Biochemistry (Moscow) (v.76, #7)

Microbial carbohydrates by Yuriy A. Knirel (727-728).

The biosynthesis of glycoconjugates is remarkably conserved in all types of cells since the biochemical reactions involved exhibit similar characteristics, which can be summarized as follows: (a) the saccharide moiety is formed as a lipidlinked, membrane-associated glycan; (b) the lipid component in most cases is a polyisoprenoid phosphate; (c) the assembly of the lipid-linked saccharide intermediate depends on reactions taking place at both sides of the cell membrane, which requires the obligatory transmembrane movement of amphipathic molecules across the lipid bilayer. These general characteristics are present in the biosynthesis of the O-antigen component of the bacterial lipopolysaccharide, which serves as a model system to investigate the molecular and mechanistic basis of glycoconjugate synthesis, as summarized in this minireview.
Keywords: lipopolysaccharide; undecaprenyl phosphate; ligase; glycosylphosphate transferase; outer membrane

Teichuronic and teichulosonic acids of actinomycetes by E. M. Tul’skaya; A. S. Shashkov; G. M. Streshinskaya; S. N. Senchenkova; N. V. Potekhina; Yu. I. Kozlova; L. I. Evtushenko (736-744).
The subject of the present review is the structural diversity and abundance of cell wall teichuronic and teichulosonic acids of representatives of the order Actinomycetales. Recently found teichulosonic acids are a new class of natural glycopolymers with ald-2-ulosonic acid residues: Kdn (3-deoxy-D-glycero-D-galacto-non-2-ulosonic acid) or di-N-acyl derivatives of Pse (5,7-diamino-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic or pseudaminic acid) as the obligatory component. The structures of teichuronic and teichulosonic acids are presented. Data are summarized on the occurrence of the glycopolymers of different nature in the cell wall of the studied actinomycetes. The biological role of the glycopolymers and their possible taxonomic implication are discussed. The comprehensive tables given in the Supplement show 13C NMR spectroscopic data of teichuronic and teichulosonic acids obtained by the authors.
Keywords: cell wall; actinomycetes; teichuronic acid; teichulosonic acid; NMR spectroscopy

Phosphate-containing cell wall polymers of bacilli by N. V. Potekhina; G. M. Streshinskaya; E. M. Tul’skaya; Yu. I. Kozlova; S. N. Senchenkova; A. S. Shashkov (745-754).
Anionic phosphate-containing cell wall polymers of bacilli are represented by teichoic acids and poly(glycosyl 1-phosphates). Different locations of phosphodiester bonds in the main chain of teichoic acids as well as the nature and combination of the constituent structural elements underlie their structural diversity. Currently, the structures of teichoic acids of bacilli can be classified into three types, viz. poly(polyol phosphates) with glycerol or ribitol as the polyol; poly(glycosylpolyol phosphates), mainly glycerol-containing polymers; and poly(acylglycosylglycerol phosphate), in which the components are covalently linked through glycosidic, phosphodiester, and amide bonds. In addition to teichoic acids, poly(glycosyl 1-phosphates) with mono- and disaccharide residues in the repeating units have been detected in cell walls of several Bacillus subtilis and Bacillus pumilus strains. The known structures of teichoic acids and poly(glycosyl 1-phosphates) of B. subtilis, B. atrophaeus, B. licheniformis, B. pumilus, B. stearothermophilus, B. coagulans, B. cereus as well as oligomers that link the polymers to peptidoglycan are surveyed. The reported data on the structures of phosphate-containing polymers of different strains of B. subtilis suggest heterogeneity of the species and may be of interest for the taxonomy of bacilli to allow differentiation of closely related organisms according to the “structures and composition of cell wall polymers” criterion
Keywords: Bacillus ; cell wall; teichoic acids; poly(glycosyl 1-phosphates)

Pseudomonas aeruginosa is a Gram-negative bacterium that is ubiquitous in the environment and generally considered to be a saprophyte, but it is also an important opportunistic human pathogen. Pseudomonas aeruginosa elaborates a variety of virulence factors, one of which is lipopolysaccharide (LPS). LPS of P. aeruginosa is composed of three distinct regions: lipid A, core oligosaccharide (OS), and the long-chain O antigen. The core OS of P. aeruginosa is composed of L-glycero-D-manno-heptose, 3-deoxy-D-manno-oct-2-ulosonic acid, D-galactosamine, D-glucose, and L-rhamnose. Noncarbohydrate substituents are also found in the core OS including phosphate, 2-aminoethyl (di)phosphate, acetyl, alanyl and carbamoyl groups. Pseudomonas aeruginosa simultaneously synthesizes two core glycoforms, namely, capped and uncapped core. The capped core is covalently attached to an O antigen, whereas the uncapped core is devoid of O antigen. Although the core of P. aeruginosa LPS is relatively conserved, strain-to-strain variability of its structure exists. This includes phosphorylation pattern, the level of O-acetylation, and the presence or absence of a fourth glucose residue at the distal end of the uncapped core. A number of studies have been reported on the structures of unique truncated core OS with unusual modifications. This mini-review summarizes the diversity of P. aeruginosa complete and truncated core OS structures published over the past fifteen years
Keywords: lipopolysaccharide; core oligosaccharide; Pseudomonas aeruginosa

Leishmania are a genus of sandfly-transmitted protozoan parasites that cause a spectrum of debilitating and often fatal diseases in humans throughout the tropics and subtropics. During the parasite life cycle, Leishmania survive and proliferate in highly hostile environments. Their survival strategies involve the formation of an elaborate and dense cell-surface glycocalyx composed of diverse stage-specific glycoconjugates that form a protective barrier. Phosphoglycans constitute the variable structural and functional domain of major cell-surface lipophosphoglycan and secreted proteophosphoglycans. In this paper, we discuss structural aspects of various phosphoglycans from Leishmania with the major emphasis on the chemical preparation of neoglycoconjugates (neoglycoproteins and neoglycolipids) based on Leishmania lipophosphoglycan structures as well as the immunological evaluation for some of them as potential anti-leishmaniasis vaccines.
Keywords: Leishmania ; lipophosphoglycan; carbohydrates; neoglycoconjugates; glycoconjugate vaccines; chemical synthesis

Structure elucidation of the O-antigen of Salmonella enterica O51 and its structural and genetic relation to the O-antigen of Escherichia coli O23 by A. V. Perepelov; Bin Liu; Dan Guo; S. N. Senchenkova; A. S. Shahskov; Lu Feng; Lei Wang; Y. A. Knirel (774-779).
The O-polysaccharide (O-antigen) of Salmonella enterica O51 was isolated by mild acid degradation of the lipopolysaccharide and its structure was established using sugar analysis and NMR spectroscopy. The O-antigen of Escherichia coli O23, whose structure was elucidated earlier, possesses a similar structure and differs only in the presence of an additional lateral α-D-Glcp residue at position 6 of the GlcNAc residue in the main chain. Sequencing of the O-antigen gene clusters of S. enterica O51 and E. coli O23 revealed the same genes with a high-level similarity. By comparison with opened gene databases, all genes expected for the synthesis of the common structure of the two O-antigens were assigned functions. It is suggested that the gene clusters of both bacteria originated from a common ancestor, whereas the O-antigen modification in E. coli O23, which, most probably, is induced by prophage genes outside the gene cluster, could be introduced after the species divergence.
Keywords: Salmonella enterica ; Escherichia coli ; lipopolysaccharide; bacterial polysaccharide; structure; gene cluster; O-antigen

Heterogeneous structure of O-antigenic part of lipopolysaccharide of Salmonella telaviv (Serogroup O:28) containing 3-acetamido-3,6-dideoxy-D-glucopyranose by J. Kumirska; H. Dziadziuszko; M. Czerwicka; E. A. Lubecka; D. Kunikowska; E. M. Siedlecka; P. Stepnowski (780-790).
The O-polysaccharide of Salmonella Telaviv was obtained by mild acid degradation of the lipopolysaccharide and studied by chemical methods (sugar and methylation analyses, Smith degradation, de-O-acetylation) and NMR spectroscopy. The structure of the O-polysaccharide was established. The repeating units that are proximal to the lipopolysaccharide core region mostly have a digalactose side chain and lack glucose, whereas those at the other end of the chain mostly do bear glucose but are devoid of the disaccharide side chain. This is the first structure established for the O-polysaccharide of a Salmonella serogroup O:28 (formerly M) strain characterized by subfactors O281 and O282. Knowledge of this structure and the structure of the O-polysaccharide of Salmonella Dakar (O281, O283) established earlier is crucial for determination of the exact structures associated with subfactors O281, O282, and O283 and elucidation of the genetic basis of the close relationship between Escherichia coli O71 and S. enterica O:28 O-antigens.
Keywords: Salmonella Telaviv; O-antigen; structural analysis; 3-acetamido-3,6-dideoxy-D-glucose

Structure of the O-specific polysaccharide from Shewanella japonica KMM 3601 containing 5,7-diacetamido-3,5,7,9-tetradeoxy-d-glycero-d-talo-non-2-ulosonic acid by E. L. Nazarenko; A. V. Perepelov; L. S. Shevchenko; E. D. Daeva; E. P. Ivanova; A. S. Shashkov; G. Widmalm (791-796).
Structure of the O-specific polysaccharide chain of the lipopolysaccharide (LPS) of Shewanella japonica KMM 3601 was elucidated. The initial and O-deacylated LPS as well as a trisaccharide representing the O-deacetylated repeating unit of the O-specific polysaccharide were studied by sugar analysis along with 1H and 13C NMR spectroscopy. The polysaccharide was found to contain a rare higher sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-d-glycero-d-talo-non-2-ulosonic acid (a derivative of 4-epilegionaminic acid, 4eLeg). The following structure of the trisaccharide repeating unit was established: →4)-α-4eLegp5Ac7Ac-(2→4)-β-d-GlcpA3Ac-(1→3)-β-d-GalpNAc-(1→.
Keywords: Shewanella japonica ; O-specific polysaccharide; lipopolysaccharide; marine bacteria; NMR spectroscopy; 5,7-diacetamido-3,5,7,9-tetradeoxy-d-glycero-d-talo-non-2-ulosonic acid

Structural peculiarities of the O-specific polysaccharides of Azospirillum bacteria of serogroup III by Yu. P. Fedonenko; A. S. Boiko; E. L. Zdorovenko; S. A. Konnova; A. S. Shashkov; V. V. Ignatov; Yu. A. Knirel (797-802).
Lipopolysaccharides and O-specific polysaccharides were isolated from the outer membrane of bacterial cells of three strains belonging to two Azospirillum species, and their structures were established by monosaccharide analysis including determination of the absolute configurations, methylation analysis, and one- and two-dimensional NMR spectroscopy. It was shown that while having the identical composition, the O-polysaccharides have different branched tetrasaccharide repeating units. Two neutral polysaccharides were found in the lipopolysaccharide of A. brasilense 54, and the structure for the predominant O-polysaccharide was determined. The structural data, together with results of serological studies, enabled assignment of strains examined to a novel serogroup, III. The chemical basis for the serological relatedness among the azospirilla of this serogroup is presumably the presence of a common →3)-α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-α-L-Rhap-(1→oligosaccharide motif in their O-polysaccharides.
Keywords: Azospirillum ; lipopolysaccharide; O-antigen; O-polysaccharide structure; serological characterization

The structure of the core part of the lipopolysaccharide from Proteus mirabilis genomic strain HI4320 was studied. Core oligosaccharide was isolated by mild acid hydrolysis of the lipopolysaccharide and analyzed by NMR spectroscopy and mass spectrometry as well as chemical methods. The structure of the oligosaccharide was established.
Keywords: lipopolysaccharide; Proteus mirabilis ; core structure; NMR spectroscopy

Functional characterization and biological significance of Yersinia pestis lipopolysaccharide biosynthesis genes by S. V. Dentovskaya; A. P. Anisimov; A. N. Kondakova; B. Lindner; O. V. Bystrova; T. E. Svetoch; R. Z. Shaikhutdinova; S. A. Ivanov; I. V. Bakhteeva; G. M. Titareva; Yu. A. Knirel (808-822).
In silico analysis of available bacterial genomes revealed the phylogenetic proximity levels of enzymes responsible for biosynthesis of lipopolysaccharide (LPS) of Yersinia pestis, the cause of plague, to homologous proteins of closely related Yersinia spp. and some other bacteria (Serratia proteamaculans, Erwinia carotovora, Burkholderia dolosa, Photorhabdus luminescens and others). Isogenic Y. pestis mutants with single or double mutations in 14 genes of LPS biosynthetic pathways were constructed by site-directed mutagenesis on the base of the virulent strain 231 and its attenuated derivative. Using high-resolution electrospray ionization mass spectrometry, the full LPS structures were elucidated in each mutant, and the sequence of monosaccharide transfers in the assembly of the LPS core was inferred. Truncation of the core decreased significantly the resistance of bacteria to normal human serum and polymyxin B, the latter probably as a result of a less efficient incorporation of 4-amino-4-deoxyarabinose into lipid A. Impairing of LPS biosynthesis resulted also in reduction of LPS-dependent enzymatic activities of plasminogen activator and elevation of LD50 and average survival time in mice and guinea pigs infected with experimental plague. Unraveling correlations between biological properties of bacteria and particular LPS structures may help a better understanding of pathogenesis of plague and implication of appropriate genes as potential molecular targets for treatment of plague.
Keywords: Yersinia pestis ; lipopolysaccharide biosynthesis; virulence; serum resistance; antimicrobial-peptide resistance; fibrinolytic activity; coagulase activity

The lipopolysaccharide (LPS) of strains representing various serotypes of Yersinia enterocolitica and Y. enterocolitica-like bacteria was studied by deoxycholate-PAGE and silver staining analysis. Four main types of LPS were detected based on the O-polysaccharide (O-PS): (i) LPS with homopolymeric O-PS, (ii) LPS with ladder-forming heteropolymeric O-PS, (iii) LPS with single-length O-PS, and (iv) semi-rough LPS without O-PS. Within the first three types, several subvariants were detected. Selected serotypes representing all above LPS types are sensitive to bacteriophage ϕR1-37 indicating that they share the phage receptor, a hexasaccharide called outer core in Y. enterocolitica O:3. Whereas phage ϕR1-37-resistant mutants of homopolymeric O-PS have lost only the outer core, those of ladder-forming or single-length O-PS have lost also the O-PS suggesting that in the latter ones the outer core is bridging between O-PS and lipid A-core. This work forms a basis of further structural, biochemical and genetic studies of these LPSs.
Keywords: Yersinia enterocolitica ; lipopolysaccharide; silver staining; deoxycholate-PAGE

Characterization of anti-ECA antibodies in rabbit antiserum against rough Yersinia enterocolitica O:3 by K. Rabsztyn; K. Kasperkiewicz; K. A. Duda; C. -M. Li; M. Łukasik; J. Radziejewska-Lebrecht; M. Skurnik (832-839).
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 (ECAPG) and at the same time in some special cases via a linkage to lipopolysaccharide (ECALPS); the latter form is immunogenic. Yersinia enterocolitica O:3 expresses both ECAPG and ECALPS. To study whether ECA-immunogenicity of Y. enterocolitica O:3 is temperature-regulated, rabbits were immunized with ECA-expressing Y. enterocolitica O:3 bacteria grown at 22 and 37°C. To induce minimal amount of anti-LPS antibodies, immunization was performed with YeO3-c-trs8-R, an LPS mutant missing both O-polysaccharide and the outer core hexasaccharide. However, abundant antibodies specific for LPS core were still present in the obtained antisera such that the reactivity of ECA-specific antibodies could not be detected. To obtain “monovalent” anti-ECA antisera, the sera were absorbed with ECA-negative bacteria. Absorption with live bacteria removed efficiently the anti-LPS antibodies, whereas this was not the case with boiled bacteria. Western blotting revealed that the specificity of the monovalent anti-ECA antiserum was different from that of a monoclonal anti-ECA antibody (mAb 898) as it did not react with ECAPG, and this suggested that in Y. enterocolitica O:3 ECALPS only one or two ECA repeat unit(s) is/are linked to LPS. Both ECAPG and ECALPS expression were found to be regulated by temperature and repressed at 37°C.
Keywords: Yersinia enterocolitica O:3; enterobacterial common antigen; lipopolysaccharide; monovalent antiserum

Biological activity of (Lipo)polysaccharides of the exopolysaccharide-deficient mutant Rt120 derived from Rhizobium leguminosarum bv. trifolii strain TA1 by J. Kutkowska; A. Turska-Szewczuk; M. Janczarek; R. Paduch; T. Kaminska; T. Urbanik-Sypniewska (840-850).
Lipopolysaccharides (LPS) from Rhizobium leguminosarum biovar trifolii TA1 (RtTA1) and its mutant Rt120 in the pssB-pssA intergenic region as well as degraded polysaccharides (DPS) derived from the LPS were elucidated in terms of their chemical composition and biological activities. The polysaccharide portions were examined by methylation analysis, MALDI-TOF mass spectrometry, and 1H NMR spectroscopy. A high molecular mass carbohydrate fraction obtained from Rt120 DPS by Sephadex G-50 gel chromatography was composed mainly of L-rhamnose, 6-deoxy-L-talose, D-galactose, and D-galacturonic acid, whereas that from RtTA1 DPS contained L-fucose, 2-acetamido-2,6-dideoxy-D-glucose, D-galacturonic acid, 3-deoxy-3-methylaminofucose, D-glucose, D-glucuronic acid, and heptose. Relative intensities of the major 1H NMR signals for O-acetyl and N-acetyl groups were 1: 0.8 and 1: 1.24 in DPS of Rt120 and RtTA1, respectively. The intact mutant LPS exhibited a twice higher lethal toxicity than the wild type LPS. A higher in vivo production of TNFα and IL-6 after induction of mice with Rt120 LPS correlated with the toxicity, although the mutant LPS induced the secretion of IL-1β and IFNγ more weakly than RtTA1 LPS. A polysaccharide obtained by gel chromatography on Bio-Gel P-4 of the high molecular mass material from Rt120 had a toxic effect on tumor HeLa cells but was inactive against the normal human skin fibroblast cell line. The polysaccharide from RtTA1 was inactive against either cell line. The potent inhibitory effect of the mutant DPS on tumor HeLa cells seems to be related with the differences in sugar composition.

Serotyping of Proteus mirabilis clinical strains based on lipopolysaccharide O-polysaccharide and core oligosaccharide structures by W. Kaca; J. Glenska; L. Lechowicz; S. Grabowski; A. Brauner; M. Kwinkowski (851-861).
The aim of this work was to serotype Proteus mirabilis urinary tract infection (UTI) strains based on chemically defined O-antigens with the use of two clinical collections from Sweden and Poland consisting of 99 and 24 UTI strains, respectively. A simple two-step serotyping scheme was proposed using enzyme immunoassay with heat-stable surface antigens of Proteus cells and immunoblotting with isolated lipopolysaccharides (LPSs). Using polyclonal anti-P. mirabilis rabbit antisera, 50 Swedish and 8 Polish strains were classified into serogroups O10, O38, O36, O30, O17, O23, O9, O40, O49, O27, O5, O13, O24, O14, and O33. From the Swedish strains, 10 belonged to serogroup O10 and five to each of serogroups O38, O36, and O9. Therefore, none of the O-serogroups was predominant. The majority of the serotyped clinical strains possess acidic O-antigens containing uronic acids and various acidic non-carbohydrate substituents. In immunoblotting, antisera cross-reacted with both O-antigen and core of LPSs. The core region of 19 LPSs bound a single serum, and that of 12 LPSs bound more than two sera. Following bioinformatic analysis of the available sequences, a molecular approach to the prediction of Proteus core oligosaccharide structures was proposed. The identification of the core type of P. mirabilis R110, derived from a serogroup O3 wild strain, using restriction fragments length polymorphism analysis of galacturonic acid transferase is shown as an example. In summary, the most frequent O-serogroups among P. mirabilis UTI stains were identified. The diversity of serological reactions of LPSs is useful for serotyping of P. mirabilis clinical isolates. A possible role of the acidic components of O-antigens in UTI is discussed.
Keywords: lipopolysaccharide; O-antigen; Proteus mirabilis ; serology; serotyping; glycosyl transferase

Changes in the repertoire of natural antibodies caused by immunization with bacterial antigens by N. V. Shilova; M. J. Navakouski; M. Huflejt; A. Kuehn; R. Grunow; O. Blixt; N. V. Bovin (862-866).
The repertoire of natural anti-glycan antibodies in naive chickens and in chickens immunized with bacteria Burkholderia mallei, Burkholderia pseudomallei, and Francisella tularensis as well as with peptides from an outer membrane protein of B. pseudomallei was studied. A relatively restricted pattern of natural antibodies (first of all IgY against bacterial cell wall peptidoglycan fragments, L-Rha, and core N-acetyllactosamine) shrank and, moreover, the level of detectable antibodies decreased as a result of immunization.
Keywords: bacterial polysaccharides; natural antibodies; Burkholderia mallei ; Burkholderia pseudomallei ; Francisella tularensis