Chemical composition and immunological properties of glycoproteins of Sporothrix species

Glycoproteins of 11Sporothrix species were purified from their respective culture filtrates by use of DEAE-Sephadex A-50 and QAE-Sephadex A-25 column chromatography and investigated for their chemical and immunological properties. On the basis of sugar composition, the glycoproteins of the 11Sporothrix species could be divided into two groups, i.e., rhamnose containing (i.e., Rha⁺), and non rhamnose containing (i.e., Rha^–) groups. The species in the former group wereS. curviconia, S. inflata, S. schenckii andS. schenckii var. luriei, and those in the latter group wereS. cyanescens, S. foliorum, S. fungorum, S. ghanensis, S. imectorum, S. luteoalba andS. ramosissima. The glycoproteins of four of the (Rha⁺) species were relatively similar in elution patterns of DEAE-Sephadex A-50 chromatograms, sugar and amino acid compositions, serological reactivity with rabbit andS. schenckii serum and rabbit antiKlebsiella pneumoniae K47 serum, and cutaneous delayed hypersensitivity. In the case of the (Rha^–) species, the glycoproteins of five species cross-reacted with rabbit antiS. schenckii serum and all, but theS. cyanescens, glycoprotein were reactive to some degree in skin tests in sporotrichotic patients. These results strongly suggest that the chemical and immunological properties of these glycoproteins correspond with the morphological observations amongSporothrix species.     

Chemical composition and immunological properties of glycoproteins of Sporothrix species

Glycoproteins of 11Sporothrix species were purified from their respective culture filtrates by use of DEAE-Sephadex A-50 and QAE-Sephadex A-25 column chromatography and investigated for their chemical and immunological properties. On the basis of sugar composition, the glycoproteins of the 11Sporothrix species could be divided into two groups, i.e., rhamnose containing (i.e., Rha⁺), and non rhamnose containing (i.e., Rha^?) groups. The species in the former group wereS. curviconia, S. inflata, S. schenckii andS. schenckii var. luriei, and those in the latter group wereS. cyanescens, S. foliorum, S. fungorum, S. ghanensis, S. imectorum, S. luteoalba andS. ramosissima. The glycoproteins of four of the (Rha⁺) species were relatively similar in elution patterns of DEAE-Sephadex A-50 chromatograms, sugar and amino acid compositions, serological reactivity with rabbit andS. schenckii serum and rabbit antiKlebsiella pneumoniae K47 serum, and cutaneous delayed hypersensitivity. In the case of the (Rha^?) species, the glycoproteins of five species cross-reacted with rabbit antiS. schenckii serum and all, but theS. cyanescens, glycoprotein were reactive to some degree in skin tests in sporotrichotic patients. These results strongly suggest that the chemical and immunological properties of these glycoproteins correspond with the morphological observations amongSporothrix species.     

Serological cross-reactivity between sporothrix schenckii and various unrelated fungi

The serological cross-reactivity ofSporothrix schenckii with various unrelated fungi was investigated by use of immunodiffusion tests. A rabbit antiS. schenckii serum was obtained, which reacted withCladosporium werneckii, C. carrionii, C. bantianum, Coccidioides immitis, Phialophora jeanselmei, P. gougerotii, P. dermatitidis, Fonsecaea pedrosoi, Aspergillus fumigatus, Histoplasma capsulatum andTrichophyton mentagrophytes, but not withSaccharomyces cerevisiae antigens. The serological determinants responsible for the cross-reactions were suggested to be D-galactosyl residues.     

Differentiation of Capsular Polysaccharides from Acetobacter diazotrophicus Strains Isolated from Sugarcane

Capsular polysaccharides (CPSs) from six representative strains of Acetobacter diazotrophicus were isolated and fractionated by gel filtration and anion-exchange chromatography. Purified CPSs obtained in the non-adsorbed fraction of a DEAE-Sephadex A-25 column were qualitatively and quantitatively analyzed for sugar composition. Uronic acid and amino sugars were not detected in all purified CPSs. Basically the CPSs of A. diazotrophicus are composed of rhamnose, mannose, galactose and glucose. The presence of fucose was only observed in the CPS of strains PR2 and PAL3. Based on these results, the six strains of A. diazotrophicus could be divided into four groups according to the sugar content of their capsules: (i) fucose-containing capsules (PR2 and PAL3, localized in roots), (ii) mannose-rich capsule (PAL5, localized in root), (iii) capsules with a high ratio of hexose to rhamnose (PR4 and PR20, localized in stems) and (iv) capsules with a low ratio of hexose to rhamnose (PR14, localized in rhizosphere). For all CPSs, sodium dodecy sulfate-polyacrylamide gel electrophoresis showed diffuse bands of slow mobility in silver-stained gels. The different CPS migration patterns could not be correlated with sugar composition. The purified CPS of strain PAL3 was found to be immunogenic and immunochemically similar to the CPS of strain PR2. The serological specificity to CPS of strains PAL3 and PR2 correlated well with the presence of fucose, indicating that this deoxyhexose is immunodominant. These findings demonstrated the feasibility of preparing specific antibodies to fucose-containing CPS of A. diazotrophicus, indicating the possibility of utilization of this antiserum for future taxonomic studies or to select strains with chemically related capsular polysaccharides from their natural habitat.     

Cell wall formation in zoospores of Allomyces arbuscula

Carbohydrate composition was determined in isolated cell walls of meiospores of Allomyces arbuscula after incubation for 15 min (encysted meiospores: cysts), 150 min (germlings: cysts + rhizoids) and 24 h (cysts + rhizoids + hyphae). The principal constituent in all cell wall samples is chitin, accounting for about 75% of the recovered carbohydrates. In addition, cell walls of all stages examined contain polysaccharides which release galactose, glucose, mannose, arabinose, xylose, fucose, and rhamnose on acid hydrolysis. While different developmental stages show minor quantitative changes in chitin, the ratio of galactose to glucose decreases sharply during differentiation of ungerminated cysts into germlings with rhizoids and hyphae. The increase in glucose is accompanied by a decrease in the amount of xylose and/or fucose and of galactose.List of Abbreviation TFA trifluoroacetic acid     

Serological cross-reactivity betweenKlebsiella pneumoniae K47 andSporothrix species

The serological cross-reactivity ofKlebsiella pneumoniae K47 antiserum with antigens of 11Sporothrix species was investigated by use of immunodiffusion. Cross-reactions occurred withK. pneumoniae K47 and theSporothrix speciesS. schenckii, S. schenckii var.luriei, S. curviconia, andS. inflata.     

Water-soluble endopolysaccharides from the fruiting bodies of <Emphasis Type="Italic">Laetiporus sulphureus</Emphasis> (Bull.:Fr.) Murr.

Endopolysaccharides represented by glucans, galactans, and glycoproteins were isolated from the fruiting bodies of the sulfur shelf (Laetiporus sulphureus (Bull.:Fr.) Murr.), which were obtained by the naturalplantation method. The study of the major 56-kDa polysaccharide, laetiporan A, the content of which accounted for 0.28% of the fruiting body weight, showed that it is a β-1,3-glucan containing mannose, galactose, fucose, xylose, and rhamnose residues at position C-6. An antioxidant effect of laetiporan A was discovered.     

Production and composition of extracellular polysaccharide from cell suspension cultures of Mentha

A suspension culture of Mentha was established from callus which formed on the tips of young shoots of a Mentha hybrid (M. arvenis × M. spicata). Changes in growth parameters during a culture cycle were recorded. The general appearance of cells during division and growth, including the changes in cell form, was also represented.Suspension-cultured cells of Mentha hybrid released a large amount of extracellular polysaccharides (ECP) mainly at the logarithmic phase of the growth cycle. The ECP contained galacturonic acid as major components and arabinose, galactose, glucose, xylose, rhamnose and mannose as minor components. The ratio of the uronic acid content to total sugar content in the ECP was below 40% at day 7, but increased up to 90% at day 21. The relative contents of xylose and glucose in the ECP decreased during the culture period, while the arabinose content increased and those of rhamnose, mannose and galactose remained constant.The IR spectrum suggested that the ECP were low-methoxylated pectic polysaccharides. The presence of lignin and related compounds in the ECP was not detected. The protein content of the ECP was about 10% and the main amino acids were alanine, proline, hydroxyproline, valine, asparticacid and serine, in that order.     

Isolation and Sugar Composition of Blood Group H-Like Substance from Seeds of Euonymus Sieboldiana

Blood group H-like polysaccharides were isolated from seeds of Euonymus Sieboldiana by a procedure that included fractionation with (NH₄)SO₄, heat treatment, chromatography on DEAE-cellulose and gel filtration on Bio-Gel P-150. One of the highly purified polysaccharide fractions was composed of arabinose, mannose, glucose, rhamnose, galactose and fucose. Arabinose and mannose were the main components, and their molar ratio was calculated as about 3: 1 by gas chromatographic analysis. An analytical ultracentrifugal experiment revealed that the finally purified H-like substance was close to an homogeneous preparation with a small disperse fraction. This heteropolysaccharide inhibited the haemagglutination of human group O red blood cells and eel anti-H serum minimally at 0.004 mg/ml, reacted also with the eel anti-H serum on an immunodiffusion plate to form sharp precipitin line(s).     

Chemical characterisation of the released polysaccharide from the cyanobacterium Aphanothece halophytica GR02

The released polysaccharide from the halophilic cyanobacterium Aphanothece halophytica GR02 was separated into two main fractions byanion-exchange chromatography. The major fraction consisted of glucose,fucose, mannose, arabinose and glucuronic acid. Judging from thechromatography on Sepharose 2B, the major fraction was not furtherfractionated, and its apparent molecular weight was above 2.0 × 10⁶ Da.The minor fraction consisted of rhamnose, mannose, fucose,glucose, galactose and glucuronic acid, with traces of arabinose.Methylation and GC-MS spectrometry analyses of the major fractionrevealed the presence of 1-linked glucose, 1,3-linked glucose, 1,3-linkedfucose, 1,4-linked fucose, 1,3-linked arabinose, 1,2,4-linked mannose,1,3,6-linked mannose, 1-linked glucuronic acid and 1,3-linked glucuronicacid residues. The major fraction was thought to originate from capsularpolysaccharide. The released polysaccharides, obtained from cultures atdifferent age of culture, showed no striking variations in themonosaccharide composition and the relative proportions of themonosaccharides. However, the proportions of galactose and rhamnose inthe released polysaccharides, obtained from cultures under different salinity,were significantly different. The released polysaccharide also exhibitedgelling properties and strong affinity for metal ions.     

Camparison of mucilage polysaccharides extracted from sewage activated sludge

The sugar composition of mucilage polysaccharides extracted from activated sludge from five different sewage treatment plats were compared. All the polysaccharides contained rhamnose, fucose, arabisone, xylose, mannose, galactose, glucose, amino sugars, and uronic acids in similar proportions, especially in the neutral sugar fraction. The main components were rhamnose (12–18%), mannose (14–21%), galactose (16–19%), and glucose (15–23%). No significant changes was observed in the sugar composition of activated sludge from a sewage treatment plant over a period of more than one year. Recovery of the mucilage polysaccharides fell to 46% of the initial amount when activated sludge was digested aerobically for 10 days, but the sugar composition was not affected.     

Immunochemical investigations of antigens isolated fromBacteroides ovatus strain ATCC 8483

A saline extract (SE) and a phenol/water extract (WL) were prepared fromBacteroides ovatus strain ATCC 8483. A fraction CS was isolated from the culture supernatant. WL was further split by ultracentrifugation into lipopolysaccharide (LPS) and supernatant (L₁). Fractions SE, WL, LPS and L₁ reacted serologically with homologous antiserum but did not cross-react with antisera against heterologousBacteroides serotypes. Fraction CS was inactive in haemagglutination, haemagglutination inhibition and immunoelectrophoresis tests. SE, WL, LPS and L₁ proved to be serologically heterogeneous. A distinct serological specificity for SE was demonstrated. The serological reactivity in SE and WL was not altered after treatment with proteolytic enzymes yet completely destroyed in WL and partially in SE by sodium metaperiodate. SE, WL, LPS and L₁ contained the sugar components rhamnose, fucose, ribose, mannose, galactose, glucose and glucosamine in different molar ratios for each fraction. Galactosamine was found in WL and LPS, uronic acid in WL and L₁. Two unidentified aminohexoses were detected in WL, one of which was also detectable in L₁ and SE. 2-Keto-3-deoxyaldonic acid was demonstrated in LPS and L₁ after strong acid hydrolysis.     

Chemical composition and immunochemical characteristics of the lipopolysaccharide of nitrogen-fixing rhizobacterium Azospirillum brasilense CD

The chemical composition of the lipopolysaccharide of the associative diazotrophic rhizobacterium Azospirillum brasilense Cd has been studied. Among the main components of the hydrophobic part of the lipopolysaccharide, we identified 3-hydroxytetradecanoic, hexadecenoic, 3-hydroxyhexadecanoic, hexadecanoic, octadecenoic, and nanodecanoic fatty acids; the carbohydrate part contained rhamnose, galactose, and mannose. Polyclonal antibodies against the preparation under study were raised in rabbits. Serological relations between A. brasilense Cd and other strains of Azospirillum spp. were studied using double radial immunodiffusion and enzyme-linked immunosorbent assay.     

Neutral sugars in lipopolysaccharides ofRhizobium trifolii and its non-nodulating mutant

Summary The nodulatingRhizobium trifolii strain 24 and its non-nodulating mutant 24 nod^–3 have been examined. The exopolysaccharides of both cultures studied contained mannose, galactose and glucose at similar molar ratios. On the other hand some quantitative differences have been found between the lipopolysaccharides in respect of the composition of neutral sugars. Glucose and rhamnose were the main constituents of the nodulating strain 24, whereas rhamnose and galactose in non-nodulating mutant 24 nod^–3 deprived of the plasmid pWZ2.     

Intracellular hydroxyproline-rich glycoprotein of suspension-cultured tobacco cells

Two soluble glycoproteins containing hydroxyproline were extractedfrom cultured tobacco cells (cell line XD-6S) and purified byion-exchange and gel-filtration chromatography. On DEAR-cellulosecolumn chromatography in the final step of the purification,one was eluted at 90 mM NaCl and the other at 120 mM as singlepeak. Both purified glycoproteins were also sedimented as singlepeak with an ultracentrifugation. The S_20,w values were 6.1for the former and 7.0 for the latter. These glycoproteins were composed of 94% polysaccharide and6% protein in the former, and 87% polysaccharide and 13% proteinin the latter. The sugar moiety consisted of galactose, arabinose,rhamnose, and uronic acid in both. Hydroxyproline accountedfor 12% in the former and 20% in the latter amino acid composition.A high content of alanine in both (14 and 15%) was one of thedistinctive characteristics of these soluble glycoproteins. These intracellular soluble hydroxyproline-containing glycoproteinswere not labelled within 30 min of incubation with ₃H-proline,although the radioactivity was rapidly incorporated (within15 min) into the intracellular macromolecules. (Received February 21, 1978; )     

Production of a Novel Extracellular Polysaccharide by a Bacillus Strain Isolated from Soil

A bacterium that was isolated from soil and identified as Bacillus circulans was found to produce a highly viscous extracellular polysaccharide when it was grown aerobically in a medium containing glucose as a sole source of carbon. The product was characterized by TLC and GC analyses as a novel heteropolysaccharide consisted of rhamnose, mannose, galactose, and mannuronic acid as sugar components. A maximal yield of polysaccharide reached about 2 g/liter by jar-fermentor culture at 30°C for 48 hr with a medium containing 1% glucose, 0.05% asparagine, 0.005% yeast extract, and small amounts of inorganic salts. Some culture conditions for the production of polysaccharide were investigated with flask culture; an optimal production was attained with a medium containing 0.1–1 % glucose and 0.01–0.05% asparagine, pH 7–8, at 30°C under aerobic conditions.     

Cell Wall Glycoproteins Participate in the Adhesion of <Emphasis Type="Italic">Sporothrix schenckii</Emphasis> to Epithelial Cells

Sporothrix schenckii is the etiologic agent of sporotrichosis. This fungal infection is an emerging disease potentially fatal in immunocompromised patients. The adhesion to host cells is a crucial early event related with the dissemination of pathogens. In order to clarify the mechanisms of adhesion of S. schenckii yeast cell to epithelial cells, we studied the biochemical basis of this process. The electrophoretic analysis of cell wall protein from S. schenckii coupled at ConA and stained with HRP, revealed nine different proteins with MW ≥ 180, 115, 90, 80, 58, 40, 36, 22 and 18 kDa. Using ligand-like assay with biotinylated S. schenckii surface proteins, five proteins with MW ≥ 190, 180, 115, 90 and 80 kDa which have affinity to epithelial cells were identified. The adhesion of yeast to epithelial monolayer was significantly inhibited when S. schenckii was pretreated with concanavalinA (ConA) and wheat germ agglutinin (WGA) lectins, alkali, periodate, trypsin, endoglycosidase H (EndoH), salt solutions and detergents. The ability of adhesion of S. schenckii yeast was recovered by blocking the lectin with sugar complementary. These data suggest that surface glycoprotein with mannose and glucose residue could be participate in the process of fungal adhesion to epithelial cells.     

Sugar composition of biofilms produced by paper mill bacteria

Biofilms of paper mill bacteria were cultivated in paper mill white water-simulating conditions on glass slides or stainless steel coupons in a laboratory culture system. The sugar content and composition of the biofilms were analysed and compared with the sugar composition of paper mill slimes. Acid methanolysis followed by gas chromatography revealed that Burkholderia was the major biofilm producer in pure culture, producing up to 50 microg of biofilm sugar cm(-2) in 5 days in rich medium and 10 microg in paper mill simulating medium. A mixture of simulated paper mill water with a culture medium yielded more biofilm (100 microg cm(-2)) than either of the media alone, so the biofilm accumulation was not proportional to the available substrate. More biofilm accumulated on stainless steel coupons than on glass slides, and the steel-coupon biofilms contained slightly more uronic acids. The biofilm sugars contained mainly galactose, glucose, mannose, and rhamnose. In paper mill medium, the Burkholderia biofilm contained more galactose and glucose, and less rhamnose, than in rich laboratory medium. The sugar composition of paper mill slimes was quite similar to those of steel-cultured Burkholderia cepacia biofilms. This suggests that Burkholderia cepacia is responsible for much of the slime in the paper mill.     

Comparative studies of flocculation and deflocculation of Saccharomyces uvarum and Kluyveromyces bulgaricus

Summary Flocculation of Kluyveromyces bulgaricus and Saccharomyces uvarum occurred when these yeasts were grown in a peptone glucose medium enriched with calcium ions. K. bulgaricus and S. uvarum flocculated at the beginning and at the end, respectively, of the exponential growth phase. After growth, both yeasts were washed with an EDTA solution, flocculated again in an acetate buffer, and optimum flocculation was obtained at pH 4.5 in the presence of 3.75 mM Ca⁺⁺. K. bulgaricus flocculation was irreversibly suppressed by incubation at 80° C for 6 min. S. uvarum needed an incubation at 100° C for 20 min to be irreversibly deflocculated. For both yeasts, flocculation stability depended on the presence of sugars. Mannose, mannose 6P and oligosaccharides bearing a mannose in a terminal non-reducing position reversed flocculation of S. uvarum, while galactose, galactose 6P and oligosaccharides bearing a galactose in a terminal nonreducing position reversed flocculation of K. bulgaricus. It is suggested that sugars specifically reverse flocculation because cell-to-cell aggregation of these yeasts is a lectin-carbohydrate-linked mechanism; not any sugar is capable of deflocculating any yeast, but the mechanism is specific.     

Short Communication: Lipopolysaccharide composition of Desulfovibrio cell wall

The lipopolysaccharide of the sulphate-reducing bacterium Desulfovibrio desulfuricans was analysed by GC, combined either with flame-ionization detection or with MS, and by standard chemical tests. The major sugar of the polysaccharide portion of the macromolecule was glucose (56%). Low amounts of mannose, galactose, rhamnose and amino sugar were also found. Six fatty acids were identified in the lipid A fraction: 9-octadecenoic, tetracosenoic, heptadecenoic, 10-octadecenoic, eicosenoic and 8-octadecynoic.