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A recently described water-soluble exopolysaccharide of Burkholderia pseudomallei recognized by the IgG 1 monoclonal antibody 3015 [Steinmetz, I., Rohde, M. & Brenneke, B. (1995) Infect. Immun. 63, 3959Ϫ3965] was isolated by repetitive ethanol-precipitation steps and by anion-exchange chromatography. The structure of the polysaccharide was determined by a combination of chemical-derivatization and mass-spectrometric techniques (compositional and methylation analysis, GC/MS, and electrosprayionization-MS/MS of reduced and permethylated hydrolytic fragments), and two-dimensional 1 H-NMR methods (COSY, TOCSY and NOESY) and confirmed by isolation and structural characterization of the depolymerized repeating unit of the polysaccharide. The combined structural data established a linear tetrasaccharide repeating unit consisting of three galactose residues, one bearing a 2-linked O-acetyl group, and a 3-deoxy-D-manno-2-octulosonic acid residue.Keywords : Burkholderia pseudomallei; exopolysaccharide.The gram-negative saprophytic rod Burkholderia pseu-described previously [3] but with a modified final analytical purification step. Anion-exchange chromatography using a MonoQ domallei is the causative organism of melioidosis, an infectious disease of humans and animals, which is known to be a major HR 5/5 column coupled to a FPLC sytem (Pharmacia) was used instead of affinity chromatography based on mAb 3015 [3]. The public health problem in certain areas of the tropics [1]. Melioidosis is likely to be underdiagnosed and the true worldwide exopolysaccharide was eluted by a linear gradient from 0 to 1.5 M NaCl in 20 mM Tris/HCl, pH 7.4, with a flow rate of importance remains to be determined. Clinical manifestations of human melioidosis are extremly variable and range from sub-1 ml/min. Each fraction was dialyzed and tested in an ELISA using mAb 3015 [3] and by carbohydrate compositional analysis clinical infections to fulminant septicemias with high mortality rates [2]. In contrast to the growing knowledge on the epidemi-for the presence of exopolysaccharide.Deacetylation of the polysaccharide and isolation of the ology of melioidosis, investigations on the contribution of single bacterial structures to the virulence of B. pseudomallei have only repeating tetrasaccharide. Native polysaccharide material was dissolved in 0.1 M NaOH and maintained at 50°C for 2 h. After been carried out on a very limited scale.We have purified and characterized recently a high-molecu-neutralization, the deacylated product was dialyzed against distilled water and the solvent removed at reduced pressure and lar-mass exopolysaccharide of B. pseudomallei recognized by a monoclonal antibody (mAb) [3]. Immunoelectron microscopy room temperature. The repeating tetrasaccharide was isolated after partial hydrolysis of the deacetylated polysaccharide by treatdemonstrated its surface location to be mostly on the outer of the outer membrane of the cell [3]. This exopolysaccharide is ment with 0.5 M trifluoroacetic acid at 100°C for 1 h. The resulting...

The antifungal and cytotoxic metabolites vioprolides A-D ( 1 -4) were obtained from the myxobacterium Cystobacter violaceus strain Cb vi35 by fermentation in the presence of the adsorber resin XAD-1180. Peptolide structures 1-4 composed of eight amino acids and a glyceric acid were determined by spectroscopic methods and total hydrolysis. The absolute configurations of the building blocks were established by GC analysis on chiral stationary phases. Common build- . Two defined isomers of 1 were detected in CDC13 in a ratio of 3 : 2 (2D-'H NOESY) and assigned to rotational isomers at the amide bond of Thr-MeVal.In a screening of myxobacteria for antifungal metabolites the Cystobacter violaceus strain Cb vi35 was selected for its activity against Pythium debaryanum. After cultivation in the presence of the adsorber resin XAD 1180, the active principle was found exclusively in the methanol eluate of the adsorber resin. TLC of the extract, in combination with bioautography on Pytlzium debaryanum plates, indicated a single activity which could also be detected by UV absorption at h= 254 nm. The antibiotic was isolated by preparative TLC on silica gel plates. On the basis of the chromatographic behavior and the UV spectrum, the product was not related to the antifungals myxalamid[2], pyrrolnitrinr'l, and ~tigmatellin[~I previously isolated from other strains of Cystobacter. Analytical HPLC showed that the product isolated by TLC consisted of four related components later called vioprolides A-D (1-4).After the production had been optimized, two 651 fermentations in the presence of the adsorber resin XAD 1180 were performed [5]. The resin was eluted with methanol and the bulk of polar contaminants eliminated by liquid-liquid partition between water and ethyl acetate (Figure 1). The concentrated organic phase was separated by RP-18 chromatography with the solvent system methanollwater to yield a mixture of 1 and 2 as well as a mixture of 3 and 4. Further separation by Si 60 chromatography with the solvent system petroleum etherldiethyl etherlmethanol yielded the pure vioprolides A-D (1-4) as colorless amorphous solids.

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease, which is increasingly recognized as an important public health problem in various tropical regions. This study describes the identification and characterization of a heat-stable extracellular toxin of B. pseudomallei. After cultivation of B. pseudomallei in liquid media, the heated cell-free supernatant was concentrated by ultrafiltration. The concentrate exhibited a cytotoxic and hemolytic activity which showed remarkable resistance against alkaline and acidic treatments. For further purification, reversed-phase chromatography using a fast-performance liquid chromatography system was performed. After elution with an acetonitrile gradient, a single cytotoxic and hemolytic peak was detected. Structural characterization of the toxin was performed by a combination of mass spectrometric and nuclear magnetic resonance spectroscopic techniques. A highly purified glycolipid, 2-O-α-l-rhamnopyranosyl-α-l-rhamnopyranosyl-β-hydroxytetradecanoyl-β-hydroxytetradecanoate (Rha-Rha-C14-C14), with a molecular mass of 762 Da was identified. The purified exolipid showed a time- and dose-dependent cytotoxic effect on phagocytic (HL60) and nonphagocytic (HeLa) cell lines. In addition, a time- and dose-dependent hemolysis of erythrocytes from various species was observed. The toxin structure makes a detergentlike action most probable. Interestingly, the cytotoxic and hemolytic activities of the glycolipid could be neutralized by albumin. Future studies will concentrate on the role of this exolipid as a virulence factor in the pathogenesis of melioidosis.

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