Sof'ya N. Senchenkova

Structural and Genetic Characterization of the Shigella boydii Type 13 O Antigen

Shigella is an important human pathogen. It is generally agreed that Shigella and Escherichia coli constitute a single species; the only exception is Shigella boydii type 13, which is more distantly related to E. coli and other Shigella forms and seems to represent another species. This gives S. boydii type 13 an important status in evolution. O antigen is the polysaccharide part of the lipopolysaccharide in the outer membrane of gram-negative bacteria and plays an important role in pathogenicity. The chemical structure and genetic organization of the S. boydii type 13 O antigen were investigated. The O polysaccharide was found to be acid labile owing to the presence of a glycosyl phosphate linkage in the main chain. The structure of the linear pentasaccharide phosphate repeating unit (O unit) was established by nuclear magnetic resonance spectroscopy, including two-dimensional COSY, TOCSY, ROESY, and H-detected 1H, 13C and 1H, 31P HMQC experiments, along with chemical methods. The O antigen gene cluster of S. boydii type 13 was located and sequenced. Genes for synthesis of UDP-2-acetamido-2,6-dideoxy-L-glucose and genes that encode putative sugar transferases, O unit flippase, and O antigen polymerase were identified. Seven genes were found to be specific to S. boydii type 13. The S. boydii type 13 O antigen gene cluster has higher levels of sequence similarity with Vibrio cholerae gene clusters and may be evolutionarily related to these gene clusters.

Structure of a phosphorylated polysaccharide from Shewanella putrefaciens strain S29

A phosphorylated polysaccharide was isolated from the aqueous layer of the phenol-water extract of a non-halophilic bacterium Shewanella putrefaciens strain S29. The glycosyl phosphate linkage in the polysaccharide was split under mild acid conditions to give, after borohydride reduction, a phosphorylated oligosaccharide-alditol. On the basis of sugar analysis and 1H, 13C and 31P NMR spectroscopy, including 2D COSY, relayed COSY, rotating-frame NOE spectroscopy (ROESY), heteronuclear 13C,1H COSY, and H-detected heteronuclear 1H,31P multiple-quantum coherence (HMQC), it was concluded that the polysaccharide is built up of tetrasaccharide-phosphate repeating units having the following structure: [sequence: see text] where QuiNAc and Qui4NAc are 2-acetamido-2,6-dideoxyglucose and 4-acetamido-4,6-dideoxyglucose, respectively.

Structure of the O-polysaccharide of Aeromonas hydrophila O:34; a case of random O-acetylation of 6-deoxy-L-talose

The O-polysaccharide of Aeromonas hydrophila O:34 was obtained by mild-acid degradation of the lipopolysaccharide and studied by chemical methods and NMR spectroscopy before and after O-deacetylation. The polysaccharide was found to contain D-Man, D-GalNAc and 6-deoxy-L-talose (L-6dTal), and the following structure of the tetrasaccharide repeating unit was established [carbohydrate structure see text] where 6dTal(I) is O-acetylated stoichiometrically at position-2 and 6dTal(II) carries no, one or two O-acetyl groups at any positions.

Structures of the O-antigens of Proteus bacilli belonging to OX group (serogroups O1–O3) used in Weil-Felix test

Structures of the O‐specific polysaccharide chains of lipopolysaccharides from Proteus group OX strains (serogroups O1–O3) used as antigens in Weil‐Felix test for diagnosis of rickettsiosis, were established. From them, the acid‐labile polysaccharide of Proteus vulgaris OX19 (O1) is built up of the following branched pentasaccharide repeating units connected via a phosphate group: where QuiNAc stands for 2‐acetamido‐2,6‐dideoxyglucose (N‐acetylquinovosamine). The basis of serospecificity of the Proteus group OX antigens and their cross‐reactivity with human anti‐rickettsial antibodies is discussed.

Structure of a new 6-deoxy-α-d-talan from Burkholderia (Pseudomonas) plantarii strain DSM 6535, which is different from the O-chain of the lipopolysaccharide☆

An O-acetylated homopolysaccharide of 6-deoxy-D-talose (6-deoxy-alpha-D-talan polymer) was isolated from Burkholderia (Pseudomonas) plantarii strain DSM 6535 by extraction with 2-propanol. The structure (1) of the trisaccharide repeating unit of the polysaccharide was established by studies of the intact and O-deacetylated polysaccharides using methanolysis, methylation analysis, 1H and 13C NMR spectroscopy, including 2D COSY, heteronuclear 13C, 1H COSY, 1D NOE, and computer-assisted analysis of 1D 13C NMR spectra. The remaining material after extraction of the biomass with 2-propanol showed to be a lipopolysaccharide with an O-specific polysaccharide chain having a different structure (2), which has been found previously in lipopolysaccharides of a number of other Gram-negative bacteria. [formula: see text]

Relationship of the Lipopolysaccharide Structure of Yersinia bpestis 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 two polysaccharides of Campylobacter jejuni 81116.

Campylobacter jejuni 81116 has been extensively investigated in studies on genes associated with the synthesis of Campylobacter lipopoly/lipooligosaccharides (LPS/LOS). Despite these investigations, data on the chemical structure of polysaccharides from C. jejuni 81116 have been absent. The present study was undertaken to fill that void. Biomass was grown in large quantities on agar medium, harvested and extracted by hot phenol-water extraction. Subsequently, extracts were treated by DNase, RNase and proteinase K to remove contaminants. After mild acid treatment, followed by preparative gel-permeation and anion-exchange chromatography, fractions were isolated and studied by 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, 1H,(13)C HMQC and HMBC experiments. These advanced investigations revealed the occurrence of two different polysaccharides in the approximate ratio of 3:1, each having a tetrasaccharide repeating unit. Polysaccharide A contained glucose, glucuronic acid and mannose, and is O-acetylated. Polysaccharide B contained glucose, galactose and N-acetylglucosamine. Importantly, polysaccharide A is acidic, whereas polysaccharide B is neutral. [carbohydrate structure: see text]

Pleiotropic effects of the lpxM mutation in Yersinia pestis resulting in modification of the biosynthesis of major immunoreactive antigens

Deletion mutants in the lpxM gene in two Yersinia pestis strains, the live Russian vaccine strain EV NIIEG and a fully virulent strain, 231, synthesise a less toxic penta-acylated lipopolysaccharide (LPS). Analysis of these mutants revealed they possessed marked reductions in expression and immunoreactivity of numerous major proteins and carbohydrate antigens, including F1, Pla, Ymt, V antigen, LPS, and ECA. Moreover, both mutants demonstrated altered epitope specificities of the antigens as determined in immunodot-ELISAs and immunoblotting analyses using a panel of monoclonal antibodies. The strains also differed in their susceptibility to the diagnostic plague bacteriophage L-413C. These findings indicate that the effects of the lpxM mutation on reduced virulence and enhanced immunity of the Y. pestis EV DeltalpxM is also associated with these pleiotropic changes and not just to changes in the lipid A acylation.

Structure of the O-specific polysaccharide of Proteus vulgaris O4 containing a new component of bacterial polysaccharides, 4,6-dideoxy-4-{N-[(R)-3-hydroxybutyryl]-l-alanyl}amino-d-glucose

Abstract A high-molecular-mass O-specific polysaccharide was obtained by mild acid degradation of Proteus vulgaris O4 lipopolysaccharide followed by GPC. The polysaccharide was studied by chemical methods along with 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, H-detected 1H,13C HMQC, and 1H,13C HMBC experiments. Solvolysis of the polysaccharide with trifluoromethanesulfonic (triflic) acid resulted in a GlcpA-(1→3)-GlcNAc disaccharide and a novel amino sugar derivative, 4,6-dideoxy-4-{N-[(R)-3-hydroxybutyryl]- l -alanyl}amino- d -glucose [Qui4N(HbAla)]. On the basis of the data obtained, the following structure of the tetrasaccharide repeating unit of the O-specific polysaccharide was established: →4 )- β - d - Glc p A -(1 →3 )- β - d - Glc p NAc -(1 →2 )- β - d - Qui p4 N ( HbAla )-(1 →3 )- α - d - Gal p-(1 → This structure is unique among the O-specific polysaccharides, which is in accordance with classification of the strain studied in a separate Proteus serogroup.

Structure elucidation of the capsular polysaccharide of Acinetobacter baumannii AB5075 having the KL25 capsule biosynthesis locus

Capsular polysaccharide was isolated by the phenol-water extraction of Acinetobacter baumannii AB5075 and studied by 1D and 2D (1)H and (13)C NMR spectroscopy. The following structure of the linear trisaccharide repeating unit was established: → 3)-β-D-ManpNAcA-(1 → 4)-β-D-ManpNAcA-(1 → 3)-α-D-QuipNAc4NR-(1 → where R indicates (S)-3-hydroxybutanoyl or acetyl in the ratio ∼ 2.5:1. The genes in the polysaccharide biosynthesis locus designated KL25 are appropriate to the established CPS structure.