Specific Interaction of Novel Friunavirus Phages Encoding Tailspike Depolymerases with Corresponding Acinetobacter baumannii Capsular Types.

Acinetobacter baumannii is one of the most clinically important nosocomial pathogens. The World Health Organisation refers it to its «critical priority¼ category to develop new strategies for effective therapy. This microorganism is capable of producing structurally diverse capsular polysaccharides (CPSs), which serve as primary receptors for A. baumannii bacteriophages carrying polysaccharide-depolymerasing enzymes. In this study, eight novel bacterial viruses that specifically infect A. baumannii strains belonging to K2/K93, K32, K37, K44, K48, K87, K89 and K116 capsular types were isolated and characterized. The overall genomic architecture demonstrated that these viruses are representatives of the Friunavirus genus of the family Autographiviridae The linear double-stranded DNA phage genomes of 41,105-42,402 bp share high nucleotide sequence identity, except for genes encoding structural depolymerases or tailspikes which determine the host specificity. Deletion mutants lacking N-terminal domains of tailspike proteins were cloned, expressed and purified. The structurally defined CPSs of the phage bacterial hosts were cleaved with the specific recombinant depolymerases, and the resultant oligosaccharides that corresponded to monomers or/and dimers of the CPS repeats (K-units) were isolated. Structures of the derived oligosaccharides were established by nuclear magnetic resonance spectroscopy and high-resolution electrospray ionization mass spectrometry. The data obtained showed that all depolymerases studied were glycosidases that cleave specifically the A. baumannii CPSs by the hydrolytic mechanism, in most cases, by the linkage between the K-units.IMPORTANCE Acinetobacter baumannii, a nonfermentative, Gram-negative, aerobic bacterium, is one of the most significant nosocomial pathogens. The pathogenicity of A. baumannii is based on the cooperative action of many factors, one of them being the production of capsular polysaccharides (CPSs) that surround bacterial cells with a thick protective layer. Polymorphism of the chromosomal capsule loci is responsible for the observed high structural diversity of the CPSs. In this study, we describe eight novel lytic phages which have different tailspike depolymerases (TSDs) determining the interaction of the viruses with corresponding A. baumannii capsular types (K-types). Moreover, we elucidate the structures of oligosaccharide products obtained by cleavage of the CPSs by the recombinant depolymerases. We believe that as the TSDs determine phage specificity, the diversity of their structures should be taken into consideration as selection criteria for inclusion of certain phage candidate to the cocktail designed to control A. baumannii with different K-types.

Two Glycosyl 1-Phosphate Polymers and Teichulosonic Acid from Glutamicibacter protophormiae VKM Ac-2104T Cell Wall.

Two glycosyl 1-phosphate polymers containing monoglycosyl 1-phosphate, -6)-α-D-Glcp-(1-P-, and diglycosyl 1-phosphate, -6)-α-D-GalpNAc-(1→6)-α-D-GlcpNAc-(1-P-, in the repeating unit were identified in the cell wall of Glutamicibacter protophormiae VKM Ac-2104T (formerly, Arthrobacter protophormiae). The structures of these polymers were described for the first time in prokaryotes. Teichulosonic acid, the third identified polymer, with 3-deoxy-D-glycero-α-D-galacto-non-2-ulopyranosonic acid (Kdn) and ß-D-glucopyranose residues in the main chain, →6)-ß-D-Glcp-(1→8)-α-Kdn-(2→, has been previously detected in a number of actinobacteria. The structures of these glycopolymers were established based on the results of chemical analysis and one-dimensional 1H, 13C, and 31P NMR spectroscopy using two-dimensional homonuclear (1H,1H COZY, TOCSY, ROESY) and heteronuclear (1H,13C HSQC, HSQC-TOCSY, HMBC, and 1H,31P HMBC) techniques.

Mechanisms of Acinetobacter baumannii Capsular Polysaccharide Cleavage by Phage Depolymerases.

Aerobic gram-negative bacterium Acinetobacter baumannii has recently become one of the most relevant pathogens associated with hospital-acquired infections worldwide. A. baumannii produces a capsule around the cell, which represents a thick viscous layer of structurally variable capsular polysaccharide (CPS). The capsule protects the bacteria against unfavorable environmental factors and biological systems, including bacteriophages and host immune system. Many A. baumannii phages have structural depolymerases (tailspikes) that specifically recognize and digest bacterial CPS. In this work, we studied the interaction of tailspike proteins of four lytic depolymerase-carrying phages with A. baumannii CPS. Depolymerases of three bacteriophages (Fri1, AS12, and BS46) were identified as specific glycosidases that cleave the CPS of A. baumannii strains 28, 1432, and B05, respectively, by the hydrolytic mechanism. The gp54 depolymerase from bacteriophage AP22 was characterized as a polysaccharide lyase that cleaves the CPS of A. baumannii strain 1053 by ß-elimination at hexuronic acid (ManNAcA) residues.

Elucidation of the K32 Capsular Polysaccharide Structure and Characterization of the KL32 Gene Cluster of Acinetobacter baumannii LUH5549.

Capsular polysaccharide (CPS), isolated from Acinetobacter baumannii LUH5549 carrying the KL32 capsule biosynthesis gene cluster, was studied by sugar analysis, Smith degradation, and one- and two-dimensional 1H and 13C NMR spectroscopy. The K32 CPS was found to be composed of branched pentasaccharide repeats (K units) containing two residues of ß-D-GalpNAc and one residue of ß-D-GlcpA (ß-D-glucuronic acid) in the main chain and one residue each of ß-D-Glcp and α-D-GlcpNAc in the disaccharide side chain. Consistent with the established CPS structure, the KL32 gene cluster includes genes for a UDP-glucose 6-dehydrogenase (Ugd3) responsible for D-GlcA synthesis and four glycosyltransferases that were assigned to specific linkages. Genes encoding an acetyltransferase and an unknown protein product were not involved in CPS biosynthesis. Whilst the KL32 gene cluster has previously been found in the global clone 2 (GC2) lineage, LUH5549 belongs to the sequence type ST354, thus demonstrating horizontal gene transfer between these lineages.

Rhamnomannans and Teichuronic Acid from the Cell Wall of Rathayibacter tritici VKM Ac-1603T.

The structures of three cell wall glycopolymers of the phytopathogen Rathayibacter tritici VKM Ac-1603T (family Microbacteriaceae, order Micrococcales, class Actinobacteria) were established by chemical methods and NMR spectroscopy. Polymer 1 is a branched rhamnomannan with the repeating unit →3)-α-[ß-D-Xylp-(1→2)]-D-Manp-(1→2)-α-D-Rhap-(1→3)-α-D-Manp-(1→2)-α-D-Rhap-(1→; polymer 2 is a linear rhamnomannan with the repeating unit →2)-α-D-Manp-(1→2)-α-D-Rhap-(1→3)-α-D-Manp-(1→2)-α-D-Rhap-(1→; polymer 3 is a branched teichuronic acid containing monosaccharide residues GlcA, Gal, Man, and Glc at a 1 : 1 : 1 : 5 ratio (see the text for the structures). It has been demonstrated that representatives of four Rathayibacter species studied to date (R. tritici VKM Ac-1603T, R. iranicus VKM Ac-1602 T, R. toxicus VKM Ac-1600 and "Rathayibacter tanaceti" VKM Ac-2596) contain differing patterns of phosphate-free glycopolymers. At the same time, the above Rathayibacter strains have a common property - the presence of rhamnomannans with D-rhamnose. These rhamnomannans may be linear or branched and differing in the positions of glycosidic bonds and side substituents. The presence in the cell wall of rhamnomannans with D-rhamnose may serve as useful chemotaxonomic marker of the genus Rathayibacter.

Synthesis of oligosaccharides related to galactomannans from Aspergillus fumigatus and their NMR spectral data.

The synthesis of model oligosaccharides related to antigenic galactomannans of the dangerous fungal pathogen Aspergillus fumigatus has been performed employing pyranoside-into-furanoside (PIF) rearrangement and controlled O(5) → O(6) benzoyl migration as key synthetic methods. The prepared compounds along with some previously synthesized oligosaccharides were studied by NMR spectroscopy with the full assignment of 1H and 13C signals and the determination of 13C NMR glycosylation effects. The obtained NMR database on 13C NMR chemical shifts for oligosaccharides representing galactomannan fragments forms the basis for further structural analysis of galactomannan related polysaccharides by a non-destructive approach based on the calculation of the 13C NMR spectra of polysaccharides by additive schemes.

Rhamnose-Containing Cell Wall Glycopolymers from Rathayibacter toxicus VKM Ac-1600 and "Rathayibacter tanaceti" VKM Ac-2596.

Structures of the cell wall glycopolymers from two representatives of the genus Rathayibacter were investigated using chemical, NMR spectroscopy, and optical methods. The R. toxicus VKM Ac-1600 strain contains two neutral glycopolymers - a linear rhamnomannan →2)-α-D-Rhap-(1→3)-α-D-Manp-(1→ and a branched polysaccharide containing in the repeating unit the residues of D-Manp, D-Glcp, and L-Rhap in the ratios of 2 : 4 : 1, respectively (the structure is presented in the text). The "Rathayibacter tanaceti" VKM Ac-2596 contains a rhamnomannan that is different from the above-described one by localization of glycosidic bonds on the residues of α-Rhap and α-Manp, i.e. →3)-α-D-Rhap (1→2)-α-D-Manp-(1→. The structures of all identified glycopolymers are described for the first time in actinobacteria. The data obtained make it possible to characterize representatives of the studied actinobacteria more fully and can be used to differentiate Rathayibacter species at the phenotype level.

Structure of the K82 Capsular Polysaccharide from Acinetobacter baumannii LUH5534 Containing a d-Galactose 4,6-Pyruvic Acid Acetal.

Type K82 capsular polysaccharide (CPS) was isolated from Acinetobacter baumannii LUH5534. The structure of a linear tetrasaccharide repeating unit of the CPS was established by sugar analysis along with one- and two-dimensional 1H and 13C NMR spectroscopy. Proteins encoded by the KL82 capsule gene cluster in the genome of LUH5534 were assigned to roles in the synthesis of the K82 CPS. In particular, functions were assigned to two new glycosyltransferases (Gtr152 and Gtr153) and a novel pyruvyltransferase, Ptr5, responsible for the synthesis of d-galactose 4,6-(R)-pyruvic acid acetal.

Erratum to: "Rhamnose-Containing Cell Wall Glycopolymers from Rathayibacter toxicus VKM Ac-1600 and "Rathayibacter tanaceti" VKM Ac-2596" [Biochemistry (Moscow), 83, 717 (2018)].

This corrects the article DOI: 10.1134/S0006297918060093.

Erratum to: "Structural Relationships Between Genetically Closely Related O-Antigens of Escherichia coli and Shigella spp." [Biochemistry (Moscow), 81, 600 (2016)].

This corrects the article DOI: 10.1134/S0006297916060067.

O-Antigens of Escherichia coli Strains O81 and HS3-104 Are Structurally and Genetically Related, Except O-Antigen Glucosylation in E. coli HS3-104.

Glycerophosphate-containing O-specific polysaccharides (OPSs) were obtained by mild acidic degradation of lipopolysaccharides isolated from Escherichia coli type strain O81 and E. coli strain HS3-104 from horse feces. The structures of both OPSs and of the oligosaccharide derived from the strain O81 OPS by treatment with 48% HF were studied by monosaccharide analysis and one- and two-dimensional 1H- and 13C-NMR spectroscopy. Both OPSs had similar structures and differed only in the presence of a side-chain glucose residue in the strain HS3-104 OPS. The genes and the organization of the O-antigen biosynthesis gene cluster in both strains are almost identical with the exception of the gtr gene cluster responsible for glucosylations in the strain HS3-104, which is located elsewhere in the genome.

Structure and Gene Cluster of the K93 Capsular Polysaccharide of Acinetobacter baumannii B11911 Containing 5-N-Acetyl-7-N-[(R)-3-hydroxybutanoyl]pseudaminic Acid.

Capsular polysaccharide (CPS) assigned to the K93 type was isolated from the bacterium Acinetobacter baumannii B11911 and studied by sugar analysis along with one- and two-dimensional 1H and 13C NMR spectroscopy. The CPS was found to contain a derivative of pseudaminic acid, and the structure of the branched tetrasaccharide repeating unit was established. Genes in the KL93 capsule biosynthesis locus were annotated and found to be consistent with the CPS structure established. The K93 CPS has the α-d-Galp-(1→6)-ß-d-Galp-(1→3)-d-GalpNAc trisaccharide fragment in common with the K14 CPS of Acinetobacter nosocomialis LUH 5541 and A. baumannii D46. It also shares the ß-d-Galp-(1→3)-d-GalpNAc disaccharide fragment and the corresponding predicted Gal transferase Gtr5, as well as the initiating GalNAc-1-P transferase ItrA2, with a number of A. baumannii strains.

Structure and Biosynthesis Gene Cluster of the O-Antigen of Escherichia coli O12.

Two polysaccharides were isolated from Escherichia coli O12, the major being identified as the O12-antigen and the minor as the K5-antigen. The polysaccharides were studied by sugar analysis, Smith degradation, and one- and two-dimensional (1)H and (13)C NMR spectroscopy. As a result, the following structure of the O12-polysaccharide was elucidated, which, to our knowledge, has not been hitherto found in bacterial carbohydrates: →2)-ß-d-Glcp-(1→6)-α-d-GlcpNAc-(1→3)-α-l-FucpNAc-(1→3)-ß-d-GlcpNAc-(1→. The →4)-ß-d-GlcpA-(1→4)-α-d-GlcpNAc-(1→ structure established for the K5-polysaccharide (heparosan) is previously known. Functions of genes in the O-antigen biosynthesis gene cluster of E. coli O12 were assigned by comparison with sequences in the available databases and found to be consistent with the O12-polysaccharide structure.

Structural Relationships Between Genetically Closely Related O-Antigens of Escherichia coli and Shigella spp.

Gene clusters for biosynthesis of 24 of 34 basic O-antigen forms of Shigella spp. are identical or similar to those of the genetically closely related bacterium Escherichia coli. For 18 of these relatedness was confirmed chemically by elucidation of the O-antigen (O-polysaccharide) structures. In this work, structures of the six remaining O-antigens of E. coli O32, O53, O79, O105, O183 (all related to S. boydii serotypes), and O38 (related to S. dysenteriae type 8) were established using (1)H and (13)C NMR spectroscopy. They were found to be identical to the Shigella counterparts, except for the O32- and O38-polysaccharides, which differ in the presence of O-acetyl groups. The structure of the E. coli O105-related O-polysaccharide of S. boydii type 11 proposed earlier is revised. The contents of the O-antigen gene clusters of the related strains of E. coli and Shigella spp. and different mechanisms of O-antigen diversification in these bacteria are discussed in view of the O-polysaccharide structures established. These data illustrate the value of the O-antigen chemistry and genetics for elucidation of evolutionary relationships of bacteria.

O-antigen modifications providing antigenic diversity of Shigella flexneri and underlying genetic mechanisms.

O-Antigens (O-specific polysaccharides) of Shigella flexneri, a primary cause of shigellosis, are distinguished by a wide diversity of chemical modifications following the oligosaccharide O-unit assembly. The present review is devoted to structural, serological, and genetic aspects of these modifications, including O-acetylation and phosphorylation with phosphoethanolamine that have been identified recently. The modifications confer the host with specific immunodeterminants (O-factors or O-antigen epitopes), which accounts for the antigenic diversity of S. flexneri considered as a virulence factor of the pathogen. Totally, 30 O-antigen variants have been recognized in these bacteria, the corresponding O-factors characterized using specific antibodies, and a significant extension of the serotyping scheme of S. flexneri on this basis is suggested. Multiple genes responsible for the O-antigen modifications and the resultant serotype conversions of S. flexneri have been identified. The genetic mechanisms of the O-antigen diversification by acquisition of mobile genetic elements, including prophages and plasmids, followed occasionally by gene mobilization and inactivation have been revealed. These findings further our understanding of the genetics and antigenicity of S. flexneri and assist control of shigellosis.

Disaccharide 1-phosphate polymers of some representatives of the Bacillus subtilis group.

Disaccharide 1-phosphate polymers as well as teichoic acids of various structures have been found in the cell walls of the representatives of the Bacillus subtilis group, namely Bacillus subtilis subsp. spizizenii VKM B-720 and VKM B-916, B. subtilis VKM B-517, and Bacillus vallismortis VKM B-2653(T). Disaccharide 1-phosphate polymers are composed of repeating units of the following structure: -P-4)-ß-D-GlcpNAc-(1→6)-α-D-Galp-(1-, the N-acetylglucosamine residues are partially acetylated at positions O3 and O6 (VKM B-720 and VKM B-916); -P-4)-ß-D-Glcp-(1→6)-α-D-GlcpNAc-(1-, the glucopyranose residues are partially acetylated at positions O2 or O3 (VKM B-517); -P-6)-α-D-GlcpNH3(+)/α-D-GlcpNAc-(1→2)-α-D-Glcp-(1-, the N-acetylglucosamine residues are partially deacetylated (VKM B-2653(T)). The structures of the two last disaccharide 1-phosphate polymers have not been reported so far for Gram-positive bacteria. The teichoic acids in the studied strains are O-D-alanyl-1,5-poly(ribitol phosphates) substituted with ß-D-glucopyranose (VKM B-517, VKM B-720, VKM B-916) or 2-acetamido-2-deoxy-ß-D-glucopyranose (VKM B-2653(T)). The structures of the phosphate-containing polymers have been studied by chemical methods and by NMR spectroscopy.

Structure of hexasaccharide 1-phosphate polymer from Arthrobacter uratoxydans VKM Ac-1979(T) cell wall.

A hexasaccharide 1-phosphate polymer of original structure and two teichoic acids (TA) belonging to different structural types were found in Arthrobacter uratoxydans VKM Ac-1979(T) cell wall. The poly(hexasaccharide 1-phosphate) combines features of teichuronic acids and glycosyl 1-phosphate polymers, and its structure has never been reported earlier. Its composition includes residues of α- and ß-D-glucuronic acid as well as α-D-galacto-, ß-D-gluco-, α-D-mannopyranose, and 6-O-acetylated 2-acetamido-2-deoxy-α-D-glucopyranose. The phosphodiester bond in the polymer joins the glycoside hydroxyl of α-D-glucuronic acid and O6 of α-D-galactopyranose. TA 1 is ß-D-glucosylated 1,3-poly(glycerol phosphate), and TA 2 is 3,6-linked poly[α-D-glucosyl-(1→2)-glycerol phosphate]. The phosphate-containing polymers were studied by chemical methods and on the basis of one-dimensional 1H-, 13C-, and (31)P-NMR spectra, homonuclear two-dimensional (1)H/(1)H COSY, TOCSY, ROESY, and heteronuclear (1)H/(13)C HSQC, HSQC-TOCSY, HMBC, and (1)H/(31)P HMBC experiments. The set and structure of the polymers revealed as well as the cell wall sugars (galactose, glucose, mannose, glucosamine) and glycerol can be used in microbiological practice for taxonomic purposes.

Structure of a peptidoglycan-related polysaccharide from Providencia alcalifaciens O45.

A polysaccharide was isolated from the opportunistic human pathogen Providencia alcalifaciens O45:H26 by extraction with aqueous phenol and studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including two-dimensional ROESY and H-detected (1)H,(13)C HSQC experiments. The polysaccharide contains N-acetylglucosamine and N-acetylmuramic acid (D-GlcpNAc3Rlac) amidated with L-alanine and has the following structure: →4)-ß-D-GlcpNAc-(1→4)-ß-D-GlcpNAc3(Rlac-L-Ala)-(1→. The polysaccharide possesses a remarkable structural similarity to the bacterial cell wall peptidoglycan. It is not unique to the strain studied but is common to strains of at least four P. alcalifaciens O-serogroups (O3, O24, O38, and O45). No evidence was obtained that the polysaccharide is associated with the LPS, and hence it might represent a bacterial capsule component.

Teichulosonic acid, an anionic polymer of a new class from the cell wall of Actinoplanes utahensis VKM Ac-674(T).

The cell wall of Actinoplanes utahensis VKM Ac-674(T) contains two anionic polymers: teichoic acid 1,3-poly(glycerol phosphate) that is widespread in cell walls of Gram-positive bacteria; and a unique teichulosonic acid belonging to a new class of bioglycans described only in microorganisms of the Actinomycetales order. The latter polymer contains residues of di-N-acyl derivative of sialic acid-like monosaccharide - 5,7-diamino-3,5,7,9-tetradeoxy-L-glycero-ß-L-manno-non-2-ulosonic or pseudaminic acid (Pse) which bears the N-(3,4-dihydroxybutanoyl) group (Dhb) at C7. This polymer has irregular structure and consists of fragments of two types, which differ in substitution of the Dhb residues at O4 either with ß-D-glucopyranose or with ß-Pse residues. Most of the ß-Pse residues (~80%) are glycosylated at position 4 with α-D-galactopyranose residues in both types of fragments. The glucose, galactose, and Dhb residues are partly O-acetylated. The structures of the polymers were established by chemical and NMR spectroscopy methods.

Structure of the O-polysaccharide of Providencia alcalifaciens O25 containing an amide of D-galacturonic acid with N(ε)-[(R)-1-carboxyethyl]-L-lysine.

An acidic O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O25 followed by gel-permeation and anion-exchange chromatography. The O-polysaccharide was studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including two-dimensional correlation (1)H,(13)C HMBC, and (1)H,(1)H ROESY experiments both in D(2)O and, to detect correlations for NH protons, in a 9 : 1 H(2)O/D(2)O mixture. An amino acid was isolated from the polysaccharide by acid hydrolysis and identified as N(ε)-[(R)-1-carboxyethyl]-L-lysine ("alaninolysine", 2S,8R-alaLys) by determination of the specific optical rotation and (13)C NMR spectroscopy, using the authentic synthetic diastereomers 2S,8R-alaLys and 2S,8S-alaLys for comparison. The structure of the branched tetrasaccharide repeating unit of the O-polysaccharide was established.