Serological characterization of the enterobacterial common antigen substitution of the lipopolysaccharide of Yersinia enterocolitica O : 3.

Enterobacterial common antigen (ECA) is a polysaccharide present in all members of Enterobacteriaceae anchored either via phosphatidylglycerol (PG) or LPS to the outer leaflet of the outer membrane (ECAPG and ECALPS, respectively). Only the latter form is ECA-immunogenic. We previously demonstrated that Yersinia enterocolitica O : 3 and its rough (O-specific polysaccharide-negative) mutants were ECA-immunogenic, suggesting that they contained ECALPS; however, it was not known which part of the LPS core region was involved in ECA binding.

Structure and genetic basis of Yersinia similis serotype O:9 O-specific polysaccharide.

The O-polysaccharide (OPS, O-Ag) cap of LPS is a major virulence factor of Yersinia species and also serves as a receptor for the binding of lytic bacteriophage φR1-37. Currently, the OPS-based serotyping scheme for the Yersinia pseudotuberculosis complex includes 21 known O-serotypes that follow three distinct lineages: Y. pseudotuberculosis sensu stricto, Y.

Enterobacterial common antigen and O-specific polysaccharide coexist in the lipopolysaccharide of Yersinia enterocolitica serotype O:3.

Yersinia enterocolitica serotype O : 3 produces two types of lipopolysaccharide (LPS) molecules to its surface. In both types the lipid A (LA) structure is substituted by inner core (IC) octasaccharide to which either outer core (OC) hexasaccharide or homopolymeric O-polysaccharide (OPS) is linked. In addition, enterobacterial common antigen (ECA) can be covalently linked to LPS, however, via an unknown linkage.

Bacterial cell surface structures in Yersinia enterocolitica.

Yersinia enterocolitica is a widespread member of the family of Enterobacteriaceae that contains both non-virulent and virulent isolates. Pathogenic Y. enterocolitica strains, especially belonging to serotypes O:3, O:5,27, O:8 and O:9 are etiologic agents of yersiniosis in animals and humans.

Characterization of anti-ECA antibodies in rabbit antiserum against rough Yersinia enterocolitica O:3.

Enterobacterial common antigen (ECA) is a characteristic surface component in bacteria belonging to the Enterobacteriaceae family. It is generally integrated in the outer membrane via a linkage to phosphatidylglycerol (ECA(PG)) and at the same time in some special cases via a linkage to lipopolysaccharide (ECA(LPS)); the latter form is immunogenic. Yersinia enterocolitica O:3 expresses both ECA(PG) and ECA(LPS).

Apolipoprotein A-I exerts bactericidal activity against Yersinia enterocolitica serotype O:3.

Apolipoprotein A-I (apoA-I), the main protein component of high density lipoprotein (HDL), is well recognized for its antiatherogenic, antioxidant, and antiinflammatory properties. Here, we report a novel role for apoA-I as a host defense molecule that contributes to the complement-mediated killing of an important gastrointestinal pathogen, Gram-negative bacterium Yersinia enterocolitica. We specifically show that the C-terminal domain of apoA-I is the effector site providing the bactericidal activity.

Characterization of the six glycosyltransferases involved in the biosynthesis of Yersinia enterocolitica serotype O:3 lipopolysaccharide outer core.

Yersinia enterocolitica (Ye) is a gram-negative bacterium; Ye serotype O:3 expresses lipopolysaccharide (LPS) with a hexasaccharide branch known as the outer core (OC). The OC is important for the resistance of the bacterium to cationic antimicrobial peptides and also functions as a receptor for bacteriophage phiR1-37 and enterocoliticin. The biosynthesis of the OC hexasaccharide is directed by the OC gene cluster that contains nine genes (wzx, wbcKLMNOPQ, and gne).

Identification and role of a 6-deoxy-4-keto-hexosamine in the lipopolysaccharide outer core of Yersinia enterocolitica serotype O:3.

The outer core (OC) region of Yersinia enterocolitica serotype O:3 lipopolysaccharide is a hexasaccharide essential for the integrity of the outer membrane. It is involved in resistance against cationic antimicrobial peptides and plays a role in virulence during early phases of infection. We show here that the proximal residue of the OC hexasaccharide is a rarely encountered 4-keto-hexosamine, 2-acetamido-2,6-dideoxy-D-xylo-hex-4-ulopyranose (Sugp) and that WbcP is a UDP-GlcNAc-4,6-dehydratase enzyme responsible for the biosynthesis of the nucleotide-activated form of this rare sugar converting UDP-2-acetamido-2-deoxy-D-glucopyranose (UDP-D-GlcpNAc) to UDP-2-acetamido-2,6-dideoxy-D-xylo-hex-4-ulopyranose (UDP- Sugp).

ECA-immunogenicity of Proteus mirabilis strains.

Introduction: Bacteria of the genus Proteus are opportunistic pathogens and cause mainly urinary tract infections. They also play a role in the pathogenesis of reactive arthritis (RA). Patients suffering from Yersinia-triggered RA often carry high titers of antibodies specific to enterobacterial common antigen (ECA).

Lipopolysaccharides from Serratia marcescens possess one or two 4-amino-4-deoxy-L-arabinopyranose 1-phosphate residues in the lipid A and D-glycero-D-talo-oct-2-ulopyranosonic acid in the inner core region.

The carbohydrate backbones of the core-lipid A region were characterized from the lipopolysaccharides (LPSs) of Serratia marcescens strains 111R (a rough mutant strain of serotype O29) and IFO 3735 (a smooth strain not serologically characterized but possessing the O-chain structure of serotype O19). The LPSs were degraded either by mild hydrazinolysis (de-O-acylation) and hot 4 M KOH (de-N-acylation), or by hydrolysis in 2 % aqueous acetic acid, or by deamination. Oligosaccharide phosphates were isolated by high-performance anion-exchange chromatography.

The structure of the O-specific polysaccharide from Salmonella cerro (serogroup K, O:6,14,18).

The following structure of the Salmonella cerro LPS O-chain repeating unit has been determined using NMR and chemical methods: -->4)-alpha-D-Man(1-->2)-alpha-D-Man(1-->2)-beta-D-Man(1-->3)-alpha-D-GalNAc-(1-->.

The structure of the polysaccharide part of the LPS from Serratia marcescens serotype O19, including linkage region to the core and the residue at the non-reducing end.

The structure of the LPS from Serratia marcescens serotype O19 was investigated. Deamination of the LPS released the O-chain polysaccharide together with a fragment of the core oligosaccharide. The following structure of the product was determined by NMR spectroscopy, mass spectrometry, and chemical methods: [carbohydrate structure: see text] The main polymer consists of a repeating disaccharide V-U and is present on average of 18 units per chain as estimated by integration of signals in the NMR spectra.

Structural investigation of the O-specific polysaccharides of Morganella morganii consisting of two higher sugars.

The lipopolysaccharide of the bacterium Morganella morganii (strain KF 1676, RK 4222) yielded two polysaccharides, PS1 and PS2, when subjected to mild acid degradation followed by GPC. The polysaccharides were studied by 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, 1H,(13)C HMQC, and HMBC experiments. Each polysaccharide was found to contain a disaccharide repeating unit consisting of two higher sugars, 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-L-glycero-D-galacto-non-2-ulosonic acid (a derivative of 8-epilegionaminic acid, 8eLeg5Am7Ac) and 2-acetamido-4-C-(3'-carboxamide-2',2'-dihydroxypropyl)-2,6-dideoxy-D-galactose (shewanellose, She).

Functional characterization of Gne (UDP-N-acetylglucosamine-4-epimerase), Wzz (chain length determinant), and Wzy (O-antigen polymerase) of Yersinia enterocolitica serotype O:8.

The lipopolysaccharide (LPS) O-antigen of Yersinia enterocolitica serotype O:8 is formed by branched pentasaccharide repeat units that contain N-acetylgalactosamine (GalNAc), L-fucose (Fuc), D-galactose (Gal), D-mannose (Man), and 6-deoxy-D-gulose (6d-Gul). Its biosynthesis requires at least enzymes for the synthesis of each nucleoside diphosphate-activated sugar precursor; five glycosyltransferases, one for each sugar residue; a flippase (Wzx); and an O-antigen polymerase (Wzy). As this LPS shows a characteristic preferred O-antigen chain length, the presence of a chain length determinant protein (Wzz) is also expected.

The structure of the carbohydrate backbone of the core-lipid A region of the lipopolysaccharide from Proteus mirabilis serotype O28.

The following structure of the lipid A-core region of the lipopolysaccharide (LPS) from Proteus mirabilis serotype O28 was determined using NMR, MS, and chemical analysis of the core oligosaccharide, obtained by mild acid hydrolysis of LPS, and of the products of alkaline deacylation of the LPS: carbohydrate sequence [see text] where S = beta-GalALys (amide of beta-D-galactopyranosyluronic acid with the alpha-amino group of L-lysine) or beta-GalALys4PEtN are present in the ratio of approximately 1:1. beta-GalA and Ara4N (indicated by bold italics) are present in non stoichometric amount. All sugars are present in the pyranose form and all except L-Ara4N have the D configuration.

The structure of the carbohydrate backbone of core-lipid A region ofthe lipopolysaccharides from Proteus mirabilis wild-type strain S1959 (serotype O3) and its Ra mutant R110/1959.

The following structure of core-lipid A region of the lipopolysaccharide (LPS) from Proteus mirabilis strain 1959 (serotype O3) and its rough mutant R110/1959 (Proteus type II core) was determined using NMR and chemical analysis of the core oligosaccharide, obtained by mild acid hydrolysis of LPS, and of the products of alkaline deacylation of the LPS: Incomplete substitutions are indicated by italics. All sugars are in pyranose form, alpha-Hep is the residue Lglycero-alpha-Dmanno-Hep, alpha-DD-Hep is the residue Dglycero-alpha-Dmanno-Hep. The differences with the previously reported structures are discussed.

Immunochemical studies on R mutants of Yersinia enterocolitica O:3.

Three mutants of Yersinia enterocolitica O:3, namely: YeO3-R1, YeO3-RfbR7 and YeO3-c-trs8-R were classified on the basis of sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE) profile of isolated lipopolysaccharides (LPS) as belonging to the Ra- (the first) and the Rc-type (the other two mutants). Methylation analysis, in addition to 13C and 1H NMR studies of purified core oligosaccharides revealed structures similar to those established previously for the full core of Y. enterocolitica O:3 in the case of the Ra mutant, and identical to that reported for the Rc mutant Ye75R, in the case of the two other mutants.

The occurrence of 4-amino-4-deoxy-arabinose in LPS of supersusceptible strain Pseudomonas aeruginosa Z61: noncorrelation with polymyxin resistance.

Lipopolysaccharides (LPS) extracted from the supersusceptible strain Pseudomonas aeruginosa Z61 were compared with LPS from other strains with varying antimicrobial susceptibilities. The presence of 4-amino-4-deoxy-arabinose (4-AraN) in P. aeruginosa Z61 LPS was confirmed by gas-liquid chromatography/mass spectrometry (GLC-MS) and quantitated by high-performance liquid chromatography (HPLC).

Molecular and chemical characterization of the lipopolysaccharide O-antigen and its role in the virulence of Yersinia enterocolitica serotype O:8.

The Y. enterocolitica O:8(YeO8) O-antigen repeat units consist of five sugar residues: N-acetyl-D-galactosamine (GalNAc), D-galactose (Gal), D-mannose (Man), L-fucose (Fuc), and 6-deoxy-D-gulose (6d-Gul). The nucleotide sequence of the O-antigen gene cluster of the YeO8 strain 8081-c was determined.

Structural and immunochemical studies on the lipopolysaccharide of the 'T-antigen'-containing mutant Proteus mirabilis R14/1959.

In DOC-PAGE, lipopolysaccharide (LPS) of Proteus mirabilis R14/1959 (Rb-type) mutant showed a ladder-like migration pattern indicating the presence of a high molecular weight polysaccharide chain. The isolated polysaccharide, called T-antigen because of similarity with the T1 chain of Salmonella friedenau LPS, contained D-glucose, D-galacturonic acid (D-GalA), and D-GlcNAc in molar ratios 2:1:1 and was structurally different from the O-antigen of the parental S-strain P. mirabilis S1959 but identical to the O-antigen of another S-strain Proteus penneri 42.

Structure and epitope characterisation of the O-specific polysaccharide of Proteus mirabilis O28 containing amides of D-galacturonic acid with L-serine and L-lysine.

The O-specific polysaccharide of Proteus mirabilis O28 was found to contain D-galactose, D-galacturonic acid (GalA), 2-acetamido-2-deoxy-D-glucose, L-serine, L-lysine, and O-acetyl groups in molar ratios 1:2:1:1:1:1, the amino acids being linked via their alpha-amino group to the carboxyl group of GalA. The polysaccharide was studied using 1H- and 13C-NMR spectroscopy, including selective spin-decoupling, one-dimensional total correlation spectroscopy, two-dimensional homonuclear correlation spectroscopy (COSY), heteronuclear 13C,1H COSY, one-dimensional NOE, and two-dimensional rotating-frame NOE spectroscopy and partial acid hydrolysis followed by borohydride reduction, methylation, and GLC/MS analysis of the derived glycosyl alditols. The following structure of the repeating unit was established: [formula: see text] Epitope specificity of the P.

4-Amino-4-deoxy-L-arabinose in LPS of enterobacterial R-mutants and its possible role for their polymyxin reactivity.

The content of 4-amino-4-deoxy-L-arabinopyranose (L-Arap4N) and the phosphate substitution pattern of the LPS of various strains from Salmonella minnesota, Yersinia enterocolitica and Proteus mirabilis was determined by GC/MS, HPLC and 31P-NMR. These data allowed us to examine the possible role of these components for the polymyxin B-binding capacity of LPS and for the minimal inhibiting concentration (MIC) and the minimal bactericidal concentration (MBC) of polymyxins B and E towards the respective R-mutants. Contrary to other investigated Re-, Rd- and Rc-mutants of S.

The inner core region of Yersinia enterocolitica Ye75R (0:3) lipopolysaccharide.

The inner-core region of the lipopolysaccharide of an UDPGalNAc-4-epimerase-deficient mutant of Yersinia enterocolitica 0:3, designated as Ye75R, was investigated using methylation analysis, 1D-13C-NMR and 2D-13C-NMR and 1H-NMR, as well as 31P-NMR, fast-atom-bombardment mass spectrometry (FAB MS) and FAB MS/MS in positive and negative modes. The isolated core heptasaccharide (OS) was composed of 2 units D-glucose, 3 units LD-heptose and 1 unit each of DD-heptose and 3-deoxy-D-manno-octulosonic acid. Methylation analysis indicated that OS was highly branched with terminal location of the two glucoses and the DD-heptose unit, which was partially (to about 40%) phosphorylated at C7.

[Further types of core lipopolysaccharides in Proteus].

Comparative analysis of the chemical composition of 11 core oligosaccharides isolated from lipopolysaccharides of the wild (S) and phenotypically rough (R) strains Proteus mirabilis (nine) and Proteus vulgaris (two) allowed to recognize three new types Proteus core, classified as IV, V, VI. All of them contained D-galactose and D-galactosamine in addition to common core constituents: D-glucose, D-galacturonic acid, L-glycero-D-manno-heptose, KDO, EtN described for Proteus core types I, II, III (6, 7, 8). D-glucosamine was characteristic for Proteus core type VI whereas D-glycero-D-manno-heptose for types V and VI.