Purification and characterization of a cytotoxic exolipid of Burkholderia pseudomallei

Abstract

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-alpha-L-rhamnopyranosyl-alpha-L-rhamnopyranosyl-beta-hydroxytetradec anoyl-beta-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.

FIG. 1

Time course of production of the exotoxin into the supernatant of a B. pseudomallei culture grown with glycerol. Bacteria were inoculated at high density in 0.15 M NaCl containing 4% (vol/vol) glycerol at pH 7 and incubated for 6 days. Symbols: •, CFU per ml; ▾, HU.

FIG. 2

Positive-ion mode ESI-MS/MS of the molecular ion (sodium adduct) of the exotoxin isolated from B. pseudomallei. The detected fragments and their m/z values are shown in the fragmentation scheme at the top of the figure. Detailed information on the fragmentation of glycoconjugates can be found in reference .

FIG. 3

Proposed structure of the rhamnolipid produced by B. pseudomallei.

FIG. 4

Time- and dose-dependent hemolysis of human erythrocytes by the rhamnolipid at various levels. Symbols: •, 4 HU per ml; ▪, 2 HU per ml; ▴, 1 HU per ml.

FIG. 5

Neutralization of hemolytic activity (64 HU) of the B. pseudomallei rhamnolipid by bovine serum albumin (•). Bovine gamma globulin (▪) showed no neutralizing effect. The same results were obtained with human albumin and gamma globulin.

FIG. 6

Time- and dose-dependent cytotoxicity of the B. pseudomallei rhamnolipid on HL60 cells (A) or HeLa cells (B). Symbols: •, 80 HU per ml; ▪, 40 HU per ml; ▴, 20 HU per ml.