Filip V. Toukach

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.

Computer-assisted structural analysis of regular glycopolymers on the basis of 13C NMR data

A computer-assisted approach to the prediction of the primary structures of regular glycopolymers is described. The analysis is based on comparing the calculated 13C NMR spectra of all the possible structures of the repeating unit (for the given monomeric composition) to an experimental 13C NMR spectrum. The spectra generation is based on the spectral database containing information on the 13C chemical shifts of monomers, di- and trimeric fragments. If the required data are missing from this database, the special database for average glycosylation effects is used. The analysis reveals those structures with the calculated 13C NMR spectrum most close to observed. The structures of repeating units of any topology containing up to six residues linked by glycosidic, amidic or phospho-diester bridges can be predicted. Unambiguous selection of the proper structure from the output list of possible structures may require additional experimental data. Testing the created program and databases on bacterial polysaccharides and their derivatives containing up to three non-sugar residues (alditols, amino acids, phosphate groups etc.) per repeating unit revealed the good convergence of prediction with independently obtained structural data.

STRUCTURES OF NEW ACIDIC O-SPECIFIC POLYSACCHARIDES OF THE BACTERIUM PROTEUS MIRABILIS SEROGROUPS O26 AND O30

The polysaccharide chains of the lipopolysaccharides of the Proteus mirabilis serogroups O26 and O30 were studied using sugar and methylation analysis and 1H and 13C NMR spectroscopy, including two‐dimensional correlation spectroscopy and rotating‐frame NOE spectroscopy. The polysaccharides were found to be acidic due to the presence of d‐galacturonic acid and its amide with l‐lysine in serogroup O26 or d‐glucuronic acid in serogroup O30, and the structures of their tetrasaccharide repeating units were established. The O26‐specific polysaccharide is structurally and serologically related to the O‐specific polysaccharide of P. mirabilis O28, which includes amides of d‐GalA with l‐lysine and l‐serine [Radziejewska‐Lebrecht, J. et al. (1995) Eur. J. Biochem. 230, 705–712].

New structures of the O-specific polysaccharides of bacteria of the genus Proteus. 1. Phosphate-containing polysaccharides.

The O-specific polysaccharide chains (O-antigens) of the lipopolysaccharides of five Proteus strains, P. vulgaris O17, P. mirabilis O16 and O33, and P. penneri 31 and 103, were found to contain phosphate groups that link the non sugar components, e.g., ethanolamine and ribitol. The polysaccharides of P. mirabilis O16 and P. penneri 103 include ribitol phosphate in the main chain and thus resemble ribitol teichoic acids of Gram-positive bacteria. The structures of the polysaccharides were elucidated using NMR spectroscopy, including two-dimensional 1H, 1H correlation spectroscopy (COSY and TOCSY), nuclear Overhauser effect spectroscopy (NOESY or ROESY), and H-detected 1H, 13C and 1H, 31P heteronuclear multiple-quantum coherence spectroscopy (HMQC), along with chemical methods. The structures determined are unique among the bacterial polysaccharides and, together with the data obtained earlier, represent the chemical basic for classification of Proteus strains. Based on structural similarities of the O-specific polysaccharides and serological relationships between the O-antigens, we propose to extend Proteus serogroups O17 and O19 by including P. penneri strains 16 and 31, respectively.

Structural and serological studies of the O-specific polysaccharide of the bacterium Proteus mirabilis O10 containing L-altruronic acid, a new component of O-antigens.

An acidic O‐specific polysaccharide from the lipopolysaccharide of Proteus mirabilis O10 contains 2‐acetamido‐2deoxy‐d‐glucose, 2‐acetamido‐2‐deoxy‐d‐galactose, d‐galacturonic acid, and l‐altruronic acid, the last‐named sugar having not been found hitherto in O‐antigens. Structure of a branched tetrasaccharide repeating unit of the polysaccharide was established by 1H and 13C NMR spectroscopy, including two‐dimensional COSY and rotating‐frame NOE spectroscopy. The lateral l‐altruronic acid residue plays the immunodominant role in manifestation of the O10 specificity of Proteus, whereas a disaccharide fragment of the main chain in common with the O‐specific polysaccharide of P. mirabilis O43 provides the one‐way serological cross‐reactivity between anti‐O10 serum and O43‐antigen.

New structures of the O-specific polysaccharides of Proteus. 2. Polysaccharides containing O-acetyl groups

Structures of five new O-specific polysaccharides of Proteus bacteria were established. Four of them, Proteus penneri 4 (O72), Proteus vulgaris 63/57 (O37), Proteus mirabilis TG 277 (O69), and Proteus penneri 20 (O17), contain O-acetyl groups in non-stoichiometric quantities, and the polysaccharide of P. penneri 1 is structurally related to that of P. penneri 4. The structures were elucidated using NMR spectroscopy, including one dimensional 1H- and 13C-NMR spectroscopy, two-dimensional 1H, 1H correlation (COSY, TOCSY), H-detected 1H, 13C heteronuclear multiple-quantum coherence (HMQC), heteronuclear multiple-bond correlation (HMBC), and nuclear Overhauser effect spectroscopy (NOESY or ROESY), along with chemical methods. The structural data obtained are useful as the chemical basis for the creation of the classification scheme for Proteus strains.

New structures of the O-specific polysaccharides of Proteus. 3. Polysaccharides containing non-carbohydrate organic acids.

Four new Proteus O-specific polysaccharides were isolated by mild acid degradation from the lipopolysaccharides of P. penneri 28 (1), P. vulgaris O44 (2), P. mirabilis G1 (O3) (3), and P. myxofaciens (4), and their structures were elucidated using NMR spectroscopy and chemical methods. They were found to contain non-carbohydrate organic acids, including ether-linked lactic acid and amide-linked amino acids, and the following structures of the repeating units were established: →3)-α-L-QuipNAc-(1→3)-α-D-GlcpNAc-(1→6)-α-D-GlcpNAc-(1→ (S)-Lac-(2–3)⌋ (1) →4)-β-D-GlcpA-(1→3)-β-D-GalpNAc-(1→4)-β-D-Glcp-(1→3)-α-D-Galp-(1→4)-β-D-GalpNAc-(1→ L-Ala-(2–6)⌋ (2) →3)-β-D-GalpNAc-(1→6)-β-D-GalpNAc-(1→4)-β-D-GlcpA-(1→ L-Lys-(2–6)-α-D-GalpA-(1→4)⌋ (3) →4)-β-D-GlcpA-(1→6)-α-D-GalpNAc-(1→6)-β-D-GlcpNAc-(1→3)-β-D-GlcpNAc-(1→ (R)-aLys-(2–6)⌋ (4) where (S)-Lac and (R)-aLys stand for (S)-1-carboxyethyl (residue of lactic acid) and Nε-[(R)-1-carboxyethyl]-L-lysine (“alaninolysine”), respectively. The data obtained in this work and earlier serve as the chemical basis for classification of the bacteria Proteus.

Structure of a new neutral O-specific polysaccharide of Proteus penneri 34

The O-specific polysaccharide of Proteus penneri strain 34 was studied using 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, and H-detected 1H, 13C HMQC experiments. The following structure was established, which is unique among the known structures of Proteus O-antigens:-->4)-beta-D-Glcp-(1-->3)-beta-D-GalpNAc-(1-->4)-beta- D-GalpNAc-(1-->4)-beta-D-Galp-(1-->. Accordingly, no cross-reaction was observed between P. penneri 34 O-antiserum and O-antigens of other Proteus strains. Therefore, the strain studied should belong to a new Proteus serogroup O65.

Structure of the O-polysaccharide of Providencia alcalifaciens O8 containing (2S,4R)-2,4-dihydroxypentanoic acid, a new non-sugar component of bacterial glycans.

A glycerol teichoic acid-like O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O8 and studied by chemical methods and NMR spectroscopy, including 2D ROESY, {(1)H,(13)C} HSQC, and HMQC-TOCSY experiments. It was found that the compound contains a new component of bacterial lipopolysaccharides: ether-linked (2S,4R)-2,4-dihydroxypentanoic acid (Dhpa), which was identified by NMR spectroscopy. The following structure of the repeating unit of the polysaccharide was established: [structure: see text]

Structure of the O-specific polysaccharide of Proteus mirabilis O16 containing ethanolamine phosphate and ribitol phosphate

The O-specific polysaccharide of Proteus mirabilis O16 was studied by 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, H-detected 1H,13C HMQC, HMQC-TOCSY, and 1H,31P HMQC experiments, along with chemical methods. The polysaccharide was found to be a ribitol teichoic acid-like polymer having the following structure [structure: see text].