20 was found to be less active in both assays than the LPS of P. mirabilis O14a,14c and, therefore, the structurally variable O-polysaccharide may influenced the biological activity of the conserved lipid A moiety of the LPS.     

Structure of the oligosaccharide chain of the SR-type lipopolysaccharide of Ralstonia solanacearum Toudk-2

Aiming at improving classification and taxonomy of Gram-negative phytopathogenic bacteria, we studied the structure of the lipopolysaccharide of Ralstonia solanacearum. Mild acid hydrolysis of the lipopolysaccharide of strain Toudk-2 followed by gel chromatography resulted in an O-polysaccharide and two oligosaccharide fractions. The smallest-size oligosaccharide fraction was studied by sugar analysis, high-resolution electrospray ionization mass spectrometry, and, after fractionation by anion-exchange chromatography on HiTrap Q, by one- and two-dimensional (1)H and (13)C NMR spectroscopy. It was found that the isolated oligosaccharides consist of the lipopolysaccharide core with one O-polysaccharide repeat (O-unit) attached. The core exists in two major glycoforms differing from each other in a lateral octulosonic acid residue, which is either D-glycero-D-talo-oct-2-ulosonic acid or 3-deoxy-D-manno-oct-2-ulosonic acid. A peculiar feature of the core is the occurrence of 4-amino-4-deoxy-L-arabinose nonstoichiometrically linked to a heptose residue. The full structures of the core and the biological O-unit as well as the site of the attachment of the O-unit to the core were established.     

Structure of the O-polysaccharide of Proteus penneri 28 and Proteus vulgaris O31 and classification of P. penneri 26 and 28 in Proteus serogroup O31

The lipopolysaccharides (LPS) of Proteus penneri 28 and Proteus vulgaris O31 (PrK 55/57) were degraded with dilute acetic acid and structurally identical high-molecular-mass O-polysaccharides were isolated by gel-permeation chromatography. Sugar analysis and nuclear magnetic resonance (NMR) spectroscopic studies showed that both polysaccharides contain D-GlcNAc, 2-acetamido-2,6-dideoxy-L-glucose (L-2-acetamido-2,6-dideoxyglucose (N-acetylquinovosamine)) and 2-acetamido-3-O-[(S)-1-carboxyethyl]-2-deoxy-D-glucose (N-acetylisomuramic acid) and have the following structure: [carbohydrate structure: see text] where (S)-1-carboxyethyl [a residue of (S)-lactic acid] (S-Lac) is an ether-linked residue of (S)-lactic acid. The O-polysaccharide studied is structurally similar to that of P. penneri 26, which differs only in the absence of S-Lac from the GlcNAc residue. Based on the O-polysaccharide structures and serological data of the LPS, it was suggested classifying these strains in one Proteus serogroup, O31, as two subgroups: O(31a), 31b for P. penneri 28 and P. vulgaris PrK 55/57 and O31a for P. penneri 26. A serological relatedness of the LPS of Proteus O(31a), 31b and P. penneri 62 was revealed and substantiated by sharing epitope O31b, which is associated with N-acetylisomuramic acid. It was suggested that a cross-reactivity of P. penneri 28 O-antiserum with the LPS of several other P. penneri strains is due to a common epitope(s) on the LPS core.     

Detoxification of lipopolysaccharide (LPS) by egg white lysozyme

Abstract Recent studies carried out by our group suggest that lysozyme binds to bacterial lipopolysaccharide with a high affinity to produce a complex, and inhibits various biological activities of lipopolysaccharide. Although the basic structure of lipopolysaccharide is independent of the species and strains of Gram-negative bacteria, many structural factors such as O-antigenic polysaccharide, lipid A, substituted groups, and associated molecules, affect the biological activities of lipopolysaccharide. In this study, we prepared lysozyme/lipopolysaccharide complexes using various structures of lipopolysaccharide and compared the activity and physiochemical properties. Native and dansylated lysozyme were found to bind to all tested lipopolysaccharides. The mitogenic activity and TNF production by all tested lipopolysaccharides were significantly reduced by complex formation in vitro. Administration of the complex prepared by various lipopolysaccharides produced significantly less quantities of TNF in the septic shock model. These results suggested that binding of lysozyme to lipopolysaccharide is important for the host both in pathophysiological responses to lipopolysaccharides and in the modification of lipopolysaccharide biological activity.     

Relationships between physicochemical characteristics and biological activity of lipopolysaccharides

The review covers current notions and experimental data on relationships between biological activity of lipopolysaccharides (LPSs) and their physicochemical characteristics: chemical composition and molecular conformation of lipid A, LPS glycoforms and their supramolecular structures, cationic composition and charges of LPS molecules.     

Structures and genetics of Kdo-containing O-antigens of Cronobacter sakazakii G2706 and G2704, the reference strains of serotypes O5 and O6

Cronobacter sakazakii G2706 and G2704 are the reference strains of serotypes O5 and O6 in the serological classification of this species proposed recently. Mild acid degradation of the lipopolysaccharides of both strains resulted in cleavage of the O-polysaccharide chains at the acid-labile linkage of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) to yield oligosaccharides representing repeating units of the O-polysaccharides. The oligosaccharides and alkali-degraded lipopolysaccharides were studied by sugar analysis along with 1D and 2D ¹H and ¹³C NMR spectroscopy, and the following O-polysaccharide structures were established:The structure of strain G2706 is unique among the known bacterial polysaccharide structures, whereas that of strain G2704 is identical to the structure of Cronobacter malonaticus 3267 [MacLean, L. L.; Vinogradov, E.; Pagotto, F.; Farber, J. M.; Perry, M. B. Biochem. Cell Biol.2009, 87, 927–932], except for that the latter lacks O-acetylation. Putative functions of the genes in the O-antigen gene clusters of C. sakazakii strains studied are in agreement with the O-polysaccharide structures.     

Structure of the O-polysaccharide and serological studies of the lipopolysaccharide of Proteus penneri 60 classified into a new Proteus serogroup O70

An alkali-treated lipopolysaccharide of Proteus penneri strain 60 was studied by chemical analyses and 1H, 13C and 31P NMR spectroscopy, and the following structure of the linear pentasaccharide-phosphate repeating unit of the O-polysaccharide was established: 6)-alpha-D-Galp-(1-->3)-alpha-L-FucpNAc-(1-->3)-alpha-D-GlcpNAc-(1-->3)-beta-D-Quip4NAc-(1-->6)-alpha-D-Glcp-1-P-(O--> Rabbit polyclonal O-antiserum against P. penneri 60 reacted with both core and O-polysaccharide moieties of the homologous LPS. Based on the unique O-polysaccharide structure and serological data, we propose to classify P. penneri 60 into a new, separate Proteus serogroup O70. A weak cross-reactivity of P. penneri 60 O-antiserum with the lipopolysaccharide of Proteus vulgaris O8, O15 and O19 was observed and discussed in view of the chemical structures of the O-polysaccharides.     

Identification of the A and M antigens of Brucella as the O-polysaccharides of smooth lipopolysaccharides

Rabbit antisera, cross-absorbed serotype-specific for the Brucella A and M antigens, precipitated respectively the smooth lipopolysaccharides from B. abortus 1119-3 and B. melitensis 16M. The antigenic A and M activity of these lipopolysaccharides was shown to reside within the O-chain region of the smooth lipopolysaccharides by inhibition experiments. Homologous O-polysaccharides showed the highest inhibitory activity in ELISA, although both the A and M antigens were active as heterologous inhibitors, showing that the antigenic determinants of the classical A and M antigens are therefore within the respective O-polysaccharide structures. Their cross-serological activity may be explained in terms of the distinct but related chemical structures of these polysaccharides.