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.

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.

Variations in the Expression of Terminal Oligosaccharide Units and Glycosylation of Poly(N-acetyllactosamine) Chain in the Helicobacter pylori Lipopolysaccharide upon Colonization of Rhesus Macaques.

Helicobacter pylori is an important human pathogen that causes gastritis, gastric and duodenal ulcers, and gastric cancer. O-polysaccharides of H. pylori lipopolysaccharide (LPS) are composed of (ß1→3)-poly(N-acetyllactosamine) (polyLacNAc) decorated with multiple α-L-fucose residues. In many strains, their terminal LacNAc units are mono- or di-fucosylated to mimic Lewis X (Lex) and/or Lewis Y (Ley) oligosaccharides. The studies in rhesus macaques as a model of human infection by H. pylori showed that this bacterium adapts to the host during colonization by expressing host Lewis antigens. Here, we characterized LPS from H. pylori strains used in the previous study, including the parental J166 strain and the three derivatives (98-149, 98-169, and 98-181) isolated from rhesus macaques after long-term colonization. Chemical and NMR spectroscopic analyses of the LPS showed that the parent strain expressed Lex, Ley, and H type 1 terminal oligosaccharide units. The daughter strains were similar to the parental one in the presence of the same LPS core and fucosylated polyLacNAc chain of the same length but differed in the terminal oligosaccharide units. These were Lex in the isolates 98-149 and 98-169, which corresponded to the Lea phenotype of the host animals, and Ley was found in the 98-181 isolate from the macaque characterized by the Leb phenotype. As Lea and Leb are isomers of Lex and Ley, respectively, the observed correlation confirmed adaptation of the expression of terminal oligosaccharide units in H. pylori strains to the properties of the host gastric mucosa. The 98-181 strain also acquired glucosylation of the polyLacNAc chain and was distinguished by a lower expression of fucosylated internal LacNAc units (internal Lex) as a result of decoration of polyLacNAc with ß-glucopyranose, which may also play a role in the bacterial adaptation.

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.

The O-polysaccharide of Escherichia coli F5, which is structurally related to that of E. coli O28ab, provides cells only weak protection against bacteriophage attack.

Several types of Escherichia coli O-antigens form highly effective shields protecting the bacterial cell surface and preventing bacteriophages from interacting directly with their secondary (terminal) receptors. However, it is not clear if O-antigens of various types (O-serotypes) differ in their anti-phage protection efficacy. Here, we describe a new E. coli strain, F5, which has an E. coli O28ab-related O-antigen. Although the amount of O-antigen produced by this strain is comparable to that produced by other E. coli strains we tested, it appears to give the cells significantly lower protection against phage attack than other O-antigen types, such as the O-polysaccharide of E. coli F17, which we studied earlier.

Structure and gene cluster of the O-polysaccharide of Yersinia rohdei H274-36/78.

A branched O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Yersinia rohdei H274-36/78 and found to contain d-rhamnose, d-mannose, and 3,6-dideoxy-4-C-[(S)-1-hydroxyethyl]-d-xylo-hexose called yersiniose A (Yer). Partial acid hydrolysis of the O-polysaccharide eliminated Yer residues to give a modified linear polysaccharide. Studies by sugar analysis and 1H and 13C NMR spectroscopy, including computational NMR analysis, enabled structure elucidation of a hexasaccharide repeating unit of the O-polysaccharide having two Yer residues attached as monosaccharide side chains. The O-antigen gene cluster of Y. rohdei H274-36/78 located between JUMPStart and galF genes contained putative genes for synthesis of precursors of two O-antigen constituents, GDP-d-Man and GDP-d-Rha, whereas genes responsible for synthesis of CDP-Yer were within the chromosome outside the O-antigen gene cluster. Glycosyltransferase genes and ABC 2 transporter genes were present in the O-antigen gene cluster, and hence the structure established is consistent with the polysaccharide synthesis gene content of the genome.

Structural studies of O-specific polysaccharide(s) and biological activity toward plants of the lipopolysaccharide from Azospirillum brasilense SR8.

Lipopolysaccharide (LPS) was extracted from dry bacterial cells of plant-growth-promoting bacterium Azospirillum brasilense SR8 (IBPPM 5). The O-specific polysaccharide (OPS) was obtained by mild acid hydrolysis of the lipopolysaccharide and studied by sugar analysis, 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, and 1H,13C HSQC and HMBC experiments, computational NMR-based structure analysis, and Smith degradation. The OPS was shown to contain two types of repeating units of the following structure: Both OPS structures are present in A. brasilense 54, from which structure 1 has been reported earlier (Fedonenko et al., 2011), whereas to our knowledge structure 2 has not been hitherto found in bacterial saccharides. Treatment of wheat seedling roots with LPS of A. brasilense SR8 increased the number of root hair deformations as compared to seedlings grown without LPS, but had no effect on adsorption of the bacteria to the root surface. A. brasilense SR8 was able to utilize LPS of several structurally related Azospirillum strains.

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.

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 a tyvelose-containing O-polysaccharide of an entomopathogenic bacterium Yersinia entomophaga MH96T related to Yersinia pseudotuberculosis.

An O-polysaccharide was isolated from the lipopolysaccharide of an entomopathogenic bacterium Yersinia entomophaga MH96T by mild acid hydrolysis and studied by 2D NMR spectroscopy. The following structure of the branched tetrasaccharide repeating unit of the polysaccharide was established: where Tyv indicates 3,6-dideoxy-d-arabino-hexose (tyvelose). The structure established is consistent with the gene content of the O-antigen gene cluster. The O-polysaccharide structure and gene cluster of Y. entomophaga are related to those of some Y. pseudotuberculosis serotypes.

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.

Isolation and structural identification of glycopolymers of Bifidobacterium bifidum BIM B-733D as putative players in pathogenesis of autoimmune thyroid diseases.

Bifidobacterium bifidum 791 (commercially available as B. bifidum BIM B-733D) cell-surface biopolymers (BPs) interact selectively with human serum thyroid peroxidase (TPO) and thyroglobulin (Tg) autoantibodies (anti TPO and anti Tg, respectively). BPanti-TPO and BPanti-Tg were isolated from the soluble fraction of B. bifidum BIM B-733D by affinity chromatography with anti-TPO or anti-Tg, respectively. Homogeneity of affinity eluates (AEanti-TPO and AEanti-Tg) was tested by size exclusion chromatography. For each AE, the elution profiles generated on the basis of absorbance at 280 nm do not conform to ELISA data for functional activity characteristic of BPs. Moreover, high functional activity was detected in chromatographic fractions that had significantly different molecular weights and no absorbance at 280 nm, which suggests a non-protein (carbohydrate) nature of BPanti-TPO and BPanti-Tg. The semi-preparative size exclusion chromatography of AEanti-TPO and AEanti-Tg with detection by refractometer gave 5,000-7,000 Da fractions containing substances that interact selectively with either anti TPO (BPanti-TPO) or anti-Tg (BPanti-Tg) according to ELISA data. Analysis by two-dimensional NMR spectroscopy including a 1H, 13C-heteronuclear single-quantum coherence experiment indicated that both substances are linear α-1,6-glucans. For the first time, an immunological similarity (molecular mimicry) of glycopolymers of B. bifidum BIM B-733D and human thyroid proteins, TPO and Tg, was shown. On the whole, our data point to a possible role of bifidobacteria in the pathogenesis of autoimmune thyroid diseases (ATD). The main requirements for triggering/acceleration or prevention/abrogation of ATD by bifidobacteria through molecular mimicry mechanism are hypothesised to be (1) genetic predisposition to ATD and (2) intestinal epithelium penetration by α-1,6-glucan.

O-antigens of bacteria of the genus providencia: structure, serology, genetics, and biosynthesis.

The genus Providencia consists of eight species of opportunistic pathogenic enterobacteria that can cause enteric diseases and urinary tract infections. The existing combined serological classification scheme of three species, P. alcalifaciens, P. stuartii, and P. rustigianii, is based on the specificity of O-antigens (O-polysaccharides) and comprises 63 O-serogroups. Differences between serogroups are related to polymorphism at a specific genome locus, the O-antigen gene cluster, responsible for O-antigen biosynthesis. This review presents data on structures of 36 O-antigens of Providencia, many of which contain unusual monosaccharides and non-carbohydrate components. The structural data correlate with the immunospecificity of the O-antigens and enable substantiation on a molecular level of serological relationships within the genus Providencia and between strains of Providencia and bacteria of the genera Proteus, Escherichia, and Salmonella. Peculiar features of the O-antigen gene cluster organization in 10 Providencia serogroups and biosynthetic pathways of nucleotide precursors of specific monosaccharide components of the O-antigens also are discussed.

Lipopolysaccharide of Yersinia pestis, the Cause of Plague: Structure, Genetics, Biological Properties.

The present review summarizes data pertaining to the composition and structure of the carbohydrate moiety (core oligosaccharide) and lipid component (lipid A) of the various forms of lipopolysaccharide (LPS), one of the major pathogenicity factors ofYersinia pestis, the cause of plague. The review addresses the functions and the biological significance of genes for the biosynthesis of LPS, as well as the biological properties of LPS in strains from various intraspecies groups ofY. pestis and their mutants, including the contribution of LPS to the resistance of bacteria to factors of the innate immunity of both insect-vectors and mammal-hosts. Special attention is paid to temperature-dependent variations in the LPS structure, their genetic control and roles in the pathogenesis of plague. The evolutionary aspect is considered based on a comparison of the structure and genetics of the LPS ofY. pestis and other enteric bacteria, including otherYersinia species. The prospects of development of live plague vaccines created on the basis ofY. pestis strains with the genetically modified LPS are discussed.

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.

Structure elucidation of the O-Antigen of Salmonella enterica O51 and its structural and genetic relation to the O-Antigen of Escherichia coli O23.

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.

The role of components of Bifidobacterium and Lactobacillus in pathogenesis and serologic diagnosis of autoimmune thyroid diseases.

During recent years, researchers have been focusing on the concept of an infectious etiology of autoimmune diseases. The most discussed theory is molecular mimicry, i.e. the emergence of autoreactive clones of T- and B-lymphocytes as a result of cross-immune response to homologous bacterial or viral antigen. Information on the role of probiotic microorganisms (PM) in the molecular mechanisms of autoimmune thyroid diseases (ATD) is limited. Using proteins and immunogenic peptides databanks and relevant computer programs, the homology between the amino acid sequences of thyroid peroxidase (TPO) and thyroglobulin (Tg), which are potential B- and T-cell epitopes of these antigens, and proteins of bifidobacteria and lactobacilli was established. Moreover, we have found components of cells of Bifidobacterium bifidum 791, Bifidobacterium adolescentis 94 BIM, Bifidobacterium longum B379M and Lactobacillus plantarum B-01 that selectively bind human antibodies to TPO (anti-TPO) and antibodies to Tg (anti-Tg) and compete with natural antigens for the binding of anti-TPO and anti-Tg in ELISA. Additionally, a three-fold difference was observed between the probability of detecting antibodies (Abs) to the antigens of L. plantarum B-01 and B. bifidum 791 in serum samples containing and those not containing anti-TPO. On the whole, our data are arguments in favour of the assumption of the possible role of PM of the genera Bifidobacterium and Lactobacillus in triggering ATD by the mechanism of molecular mimicry. The data obtained in silico and in vitro should be proven by use of animal models and clinical studies for extrapolations to the whole body. Possible antigenic properties of components/proteins of bifidobacteria and lactobacilli, selectively binding anti-TPO and anti-Tg should be taken into consideration. Natural human Abs to these bacterial components are probably able to cross-react with the TPO and Tg in the ELISA for detection of anti-TPO and anti-Tg, which are serologic markers of ATD. It can lead to unspecific false positive results and, hence, to an incorrect diagnosis.

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.

Structure of the O-antigen and characterization of the O-antigen gene cluster of Escherichia coli O108 containing 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-D-galacto-non-2-ulosonic (8-epilegionaminic) acid.

On mild acid degradation of the lipopolysaccharide of Escherichia coli O108, the O-polysaccharide was isolated and studied by sugar analysis and one- and two-dimensional 1H- and 13C-NMR spectroscopy. The polysaccharide was found to contain an unusual higher sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-d-galacto-non-2-ulosonic acid (di-N-acetyl-8-epilegionaminic acid, 8eLeg5Ac7Ac). The following structure of the tetrasaccharide repeating unit of the polysaccharide was established: -->4)-alpha-8eLegp5Ac7Ac-(2-->6)-alpha-D-Galp-(1-->3)-alpha-L-FucpNAc-(1-->3)-alpha-D-GlcpNAc-(1-->. Functions of the E. coli O108 antigen biosynthetic genes, including seven putative genes for synthesis of 8eLeg5Ac7Ac, were assigned by sequencing the O-antigen gene cluster along with comparison with gene databases and known biosynthetic pathways for related nonulosonic acids.