Lipopolysaccharide of the Yersinia pseudotuberculosis Complex.

Lipopolysaccharide (LPS), localized in the outer leaflet of the outer membrane, serves as the major surface component of the Gram-negative bacterial cell envelope responsible for the activation of the host's innate immune system. Variations of the LPS structure utilized by Gram-negative bacteria promote survival by providing resistance to components of the innate immune system and preventing recognition by TLR4. This review summarizes studies of the biosynthesis of Yersinia pseudotuberculosis complex LPSs, and the roles of their structural components in molecular mechanisms of yersiniae pathogenesis and immunogenesis.

Hypoacylated LPS from Foodborne Pathogen Campylobacter jejuni Induces Moderate TLR4-Mediated Inflammatory Response in Murine Macrophages.

Toll-like receptor 4 (TLR4) initiates immune response against Gram-negative bacteria upon specific recognition of lipid A moiety of lipopolysaccharide (LPS), the major component of their cell wall. Some natural differences between LPS variants in their ability to interact with TLR4 may lead to either insufficient activation that may not prevent bacterial growth, or excessive activation which may lead to septic shock. In this study we evaluated the biological activity of LPS isolated from pathogenic strain of Campylobacter jejuni, the most widespread bacterial cause of foodborne diarrhea in humans. With the help of hydrophobic chromatography and MALDI-TOF mass spectrometry we showed that LPS from a C. jejuni strain O2A consists of both hexaacyl and tetraacyl forms. Since such hypoacylation can result in a reduced immune response in humans, we assessed the activity of LPS from C. jejuni in mouse macrophages by measuring its capacity to activate TLR4-mediated proinflammatory cytokine and chemokine production, as well as NFκB-dependent reporter gene transcription. Our data support the hypothesis that LPS acylation correlates with its bioactivity.

Full structure and insight into the gene cluster of the O-specific polysaccharide of Yersinia intermedia H9-36/83 (O:17).

Lipopolysaccharide was isolated from bacteria Yersinia intermedia H9-36/83 (O:17) and degraded with mild acid to give an O-specific polysaccharide, which was isolated by GPC on Sephadex G-50 and studied by sugar analysis and 1D and 2D NMR spectroscopy. The polysaccharide was found to contain 3-deoxy-3-[(R)-3-hydroxybutanoylamino]-d-fucose (d-Fuc3NR3Hb) and the following structure of the heptasaccharide repeating unit was established: The structure established is consistent with the gene content of the O-antigen gene cluster. The O-polysaccharide structure and gene cluster of Y. intermedia are related to those of Hafnia alvei 1211 and Escherichia coli O:103.

Changes in the lipopolysaccharide of Proteus mirabilis 9B-m (O11a) clinical strain in response to planktonic or biofilm type of growth.

The impact of planktonic and biofilm lifestyles of the clinical isolate Proteus mirabilis 9B-m on its lipopolysaccharide (O-polysaccharide, core region, and lipid A) was evaluated. Proteus mirabilis bacteria are able to form biofilm and lipopolysaccharide is one of the factors involved in the biofilm formation. Lipopolysaccharide was isolated from planktonic and biofilm cells of the investigated strain and analyzed by SDS-PAGE with silver staining, Western blotting and ELISA, as well as NMR and matrix-assisted laser desorption ionization time-of-flight mass spectrometry techniques. Chemical and NMR spectroscopic analyses revealed that the structure of the O-polysaccharide of P. mirabilis 9B-m strain did not depend on the form of cell growth, but the full-length chains of the O-antigen were reduced when bacteria grew in biofilm. The study also revealed structural modifications of the core region in the lipopolysaccharide of biofilm-associated cells-peaks assigned to compounds absent in cells from the planktonic culture and not previously detected in any of the known Proteus core oligosaccharides. No differences in the lipid A structure were observed. In summary, our study demonstrated for the first time that changes in the lifestyle of P. mirabilis bacteria leads to the modifications of their important virulence factor-lipopolysaccharide.

The O-antigen structure of bacterium Comamonas aquatica CJG.

Genus Comamonas is a group of bacteria that are able to degrade a variety of environmental waste. Comamonas aquatica CJG (C. aquatica) in this genus is able to absorb low-density lipoprotein but not high-density lipoprotein of human serum. Using 1H and 13C NMR spectroscopy, we found that the O-polysaccharide (O-antigen) of this bacterium is comprised of a disaccharide repeat (O-unit) of d-glucose and 2-O-acetyl-l-rhamnose, which is shared by Serratia marcescens O6. The O-antigen gene cluster of C. aquatica, which is located between coaX and tnp4 genes, contains rhamnose synthesis genes, glycosyl and acetyl transferase genes, and ATP-binding cassette transporter genes, and therefore is consistent with the O-antigen structure determined here.

Structures and serospecificity of threonine-containing O polysaccharides of two clinical isolates belonging to the genus Proteus and their classification into O11 subserogroups.

Two clinical isolates from Polish patients, Proteus mirabilis 9B-m and Proteus genomospecies 3J-r, were found to be serologically related to P mirabilis O11. However, serological studies involving ELISA and Western blotting methods, using lipopolysaccharides (LPSs) extracted from the strains as antigens and native or adsorbed rabbit polyclonal O antisera, specific to the studied strains, revealed slight differences in the cross-reactivity and specificity of the two studied Proteus isolates, when compared to P. mirabilis O11. Two different O polysaccharides containing N-(d-galacturonoyl)-l-threonine were isolated from the LPSs of the isolates. Their structures were determined by chemical analysis and NMR spectroscopy and found to be related to the P. mirabilis O11 antigen structure established earlier, the 9B-m structure differing in the absence of the lateral glucose residue and the 3J-r structure in non-stoichiometric O-acetylation of the threonine residue only. Thus, the Proteus O11 serogroup should be divided into two subgroups: O11a, represented by the 9B-m isolate and O11a, b possessing the additional b epitope, containing the lateral residue of glucose and formed by the 3J-r isolate as well as P. mirabilis 25/57 belonging to O11 serogroup so far. O11a is the sixth new serotype found in Proteus spp. strains recently isolated from patients in central Poland.

Structural Relationship of the Lipid A Acyl Groups to Activation of Murine Toll-Like Receptor 4 by Lipopolysaccharides from Pathogenic Strains of Burkholderia mallei, Acinetobacter baumannii, and Pseudomonas aeruginosa.

Toll-like receptor 4 (TLR4) is required for activation of innate immunity upon recognition of lipopolysaccharide (LPS) of Gram-negative bacteria. The ability of TLR4 to respond to a particular LPS species is important since insufficient activation may not prevent bacterial growth while excessive immune reaction may lead to immunopathology associated with sepsis. Here, we investigated the biological activity of LPS from Burkholderia mallei that causes glanders, and from the two well-known opportunistic pathogens Acinetobacter baumannii and Pseudomonas aeruginosa (causative agents of nosocomial infections). For each bacterial strain, R-form LPS preparations were purified by hydrophobic chromatography and the chemical structure of lipid A, an LPS structural component, was elucidated by HR-MALDI-TOF mass spectrometry. The biological activity of LPS samples was evaluated by their ability to induce production of proinflammatory cytokines, such as IL-6 and TNF, by bone marrow-derived macrophages. Our results demonstrate direct correlation between the biological activity of LPS from these pathogenic bacteria and the extent of their lipid A acylation.

Structure of a zwitterionic O-polysaccharide from Photorhabdus temperata subsp. cinerea 3240.

A phosphorylated O-polysaccharide was isolated from the lipopolysaccharide of an entomopathogenic bacterium Photorhabdus temperata subsp. cinerea 3240 and studied by sugar analysis, dephosphorylation, and (1)H and (13)C NMR spectroscopy. The following structure of the linear trisaccharide repeating unit of the O-polysaccharide was established: →3)-ß-D-GalpNAc4PEtN-(1→4)-ß-D-GlcpA-(1→3)-ß-D-FucpNAc4N-(1→ where GlcA indicates glucuronic acid, FucNAc4N 2-acetamido-4-amino-2,4,6-trideoxygalactose, and PEtN 2-aminoethyl phosphate.

Structure of the O-polysaccharide of Photorhabdus temperata subsp. temperata XlNach(T) containing a novel branched monosaccharide, 3,6-dideoxy-4-C-[(S)-1,2-dihydroxyethyl]-d-xylo-hexose.

O-Polysaccharide was isolated from the lipopolysaccharide of an entomopathogenic bacterium Photorhabdus temperata subsp. temperata XlNach(T). Sugar analysis after full acid hydrolysis of the polysaccharide revealed D-glucose, D-mannose, D-galactose, D-GalNAc, and a branched monosaccharide, 3,6-dideoxy-4-C-[(S)-1',2'-dihydroxyethyl]-D-xylo-hexose (Sug), which was isolated as a 1,2'-anhydro furanose derivative. The following structure of the polysaccharide was established by 1D and 2D 1H and 13C NMR spectroscopy:

Structure of the O-polysaccharide of Pseudomonas mandelii CYar1 containing 3,6-dideoxy-4-C-[(S)-1-hydroxyethyl]-D-xylo-hexose (yersiniose A).

The O-polysaccharide isolated by mild acid hydrolysis of the lipopolysaccharide of Pseudomonas mandelii CYar1 was studied by sugar analysis and 1D and 2D (1)H and (13)C NMR spectroscopies. The following structure of the O-polysaccharide was established:

Structure of the alanopine-containing O-polysaccharide and serological cross-reactivity of the lipopolysaccharide of Proteus vulgaris HSC 438 classified into a new Proteus serogroup, O76.

The O-polysaccharide was isolated by mild acid hydrolysis of the lipopolysaccharide of Proteus vulgaris HSC 438, and the following structure was established by chemical methods and one- and two-dimensional (1)H and (13)C NMR spectroscopy: →3)-ß-d-Quip4NAlo-(1→3)-α-d-Galp6Ac-(1→6)-α-d-Glcp-(1→3)-α-l-FucpNAc-(1→3)-ß-d-GlcpNAc-(1→, where d-Qui4N stands for 4-amino-4,6-dideoxy-d-glucose and Alo for N-((S)-1-carboxyethyl)-l-alanine (alanopine); only about half of the Gal residues are O-acetylated. This structure is unique among the Proteus O-polysaccharides, and therefore it is proposed to classify P. vulgaris HSC 438 into a new Proteus serogroup, O76. A serological cross-reactivity of HSC 438 O-antiserum and lipopolysaccharides of some other Proteus serogroups was observed and accounted for by shared epitopes on the O-polysaccharides or lipopolysaccharide core regions, including that associated with d-Qui4NAlo.

Structure of the O-specific polysaccharide from the lipopolysaccharide of Psychrobacter cryohalolentis K5(T) containing a 2,3,4-triacetamido-2,3,4-trideoxy-L-arabinose moiety.

A novel constituent of bacterial polysaccharides, 2,3,4-triacetamido-2,3,4-trideoxy-L-arabinose, was found in the O-specific polysaccharide from the lipopolysaccharide of Psychrobacter cryohalolentis K5(T) and identified by 1D and 2D (1)H and (13)C NMR studies of the polysaccharide and a disaccharide obtained by solvolysis of the polysaccharide with triflic acid. The following structure of the branched polysaccharide was established by sugar analysis, triflic acid solvolysis, Smith degradation, and 2D NMR spectroscopy.

Structure of the O-polysaccharide of Providencia alcalifaciens O3 containing 3,6-dideoxy-3-formamido-D-glucose and D-galacturonamide.

Mild acid degradation of the lipopolysaccharide (LPS) of Providencia alcalifaciens O3 followed by GPC on Sephadex G-50 and anion-exchange chromatography on DEAE-Trisacryl M afforded neutral and acidic polysaccharides, LPS core oligosaccharide, and an oligosaccharide composed of one repeat of the neutral polysaccharide (O-unit) linked to the LPS core. The following structure of the pentasaccharide O-unit was established by sugar and methylation analyses, 2D (1)H and (13)C NMR spectroscopy and ESI MS: [formula: see text] where Qui3NFo stands for 3,6-dideoxy-3-formamidoglucose and GalAN for galacturonamide. The LPS core is represented by the Glc(3)Gal(1)GalA(1)Hep(3)Kdo(1)Ara4N(1)P(3)EtN(2) glycoform reported earlier for P. alcalifaciens O9, O34, and O19. The acidic polysaccharide had the same peptidoglycan-like structure as a polysaccharide isolated earlier from P. alcalifaciens O24, O38, and O45, and, most likely, represents bacterial capsule material.

Structure of an acidic polysaccharide isolated from Psychrobacter maritimus 3pS containing a bacillosamine derivative.

An acidic polysaccharide was obtained from Psychrobacter maritimus 3pS isolated from a Siberian cryopeg sample (Kolyma lowland). The following structure of the tetrasaccharide repeating unit of the polysaccharide was established by sugar analysis along with (1)H and (13)C NMR spectroscopy: →2)-α-L-Rhap-(1→4)-α-D-GalpNAcA-(1→3)-α-D-QuipNAc4NHb-(1→3)-ß-D-QuipNAc4NHb-(1→ where D-GalNAcA indicates 2-acetamido-2-deoxy-D-galacturonic acid and d-QuiNAc4NHb indicates 2-acetamido-2,4,6-trideoxy-4-[(S)-3-hydroxybutanoyl]amino-D-glucose.

Structure of the O-polysaccharide of Photorhabdus luminescens subsp. laumondii containing D-glycero-D-manno-heptose and 3,6-dideoxy-3-formamido-D-glucose.

The O-polysaccharide from the lipopolysaccharide of a symbiotic bacterium Photorhabdus luminescens subsp. laumondii TT01 from an insect-pathogenic nematode was studied by sugar analysis and (1)H and (13)C NMR spectroscopy and found to contain D-glycero-D-manno-heptose (DDHep) and 3,6-dideoxy-3-formamido-D-glucose (D-Qui3NFo). The following structure of the pentasaccharide repeating unit of the O-polysaccharide was established:

Revision of the O-polysaccharide structure of Yersinia pseudotuberculosis O:1a; confirmation of the function of WbyM as paratosyltransferase.

An O-polysaccharide was isolated by mild acid degradation at pH 4.5 of the long-chain lipopolysaccharide of Yersinia pseudotuberculosis PB1 (serotype O:1a) and studied using 2D NMR spectroscopy. It was found to contain two uncommon monosaccharides: paratose (3,6-dideoxy-d-ribo-hexose, Par) in the furanose form and 6-deoxy-d-manno-heptose (d-6dmanHep). The following structure of a branched tetrasaccharide repeat (O-unit) with a disaccharide side chain was established: This structure is at variance with the O-polysaccharide structure of Y. pseudotuberculosis O:1a reported earlier (Komandrova, N. A.; Gorshkova, R. P.; Isakov, V. V.; Ovodov, Y. S. Bioorg. Khim.1984, 10, 232-237). A comparative study by high-resolution ESI MS of the short-chain lipopolysaccharides from strain PB1 and a wbyM mutant thereof confirmed the function of wbyM as the paratosyltransferase gene.

Structure of the O-polysaccharide chain of the lipopolysaccharide of Psychrobacter muricolla 2pS(T) isolated from overcooled water brines within permafrost.

Psychrotrophic bacteria of the genus Psychrobacter have not been studied in respect to lipopolysaccharide structure. In this work, we determined the structure of the O-specific polysaccharide of the lipopolysaccharide of Psychrobacter muricolla 2pS(T) isolated from overcooled (-9°C) water brines within permafrost. The polysaccharide was found to be acidic due to the presence of an amide of 2-acetamido-2-deoxy-l-guluronic acid with glycine (l-GulNAcA6Gly), which has not been hitherto found in nature. The following structure of the disaccharide repeating unit of the polysaccharide was established using composition analysis along with 1D and 2D (1)H and (13)C NMR spectroscopy: →4)-α-l-GulpNAcA6Gly-(1→3)-ß-d-GlcpNAc-(1→

Structure of the O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O28.

An O-polysaccharide and oligosaccharides were isolated by GPC following mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O28. The O-polysaccharide was studied by sugar and methylation analyses, (1)H and (13)C NMR spectroscopy, including 2D ROESY and H-detected (1)H,(13)C HSQC and HMBC experiments, and the following structure of the branched pentasaccharide repeating unit was established: [see formula in text]. This structure was confirmed by ESI MS of the isolated tridecasaccharide consisting of the lipopolysaccharide core and one O-polysaccharide repeat. The ESI mass spectrum also enabled inferring the composition of the core oligosaccharide.

Low structural diversity of the O-polysaccharides of Photorhabdus asymbiotica subspp. asymbiotica and australis and their similarity to the O-polysaccharides of taxonomically remote bacteria including Francisella tularensis.

The O-polysaccharides were isolated from the lipopolysaccharides of emerging human pathogens Photorhabdus asymbiotica subsp. asymbiotica US-86 and US-87 and subsp. australis AU36, AU46, and AU92. Studies by sugar analysis and (1)H and (13)C NMR spectroscopy before and after O-deacetylation showed that the O-polysaccharide structures are essentially identical within, and only slightly different between, the subspecies. The following structures of the repeating units of the O-polysaccharides were established: →3)-ß-d-Quip4NGlyFo-(1→4)-α-d-GalpNAcAN3Ac-(1→4)-α-d-GalpNAcA3R-(1→3)-α-d-QuipNAc-(1→ where GalNAcA stands for 2-acetamido-2-deoxygalacturonic acid, GalNAcAN for amide of GalNAcA, QuiNAc for 2-acetamido-2,6-dideoxyglucose, and Qui4NGlyFo for 4,6-dideoxy-4-(N-formylglycyl)aminoglucose; R=Ac in subsp. asymbiotica or H in subsp. australis. The structures established resemble those of a number of taxonomically remote bacteria including Francisella tularensis (Vinogradov, E. V.; Shashkov, A. S.; Knirel, Y. A.; Kochetkov, N. K.; Tochtamysheva, N. V.; Averin, S. P.; Goncharova, O. V.; Khlebnikov, V. S. Carbohydr. Res.1991, 214, 289-297), which differs in (i) the presence of a formyl group on Qui4N rather than the N-formylglycyl group, (ii) the mode of the linkage between the repeating units (ß1→2 vs α1→3), (iii) amidation of both GalNAcA residues rather than one residue, and iv) the lack of O-acetylation.

Lipopolysaccharide core structures and their correlation with genetic groupings of Shigella strains. A novel core variant in Shigella boydii type 16.

Bacteria Shigella, the cause of shigellosis, evolved from the intestinal bacteria Escherichia coli. Based on structurally diverse O-specific polysaccharide chains of the lipopolysaccharides (LPSs; O-antigens), three from four Shigella species are subdivided into multiple serotypes. The central oligosaccharide of the LPS called core is usually conserved within genus but five core types called R1-R4 and K-12 have been recognized in E. coli. Structural data on the Shigella core are limited to S. sonnei, S. flexneri and one S. dysenteriae strain, which all share E. coli core types. In this work, we elucidated the core structure in 14 reference strains of S. dysenteriae and S. boydii. Core oligosaccharides were obtained by mild acid hydrolysis of the LPSs and studied using sugar analysis, high-resolution mass spectrometry and two-dimensional NMR spectroscopy. The R1, R3 and R4 E. coli core types were identified in 8, 3 and 2 Shigella strains, respectively. A novel core variant found in S. boydii type 16 differs from the R3 core in the lack of GlcNAc and the presence of a D-glycero-D-manno-heptose disaccharide extension. In addition, the structure of an oligosaccharide consisting of the core and one O-antigen repeat was determined in S. dysenteriae type 8. A clear correlation of the core type was observed with genetic grouping of Shigella strains but not with their traditional division to four species. This finding supports a notion on the existing Shigella species as invalid taxa and a suggestion of multiple independent origins of Shigella from E. coli clones.