The potential lipopolysaccharide (LPS)-degrading capability of three different systems was investigated. The acellular slime mould Physarum polycephalum was chosen as an example of a phagotrophic microorganism utilizing bacteria as food. Gut juice from the snail Helix pomatia was used because this animal is an example of an invertebrate ingesting materials rich in bacteria. Also the gut juice is a known source of many different enzyme activities. The fate of LPS when incubated with samples of marine mud and sand was investigated because of the abundance of gram-negative bacteria in marine environments, particularly muds. The main methods used to detect and measure LPS and to obtain evidence of its degradation were: Ketodeoxyoctonate analysis, because of the presence of this substance in almost all LPS preparations; gas-liquid chromatography (GLC) to detect long chain fatty acids; serological methods, including the ability of LPS to sensitize red cells, and haemagglutination inhibition (HAI) tests to demonstrate whether the immunospecific sugars in the oligosaccharide component were still present; and the ability of LPS to trigger complement, as an index of "endotoxic" activity. The anticomplementary activity of LPS was investigated in some detail. A range of LPS was assayed for anticomplementary (AC) activity against 5 HU50 of complement (C) from man, pig and guinea-pig. On average, levels of LPS about 200 times lower were detected with human C than with guinea-pig C and about two times lover than with pig C. There was little variation in C samples from different people in responsiveness to LPS AC activity. Different LPS varied in AC activity over a 10-fold range with each species of C. The rank order of their activities also varied with species of C. With human C, the most active LPS could be detected down to 2.5 ug. It is suggested that the AC effect of LPS is mediated principally via the Alternative Pathway. Physarum polycephalum appeared to attack only the lipid component of LPS, with a consequent reduction in AC activity and ability to sensitize red cells, while the KDO and HAI values were unaffected. GLC analyses indicated that the lauric, myristic and palmitic acids were split off leaving the ß-hydroxynprristic acid still attached to the diglucosamine backbone. Degradation of LPS by gut juice of the snail Helix pomatia similarly appeared to affect only the lipid component. When such degraded LPS was analysed on SDS-polyacrylamide gels. one of the characteristic bands of the original LPS was lost or reduced, reflecting loss of fatty acids. Marine sediments, at different depths and sites, were extracted with phenol-water or trichloroacetic acid (TCA) and yielded more LPS in areas of high organic pollution. KDO was not, detected in any of the sediments taken below a depth of 4 cm; AC activity decreased with increasing depth. When killed gram-negative bacteria were incubated with marine sediments, degradation of the LPS was observed. The oligosaccharide component was degraded at a faster rate than the lipid moiety as demonstrated by the loss of KDO and serological specificity. Both amide and ester linked fatty acids of the lipid A were lost. Most of the previous work on alteration of the LPS molecule has involved chemical treatment. The present studies offer the possibility of more specific procedures. Purification of the P serum and snail gut juice enzyme systems might afford highly selective modes of degradation which would be very useful for increasing knowledge of the structure of LPS molecules. Also it might be possible to produce altered molecules with different patterns of biological, physiological and immunological properties.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:471325 |
| Date | January 1978 |
| Creators | Saddler, John N. |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/4040/ |
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