Yuriy A. Knirel

A computer-assisted structural analysis of regular polysaccharides on the basis of 13C-n.m.r. data.

A computerised approach to the structural analysis of unbranched regular polysaccharides is described, which is based on an evaluation of the 13C-n.m.r. spectra for all possible primary structures within the additive scheme starting from the chemical shifts of the 13C resonances of the constituent monosaccharides and the average values of the glycosylation effects. The analysis reveals a structure (or structures), the evaluated spectrum of which resembles most closely that observed. The approach has been verified by using a series of bacterial polysaccharides of known structure and, in combination with methylation analysis data, for the determination of the presently unknown structures of the O-specific polysaccharides from Salmonella arizonae O59 and O63, and Proteus hauseri O19.

Structure and genetics of Shigella O antigens

This review covers the O antigens of the 46 serotypes of Shigella, but those of most Shigella flexneri are variants of one basic structure, leaving 34 Shigella distinct O antigens to review, together with their gene clusters. Several of the structures and gene clusters are reported for the first time and this is the first such group for which structures and DNA sequences have been determined for all O antigens. Shigella strains are in effect Escherichia coli with a specific mode of pathogenicity, and 18 of the 34 O antigens are also found in traditional E. coli. Three are very similar to E. coli O antigens and 13 are unique to Shigella strains. The O antigen of Shigella sonnei is quite atypical for E. coli and is thought to have transferred from Plesiomonas. The other 12 O antigens unique to Shigella strains have structures that are typical of E. coli, but there are considerably more anomalies in their gene clusters, probably reflecting recent modification of the structures. Having the complete set of structures and genes opens the way for experimental studies on the role of this diversity in pathogenicity.

Structure of the polysaccharide chains of Pseudomonas pseudomallei lipopolysaccharides

The pathogenic bacterium Pseudomonas pseudomallei strain 57576 produces two partially O-acetylated O-antigenic polysaccharides (PS-I and PS-II). Methylation analysis and 1H and 13C NMR spectroscopy, including NOE experiments, showed PS-I to have the structure [formula: see text] and PS-II to have the structure [formula: see text] where 6dmanHep is the unusual higher sugar 6-deoxy-D-manno-heptose. PS-II is produced also by P. pseudomallei strains 100 and 110, and PS-I and O-deacetylated PS-II by strain 97.

A Single Nucleotide Exchange in the wzy Gene Is Responsible for the Semirough O6 Lipopolysaccharide Phenotype and Serum Sensitivity of Escherichia coli Strain Nissle 1917

Structural analysis of lipopolysaccharide (LPS) isolated from semirough, serum-sensitive Escherichia coli strain Nissle 1917 (DSM 6601, serotype O6:K5:H1) revealed that this strains LPS contains a bisphosphorylated hexaacyl lipid A and a tetradecasaccharide consisting of one E. coli O6 antigen repeating unit attached to the R1-type core. Configuration of the GlcNAc glycosidic linkage between O-antigen oligosaccharide and core (beta) differs from that interlinking the repeating units in the E. coli O6 antigen polysaccharide (alpha). The wa(*) and wb(*) gene clusters of strain Nissle 1917, required for LPS core and O6 repeating unit biosyntheses, were subcloned and sequenced. The DNA sequence of the wa(*) determinant (11.8 kb) shows 97% identity to other R1 core type-specific wa(*) gene clusters. The DNA sequence of the wb(*) gene cluster (11 kb) exhibits no homology to known DNA sequences except manC and manB. Comparison of the genetic structures of the wb(*)(O6) (wb(*) from serotype O6) determinants of strain Nissle 1917 and of smooth and serum-resistant uropathogenic E. coli O6 strain 536 demonstrated that the putative open reading frame encoding the O-antigen polymerase Wzy of strain Nissle 1917 was truncated due to a point mutation. Complementation with a functional wzy copy of E. coli strain 536 confirmed that the semirough phenotype of strain Nissle 1917 is due to the nonfunctional wzy gene. Expression of a functional wzy gene in E. coli strain Nissle 1917 increased its ability to withstand antibacterial defense mechanisms of blood serum. These results underline the importance of LPS for serum resistance or sensitivity of E. coli.

A lectin S-domain receptor kinase mediates lipopolysaccharide sensing in Arabidopsis thaliana

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.

5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acids in bacterial glycopolymers: chemistry and biochemistry.

Publisher Summary This chapter provides an overview of the chemistry of 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acids in bacterial glycopolymers. Ald-2-ulosonic acids are the important components of natural glycoconjugates. Sialic acids—namely, N - and O -acyl derivatives of 5-amino-3,5-dideoxy- D -glycero- D -galacto-non-2-ulosonic acid (neuraminic acid, Neu), generally occur in glycoconjugates of vertebrates and play a significant role in their recognition, regulation, and protection. A deamino analogue of neuraminic acid—3-deoxy- D -glycero- D -galacto-non-2-ulosonic acid (Kdn)—has also been found in a variety of animal tissues. 3-Deoxy- D - manno -oct-2-ulosonic acid (Kdo) is an essential component of lipopolysaccharides (LPSs) of Gram-negative bacteria that functions to link the carbohydrate portion to the lipid moiety. In rare cases, Kdo in LPS is replaced with a 3-hydroxylated analogue— D -glycero- D -talo-oct-2-ulosonic acid. The chapter focuses on the occurrence and characterization of derivatives of 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acids and presents experimental approaches that are used to identify them and to elucidate the structures of the bacterial polysaccharides that contain the nonulosonic acids. It also presents the recent data on the biosynthesis of these sugars and discusses their role in immune recognition.

Microbial glycan microarrays define key features of host-microbial interactions

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.

Innovations in host and microbial sialic acid biosynthesis revealed by phylogenomic prediction of nonulosonic acid structure

Sialic acids (Sias) are nonulosonic acid (NulO) sugars prominently displayed on vertebrate cells and occasionally mimicked by bacterial pathogens using homologous biosynthetic pathways. It has been suggested that Sias were an animal innovation and later emerged in pathogens by convergent evolution or horizontal gene transfer. To better illuminate the evolutionary processes underlying the phenomenon of Sia molecular mimicry, we performed phylogenomic analyses of biosynthetic pathways for Sias and related higher sugars derived from 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acids. Examination of ≈1,000 sequenced microbial genomes indicated that such biosynthetic pathways are far more widely distributed than previously realized. Phylogenetic analysis, validated by targeted biochemistry, was used to predict NulO types (i.e., neuraminic, legionaminic, or pseudaminic acids) expressed by various organisms. This approach uncovered previously unreported occurrences of Sia pathways in pathogenic and symbiotic bacteria and identified at least one instance in which a human archaeal symbiont tentatively reported to express Sias in fact expressed the related pseudaminic acid structure. Evaluation of targeted phylogenies and protein domain organization revealed that the “unique” Sia biosynthetic pathway of animals was instead a much more ancient innovation. Pathway phylogenies suggest that bacterial pathogens may have acquired Sia expression via adaptation of pathways for legionaminic acid biosynthesis, one of at least 3 evolutionary paths for de novo Sia synthesis. Together, these data indicate that some of the long-standing paradigms in Sia biology should be reconsidered in a wider evolutionary context of the extended family of NulO sugars.

Conserved and variable structural features in the lipopolysaccharide of Pseudomonas aeruginosa.

The review is devoted to recent progress in the structural elucidation of the lipopolysaccharide of the bacterium Pseudomonas aeruginosa, including O-antigen biological repeats, core oligosaccharide, and lipid A. Data on biosynthesis, genetics and serology of the lipopolysaccharide isolated from various P. aeruginosa O-serogroups are discussed in relation to the chemical structures.

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.