A REAPPRAISAL OF THE BIOLOGICAL-ACTIVITY OF BACTEROIDES LPS

Lipopolysaccharides (LPS) were extracted from seven Bacteroides strain s by three different techniques: the phenol-water (PW), phenol-chlorof orm-petroleum (PCP) and Triton-Mg2+ methods. The strains selected incl uded two different B. fragilis strains, one of which was grown in two different media. Yields varied between the strains, growth media and e xtraction technique, but generally the highest yield by weight was fro m the PCP method and the lowest from the PW method. The PW method was selected for the greatest amounts of carbohydrate and KDO, and the PCP method for the least. Phosphorus levels were more uniform among all e xtraction methods. Protein contamination was found in all Bacteroides LPS extracts, with extremely low levels in PW-LPS and the highest leve ls in material extracted by the PCP and Triton-Mg2+ techniques. No pro tein contamination could be detected after proteinase K treatment. Aft er silver staining LPS PAGE profiles showed ladder patterns characteri stic of smooth LPS for B. vulgatus, B. thetaiotaomicron and the contro l Escherichia coli O18:K- strains, whereas the other Bacteroides strai ns showed mainly rough and low M(r) material only. The PCP method did not select for high M(r) material in the B. fragilis strains; otherwis e the LPS profiles for all extraction methods were identical. The biol ogical activities of native and sodium salt form LPS were investigated on a weight for weight basis and compared to that of E. coil O18:K- P W-LPS. Amongst the LPS from Bacteroides strains, those prepared by the PW method were found to have a significantly higher activity in a gal actosamine mouse lethality model, in induction of TNF and the Limulus amoebocyte lysate (LAL) assay, than LPS extracted by the PCP or Triton -Mg2+ methods. LPS from Bacteroides strains extracted by the PCP metho d had consistently low activity in all assays. Comparing PW-LPS from B acteroides strains with that from E. coli O18:K- in the galactosamine mouse model, the E. coli O18:K- LPS was c. 5000-fold more active than the most active bacteroides LPS. However, in the LAL assay native PW-L PS from both the B. fragilis strains, and B. caccae had higher activit ies (up to 30-fold) than E. coil O18:K- LPS, with the PW-LPS from the other Bacteroides spp. being up to 15-fold less active than the E. col i O18:K- PW-LPS. In the TNF induction assay, E. coli O18:K- PW-LPS was 4-50-fold more active than bacteroides PW-LPS. In the LAL assay and g alactosamine mouse model, native LPS had more activity (c. two-fold) t han sodium salt form LPS. There was no clear difference in activity be tween native and sodium salt form LPS in the TNF induction assay. The results for the LAL and TNF induction assay were re-evaluated relative to KDO concentration. In the TNF induction assay, previously low acti vities seen on a weight for weight basis were due in part to less KDO being present. However, LAL activity for PCP-LPS was still low after r e-evaluation relative to KDO concentration. The molecular basis for th e differences in biological activity of bacteroides LPS in relation to extraction methods and chemical composition is not yet understood.