The structure of the O-specific polysaccharide from Salmonella cerro (serogroup K, O:6,14,18)

The following structure of the Salmonella cerro LPS O-chain repeating unit has been determined using NMR and chemical methods: -->4)-alpha-D-Man(1-->2)-alpha-D-Man(1-->2)-beta-D-Man(1-->3)-alpha-D-GalNAc-(1-->.     

Smith degradation of the O-antigenic polysaccharide of Salmonella Dakar: structural studies of the products

Two different oligosaccharides were obtained from the Smith degradation of the O-polysaccharide isolated from the lipopolysaccharide of Salmonella Dakar. The structures of these oligosaccharides were investigated by chemical analysis, NMR spectroscopy and MALDI-TOF mass spectrometry. The following structures of these products were determined: alpha-D-GalpNAc-(1-->4)-alpha-D-Quip3NAc-(1-->3)-alpha-L-Rhap-(1-->2)-threitol and [FORMULA: SEE TEXT] where Quip3NAc is 3-acetamido-3,6-dideoxyglucose. The reaction products confirmed the structure of the repeating unit of the Salmonella Dakar O-polysaccharide reported previously [Kumirska, J.; Szafranek, J.; Czerwicka, M.; Paszkiewicz, M.; Dziadziuszko, H.; Kunikowska, D.; Stepnowski, P. Carbohydr. Res. 2007,342, 2138-2143].     

Structural determination of the O-antigenic polysaccharide from the verocytotoxin-producing Escherichia coli O176

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O176 has been determined. Component analysis together with 1H and 13C NMR spectroscopy was employed to elucidate the structure. Inter-residue correlations were determined by 1H, 1H NOESY and 1H, 13C heteronuclear multiple-bond correlation experiments. The PS is composed of tetrasaccharide repeating units with the following structure: [Formula: see text] Cross-peaks of low intensity from alpha-linked mannopyranosyl residues were present in the 1H, 1H TOCSY NMR spectra and further analysis of these showed that they originate from the terminal part of the polysaccharide. Consequently, the biological repeating unit has a 3-substituted N-acetyl-d-galactosamine residue at its reducing end. The repeating unit of the E. coli O176 O-antigen is similar to those from E. coli O17 and O77, thereby explaining the reported cross-reactivities between the strains, and identical to that of Salmonella cerro (O:6, 14, 18).     

Characterisation of the Salmonella O:8 antigen in the O-chain of the lipopolysaccharide produced by Salmonella virginia O:8.

The antigenic O-polysaccharide of the lipopolysaccharide produced by Salmonella virginia (O:8), analyzed by methylation, partial acid hydrolysis, and one- and two-dimensional nuclear magnetic resonance methods, was found to be a polymer of a repeating pentasaccharide unit composed of D-mannose, D-galactose, L-rhamnose, D-abequose, and O-acetyl (2:1:1:1:1.3) and having the following structure: [formula; see text] The disaccharide structure alpha-D-Abep-(1----3)-L-Rhap was identified as the Salmonella O:8 antigenic factor epitope, since the removal of alpha-D-Abep residues from the O-polysaccharide left a residual tetrasaccharide repeating unit backbone that did not show reaction with Salmonella type O:8 factor antiserum.     

Binding-induced stabilization and assembly of the phage P22 tail accessory factor gp4

To infect and replicate, bacteriophage P22 injects its 43 kbp genome across the cell wall of Salmonella enterica serovar Typhimurium. The attachment of phage P22 to the host cell as well as the injection of the viral DNA into the host is mediated by the virion's tail complex. This 2.8 MDa molecular machine is formed by five proteins, which include the portal protein gp1, the adhesion tailspike protein gp9, and three tail accessory factors: gp4, gp10, gp26. We have isolated the tail accessory factor gp4 and characterized its structure and binding interactions with portal protein. Interestingly, gp4 exists in solution as a monomer, which displays an exceedingly low structural stability (Tm 34 degrees C). Unfolded gp4 is prone to aggregation within a narrow range of temperatures both in vitro and in Salmonella extracts. In the virion the thermal unfolding of gp4 is prevented by the interaction with the dodecameric portal protein, which stabilizes the structure of gp4 and suppresses unfolded gp4 from irreversibly aggregating in the Salmonella milieu. The structural stabilization of gp4 is accompanied by the concomitant oligomerization of the protein to form a ring of 12 subunits bound to the lower end of the portal ring. The interaction of gp4 with portal protein is complex and likely involves the distinct binding of two non-equivalent sets of six gp4 proteins. Binding of the first set of six gp4 equivalents to dodecameric portal protein yields a gp(1)12:gp(4)6 assembly intermediate, which is stably populated at 30 degrees C and can be resolved by native gel electrophoresis. The final product of the assembly reaction is a bi-dodecameric gp(1)12:gp(4)12 complex, which appears hollow by electron microscopy, suggesting that gp4 does not physically plug the DNA entry/exit channel, but acts as a structural adaptor for the other tail accessory factors: gp10 and gp26.     

Structure of the O-polysaccharide of Salmonella enterica O41

An O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Salmonella enterica O41, and the following structure of the O-unit was determined by chemical analyses along with 1D and 2D ¹H and ¹³C NMR spectroscopy:→2)-β-d-Manp-(1→4)-α-d-Glcp-(1→3)-α-l-QuipNAc-(1→3)-α-d-GlcpNAc-(1→where QuiNAc stands for 2-acetamido-2,6-dideoxyglucose. The structure established is in agreement with the O-antigen gene cluster of S. enterica O41 and tentative assignment of the gene functions reported earlier.     

Formation of a novel surface structure encoded by Salmonella Pathogenicity Island 2

The type III secretion system (T3SS) encoded by Salmonella Pathogenicity Island 2 (SPI2) is essential for virulence and intracellular proliferation of Salmonella enterica. We have previously identified SPI2-encoded proteins that are secreted and function as a translocon for the injection of effector proteins. Here, we describe the formation of a novel SPI2-dependent appendage structure in vitro as well as on the surface of bacteria that reside inside a vacuole of infected host cells. In contrast to the T3SS of other pathogens, the translocon encoded by SPI2 is only present singly or in few copies at one pole of the bacterial cell. Under in vitro conditions, appendages are composed of a filamentous needle-like structure with a diameter of 10 nm that was sheathed with secreted protein. The formation of the appendage in vitro is dependent on acidic media conditions. We analyzed SPI2-encoded appendages in infected cells and observed that acidic vacuolar pH was not required for induction of SPI2 gene expression, but was essential for the assembly of these structures and their function as translocon for delivery of effector proteins.     

The O-antigen of Salmonella enterica O13 and its relation to the O-antigen of Escherichia coli O127

The following structure of the O-polysaccharide (O-antigen) of Salmonella enterica O13 was established by chemical analyses along with 2D ¹H and ¹³C NMR spectroscopy:→2)-α-l-Fucp-(1→2)-β-d-Galp-(1→3)-α-d-GalpNAc-(1→3)-α-d-GlcpNAc-(1→The O-antigen of S. enterica O13 was found to be closely related to that of Escherichia coli O127, which differs only in the presence of a GalNAc residue in place of the GlcNAc residue and O-acetylation. The location of the O-acetyl groups in the E. coli O127 polysaccharide was determined. The structures of the O-polysaccharides studied are in agreement with the DNA sequence of the O-antigen gene clusters of S. enterica O13 and E. coli O127 reported earlier.     

Bafoudiosbulbins A, and B, two anti-salmonellal clerodane diterpenoids from Dioscorea bulbifera L. var sativa

Two clerodane diterpenoids, Bafoudiosbulbins A 1, and B 2, together with five known compounds: tetracosanoic acid, 1-(tetracosanoyl)-glycerol, trans-tetracosanylferulate, beta-sitosterol and 3-O-beta-D-glucopyranosyl-beta-sitosterol were isolated from the tubers of Dioscorea bulbifera L. var sativa. Their structures were established by spectroscopic methods (1D and 2D-NMR, MS) and X-ray crystallographic diffraction analysis of compound 1. The CH2Cl2-soluble portion of the crude extract and the two clerodanes were screened for anti-bacterial activity using both agar diffusion and broth dilution techniques against Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Salmonella typhi, Salmonella paratyphi A and Salmonella paratyphi B. They both showed significant activities against P. aeruginosa, S. typhi, S. paratyphi A and S. paratyphi B.     

Structure of the polysaccharide O-antigen of Salmonella riogrande O:40 (group R) related to blood group A activity.

The structure of the Salmonella O:40 (Group R) antigen was determined from an analysis of the antigenic O-polysaccharide component of the lipopolysaccharide produced by Salmonella riogrande O:40. Using 1H- and 13C-NMR spectroscopy, methylation analysis, and periodate degradation methods, the O-polysaccharide was found to be a high molecular weight branched polymer of repeating pentasaccharide units having the structure: [formula: see text] The reported human blood group A activity was concluded to reside in an epitope of a terminal trisaccharide portion of the O-chain involving alpha-D-GalpNAc and beta-D-GlcpNAc residues linked (1----3) and (1----2), respectively, to beta-D-Manp branched residues in which the alpha-D-GalpNAc residue would appear to be the critical antigenic factor recognized by polyclonal blood group A antisera.     

Role of histamine and acid back-diffusion in modulation of gastric microvascular permeability and hemorrhagic ulcers in Salmonella typhimurium-infected rats

Documentation concerning the pathogenesis of gastric hemorrhagic ulcer in Salmonella typhimurium (Salmonella typhi)-infective disease is lacking. This research first proposed that alterations of mast cell histamine release, gastric acid back-diffusion and mucosal microvascular permeability are important in modulating gastric ulcer and hemorrhage in Salmonella typhi-infected rats. Additionally, effects of several histamine-related drugs on this ulcer model were evaluated. Male Wistar rats were deprived food for 36 h. Live cultures of Salmonella typhi (OU 5045, 1 x 10(10) CFU in 1.0 mL of sterilized phosphate buffer saline) were challenged, intrajejunally to rats just before withdrawal of food. Control rats received the same volume of sterilized vehicle only. Rat stomachs were irrigated for 3 h with either normal saline or simulated gastric juice. Gastric acid back-diffusion, mucosal histamine concentration, microvascular permeability as well as luminal hemoglobin content and ulcer areas were determined. Severe gastric hemorrhage and mucosal ulcerations, particularly in acidic stomachs, were observed in Salmonella typhi-infected rats. A positive correlation of histamine to gastric hemorrhage and ulcer was found in those rats with Salmonella typhi-infection. This hemorrhagic ulcer in Salmonella typhi-infected rats was effectively ameliorated by intraperitoneal ketotifen, diphenhydramine and ranitidine but was worsen by exogenous histamine or diamine oxidase. In conclusion, enhancement of acid back-diffusion, mast cell histamine release and microvascular permeability is important in modulating gastric hemorrhage and ulcer in Salmonella typhi-infected rats.     

The structure of the core region of the lipopolysaccharide from Geobacter sulfurreducens

The structure of the core part of the LPS from Geobacter sulfurreducens was analysed. The LPS contained no O-specific polysaccharide (O-side chain) and upon mild hydrolysis gave a core oligosaccharide, which was isolated by gel chromatography. It was studied by chemical methods, NMR and mass spectrometry, and the following structure was proposed. [carbohydrate structure: see text] where Q = 3-O-Me-alpha-L-QuiNAc-(1-->or H (approximately 3:2).     

Isolation of a mutant from Salmonella typhimurium producing acyl-deficient lipopolysaccharides

The present paper describes the isolation and characterization of a mutant (mutant Ts7) of Salmonella typhimurium that is conditionally defective in the incorporation of dodecanoic and tetradecanoic acid into lipopolysaccharide precursor structures. Enrichment of mutant Ts7 was achieved by free-flow electrophoresis and was based on a previous observation that at least some Salmonella mutants conditionally blocked in the synthesis of the 3-deoxy-D-manno-octulosonic acid (dOc1A)-lipid-A region exhibit higher electrophoretic mobilities than cells with intact dOc1A-lipid-A regions. Under nonpermissive conditions (42 degrees C) mutant Ts7 accumulates at least two incomplete dOc1A-lipid-A structures. One is made up of glucosamine, phosphate, dOc1A, and 3-hydroxytetradecanoic acid in a molar ratio 1.0:1.3:1.0:2.2 and is devoid of dodecanoic and tetradecanoic acid. The other structure has the same basic structure but contains hexadecanoic acid.     

Structural studies on the fucosamine-containing O-specific polysaccharide of Proteus vulgaris O19

The polysaccharide chain of Proteus vulgaris O19 lipopolysaccharide contains D-galactose, N-acetyl-D-glucosamine N-acetyl-D-galactosamine and N-acetyl-L-fucosamine in the ratio 1:1:1:1. The structure of the polysaccharide was established by full acid hydrolysis and methylation analysis, as well as by non-destructive methods, i.e. the computer-assisted evaluation of the 13C-NMR spectrum and computer-assisted evaluation of the specific optical rotation by Klyne's rule. The polysaccharide is regular and built up of tetrasaccharide repeating units of the following structure: ----3)-alpha-L-FucNAcp-(1----3)-beta-D-GlcNAcp-(1----3)-alph a-D-Galp- (1----4)-alpha-D-GalNAcp-(1---- The O19-antiserum cross-reacts with lipopolysaccharide from P. vulgaris O42, the structure of which is still unknown. No cross-reactions were observed with O-polysaccharides Pseudomonas aeruginosa O7 and Salmonella arizonae O59 in spite of some structural similarities.     

Studies on the hexose region of the lipopolysaccharide from a low virulence strain of Salmonella typhimurium

The structure of the hexose region of the lipopolysaccharide from M206 strain, a mutant of Salmonella typhimurium having reduced virulence, was partially determined. Immunological tests indicated cross-reactions of anti-(M206) antiserum with wild-type C5 and Ra mutant strains. Data obtained on chemical composition, periodate oxidation, acetolysis, methylation and analysis by gas chromatography/mass spectrometry show that M206 type lipopolysaccharide contains the common core polysaccharide of Salmonella which was substituted in position 4 of the subterminal glucose unit by a disaccharide: D-glucosyl 1----3 D-galactose. This substitution is probably related to the slight virulence of M206 strain.     

Antigenic bacterial polysaccharides. 24. The structure of the O-specific polysaccharide chain of Salmonella arizonae 063 (Arizona 08) lipopolysaccharide

The O-specific polysaccharide chain of the Salmonella arizonae O63 lipopolysaccharide is composed of D-glucose, D-galactose, N-acetyl-D-galactosamine, and 3-acetamido-3,6-dideoxy-D-galactose (Fuc3NAc) residues in the ratio 1:1:2:1. On the basis of methylation analysis and calculations of 13C-NMR-spectra of the polysaccharide and of the product of its selective cleavage with anhydrous hydrogen fluoride, the linear polymer lacking 3-acetamido-3,6-dideoxygalactose, it was concluded that the polysaccharide has the following structure: (Formula: see text).     

Heritability of susceptibility to Salmonella enteritidis infection in fowls and test of the role of the chromosome carrying the NRAMP1 gene

373 thirteen-week-old chicks issued from a commercial cross and 312 chickens from the L2 line were intravenously inoculated with 10⁶ Salmonella enteritidis and the numbers of Salmonella in the spleen, liver and genital organs were assessed 3 days later. Heritabilities of the number of Salmonella were estimated at 0.02 ± 0.04 and 0.05 ± 0.05 in the liver; at 0.29 ± 0.07 and 0.10 ± 0.06 in the spleen; and at 0.16 ± 0.05 and 0.11 ± 0.08 in the genital organs, in the first and second experiments, respectively. The difference between the two experiments could result from sampling variations and from differences in the genetic structure of the two populations possibly including both heterosis and additive effects as well as their interaction in the first experiment. Genetic correlations between the number of bacteria in the genital organs and liver (0.56 ± 0.58 and 0.76 ± 0.32 in the first and second experiments, respectively) and spleen (0.37 ± 0.24 and 0.79 ± 0.23) were positive. Moreover a significant within-sire effect of VIL1, a marker gene for NRAMP1, was observed in 117 progeny resulting from 25 informative matings. These results indicate that there are genetic differences in the resistance to visceral infection by S. enteritidis in these commercial egg-laying flocks, and suggest that these differences are at least partly due to genetic polymorphism in the NRAMP1 region.     

Comparative proteomic analysis of Salmonella tolerance to the biocide active agent triclosan

Concern has been expressed about the overuse of biocides in farm animal production and food industries. Biocide application can create selective pressures that lead to increased tolerance to one or more of these compounds and are concomitant with the emergence of cross-resistance to antibiotics. A triclosan sensitive Salmonella enterica serovar Typhimurium and the isogenic triclosan tolerant mutant were studied at the proteomic level in order to elucidate cellular mechanisms that facilitate biocide tolerance. 2-D differential fluorescent gel electrophoresis (DIGE) compared protein profiles of parent and mutant Salmonella, in the presence and absence of triclosan. Differentially expressed proteins were identified by mass spectrometry and divided into two groups: Group A describes proteins differentially expressed between susceptible and triclosan tolerant Salmonella and includes the known triclosan target FabI which contained a mutation at the triclosan target binding site. Group B identified proteins differentially expressed in response to triclosan exposure and defines a general cell defence network. Only four proteins were common to both groups highlighting the diverse range of pathways employed by Salmonella to counteract biocides. These data suggest that sub-lethal concentrations of triclosan induce discernible changes in the proteome of exposed Salmonella and provide insights into mechanisms of response and tolerance.     

Structural characterization of the O-polysaccharide antigen of Edwardsiella tarda MT 108

Edwardsiella tarda, a Gram-negative bacterium, is an important cause of hemorrhagic septicemia in fish and also of gastro- and extraintestinal infections in humans. The lipopolysaccharide produced by the fish pathogenic strain E. tarda MT 108 was isolated and the structure of its antigenic O-polysaccharide component determined by the application of chemical analyses, high-resolution 1D and 2D nuclear magnetic resonance spectroscopy, and mass spectrometry. The polysaccharide was found to be a polymer of a repeating pentasaccharide unit composed of 2-acetamido-2-deoxy-D-glucose (D-GlcNAc), 2-acetamido-2-deoxy-D-galactose (D-GalNAc), D-galactose (D-Gal), L-rhamnose (L-Rha), D-galacturonic acid (D-GalA) and (2S,3R)-threonine (1:1:1:1:1:1) having the structure: [structure: see text].