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.

Structure of the O-antigen of Yersinia pseudotuberculosis O:4a revised.

The O-specific polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Yersinia pseudotuberculosis O:4a and studied by NMR spectroscopy, including 2D ROESY and (1)H, (13)C HMBC experiments. The following structure of the pentasaccharide repeating unit of the polysaccharide was established, which differs from the structure reported earlier [Gorshkova, R. P. et al., Bioorg. Khim. 1983, 9, 1401-1407] in the linkage modes between the monosaccharides: [carbohydrate sequence see in text] where Tyv stands for 3,6-dideoxy-D-arabino-hexose (tyvelose). The structure of the Y. pseudotuberculosis O:4a antigen resembles that of Y. pseudotuberculosis O:2c, which differs in the presence of abequose (3,6-dideoxy-D-xylo-hexose) in place of tyvelose only.

Structure of the O-antigen of Yersinia pseudotuberculosis O:4b.

The structure of the long-chain O-antigen of Yersinia pseudotuberculosis O:4b containing two uncommon deoxy sugars, tyvelose (3,6-dideoxy-d-arabino-hexose, Tyv) and 6-deoxy-d-manno-heptose (D-6dmanHep), was established by 1D and 2D NMR spectroscopy as alpha-Tyvp-(1-->3)-beta-D-6dmanHepp 1-->4 -->3)-alpha-D-Galp-(1-->3)-beta-D-GlcpNac-(1-->.

Structure of the O-polysaccharide of Yersinia pseudotuberculosis O:2b.

The O-polysaccharide was isolated by hydrolysis of the lipopolysaccharide of Yersinia pseudotuberculosis O:2b, and studied by 1D and 2D NMR spectroscopy. The following structure of the polysaccharide was established: alpha-Abep. 1-->3. -->2) alpha-D-Manp-(1-->3)-alpha-L-Fucp-(1-->3)-beta-D-GalpNAc-(1-->. where Abe stands for 3,6-dideoxy-D-xylo-hexose (abequose).

Reinvestigation of the O-antigens of Yersinia pseudotuberculosis: revision of the O2c and confirmation of the O3 antigen structures.

Structures of the O-antigens of Yersinia pseudotuberculosis O2c and O3 were reinvestigated by NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY, (1)H,(13)C HSQC, and HMBC experiments. The following revised structure of the O2c tetrasaccharide repeating unit was established, which differs from the structure proposed earlier in the glycosylation pattern of the mannose residue at the branching point: [carbohydrate structure: see text] where Abe stands for 3,6-dideoxy-d-xylo-hexose. The structure of the Y. pseudotuberculosis O3 antigen reported earlier was confirmed.

Structural studies of the O-antigens of Yersinia pseudotuberculosis O:2a and mutants thereof with impaired 6-deoxy-D-manno-heptose biosynthesis pathway.

The full structure of the long- and short-chain O-antigen of Yersinia pseudotuberculosis O:2a containing two uncommon deoxy sugars, abequose and 6-deoxy-d-manno-heptose (6dmanHep), was established, for the first time, by sugar analysis, NMR spectroscopy, and high-resolution ESIMS. Similar structural studies were also performed on two O:2a mutants with single disruption of 6dmanHep synthesis pathway genes each, which synthesize modified long-chain (dmhA mutant) and short-chain (both dmhA and dmhB mutants) O-antigens with 6dmanHep replaced by its putative biosynthetic precursor, D-glycero-D-manno-heptose.

Relationship of the lipopolysaccharide structure of Yersinia pestis to resistance to antimicrobial factors.

Disruption of lipopolysaccharide (LPS) biosynthesis genes in an epidemiologically significant Yersinia pestis strain showed that the ability to synthesize the full inner core of the LPS is crucial for resistances to the bactericidal action of antimicrobial peptides and to complement-mediated serum killing. Resistance to polymyxin B also requires a high content of the cationic sugar, 4-amino-4-deoxy-L-arabinose, in lipid A.

Structures of the core oligosaccharide and O-units in the R- and SR-type lipopolysaccharides of reference strains of Pseudomonas aeruginosa O-serogroups.

Highly phosphorylated core oligosaccharides and those substituted with one O-antigen repeating unit were obtained by mild acid degradation or strong alkaline hydrolysis of lipopolysaccharide samples from 23 reference strains representing all Pseudomonas aeruginosa O-serogroups. Studies by high-resolution electrospray ionization mass spectrometry and two-dimensional NMR spectroscopy revealed both conserved and variable structural features of the lipopolysaccharides of various O-serogroups. The upstream terminal saccharide of the O-antigen, which contributes most to the immunospecificity of the bacteria, was defined in 11 from a total of 13 O-serogroups. The data obtained link together the known biosynthesis pathways, genetics and serology of the P. aeruginosa lipopolysaccharide.

Conserved and variable structural features in the lipopolysaccharide of Pseudomonas aeruginosa.

The review is devoted to recent progress in the structural elucidation of the lipopolysaccharide of the bacterium Pseudomonas aeruginosa, including O-antigen biological repeats, core oligosaccharide, and lipid A. Data on biosynthesis, genetics and serology of the lipopolysaccharide isolated from various P. aeruginosa O-serogroups are discussed in relation to the chemical structures.

Structure of the core oligosaccharide of a rough-type lipopolysaccharide of Pseudomonas syringae pv. phaseolicola.

The core structure of the lipopolysaccharide (LPS) isolated from a rough strain of the phytopathogenic bacterium Pseudomonas syringae pv. phaseolicola, GSPB 711, was investigated by sugar and methylation analyses, Fourier transform ion-cyclotron resonance ESI MS, and one- and two-dimensional 1H-, 13C- and 31P-NMR spectroscopy. Strong alkaline deacylation of the LPS resulted in two core-lipid A backbone undecasaccharide pentakisphosphates in the ratio approximately 2.5 : 1, which corresponded to outer core glycoforms 1 and 2 terminated with either L-rhamnose or 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), respectively. Mild acid degradation of the LPS gave the major glycoform 1 core octasaccharide and a minor truncated glycoform 2 core heptasaccharide, which resulted from the cleavage of the terminal Kdo residues. The inner core of P. syringae is distinguished by a high degree of phosphorylation of L-glycero-D-manno-heptose residues with phosphate, diphosphate and ethanolamine diphosphate groups. The glycoform 1 core is structurally similar but not identical to one of the core glycoforms of the human pathogenic bacterium Pseudomonas aeruginosa. The outer core composition and structure may be useful as a chemotaxonomic marker for the P. syringae group of bacteria, whereas a more conserved inner core structure appears to be representative for the whole genus Pseudomonas.

Structure of the O-polysaccharide and serological cross-reactivity of the Providencia stuartii O33 lipopolysaccharide containing 4-(N-acetyl-D-aspart-4-yl)amino-4,6-dideoxy-D-glucose.

The O-polysaccharide of Providencia stuartii O33 was obtained by mild acid degradation of the lipopolysaccharide and the following structure of the tetrasaccharide repeating unit was established: -->6)-alpha-D-GlcpNAc-(1-->4)-alpha-D-GalpA-(1-->3)-alpha-D-GlcpNAc-(1-->3)-beta-D-Quip4N(Ac-D-Asp)-(1-->, where d-Qui4N(Ac-D-Asp) is 4-(N-acetyl-D-aspart-4-yl)amino-4,6-dideoxy-D-glucose. Structural studies were performed using sugar and methylation analyses and NMR spectroscopy, including conventional 2D 1H, 1H COSY, TOCSY, NOESY and 1H, 13C HSQC experiments as well as COSY and NOESY experiments in an H2O-D2O mixture to reveal correlations for NH protons. The O-polysaccharide of P. stuartii O33 shares an alpha-D-GlcpNAc-(1-->3)-beta-D-Quip4N(Ac-D-Asp) epitope with that of Proteus mirabilis O38, which seems to be responsible for a marked serological cross-reactivity of anti-P. stuartii O33 serum with the lipopolysaccharide of the latter bacterium. P. stuartii O33 is serologically related also to P. stuartii O4, whose O-polysaccharide contains a lateral beta-D-Qui4N(Ac-L-Asp) residue.

Structure of the O-polysaccharide of Providencia stuartii O4 containing 4-(N-acetyl-L-aspart-4-yl)amino-4,6-dideoxy-D-glucose.

The O-polysaccharide of Providencia stuartii O4 was obtained by mild acid degradation of the lipopolysaccharide, and the following structure of the pentasaccharide repeating unit was established: [structure: see text] where D-Qui4N(L-AspAc) is 4-(N-acetyl-L-aspart-4-yl)amino-4,6-dideoxy-D-glucose, which has not been hitherto found in bacterial polysaccharides. Structural studies were performed using sugar and methylation analyses, Smith degradation and NMR spectroscopy, including conventional 2D 1H,1H COSY, TOCSY, NOESY and 1H,13C HSQC experiments as well as COSY and NOESY experiments run in an H(2)O-D(2)O mixture to reveal correlations for NH protons.

Structure and cross-reactivity of the O-antigen of Providencia stuartii O18 containing 3-acetamido-3,6-dideoxy-D-glucose.

The O-polysaccharide (O-antigen) of Providencia stuartii O18 was obtained by mild acid degradation of the lipopolysaccharide and studied by chemical methods and NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, NOESY and 1H,13C HSQC experiments. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established: [structure: see text] where Qui3NAc is 3-acetamido-3,6-dideoxyglucose. Anti-P. stuartii O18 serum cross-reacted with the O-antigen of Proteus genomospecies 4, which could be accounted for the marked structural similarities of the main chain.

Structure of the O-polysaccharide of Providencia alcalifaciens O19.

Studies of the O-polysaccharide chain of the lipopolysaccharide (O-antigen) of Providencia alcalifaciens O19 by sugar and methylation analyses along with NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, NOESY and 1H,13C HSQC experiments, showed that the pentasaccharide repeating unit of the polysaccharide has the following structure: [structure: see text] where Fuc3NAc is 3-acetamido-3,6-dideoxygalactose. The unique structure of the O-antigen and serological data are in consistence with classification of this bacterium in a separate Providencia serogroup.

Structure of the lipopolysaccharide of Pseudomonas aeruginosa O-12 with a randomly O-acetylated core region.

The lipopolysaccharide of Pseudomonas aeruginosa O-12 was studied by strong alkaline and mild acid degradations and dephosphorylation followed by fractionation of the products by GPC and high-performance anion-exchange chromatography and analyses by ESI FT-MS and NMR spectroscopy. The structures of the lipopolysaccharide core and the O-polysaccharide repeating unit were elucidated and the site and the configuration of the linkage between the O-polysaccharide and the core established. The core was found to be randomly O-acetylated, most O-acetyl groups being located on the terminal rhamnose residue of the outer core region.

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.

Structure of the O-polysaccharide of Providencia alcalifaciens O21 containing 3-formamido-3,6-dideoxy-D-galactose.

The O-polysaccharide (O-antigen) of Providencia alcalifaciens O21 was obtained by mild acid degradation of the lipopolysaccharide and studied by chemical methods and NMR spectroscopy. It was found that the polysaccharide is built up of branched pentasaccharide repeating units with a terminal residue of 3-formamido-3,6-dideoxy-D-galactose (D-Fuc3NFo) and has the following structure: [structure: see text]. Anti-P. alcalifaciens O21 serum cross-reacted with the O-antigen of Proteus vulgaris O47, which contains a GalNAc trisaccharide similar to that present in the P. alcalifaciens O21 O-polysaccharide.

Structure of the O-specific polysaccharide of Providencia rustigianii O14 containing N(epsilon)-[(S)-1-carboxyethyl]-N(alpha)-(D-galacturonoyl)-L-lysine.

The O-specific polysaccharide of Providencia rustigianii O14 was obtained by mild acid degradation of the LPS and studied by chemical methods and NMR spectroscopy, including 2D 1H,(1)H COSY, TOCSY, NOESY, and 1H,(13)C HSQC experiments. The polysaccharide was found to contain N (epsilon)-[(S)-1-carboxyethyl]-N(alpha)-(D-galacturonoyl)-L-lysine ('alaninolysine', 2S,8S-AlaLys). The amino acid component was isolated by acid hydrolysis and identified by 13C NMR spectroscopy and specific optical rotation, using synthetic diastereomers for comparison. The following structure of the trisaccharide repeating unit of the polysaccharide was established:Anti-P. rustigianii O14 serum was found to cross-react with O-specific polysaccharides of Providencia and Proteus strains that contains amides of uronic acid with N(epsilon)-[(R)-1-carboxyethyl]-L-lysine and L-lysine.

Structure of the O-specific polysaccharide of Providencia alcalifaciens O16 containing N-acetylmuramic acid.

The O-specific polysaccharide of Providencia alcalifaciens O16 was obtained by mild-acid degradation of the lipopolysaccharide and studied by chemical methods and NMR spectroscopy, including 2D 1H,(1)H COSY, TOCSY, NOESY, and 1H,(13)C HSQC experiments. It was found that the polysaccharide contains N-acetylmuramic acid, which was isolated by solvolysis with trifluoromethanesulfonic acid and identified by the specific optical rotation and NMR spectroscopy. The following structure of the trisaccharide repeating-unit of the polysaccharide was established:

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.