PURIFICATION AND CHARACTERIZATION OF AN EXOPOLYSACCHARIDE OF BURKHOLDERIA (PSEUDOMONAS) PSEUDOMALLEI

Burkholderia pseudomallei (basonym Pseudomonas pseudomallei) is the ca usative organism of melioidosis, a disease which is recognized as a ma jor public health problem primarily in Southeast Asia and Northern Aus tralia. In this paper, we report on the identification, purification, and characterization of a species-specific exopolysaccharide of B. pse udomallei. After immunization of mice with a B. pseudomallei strain ex hibiting mucoid growth characteristics, we isolated an immunoglobulin G1 monoclonal antibody (MAb) (3015) with specificity for a carbohydrat e structure as determined by immunoblotting following sodium dodecyl s ulfate-polyacrylamide gel electrophoresis. Electron microscopy studies with MAb 3015 revealed reactivity with an exopolysaccharide with a ca psule-like appearance in the immunizing strain. All of the mucoid and nonmucoid B, pseudomallei strains tested from geographically different tropical regions were recognized by MAb 3015 in an enzyme-linked immu nosorbent assay or immunoblot, indicating that the exopolysaccharide i s constitutively expressed among this species. Intensive testing for c ross-reactivity including members of all the Pseudomonas rRNA groups s howed no cross-reactivity except in the case of the closely related sp ecies Burkholderia mallei. A protocol for purification of the exopolys accharide which is based principally on mechanical separation from the cell surface followed by repetitive ethanol precipitation steps and f inally affinity chromatography using MAb 3015 was established. The exo polysaccharide yielded was of high purity. Gel permeation chromatograp hy was performed, and the molecular mass was estimated to be >150 k)a. Sera from patients with melioidosis were strongly reactive with the p urified exopolysaccharide, indicating its in vivo expression and immun ogenicity in natural infection, The diagnostic value of the exopolysac charide and its role in the pathogenesis of disease must still be dete rmined.