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The polysaccharide capsule surrounding bacterial cell plays an important role in pathogenesis of infections caused by the opportunistic pathogen Acinetobacter baumannii by providing protection from external factors. The structures of the capsular polysaccharide (CPS) produced by A. baumannii isolates and the corresponding CPS biosynthesis gene clusters are highly diverse, although many of them are related. Many types of A. baumannii CPSs contain isomers of 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acid (DTNA). Three of these isomers, namely acinetaminic acid (l-glycero-l-altro isomer), 8-epiacinetaminic acid (d-glycero-l-altro isomer), and 8-epipseudaminic acid (d-glycero-l-manno isomer), have not been found so far in naturally occurring carbohydrates from other species. In A. baumannii CPSs, DTNAs carry N-acyl substituents at positions 5 and 7; in some CPSs, both N-acetyl and N-(3-hydroxybutanoyl) groups are present. Remarkably, pseudaminic acid carries the (R)-isomer and legionaminic acid carries the (S)-isomer of the 3-hydroxybutanoyl group. The review addresses the structure and genetics of biosynthesis of A. baumannii CPSs containing di-N-acyl derivatives of DTNA. [ABSTRACT FROM AUTHOR]

In this report, we describe the fluorescent labeling of bacterial polysaccharides (Escherichia coli O86:B7, Escherichia coli O19ab, Pseudomonas aeruginosa O10a10b, and Shigella flexneri 2b) at the "natural" amino group of their phosphoethanolamine moiety. Two protocols for labeling are compared: 1) on a scale of a few mg of the polysaccharide, with a dialysis procedure for purification from excessive reagents; and 2) on a scale of 0.1 mg of the polysaccharide, with a simple precipitation procedure instead of dialysis. The microscale version is sufficient for comfortable cytofluorometric analysis. The resulting probes were found to specifically bind to human dendritic cells in a dose-dependent manner. The used limited set of polysaccharides did not allow us even to get close to understanding which dendritic cell-associated lectins and which cognate polysaccharide epitopes are involved in recognition, but the proposed microscale protocol allows to generate a library of fluorescent probes for further mapping of the polysaccharide specificity of the dendritic cells. [ABSTRACT FROM AUTHOR]

The impact of planktonic and biofilm lifestyles of the clinical isolate Proteus mirabilis 9B-m on its lipopolysaccharide (O-polysaccharide, core region, and lipid A) was evaluated. Proteus mirabilis bacteria are able to form biofilm and lipopolysaccharide is one of the factors involved in the biofilm formation. Lipopolysaccharide was isolated from planktonic and biofilm cells of the investigated strain and analyzed by SDS-PAGE with silver staining, Western blotting and ELISA, as well as NMR and matrix-assisted laser desorption ionization time-of-flight mass spectrometry techniques. Chemical and NMR spectroscopic analyses revealed that the structure of the O-polysaccharide of P. mirabilis 9B-m strain did not depend on the form of cell growth, but the full-length chains of the O-antigen were reduced when bacteria grew in biofilm. The study also revealed structural modifications of the core region in the lipopolysaccharide of biofilm-associated cells—peaks assigned to compounds absent in cells from the planktonic culture and not previously detected in any of the known Proteus core oligosaccharides. No differences in the lipid A structure were observed. In summary, our study demonstrated for the first time that changes in the lifestyle of P. mirabilis bacteria leads to the modifications of their important virulence factor—lipopolysaccharide. [ABSTRACT FROM AUTHOR]

Based on the O-specific polysaccharides of the lipopolysaccharides (O-polysaccharides, O-antigens), strains of a clonal species Escherichia coli are classified into 184 O serogroups. In this work, structures of the O-polysaccharides of E. coli O69 and O146 were elucidated and gene clusters for their biosynthesis were characterized. The O-polysaccharides were released from the lipopolysaccharides by mild acid hydrolysis and studied by sugar analysis and one- and two-dimensional H and C NMR spectroscopy before and after O-deacetylation. The O146 polysaccharide was also studied by Smith degradation. The O69 and O146 polysaccharides were found to contain ether conjugates of monosaccharides with lactic acid called glycolactilic acids: 2-acetamido-2-deoxy-4- O-[( R)-1-carboxyethyl]- d-glucose ( d-GlcNAc4 Rlac) and 3- O-[( S)-1-carboxyethyl]- d-glucose ( d-Glc3 Slac), respectively. Structures of the pentasaccharide repeats of the O-polysaccharides were established, and that of E. coli O69 was found to differ in the presence of d-GlcNAc4 Rlac from the structure reported for this bacterium earlier (Erbing C, Kenne L, Lindberg B. 1977. Carbohydr Res. 56:371-376). The O-antigen gene clusters of E. coli O69 and O146 between conserved genes galF and gnd were analyzed taking into account the O-polysaccharide structures established, and functions of putative genes for synthesis of d-Glc3 Slac and d-GlcNAc4 Rlac and for glycosyltransferases were assigned based on homology with O-antigen biosynthesis genes of other enteric bacteria. It was found that in E. coli and Shigella spp. predicted enolpyruvate reductases of the biosynthesis pathway of glycolactilic acids, LarR and LarS, which catalyze formation of conjugates with ( R)- or ( S)-lactic acid, respectively, are distinguished by sequence homology and size. [ABSTRACT FROM AUTHOR]

The sensing of microbe-associated molecular patterns (MAMPs) triggers innate immunity in animals and plants. Lipopolysaccharide (LPS) from Gram-negative bacteria is a potent MAMP for mammals, with the lipid A moiety activating proinflammatory responses via Toll-like receptor 4 (TLR4). Here we found that the plant Arabidopsis thaliana specifically sensed LPS of Pseudomonas and Xanthomonas. We isolated LPS-insensitive mutants defective in the bulb-type lectin S-domain-1 receptor-like kinase LORE (SD1-29), which were hypersusceptible to infection with Pseudomonas syringae. Targeted chemical degradation of LPS from Pseudomonas species suggested that LORE detected mainly the lipid A moiety of LPS. LORE conferred sensitivity to LPS onto tobacco after transient expression, which demonstrated a key function in LPS sensing and indicated the possibility of engineering resistance to bacteria in crop species. [ABSTRACT FROM AUTHOR]

Genomic approaches continue to provide unprecedented insight into the microbiome, yet host immune interactions with diverse microbiota can be difficult to study. We therefore generated a microbial microarray containing defined antigens isolated from a broad range of microbial flora to examine adaptive and innate immunity. Serological studies with this microarray show that immunoglobulins from multiple mammalian species have unique patterns of reactivity, whereas exposure of animals to distinct microbes induces specific serological recognition. Although adaptive immunity exhibited plasticity toward microbial antigens, immunological tolerance limits reactivity toward self. We discovered that several innate immune galectins show specific recognition of microbes that express self-like antigens, leading to direct killing of a broad range of Gram-negative and Gram-positive microbes. Thus, host protection against microbes seems to represent a balance between adaptive and innate immunity to defend against evolving antigenic determinants while protecting against molecular mimicry. [ABSTRACT FROM AUTHOR]

Hafnia alveiis the only species of the genus Hafnia, which belongs to the family of Enterobacteriaceae. These Gram-negative bacteria are commonly distributed in the natural environment and are often the cause of human opportunistic infections. Their lipopolysaccharides (LPSs) are important surface antigens which are responsible for the serological specificity and numerous cross-reactions with other enterobacterial genera. So far, 29 different O-polysaccharide (OPS, O-antigen) structures in Hafnias LPSs have been established and for some of them the molecular basis of the serological activity has been elucidated. OPS from H. alvei strain PCM 1219 was obtained by mild acid hydrolysis of the LPS followed by gel permeation chromatography of carbohydrate material on Sephadex G-50 column. The polysaccharide structure was determined using chemical methods as well as 13C NMR and 1H NMR spectroscopy. For serological studies, SDS-PAGE, immunoblotting, and passive hemagglutination tests were used. The serological studies revealed a cross-reactivity of the LPSs of H. alvei PCM 1219 and a group of H. alvei strains with an O-antigen containing D-glucose 1-phosphate and [ (R)-3-hydroxybutyramido]-D-glucose. The following structure of the OPS was established: where Acyl stands for ( R)-3-hydroxybutyryl and the degree of O-acetylation is ~70%. The structure of the core oligosaccharide was found to be typical of the genus Hafnia. Based on the OPS structure and serological results it was concluded that H. alvei strain PCM 1219 should be classified in the same serogroup as the H. alvei type strain ATCC 13337 and five other strains containing D-glucose 1-phosphate and 2-deoxy-2-[( R)-3-hydroxybutyramido]-D-glucose in their O-antigens. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide (endotoxin, LPS) is an important potential virulence factor of Proteus rods. The serological specificity of the bacteria is defined by the structure of the O-polysaccharide chain (O-antigen) of the LPS. Until now, 76 O-serogroups have been differentiated among Proteus strains. LPSs were isolated from Proteus mirabilis TG 83, TG 319, and CCUG 10700 (OA) strains by phenol/water extraction. Antisera were raised by immunization of rabbits with heat-killed bacteria. Serological investigations were performed using enzyme immunosorbent assay, passive immunohemolysis, inhibition of both assays, absorption of antisera, and Western blot. The cross-reactive epitope shared by these strains and P. penneri O72a,O72b is located on the O-polysaccharide and is most likely associated with an α-D-Glc p-(1→6)-β-D-Gal pNAc disaccharide fragment. The serological data indicated the occurrence of two core types in the LPSs studied, one characteristic for P. mirabilis TG 319 and CCUG 10700 (OA) and the other for P. mirabilis TG 83 and O57. The serological and structural data showed that P. mirabilis TG 83, TG 319, CCUG 10700 (OA), and O57 have the same O-antigen structure and could be qualified to the Proteus O57 serogroup. [ABSTRACT FROM AUTHOR]

Proteus rods are currently subdivided into five named species, i.e. Proteus mirabilis, P. vulgaris, P. penneri, P. hauseri, and P. myxofaciens, and three unnamed Proteus genomospecies 4 to 6. Based on the serospecificity of the lipopolysaccharide (LPS; O-antigen), strains of P. mirabilis and P. vulgaris were divided into 49 O-serogroups and 11 additional O-serogroups were proposed later. About 15 further O-serogroups have been proposed for the third medically important species, P. penneri. Here the serological classification of P. vulgaris strain TG 251, which does not belong to these serogroups, is reported. Serological investigations also allowed characterization of the epitope specificity of its LPS. Purified LPSs from five Proteus strains were used as antigens in enzyme immunosorbent assay (EIA), SDS/PAGE, and Western blot and alkali-treated LPSs in the passive immunohemolysis (PIH) test, inhibition of PIH and EIA, and absorption of the rabbit polyclonal O-antisera with the respective LPS. The serological studies of P. vulgaris TG 251 LPS indicated the identity of its O-polysaccharide with that of P. penneri O65. The antibody specificities of P. vulgaris TG 251 and P. penneri O65 O-antisera, were described. P. vulgaris TG 251 was classified to the Proteus O65 serogroup. Two disaccharide-associated epitopes present in P. vulgaris TG 251 and P. penneri O65 LPSs are suggested to be responsible for cross-reactions with three heterologous Proteus strains. [ABSTRACT FROM AUTHOR]

Bacteria of the genus Proteus are a common cause of urinary tract infections. The O-polysaccharide (OPS) chain of their lipopolysaccharide (LPS) defines the serological specificity of strains. Based on the OPS structures and the immunospecificity of the LPS, Proteus strains have been classified into 74 O-serogroups. The OPS of P. mirabilis TG 115 was obtained by mild acid degradation of the LPS and studied by 1H and 13C nuclear magnetic resonance spectroscopy. Antisera were raised by immunization of rabbits with heat-killed bacteria. Serological studies were performed using enzyme immunosorbent assay, passive immunoheamolysis, inhibition experiments, absorption of O-antisera, and Western blot. The following structure of the P. mirabilis TG 115 OPS was established: → 2)-β-D-Gal pA-(1→ 3)-α-D-Gal pNAc-(1→ 4)-α-D-Gal pA-(1→ 3)-β-D-Glc pNAc-(1→ The same structure has been reported previously for the O-polysaccharides of P. mirabilis CCUG 10701 (O74) and P. mirabilis 41/57 (O23), except that they contain O-acetyl groups in non-stoichiometric quantities. Serological studies showed the antigenic identity of the three strains and their close serological relatedness to P. vulgaris 44/57. Based on the OPS structures and serological data, it is suggested to classify P. mirabilis 41/57, TG 115, and CCUG 10701 into one subgroup and P. mirabilis 42/57 and P. vulgaris 43/57 and 44/57 into another subgroup of the Proteus O23 serogroup. [ABSTRACT FROM AUTHOR]

Bacteria of the genus Proteus are facultative pathogens which commonly cause urinary tract infections. Based on the serological specificity of the O-chain polysaccharide of the lipopolysaccharide (O-polysaccharide, O-antigen), strains of P. mirabilis and P. vulgaris have been classified into 60 serogroups. Studies on the chemical structure and serological specificity of the O-antigens aim at the elucidation of the molecular basis and improvement of the serological classification of these bacteria. The O-polysaccharide was prepared by acetic acid degradation of the lipopolysaccharide isolated from dried bacterial mass of each strain by hot phenol/water extraction. 1H- and 13C-NMR spectroscopy was used for structural studies. Serological studies were performed with rabbit O-antisera using enzyme immunosorbent assay, passive hemolysis test, and the inhibition of reactions in these assays as well DOC-PAGE and Western blot. Four Proteus strains belonging to serogroups O17 and O35 were found to possess similar O-polysaccharide structures, in particular having the same carbohydrate backbone built up of tetrasaccharide repeating units. However, they differ in the presence or absence of additional substituents, such as phosphoethanolamine in P. mirabilis O17 and glucose in P. penneri O17, as well as in the pattern and degree of O-acetylation of various monosaccharide residues. Serological studies also showed close relationships between the O-antigens studied. Based on these data it is proposed to reclassify strain P. mirabilis PrK 61/57, formerly representing the O35 serogroup, into the serogroup O17 in the Kauffman-Perch classification system of Proteus. [ABSTRACT FROM AUTHOR]