Structure of an acidic polysaccharide from the marine bacterium Pseudoalteromonas aliena type strain KMM 3562T containing two residues of L-serine in the repeating unit

The structure of an acidic polysaccharide from Pseudoalteromonas aliena type strain KMM 3562(T) has been elucidated. The polysaccharide was studied by component analysis, (1)H and (13)C NMR spectroscopy, including 2D NMR experiments. A (1)H, (13)C band-selective constant-time heteronuclear multiple-bond connectivity experiment was used to determine amide linkages, between serine and uronic acid (UA) residues, via (3)J(H,C) correlations between Ser-alphaH and UA-C-6. It was found that the polysaccharide consists of pentasaccharide repeating units with the following structure: [carbohydrate structure]; see text.     

Structural and serological characterization of the O-chain polysaccharide of Aeromonas salmonicida strains A449, 80204 and 80204-1

The O-chain polysaccharide (O-PS) of Aeromonas salmonicida was studied by a combination of compositional, methylation, CE-ESMS and one- and two-dimensional NMR analyses. It was found to be a branched polymer of trisaccharide-repeating units composed of L-rhamnose (Rha), D-glucose (Glc), 2-acetamido-2-deoxy-D-mannose (ManNAc) and O-acetyl group (OAc) and having the following structure: CE-ESMS analysis of A. salmonicida cells from strains A449, 80204 and 80204-1 grown under different conditions confirmed that the O-PS structure was conserved. ELISA-based serological study with native LPS-specific antisera performed on the native O-PS and its O-deacetylated and periodate-oxidized derivatives confirmed the importance of the O-PS backbone structure as an immunodominant determinant.     

The structure of the capsular polysaccharide of Shewanella oneidensis strain MR-4

Capsular polysaccharides were extracted from Shewanella oneidensis strain MR-4, grown on two different culture media. The polysaccharides were analyzed using 1H and 13C NMR spectroscopy, and the following structure of the repeating unit was established: [structure: see text] where the residue of 4-amino-4,6-dideoxy-D-glucose (Qui4N) was substituted with different N-acyl groups depending on the growth media. All monosaccharides are present in the pyranose form. In the PS from cells grown on enriched medium (trypticase soy broth, TSB) aerobically it was N-acylated with 3-hydroxy-3-methylbutyrate (60%) or with 3-hydroxybutyrate (40%), whereas in the PS from cells grown on minimal medium (CDM) aerobically it was acylated mostly with 3-hydroxybutyrate (>90%).     

The O-chain structure from the LPS of marine halophilic bacterium Pseudoalteromonas carrageenovora-type strain IAM 12662T

The O-chain polysaccharide of the lipopolysaccharide from the halophilic marine bacterium Pseudoalteromonas carrageenovora IAM 12662T was characterized. The structure was studied by means of chemical analysis and 2D NMR spectroscopy of the de-O-acylated lipopolysaccharide and shown to be the following:Col is colitose, 3,6-di-deoxy-L-xylo-hexose.     

Temperature-dependent variations and intraspecies diversity of the structure of the lipopolysaccharide of Yersinia pestis

Yersinia pestis spread throughout the Americas in the early 20th century, and it occurs predominantly as a single clone within this part of the world. However, within Eurasia and parts of Africa there is significant diversity among Y. pestis strains, which can be classified into different biovars (bv.) and/or subspecies (ssp.), with bv. orientalis/ssp. pestis most closely related to the American clone. To determine one aspect of the relatedness of these different Y. pestis isolates, the structure of the lipopolysaccharide (LPS) of four wild-type and one LPS-mutant Eurasian/African strains of Y. pestis was determined, evaluating effects of growth at mammalian (37 degrees C) or flea (25 degrees C) temperatures on the structure and composition of the core oligosaccharide and lipid A. In the wild-type clones of ssp. pestis, a single major core glycoform was synthesized at 37 degrees C whereas multiple core oligosaccharide glycoforms were produced at 25 degrees C. Structural differences occurred primarily in the terminal monosaccharides. Only tetraacyl lipid A was made at 37 degrees C, whereas at 25 degrees C additional pentaacyl and hexaacyl lipid A structures were produced. 4-Amino-4-deoxyarabinose levels in lipid A increased with lower growth temperatures or when bacteria were cultured in the presence of polymyxin B. In Y. pestis ssp. caucasica, the LPS core lacked D-glycero-D-manno-heptose and the content of 4-amino-4-deoxyarabinose showed no dependence on growth temperature, whereas the degree of acylation of the lipid A and the structure of the oligosaccharide core were temperature dependent. A spontaneous deep-rough LPS mutant strain possessed only a disaccharide core and a slightly variant lipid A. The diversity and differences in the structure of the Y. pestis LPS suggest important contributions of these variations to the pathogenesis of this organism, potentially related to innate and acquired immune recognition of Y. pestis and epidemiologic means to detect, classify, control and respond to Y. pestis infections.     

The structure of the O-specific polysaccharide from Ralstonia pickettii

The following structure of the Ralstonia pickettii have been determined using NMR and chemical methods: -->4)-alpha-D-Rha-(1-->4)-alpha-L-GalNAcA-(1-->3)-beta-D-BacNAc-(1-->.     

Biosynthesis of a novel 3-deoxy-D-manno-oct-2-ulosonic acid-containing outer core oligosaccharide in the lipopolysaccharide of Klebsiella pneumoniae

The core oligosaccharide region of Klebsiella pneumoniae lipopolysaccharide contains some novel features that distinguish it from the corresponding lipopolysaccharide region in other members of the Enterobacteriaceae family, such as Escherichia coli and Salmonella. The conserved Klebsiella outer core contains the unusual trisaccharide 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo)-(2,6)-GlcN-(1,4)-GalUA. In general, Kdo residues are normally found in the inner core, but in K. pneumoniae, this Kdo residue provides the ligation site for O polysaccharide. The outer core Kdo residue can also be non-stoichiometrically substituted with an l-glycero-d-manno-heptopyranose (Hep) residue, another component more frequently found in the inner core. To understand the genetics and biosynthesis of core oligosaccharide synthesis in Klebsiella, the gene products involved in the addition of the outer core GlcN (WabH), Kdo (WabI), and Hep (WabJ) residues as well as the inner core HepIII residue (WaaQ) were identified. Non-polar mutations were created in each of the genes, and the resulting mutant lipopolysaccharide was analyzed by mass spectrometry. The in vitro glycosyltransferase activity of WabI and WabH was verified. WabI transferred a Kdo residue from CMP-Kdo onto the acceptor lipopolysaccharide. The activated precursor required for GlcN addition has not been identified. However, lysates overexpressing WabH were able to transfer a GlcNAc residue from UDP-GlcNAc onto the acceptor GalUA residue in the outer core.     

Cell-surface-associated tissue transglutaminase is a target of MMP-2 proteolysis

MT1-MMP, a prototypic member of a membrane-type metalloproteinase subfamily, is an invasion promoting protease and an activator of MMP-2. In addition, MT1-MMP proteolysis regulates the functionality of cell-surface adhesion/signaling receptors including tissue transglutaminase (tTG). tTG is known to serve as an adhesion coreceptor for beta1/beta3 integrins and as an enzyme that catalyzes the cross-linking of proteins and the conjugation of polyamines to proteins. Here, we report that MMP-2, functioning in concert with MT1-MMP, hydrolyzes cell-surface-associated tTG, thereby further promoting the effect initiated by the activator of MMP-2. tTG, in return, preferentially associates with the activation intermediate of MMP-2. This event decreases the rate of MMP-2 maturation and protects tTG against proteolysis by MMP-2. Our cell culture, in vitro experiments, and in silico modeling indicate that the catalytic domain of MMP-2 directly associates with the core enzymatic domain II of tTG (the K(d) = 380 nM). The follow-up cleavage of the domain II eliminates both the receptor and the enzymatic activity of tTG. Our data illuminate the coordinated interplay involving the MT1-MMP/MMP-2 protease tandem in the regulation of the cell receptors and explain the underlying biochemical mechanisms of the extensive tTG proteolysis that exists at the normal tissue/tumor boundary. Our findings also suggest that neoplasms, which express functionally active MT1-MMP and, therefore, activate soluble MMP-2, can contribute to the degradation of tTG expressed in neighboring host cells. The loss of adhesive and enzymatic activities of tTG at the interface between tumor and normal tissue will decrease cell-matrix interactions and inhibit matrix cross-linking, causing multiple pathological alterations in host cell adhesion and locomotion.     

Nitration of protein kinase G-Iα modulates cyclic nucleotide crosstalk via phosphodiesterase 3A: Implications for acute lung injury

Phosphodiesterase 3A (PDE3A) selectively cleaves the phosphodiester bond of cAMP and is inhibited by cGMP, making it an important regulator of cAMP–cGMP signaling crosstalk in the pulmonary vasculature. In addition, the nitric oxide–cGMP axis is known to play an important role in maintaining endothelial barrier function. However, the potential role of protein kinase G-Iα (PKG-Iα) in this protective process is unresolved and was the focus of our study. We describe here a novel mechanism regulating PDE3A activity, which involves a PKG-Iα–dependent inhibitory phosphorylation of PDE3A at serine 654. We also show that this phosphorylation is critical for maintaining intracellular cAMP levels in the pulmonary endothelium and endothelial barrier integrity. In an animal model of acute lung injury (ALI) induced by challenging mice with lipopolysaccharide (LPS), an increase in PDE3 activity and a decrease in cAMP levels in lung tissue was associated with reduced PKG activity upon PKG-Iα nitration at tyrosine 247. The peroxynitrite scavenger manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin prevented this increase in PDE3 activity in LPS-exposed lungs. In addition, site-directed mutagenesis of PDE3A to replace serine 654 with alanine yielded a mutant protein that was insensitive to PKG-dependent regulation. Taken together, our data demonstrate a novel functional link between nitrosative stress induced by LPS during ALI and the downregulation of barrier-protective intracellular cAMP levels. Our data also provide new evidence that PKG-Iα is critical for endothelial barrier maintenance and that preservation of its catalytic activity may be efficacious in ALI therapy.     

Characterization of the lipopolysaccharide and beta-glucan of the fish pathogen Francisella victoria

Lipopolysaccharide (LPS) and beta-glucan from Francisella victoria, a fish pathogen and close relative of highly virulent mammal pathogen Francisella tularensis, have been analyzed using chemical and spectroscopy methods. The polysaccharide part of the LPS was found to contain a nonrepetitive sequence of 20 monosaccharides as well as alanine, 3-aminobutyric acid, and a novel branched amino acid, thus confirming F. victoria as a unique species. The structure identified composes the largest oligosaccharide elucidated by NMR so far, and was possible to solve using high field NMR with cold probe technology combined with the latest pulse sequences, including the first application of H2BC sequence to oligosaccharides. The non-phosphorylated lipid A region of the LPS was identical to that of other Francisellae, although one of the lipid A components has not been found in Francisella novicida. The heptoseless core-lipid A region of the LPS contained a linear pentasaccharide fragment identical to the corresponding part of F. tularensis and F. novicida LPSs, differing in side-chain substituents. The linkage region of the O-chain also closely resembled that of other Francisella. LPS preparation contained two characteristic glucans, previously observed as components of LPS preparations from other strains of Francisella: amylose and the unusual beta-(1-6)-glucan with (glycerol)2phosphate at the reducing end.