PURIFICATION AND CHARACTERIZATION OF A CYTOTOXIC EXOLIPID OF BURKHOLDERIA-PSEUDOMALLEI

Burkholderia pseudomallei is the causative agent of melioidosis, an in fectious disease, which is increasingly recognized as an important pub lic health problem in various tropical regions, This study describes t he identification and characterization of a heat-stable extracellular toxin of B. pseudomallei, After cultivation of B. pseudomallei in liqu id media, the heated cell-free supernatant was concentrated by ultrafi ltration, The concentrate exhibited a cytotoxic and hemolytic activity which showed remarkable resistance against alkaline and acidic treatm ents, For further purification, reversed-phase chromatography using a fast-performance liquid chromatography system was performed, After elu tion with an acetonitrile gradient, a single cytotoxic and hemolytic p eak was detected, Structural characterization of the toxin was perform ed by a combination of mass spectrometric and nuclear magnetic resonan ce spectroscopic techniques, A highly purified glycolipid, lpha-L-rham nopyranosyl-beta-hy-droxytetradecanoate (Rha-Rha-C-14-C-14), with a mo lecular mass of 762 Da was identified, The purified exolipid showed a time-and dose-dependent cytotoxic effect on phagocytic (HL60) and nonp hagocytic (HeLa) cell lines, In addition, a time-and dose-dependent he molysis of erythrocytes from various species was observed, The toxin s tructure makes a detergentlike action most probable, Interestingly, th e cytotoxic and hemolytic activities of the glycolipid could be neutra lized by albumin, Future studies sill concentrate on the role of this exolipid as a virulence factor in the pathogenesis of melioidosis.