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PubMed2005

Sugar-coated regulation of T cells.

The human gut is host to hundreds of different species of commensal bacteria that live in peaceful partnership with the host immune system. These commensal bacteria are far from neutral bystanders as they are intimately involved in the development of the immune system. Reporting in this issue of Cell, Kasper and colleagues (Mazmanian et al., 2005) reveal that a bacterial polysaccharide, PSA, produced by the commensal bacterium Bacteroides fragilis directs development of the immune system of the mouse host.

Animals↗
PubMed2006

Exopolysaccharides produced by the recently described halophilic bacteria Halomonas ventosae and Halomonas anticariensis.

We studied exopolysaccharides (EPSs) produced by Halomonas ventosae and Halomonas anticariensis, two novel species of halophilic bacteria. Under optimum environmental and nutritional conditions, H. ventosae strains Al12(T) and Al16 excreted 28.35 mg and 28.95 mg of EPS per 100 ml of culture medium (34.55 and 38.6 mg of EPS per gram of dry cell weight) respectively. The molecular masses of the polymers were about 50 kDa and their main components were glucose, mannose and galactose. They had high protein fractions and showed emulsifying activity on several hydrophobic substrates. Under optimum environmental and nutritional conditions, H. anticariensis strains FP35(T) and FP36 excreted about 29.65 and 49.95 mg of EPS per 100 ml of culture medium (43.6 and 50.95 mg of EPS per gram of dry cell weight) respectively. The molecular masses of the polymers were about 20 and 46 kDa respectively and were composed mainly of glucose, mannose and galacturonic acid. All EPSs produced solutions of low viscosity and pseudoplastic behaviour. They also had a high capacity for binding cations and incorporated considerable quantities of sulphates, which is highly unusual in bacterial polysaccharides. All strains assayed formed biofilms both in polystyrene wells and borosilicate test tubes.

Biofilms↗
PubMed2003

Molecular identification of an alpha-L-rhamnosidase from Bacillus sp strain GL1 as an enzyme involved in complete metabolism of gellan.

The genes (rhaA and rhaB) for two alpha-L-rhamnosidases of Bacillus sp. strain GL1, which assimilates a bacterial polysaccharide (gellan), were cloned from a genomic DNA library of the bacterium constructed in Escherichia coli, and the nucleotide sequences of the genes were determined. Gene rhaA (2661 bp) contained an open reading frame (ORF) encoding a protein (RhaA: 886 amino acids) with a molecular weight (MW) of 98280. Gene rhaB (2871 bp) contained an ORF encoding a protein (RhaB: 956 amino acids) with a MW of 106049. RhaA exhibited significant identity (41%) with alpha-L-rhamnosidase of Clostridium stercorarium, while RhaB showed slight homology with enzymes from other sources. An overexpression system for the two enzymes was constructed in E. coli, and the enzymes were purified and characterized. Both RhaA and RhaB were highly specific for rhamnosyl saccharides, including gellan disaccharide (rhamnosyl glucose) and naringin, and released rhamnose from substrates most efficiently at pH 6.5-7.0 and 40 degrees C. Bacillus sp. strain GL1 cells grown in a gellan medium produced only RhaB, indicating that RhaB plays a crucial role in the complete metabolism of gellan.

Amino Acid Sequence↗
PubMed1991

X-ray and computer modeling studies on gellan-related polymers: molecular structures of welan, S-657, and rhamsan.

The primary structures of the four bacterial polysaccharides gellan, welan, S-657, and rhamsan are the same with respect to their backbones, but have different side-chains. This difference has a profound influence on their behavior in aqueous media. Solutions of gellan gum form stable aqueous gels under appropriate ionic conditions. By contrast, welan, S-657, and rhamsan do not gel but give very viscous solutions over a wide range of thermal, pH, and salt conditions. X-Ray fiber diffraction analysis and computer modeling of these branched polysaccharides demonstrate that they all have the same half-staggered, double-helical conformations as in the unbranched gellan, suggesting, therefore, that the side chains are responsible for diminishing gelling behavior. Depending on the size and location, the side chains shield the carboxylate groups to varying degrees; this shielding is substantial in welan and S-657, but less in rhamsan. In all cases, side-chain-main-chain interactions within the double helix prevent the carboxylate-mediated aggregation of double helices that is necessary for the gelation.

Carbohydrate Conformation↗
PubMed2001

Computer-assisted structural analysis of regular glycopolymers on the basis of 13C NMR data.

A computer-assisted approach to the prediction of the primary structures of regular glycopolymers is described. The analysis is based on comparing the calculated 13C NMR spectra of all the possible structures of the repeating unit (for the given monomeric composition) to an experimental 13C NMR spectrum. The spectra generation is based on the spectral database containing information on the 13C chemical shifts of monomers, di- and trimeric fragments. If the required data are missing from this database, the special database for average glycosylation effects is used. The analysis reveals those structures with the calculated 13C NMR spectrum most close to observed. The structures of repeating units of any topology containing up to six residues linked by glycosidic, amidic or phospho-diester bridges can be predicted. Unambiguous selection of the proper structure from the output list of possible structures may require additional experimental data. Testing the created program and databases on bacterial polysaccharides and their derivatives containing up to three non-sugar residues (alditols, amino acids, phosphate groups etc.) per repeating unit revealed the good convergence of prediction with independently obtained structural data.

Algorithms↗
PubMed2001

Synthesis and NMR spectroscopy of nine stereoisomeric 5,7-diacetamido-3,5,7,9-tetradeoxynon-2-ulosonic acids.

Derivatives of 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acids are essential constituents of some bacterial polysaccharides and glycoproteins. In order to establish reliably the configuration of the natural sugars, nine stereoisomeric 5,7-diacetamido-3,5,7,9-tetradeoxynon-2-ulosonic acids were synthesized, including di-N-acetyl-legionaminic and -pseudaminic acids (the D-glycero-D-galacto and L-glycero-L-manno isomers, respectively) and their isomers at C-4, C-5, C-7, and C-8 having the L-glycero-D-galacto, D-glycero-D-talo, L-glycero-D-talo, D-glycero-L-altro, L-glycero-L-altro, D-glycero-L-manno, and L-glycero-L-gluco configurations. Synthesis was performed by condensation of 2,4-diacetamido-2,4,6-trideoxy-L-gulose, -D-mannose, -D-talose, and -L-allose with oxalacetic acid under basic conditions, the reaction of the last two precursors being accompanied by epimerisation at C-2. The 1H and 13C NMR data of the synthetic compounds are discussed. Acetylated methyl esters of the C-7 and C-8 isomeric nonulosonic acids were prepared and used for analysis of the side-chain conformation by NMR spectroscopy.

Gluconates↗
PubMed2003

Isolation, structural characterization, and immunological evaluation of a high-molecular-weight exopolysaccharide from Staphylococcus aureus.

Colonization of implanted medical devices by coagulase-negative staphylococci such as Staphylococcus epidermidis is mediated by the bacterial polysaccharide intercellular adhesin (PIA), a polymer of beta-(1-->6)-linked glucosamine substituted with N-acetyl and O-succinyl constituents. The icaADBC locus containing the biosynthetic genes for production of PIA has been identified in both S. epidermidis and S. aureus. Whereas it is clear that PIA is a constituent that contributes to the virulence of S. epidermidis, it is less clear what role PIA plays in infection with S. aureus. Recently, identification of a novel polysaccharide antigen from S. aureus termed poly N-succinyl beta-(1-->6)-glucosamine (PNSG) has been reported. This polymer was composed of the same glycan backbone as PIA but was reported to contain a high proportion of N-succinylation rather than acetylation. We have isolated a glucosamine-containing exopolysaccharide from the constitutive over-producing MN8m strain of S. aureus in order to prepare polysaccharide-protein conjugate vaccines. In this report we demonstrate that MN8m produced a high-molecular-weight (>300,000 Da) polymer of beta-(1-->6)-linked glucosamine containing 45-60% N-acetyl, and a small amount of O-succinyl (approx 10% mole ratio to monosaccharide units). By detailed NMR analyses of polysaccharide preparations, we show that the previous identification of N-succinyl was an analytical artifact. The exopolysaccharide we have isolated is active in in vitro hemagglutination assays and is immunogenic in mice when coupled to a protein carrier. We therefore conclude that S. aureus strain MN8m produces a polymer that is chemically and biologically closely related to the PIA produced by S. epidermidis.

Animals↗
PubMed1997

Effect of deacetylation on the synergistic interaction of acetan with locust bean gum or konjac mannan.

It has been discovered that deacetylation of the bacterial polysaccharide acetan promotes synergistic interactions with either locust bean gum (LBG) or konjac mannan (KM). Acetan is similar in structure to xanthan, and adopts a similar 5-fold conformation in the solid state. Like xanthan, it shows a thermally reversible order (helix)-disorder (coil) transition in solution. Both polymers have a cellulosic backbone with charged (anionic) sidechains attached at O-3 of alternate glucosyl residues, but the sidechains in acetan are longer (pentasaccharide rather than trisaccharide) and do not contain pyruvic substituents. Acetan has two sites of acetylation, one at O-6 of the inner mannosyl residue of the carbohydrate sidechains (as in xanthan) and the other on the polymer backbone (believed to be at O-6 of the branched glucosyl residues). Solutions of acetan or deacetylated acetan were equilibrated against 10 mM potassium chloride (to stabilise the ordered conformation) and were mixed (at 25 degrees C) with solutions of LBG or KM, also equilibrated against 10 mM potassium chloride. Unlike xanthan, native acetan showed no evidence of synergistic interaction with either LBG or KM. After deacetylation, however, large enhancements were observed in dilute-solution viscosity, and thermoreversible gels were formed at higher concentrations. With KM as co-synergist, gel melting was accompanied by an intense endotherm in differential scanning calorimetry. The magnitude of this endotherm increased with storage time at 25 degrees C, reaching a final value of delta H approximately 15.9 J/g (in comparison with delta H approximately 5.0 J/g for the order-disorder transition of deacetylated acetan alone). It is suggested that interaction occurs by formation of heterotypic junctions between the acetan backbone and unsubstituted regions of the plant polysaccharide, and that the acetate groups on native acetan promote solubility and hence inhibit association.

Acetobacter↗
PubMed2001

Elongation of alternating alpha 2,8/2,9 polysialic acid by the Escherichia coli K92 polysialyltransferase.

We have chosen E. coli K92, which produces the alternating structure alpha(2-8)neuNAc alpha(2-9)neuNAc as a model system for studying bacterial polysaccharide biosynthesis. We have shown that the polysialyltransferase encoded by the K92 neuS gene can synthesize both alpha(2-8) and alpha(2-9) neuNAc linkages in vivo by 13C-nuclear magnetic resonance analysis of polysaccharide isolated from a heterologous strain containing the K92 neuS gene. The K92 polysialyltransferase is associated with the membrane in lysates of cells harboring the neuS gene in expression vectors. Although the enzyme can transfer sialic acid to the nonreducing end of oligosaccharides with either linkage, it is unable to initiate chain synthesis without exogenously added polysialic acid. Thus, the polysialyltransferase encoded by neuS is not sufficient for de novo synthesis of polysaccharide but requires another membrane component for initiation. The acceptor specificity of this polysialyltransferase was studied using sialic acid oligosaccharides of various structures as exogenous acceptors. The enzyme can transfer to the nonreducing end of all bacteria polysialic acids, but has a definite preference for alpha(2-8) acceptors. Gangliosides containing neuNAc alpha(2-8)neuNAc are elongated, whereas monsialylated gangliosides are not. Disialylgangliosides are better acceptors than short oligosaccharides, suggesting a lipid-linked oligosaccharide may be preferred in the elongation reaction. These studies show that the K92 polysialyltransferase catalyzes an elongation reaction that involves transfer of sialic acid from CMP-sialic acid to the nonreducing end of two different acceptor substrates.

Amino Acid Motifs↗
PubMed1980

The microbial contribution to human faecal mass.

A method has been developed, based on techniques used for isolating bacteria from the rumen, that enables human faeces to be fractionated into three major components. The method requires repeated, vigorous agitation of a suspension of faecal solids with detergent, the use of a stomacher, and high-speed centrifugation. By this means the faecal microflora are separated from faecal dietary-fibre residues. These two components, with water-soluble material in the stool, comprise 98.3 +/- 0.9% of faecal solids. The purity of the microbial fraction was demonstrated by Gram and plant stains and by scanning electron microscopy. Microscopic counts of the bacteria in each fraction of the stool showed that the microbial fraction contained 95% of the total bacteria. Chemical analysis of the component sugars indicated 6-7% possible contamination by non-bacterial polysaccharides. The bacterial pellet was 6% nitrogen, and accounted for 60% of the total faecal nitrogen. When faeces from nine healthy subjects on a metabolically controlled British-type diet were studied, bacteria comprised 54.7 +/- 1.7% of the total solids, fibre 16.7 +/- 0.8% and soluble material 24.0 +/- 1.3%. Bacteria therefore represent a much larger proportion of the faecal mass than was previously thought.

Adult↗
PubMed1986

Acquisition of glucose by Rickettsia prowazekii through the nucleotide intermediate uridine 5'-diphosphoglucose.

The ability of Rickettsia prowazekii to transport potential sources of the glucose moiety of bacterial polysaccharides was determined. Transport was determined both by filtration assays and by centrifugation through nonaqueous layers. Uridine 5'-diphosphoglucose (UDPG) was transported, whereas glucose was not transported; the uptake of glucose phosphates, although greater than that for glucose, was markedly lower than the transport of UDPG. Furthermore, the activities of hexokinase and phosphoglucomutase, enzymes required for the metabolism of glucose and glucose 6-phosphate, were undetectable in rickettsial extracts. The uptake of UDPG had an extended time course and did not reach a plateau until 60 min. The maximum rate of uptake was 340 pmol/min per mg of protein, and the rate was half-maximal at a UDPG concentration of 220 microM. Measurement of true influx of UDPG was complicated by the low activity of this transport system and the metabolism of the UDPG. The uptake of labeled UDPG was markedly inhibited by a 10-fold excess of uridine monophosphate, uridine diphospho-N-acetylglucosamine, and uridine diphospho-N-acetylgalactosamine but not by a variety of other structurally related compounds.

Biological Transport, Active↗
PubMedDate not supplied

[The role of the hematopoietic microenvironment in the mechanism of the antiradiation action of prodigiozan].

Preliminary administration of a radioprotective dose (3 mg/kg) of the bacterial polysaccharide prodigiozan 24 hours before irradiation of (CBA X X C57B1) F1 hybrid mice by a dose of 8 Gr increased the efficacy of exogenous clone formation of intact and irradiated bone marrow significantly. The efficacy of exogenous clone formation increased by 44-82% in transplantation of intact bone marrow and by 180-220% in transplantation of irradiated marrow (as compared to the values in animals who did not receive prodigiozan). This phenomenon was evidently associated with intensified reproduction of the transplanted CFU in mice given prodigiozan, evidence of which is the quantitative dependence of the effect on the dose of the transplanted cells. A conclusion is drawn that one of the aspects of the hematopoiesis-stimulating effect of prodigiosin in an irradiated organism is linked with its effect on the elements of the hematopoietic stroma. It is suggested that agents of other classes may be characterized by a similar mechanism of action; the same effect may be produced by some other factors which reduce radioresistance.

Animals↗
PubMed1987

[Characteristics of the recovery of the erythron after blood loss in prodigiozan-stimulated mice].

The effect of bacterial polysaccharide prodigiozan on recovery after single acute blood loss (2.5 per cent of the body weight) was studied on female mice CBA x C57Bl (F1). When administered intravenously in a dose of 0.5 mg/kg to healthy animals without blood loss prodigiozan had no significant effect on erythropoiesis. Prophylactic administration of the drug 24 hours before blood loss promoted more rapid and intensive mobilization of erythropoiesis: increasing of the levels of 59Fe incorporation into the bone marrow erythroid cells and the number of erythroid cells in the spleen as compared to those in nonstimulated animals. This is probably connected with prodigiozan-mediated stimulation of stem hemopoietic cell proliferation.

Animals↗
PubMed1978

[Effect of gentamycin, decamethoxin and prodigiozan on the course and outcome of experimental pyocyanic infection in white mice].

The therapeutic and prophylactic effectiveness of gentamicin, decamethoxin and prodigiozan was determined on albino mice infected with two-fold lethal doses of the antibiotic-resistant strain of Ps. aeruginosa. Correlation between the effectiveness of the drug and the dose and time of its administration was found. Pronounced prophylactic and therapeutic effect of gentamicin in combination with decamethoxin was noted. Prodigiozan, a bacterial polysaccharide had no significant effect on the experimental pyocyanic infection of albino mice.

Animals↗
PubMed1979

[Prodigiozan aerosols in the treatment of nonspecific respiratory organ diseases in children].

High efficacy of prodigiozan, a bacterial polysaccharide used in the form of inhalation in combined therapy of 123 children at the age of 3 to 14 with nonspecific bronchopulmonary pathologic conditions was shown. The drug was administered by means of inhalators UI-I and PAI-I once every 4--5 days in the form of 0.02 per cent solution prepared in situ. The single and the course doses were 100--400 and 300--2000 micrograms respectively. The positive therapeutic effect was observed in 85.0 per cent of the patients: improvement of the patient state was registered 4--6 days earlier than that in the control group, the indices of the respiration mechanics and nonspecific immunity (titers of lysozyme and complement) also improved. The rate of viral respiratory diseases among the children treated with prodigiozan decreased 2.5 times. High efficacy and physiological character of the aerosol method for administration of prodigiozan used the the first time in pediatry provided its recommendation for wide use.

Adolescent↗
PubMed1983

[Evaluation of the functions of human neutrophils by their reaction to a bacterial stimulus].

The principle of functional loading, i.e. stimulation of the neutrophiles with enterobacterial inactivated vaccine in vitro or with bacterial polysaccharides in vivo followed by the NBT-test is recommended for adequate estimation of the neutrophile link in the system of the host defense from infections. 97 practically healthy persons were examined in Novosibirsk and Norilsk and it was shown that in the regions of the Far North there were differences among the population with respect to the initial functional activity of the neutrophiles and especially with respect to their reaction to the stimulation. The effect of neutrophile stimulation in vitro with an inactivated vaccine of Serratia marcescens was higher and more stable than that with a single inhalation of prodigiosan. The prevalence of the least pathway providing increased resistance of the phagocytosing cells to infection in the population of the Far North might be the cause of a significant risk of infectious diseases in them.

Adult↗