Complete structures of Bordetella bronchiseptica and Bordetella parapertussis lipopolysaccharides

The structures of the lipopolysaccharide (LPS) core and O antigen of Bordetella bronchiseptica and Bordetella parapertussis are known, but how these two regions are linked to each other had not been determined. We have studied LPS from several strains of these microorganisms to determine the complete carbohydrate structure of the LPS. LPS was analyzed using different chemical degradations, NMR spectroscopy, and mass spectrometry. This identified a novel pentasaccharide fragment that links the O chain to the core in all the LPS studied. In addition, although the O chain of these bacteria was reported as a homopolymer of 1,4-linked 2,3-diacetamido-2,3-dideoxy-alpha-galacturonic acid, we discovered that the polymer contains several amidated uronic acids, the number of which varies between strains. These new data describe the complete structure of the LPS carbohydrate backbone for both Bordetella species and help to explain the complex genetics of LPS biosynthesis in these bacteria.     

Evolution of the globin gene family in deuterostomes: lineage-specific patterns of diversification and attrition

In the Metazoa, globin proteins display an underlying unity in tertiary structure that belies an extraordinary diversity in primary structures, biochemical properties, and physiological functions. Phylogenetic reconstructions can reveal which of these functions represent novel, lineage-specific innovations, and which represent ancestral functions that are shared with homologous globin proteins in other eukaryotes and even prokaryotes. To date, our understanding of globin diversity in deuterostomes has been hindered by a dearth of genomic sequence data from the Ambulacraria (echinoderms + hemichordates), the sister group of chordates, and the phylum Xenacoelomorpha, which includes xenoturbellids, acoelomorphs, and nemertodermatids. Here, we report the results of a phylogenetic and comparative genomic analysis of the globin gene repertoire of deuterostomes. We first characterized the globin genes of the acorn worm, Saccoglossus kowalevskii, a representative of the phylum Hemichordata. We then integrated genomic sequence data from the acorn worm into a comprehensive analysis of conserved synteny and phylogenetic relationships among globin genes from representatives of the eight lineages that comprise the superphylum Deuterostomia. The primary aims were 1) to unravel the evolutionary history of the globin gene superfamily in deuterostomes and 2) to use the estimated phylogeny to gain insights into the functional evolution of deuterostome globins. Results of our analyses indicate that the deuterostome common ancestor possessed a repertoire of at least four distinct globin paralogs and that different subsets of these ancestral genes have been retained in each of the descendant organismal lineages. In each major deuterostome group, a different subset of ancestral precursor genes underwent lineage-specific expansions of functional diversity through repeated rounds of gene duplication and divergence. By integrating results of the phylogenetic analysis with available functional data, we discovered that circulating oxygen-transport hemoglobins evolved independently in several deuterostome lineages and that intracellular nerve globins evolved independently in chordates and acoelomorph worms.     

Characterization of the lipopolysaccharide from Pasteurella multocida Heddleston serovar 9: identification of a proposed bi-functional dTDP-3-acetamido-3,6-dideoxy-α-D-glucose biosynthesis enzyme

Pasteurella multocida strains are classified into 16 different lipopolysaccharide (LPS) serovars using the Heddleston serotyping scheme. Ongoing studies in our laboratories on the LPS aim to determine the core oligosaccharide (OS) structures expressed by each of the Heddleston type strains and identify the genes and transferases required for the biosynthesis of the serovar-specific OSs. In this study, we have determined the core OS of the LPS expressed by the Heddleston serovar 9 type strain, P2095. Structural information was established by a combination of monosaccharide and methylation analyses, nuclear magnetic resonance spectroscopy and mass spectrometry revealing the following structure: . The serovar 9 OS contains an inner core that is conserved among P. multocida strains with an elaborate outer core extension containing rhamnose (Rha), a D-glycero-D-manno isomer of heptose, and the unusual deoxyamino sugar, 3-acetamido-3,6-dideoxy-α-D-glucose (Qui3NAc). Genetic analyses of the LPS outer core biosynthesis locus revealed that in addition to the glycosyltransferases predicted to transfer the sugars to the nascent LPS molecule, the locus also contained the complete set of genes required for the biosynthesis of the nucleotide sugar donors dTDP-Rha and dTDP-Qui3NAc. One of the genes identified as part of the dTDP-Qui3NAc biosynthesis pathway, qdtD, encodes a proposed bi-functional enzyme with N-terminal amino acid identity to dTDP-4-oxo-6-deoxy-D-glucose-3,4-oxoisomerase and C-terminal amino acid identity to dTDP-3-oxo-6-deoxy-α-D-glucose transacetylase.     

Diversity in the Protein N-Glycosylation Pathways Within the Campylobacter Genus

The foodborne bacterial pathogen, Campylobacter jejuni, possesses an N-linked protein glycosylation (pgl) pathway involved in adding conserved heptasaccharides to asparagine-containing motifs of >60 proteins, and releasing the same glycan into its periplasm as free oligosaccharides. In this study, comparative genomics of all 30 fully sequenced Campylobacter taxa revealed conserved pgl gene clusters in all but one species. Structural, phylogenetic and immunological studies showed that the N-glycosylation systems can be divided into two major groups. Group I includes all thermotolerant taxa, capable of growth at the higher body temperatures of birds, and produce the C. jejuni-like glycans. Within group I, the niche-adapted C. lari subgroup contain the smallest genomes among the epsilonproteobacteria, and are unable to glucosylate their pgl pathway glycans potentially reminiscent of the glucosyltransferase regression observed in the O-glycosylation system of Neisseria species. The nonthermotolerant Campylobacters, which inhabit a variety of hosts and niches, comprise group II and produce an unexpected diversity of N-glycan structures varying in length and composition. This includes the human gut commensal, C. hominis, which produces at least four different N-glycan structures, akin to the surface carbohydrate diversity observed in the well-studied commensal, Bacteroides. Both group I and II glycans are immunogenic and cell surface exposed, making these structures attractive targets for vaccine design and diagnostics.In eukaryotes, glycosylated proteins are ubiquitous components of extracellular matrices and cellular surfaces. Their oligosaccharide moieties are implicated in a wide variety of essential cell-cell and cell-matrix processes ranging from immune recognition to cancer development. The first general protein glycosylation (pgl)¹ pathway was discovered in the epsilonproteobacterium Campylobacter jejuni (1). The organism transfers a conserved heptasaccharide en bloc to asparagine residues within the sequon D/E- X₁-N-X₂-S/T (X₁, X₂ ≠ P) of >60 glycoproteins (2–4). Furthermore, the pathway can be functionally transferred into Escherichia coli, and the oligosaccharyltransferase (OTase), PglB, is capable of adding foreign sugars to acceptor proteins (5–7). C. jejuni PglB also possesses hydrolase activity, influenced by the cellular growth phase and osmotic environment, releasing free oligosaccharides (fOS) into the periplasmic space in a 10:1 ratio relative to the amount of heptasaccharide N-linked to protein (8, 9).The C. jejuni N-linked heptasaccharide is conserved in structure in both C. jejuni and C. coli, the two most commonly isolated pathogenic Campylobacter species and major causes of human enteritis worldwide (10, 11). All campylobacters, but one, possess conserved pgl genes required for N-linked protein glycosylation ((12) and this study). This post-translational modification in C. jejuni influences DNA uptake, chicken and mouse colonization, epithelial cell adherence and invasion, recognition by human sera, and binding to the macrophage galactose lectin (MGL) receptor on dendritic cells (2, 13–17). Several Campylobacter species have now been recognized as emerging pathogens and causative agents of human gastroenteritis (e.g. C. upsaliensis and C. hyointestinalis), gingivitis, periodontitis, and human abortions (e.g. C. rectus, C. concisus, C. gracilis, C. showae, and C. upsaliensis) and inflammatory bowel disease in children (e.g. C. concisus) (18). Other species cause venereal disease and infertility in cattle (C. fetus subsp. venerealis; Cfv) or abortions in sheep (C. fetus subsp. fetus; Cff) (19).In this study, we used phylogenetic, immunological, structural and glycoproteomic studies to compare the N-glycosylation systems of 29 Campylobacter species and identified unexpected variations. Thus, although the pathway is a common feature within this genus, variability in the N-glycans and fOS at the species level suggests that each species possess a unique array of glycosyltransferases, which correlate with their phylogenetic relatedness.     

Clostridium difficile cell-surface polysaccharides composed of pentaglycosyl and hexaglycosyl phosphate repeating units

Clostridium difficile is a Gram-positive bacterium that is known to be a cause of enteric diseases in humans. It is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis. Recently, large outbreaks of C. difficile-associated diarrhea have been reported internationally, and there have been reports of increases in severe disease, mortality and relapse rates. At the moment, there is no vaccine against C. difficile, and the medical prevention of C. difficile infection is mostly based on the prophylactic use of antibiotics; however, this has led to an increase in the incidence of the disease. Here, we describe the chemical structure of C. difficile cell-surface polysaccharides. The polysaccharides of three C. difficile strains were structurally analyzed; ribotype 027 (North American pulsotype 1) strain was observed to express two polysaccharides, one was composed of a branched pentaglycosyl phosphate repeating unit: [-->4)-alpha-l-Rhap-(1-->3)-beta-D-Glcp-(1-->4)-[alpha-l-Rhap-(1-->3]-alpha-D-Glcp-(1-->2)-alpha-D-Glcp-(1-->P] and the other was composed of a hexaglycosyl phosphate repeating unit: [-->6)-beta-D-Glcp-(1-->3)-beta-D-GalpNAc-(1-->4)-alpha-D-Glcp-(1-->4)-[beta-D-Glcp-(1-->]-beta-D-GalpNAc-(1-->3)-alpha-D-Manp-(1-->P]. The latter polysaccharide was also observed to be produced by strains MOH900 and MOH718. The results described here represent the first literature report describing the covalent chemical structures of C. difficile cell-surface polysaccharides, of which PS-II appears to be a regular C. difficile antigen. These C. difficile teichoic-acid-like polysaccharides will be tested as immunogens in vaccine preparations in a rat and horse model.     

A polysaccharide of Alloiococcus otitidis, a new pathogen of otitis media: chemical structure and synthesis of a neoglycoconjugate thereof

Alloiococcus otitidis is a recently discovered Gram-positive bacterium that has been linked with otitis media (middle ear infections). In this study, we describe the structure of a novel capsular polysaccharide (PS) expressed by the type-strain of A. otitidis, ATCC 51267, and the synthesis of a glycoconjugate composed of the capsule PS and bovine serum albumin (BSA). The capsule PS of A. otitidis type-strain was determined to be a repeating trisaccharide composed of 3-substituted N-acetyl-D-glucosamine (GlcpNAc), 6-substituted N-acetyl-D-galactosamine (GalpNAc), and 4-substituted D-glucuronic acid (GlcpA), of which the majority was amidically decorated with L-glutamic acid (Glu): {-->6)-beta-GalpNAc-(1-->4)-[Glup-->6]-beta-GlcpA-(1-->3)-beta-GlcpNAc-(1}n. Monomeric analysis performed on other A. otitidis strains revealed that similar components were variably expressed, but Glu appeared to be a regular constituent in all the strains examined. Due to the suitable presence of GlcpA and Glu, our approach for glycoconjugate synthesis employed a carbodiimide-based strategy with activation of available carboxyl groups by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), which afforded direct coupling between the capsule PS and BSA. Analysis by mass spectrometry indicated that this A. otitidis capsule PS-BSA conjugate was composed of BSA units that carried up to seven capsule PSs. This work represents the first report in the literature describing an A. otitidis cell-surface carbohydrate and the synthesis of a glycoconjugate preparation thereof. Presently, we are formulating plans to immunologically evaluate this A. otitidis glycoconjugate vaccine in animals.     

The structure of the core region of the lipopolysaccharide from Klebsiella pneumoniae O3. 3-deoxy-alpha-D-manno-octulosonic acid (alpha-Kdo) residue in the outer part of the core, a common structural element of Klebsiella pneumoniae O1, O2, O3, O4, O5, O8, and O12 lipopolysaccharides.

The structure of lipid A-core region of the lipopolysaccharide (LPS) from Klebsiella pneumoniae serotype O3 was determined using NMR, MS and chemical analysis of the oligosaccharides, obtained by mild acid hydrolysis, alkaline deacylation, and deamination of the LPS: [carbohydrate structure see text] where P is H or alpha-Hep; J is H or beta-GalA; R is H or P (in the deacylated oligosaccharides).Screening of the LPS from K. pneumoniae O1, O2, O4, O5, O8, and O12 using deamination showed that they also contain alpha-Hep-(1-->4)-alpha-Kdo-(2-->6)-GlcN and alpha-Kdo-(2-->6)-GlcN fragments.     

Low-Molecular-Weight Glycoprotein from Cattle Blood Serum: Structure and Properties

The amino acid composition, structure, and physicochemical properties of a low-molecular-weight glycoprotein from cattle blood serum (SGP) were studied. The content of carbohydrates (represented by mannose-rich oligosaccharides) amounted to 45–50 wt %. The value of the specific partial heat of SGP, measured by differential scanning calorimetry (DSC), equaled 1.8 J/(g K), which is characteristic of unfolded proteins. Circular dichroic (CD) spectra of SGP led us to conclude that it is not highly structured and that it occurs in the shape of a statistical globule. The protein was deglycated using anhydrous trifluoromethane sulfonate (TFMS), after which its amino acid composition and the sequence of a fragment were determined. The results indicate that SGP is a protein not studied previously.     

On Photosynthesis and Organic Carbon, Carbon Dioxide, and Oxygen Flows in the Ocean

The modern concept of photosynthesis as a mechanism for utilizing the energy of solar radiation is used as the basis for assessing the scale of photosynthetic production of initial organic matter in the ocean (primary biological production), its destruction, the carbon and carbon dioxide cycles (flows) involved in this process, and the size of oil- and gas-bearing hydrocarbonaceous formations originating in sedimentary deposits.     

Specific binding of (+)-3H-SKF 10047 by mouse splenocytes

Stereoselective, saturable, reversible and temperature-dependent binding of ligand of sigma opioid receptors (+)-3H-SKF 10047 to mouse splenocytes has been demonstrated. The estimated Kd value of this interaction (20-100 nM) was similar to that of (+)-3H-SKF 10047-sigma receptor complex. The binding capacity was 100 molecules per cell. A comparatively low affinity, non-stereoselective and temperature-independent binding of (+)-3H-SKF 10047 to mouse splenocytes has also been found.