ORIGIN AND PROPERTIES OF NATURALLY OCCURRING HAPTEN FROMESCHERICHIA COLI

Abstract

Anacker , R. L. (National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratory, Hamilton, Mont.), R. A. Finkelstein, W. T. Haskins, M. Landy, K. C. Milner, E. Ribi, and P. W. Stashak . Origin and properties of naturally occurring hapten from Escherichia coli . J. Bacteriol. 88: 1705–1720. 1964.—Haptens found in preparations of endotoxin and in fractions of disrupted cells, particularly one termed “native hapten,” which appeared to be associated with the protoplasm of cells rather than with cell walls, have been further investigated with a view to establishing their origin and composition as well as their host-reactive properties. For this purpose, cells from a smooth strain of Escherichia coli O111:B4 were either extracted directly or disrupted and separated into cell-wall and protoplasmic fractions. Haptens were obtained by gel filtration of endotoxins, by trichloroacetic acid extraction of protoplasm, and by a mild acid hydrolysis of endotoxin. Several lines of evidence indicated that native hapten originated in the protoplasm rather than by autolysis or degradation of cell-wall endotoxin during procedures employed in disruption. In gel diffusion and quantitative precipitin tests, no hapten was identical with endotoxin, but native hapten was serologically the most complex of the haptens and precipitated the most antibody. Native and acid haptens, on a weight basis, fixed about 1% of the quantity of complement fixed by homologous endotoxin. Haptens did not stimulate the production of antibodies in mice or rabbits and did not elicit endotoxic host reactions. Chemically, native hapten differed from endotoxin and from acid hapten in that it lacked phosphorus, heptose, long-chain fatty acids, and 2-keto-3-deoxyoctonate. These substances did not appear to be determinants of antigenic specificity, but they may provide necessary bonds for assembling hapten-like units into fully antigenic and toxic macromolecules.

Studies of a Li‐Hapten Isolated from Cell‐Walls of the Rough Mutant Salmonella Typhimurium 395 MR10

“…Otherwise one would assume that the antibodies should be quantitatively precipitated by the hapten. h a c k e r et al (1) using purified haptenic material, arrived at the same conclusion. Our initial attempts to isolate and purify the LI-hapten from the cytoplasmic fraction were unsuccessful.…”

“…Anacker et al (1) and Rudbach et al (20) isolated from E. coli a polysaccharide, "native protoplasmic polysaccharide", which apparently was of cytoplasmic origin. This polysaccharide was isolated from the supernatant after trichloracetic acid (TCA) treatment of the protoplasmic fraction of disintegrated bacteria.…”

Immunological Responses of Mice to Native Protoplasmic Polysaccharide and Lipopolysaccharide

“…W o r k in our l a b o r a t o r y has b e e n d i r e c t e d t o w a r d i n v e s t i g a t i n g the i m m u n o l o g i c a l r e s p o n s e s of m i c e i n j e c t e d w i t h L P S (33,34) or a r e l a t e d m a t e r i a l , n a t i v e p r o t o p l a s m i c p o l y s a c c h a r i d e ( N P P ) (35). N P P , e x t r a c t e d from the p r o t o p l a s m of E s c h e r i c h i a coli (36,37), was a s i m p l e p o l y s a c c h a r i d e w i t h an a v e r a g e mQ1 wt of 163,000 d a l t o n s (38). O n c e t h o u g h t to be a p r o t o p l a s m i c p r e c u r s o r to cell wall *This investigation was supported in part by U. S. Public Health Service Training Grant 5-T01-AI00385.…”

“…A preliminary report of this work was presented, in part, at the Annual Meeting of the American Society for Microbiology, Chicago, Ill., May 17, 1974. ~Abbreviations used in this paper: LPS, lipopolysaccharide; NPP, native protoplasmic polysaccharide; RML mice, outbred mice from the Rocky Mountain Laboratory stock; nude mice, congenitally athymic nude mice; LPS-0113, LPS from E. coli 0113; LPS-0111, LPS from E. coil 0111; NPP-0113, NPP from E. coli 0113; ReGL-G30/C21, glycolipid from the Re mutant G30/C21 of S. typhimurium; PBS, phosphate-buffered saline; PFC, direct plaque-forming cells; HGG, human gamma globulin. LPS (36,37), NPP was more recently described as a product of aberrant metabolism (38). Immunochemically, the O-antigenic determinants of NPP cross-reacted completely with the O-antigenic determinants of homologous LPS (37,39).…”

“…LPS (36,37), NPP was more recently described as a product of aberrant metabolism (38). Immunochemically, the O-antigenic determinants of NPP cross-reacted completely with the O-antigenic determinants of homologous LPS (37,39). However, NPP did not contain Lipid A, 2-keto-3-deoxyoctulosonate, or heptose, and failed to elicit some of the host reactive properties associated with LPS (37,39).…”