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. To address this, we used a set of three deep-rough LPS mutants for rabbit immunization. The polyvalent antisera obtained were: (i) analysed for the presence of anti-LPS and anti-ECA antibodies; (ii) treated with caprylic acid (CA) to precipitate IgM antibodies and protein aggregates; and (iii) adsorbed with live ECA-negative bacteria to obtain specific anti-ECA antisera. We demonstrated the presence of antibodies specific for both ECAPG and ECALPS in all antisera obtained. Both CA treatment and adsorption with ECA-negative bacteria efficiently removed anti-LPS antibodies, resulting in specific anti-ECA sera. The LPS of the ECALPS-positive deepest-rough mutant contained only lipid A and 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residues of the inner core, suggesting that ECALPS was linked to the Kdo region of LPS in Y. enterocolitica O : 3.

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. similis and the Korean group of strains. Elucidation of the Y. pseudotuberculosis complex OPS structures and characterization of the OPS genetics (altogether 18 O-serotypes studied thus far) allows a better understanding of the relationships among the various O serotypes and will facilitate the analysis of the evolutionary processes giving rise to new serotypes. Here we present the characterization of the OPS structure and gene cluster of Y. similis O:9. Bacteriophage φR1-37, which uses the Y. similis O:9 OPS as a receptor, also infects a number of Y. enterocolitica serotypes, including O:3, O:5,27, O:9 and O:50. The Y. similis O:9 OPS structure resembled none of the receptor structures of the Y. enterocolitica strains, suggesting that φR1-37 can recognize several surface receptors, thus promoting broad host specificity.

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. To elucidate the relationship between ECA and LPS in Y. enterocolitica O : 3 and the effect of temperature on their expression, LPS was isolated from bacteria grown at 22 °C and 37 °C by consequent hot phenol/water and phenol-chloroform-light petroleum extractions to obtain LPS preparations free of ECA linked to glycerophospholipid. In immunoblotting, monoclonal antibodies TomA6 and 898, specific for OPS and ECA, respectively, reacted both with ladder-like bands and with a slower-migrating smear suggesting that the ECA and OPS epitopes coexist on the same molecules. These results were supported by immunoblotting with a monovalent Y. enterocolitica O : 3 ECA-specific rabbit antiserum. Also, two or three 898-positive (and monovalent-positive) TomA6-negative bands migrated at the level of the LA-IC band in LPS samples from certain OC mutants, most likely representing LA-IC molecules carrying 1-3 ECA repeat units but no OPS. These bands were also present in Y. enterocolitica O : 9 OC mutants; however, coexistence of ECA and OPS in the same molecules could not be detected. Finally, the LA-IC-ECA bands were missing from LPS of bacteria grown at 37 °C and also the general reduction in wild-type bacteria of ECA-specific monovalent-reactive material at 37 °C suggested that temperature regulates the expression of ECA. Indeed, RNA-sequencing analysis showed significant downregulation of the ECA biosynthetic gene cluster at 37 °C.

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. Y. enterocolitica cell surface structures that play a significant role in virulence have been subject to many investigations. These include outer membrane (OM) glycolipids such as lipopolysaccharide (LPS) and enterobacterial common antigen (ECA) and several cell surface adhesion proteins present only in virulent Y. enterocolitica, i.e., Inv, YadA and Ail. While the yadA gene is located on the Yersinia virulence plasmid the Ail, Inv, LPS and ECA are chromosomally encoded. These structures ensure the correct architecture of the OM, provide adhesive properties as well as resistance to antimicrobial peptides and to host innate immune response mechanisms.

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. Although the presence of the lipopolysaccharide O-antigen on the bacterial surface is absolutely required for apoA-I to kill the bacteria, apoA-I does not interact with the bacteria directly. To the contrary, exposure of the bacteria by serum proteins triggers apoA-I deposition on the bacterial surface. As our data show that both purified lipid-free and HDL-associated apoA-I displays anti-bacterial potential, apoA-I mimetic peptides may be a promising therapeutic agent for the treatment of certain Gram-negative infections.

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). In this study, we inactivated the six OC genes predicted to encode glycosyltransferases (GTase) one by one by nonpolar mutations to assign functions to their gene products. The mutants expressed no OC or truncated OC oligosaccharides of different lengths. The truncated OC oligosaccharides revealed that the minimum structural requirements for the interactions of OC with bacteriophage phiR1-37, enterocoliticin, and OC-specific monoclonal antibody 2B5 were different. Furthermore, using chemical and structural analyses of the mutant LPSs, we could assign specific functions to all six GTases and also revealed the exact order in which the transferases build the hexasaccharide. Comparative modeling of the catalytic sites of glucosyltransferases WbcK and WbcL followed by site-directed mutagenesis allowed us to identify Asp-182 and Glu-181, respectively, as catalytic base residues of these two GTases. In general, conclusive evidence for specific GTase functions have been rare due to difficulties in accessibility of the appropriate donors and acceptors; however, in this work we were able to utilize the structural analysis of LPS to get direct experimental evidence for five different GTase specificities.

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). In an aqueous environment, the 4-keto group of this sugar was present in the 4-dihydroxy form, due to hydration. Furthermore, evidence is provided that the axial 4-hydroxy group of this dihydroxy function was crucial for the biological role of the OC, that is, in the bacteriophage and enterocoliticin receptor structure and in the epitope of a monoclonal antibody.

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). The immunogenicity of ECA has not received much attention thus far and studies have focused mainly on the ECA of Escherichia coli and Yersinia enterocolitica. In this paper the ECA-immunogenicity of Proteus mirabilis is elucidated using two wild-type strains (S1959 and O28) as well as their rough (R) derivative strains R110/1959, which expresses lipopolysaccharide (LPS) with a full core, and R4/O28, which expresses LPS with only an inner core. MATERIALS AND METHODS: Rabbit polyclonal antisera were produced by immunization with boiled suspensions of the four P. mirabilis strains. The antisera were tested for the presence of antibodies specific to ECA by Western blotting using glycerophospholipid- linked ECA (ECA(PG)) of Salmonella montevideo as antigen. Lipopolysaccharide (LPS) was isolated from the four strains by the hot phenol/water procedure in which ECA(PG) is co-extracted with LPS and by the phenol/chloroform/petroleum ether extraction that results in the isolation of LPS and/or LPS-linked ECA (ECA(LPS)) free of ECA(PG). The LPS preparations were tested for the presence of ECA by Western blotting using ECA-specific antibodies. RESULTS: The results demonstrated that all four P. mirabilis strains were ECA immunogenic. The rabbit antisera immunized by the four strains all contained ECA-specific antibodies. Analysis of the LPS preparations demonstrated that the P. mirabilis wild-type strains O28 and S1959 and the Ra mutant strain R110/1959 expressed ECA(LPS), suggesting that it induced the anti-ECA antibody responses. Only the presence of ECA(PG) could be demonstrated in the Rc mutant strain R4/O28. CONCLUSIONS: These results therefore suggest that, similar to E. coli, LPS with a full core is also required as the acceptor of ECA for P. mirabilis strains to produce ECA(LPS). Since ECA(PG) is not immunogenic unless combined with some proteins, it is likely that ECA(PG)-protein complexes formed during the intravenous immunization with the Rc mutant strain R4/O28.

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. Through the use of compositional analysis, electrospray ionization Fourier transform mass spectrometry, and 1H and 13C NMR spectroscopy applying various one- and two-dimensional experiments, we identified the structures of the carbohydrate backbones that contained D-glycero-D-talo-oct-2-ulopyranosonic acid and 4-amino-4-deoxy-L-arabinose 1-phosphate residues. We also identified some truncated structures for both strains. All sugars were D-configured pyranoses and alpha-linked, except where stated otherwise.

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. The residue O corresponds to the primer, which initiates biosynthesis of the O-chain, and an oligomer of a disaccharide R-S is an insert between the primer and the main polymer. The polysaccharide has a beta-Kdo residue at the non-reducing end, a feature similar to that observed previously in the LPS from Klebsiella O12.

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). The two polysaccharides differ only in the ring size of shewanellose and have the following structures:Shewanellose has been previously identified in a phenol-soluble polysaccharide from Shewanella putrefaciens A6, which shows a close structural similarity to PS2.

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. By targeted mutagenesis, we identify within the O-antigen gene cluster the genes encoding Wzy and Wzz. We also present genetic and biochemical evidence showing that the gene previously called galE encodes a UDP-N-acetylglucosamine-4-epimerase (EC 5.1.3.7) required for the biosynthesis of the first sugar of the O-unit. Accordingly, the gene was renamed gne. Gne also has some UDP-glucose-4-epimerase (EC 5.1.3.2) activity, as it restores the core production of an Escherichia coli K-12 galE mutant. The three-dimensional structure of Gne was modeled based on the crystal structure of E. coli GalE. Detailed structural comparison of the active sites of Gne and GalE revealed that additional space is required to accommodate the N-acetyl group in Gne and that this space is occupied by two Tyr residues in GalE whereas the corresponding residues present in Gne are Leu136 and Cys297. The Gne Leu136Tyr and Cys297Tyr variants completely lost the UDP-N-acetylglucosamine-4-epimerase activity while retaining the ability to complement the LPS phenotype of the E. coli galE mutant. Finally, we report that Yersinia Wzx has relaxed specificity for the translocated oligosaccharide, contrary to Wzy, which is strictly specific for the O-unit to be polymerized.