Immunochemical and Biologic Studies of the Lipopolysaccharide of Bacteroides Melaninogenicus Subspecies Asaccharolyticus

Abstract

Lipopolysaccharide (LPS) was isolated from the outer membrane complex of Bacteroides melaninogenicus by gel chromatography by using sodium deoxycholate (NaD), an endotoxin-disaggregating detergent in the running buffer. Reaggregation occurred after removal of residual NaD. LPS was also prepared by the phenol/water method for comparison. The LPS was composed of loosely bound lipid (62%) and carbohydrate (32%), with less than 5% protein. Glucose, galactose, and glucosamine were the major sugars as detected by gas-liquid chromatography (GLC). Heptose and 2-keto-3-deoxyoctonate (KDO) were found to be absent when using colorimetric tests. Fatty acid analysis by GLC disclosed an unusual pattern; β-OH myristic acid was notably absent. Biologic activity was assessed with the dermal Shwartzman test, the Limulus lysate assay, and the chick embryo lethality test. LPS preparations gave no positive skin reactions in rabbits in doses up to 1 mg, compared to Salmonella typhi 0:901 endotoxin which was positive at 12.5 µg. Limulus lysate gelation and chick embryo lethality occurred at doses 30-fold or more different from Salmonella endotoxin controls. Hemagglutination inhibition (HAI) studies demonstrated the presence of capsular polysaccharide as a contaminant found in the phenol/water extracted LPS. This polysaccharide could be removed partially by further purifying the LPS with ultracentrifugation. The LPS of B. melaninogenicus lacks two of the core sugars characteristic of aerobic Gram-negative endotoxins, has a unique fatty acid composition, and displays low biologic potency. These findings may explain the rarity of septic shock in patients infected with this organism. The presence of capsular polysaccharide contaminating the phenol/water-extracted LPS may explain partially the serologic heterogeneity which has been described previously in studies of the LPS of this organism.