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. 1987 Mar;55(3):762–770. doi: 10.1128/iai.55.3.762-770.1987

Lipoarabinomannan and lipid-free arabinomannan antigens of Mycobacterium paratuberculosis.

E A Sugden, B S Samagh, D R Bundle, J R Duncan
PMCID: PMC260408  PMID: 3818096

Abstract

Lipoarabinomannan (LAM) and lipid-free arabinomannan (AM) were prepared from Mycobacterium paratuberculosis. Purification of LAM was done by ultracentrifugation of the phenol-water-extracted crude polysaccharide, followed by affinity and anion exchange chromatography. AM was purified from the supernatant of the ultracentrifuged polysaccharide or from alkaline-extracted material by gel filtration and anion exchange chromatography. Chemical analysis revealed arabinose and mannose in LAM (1.4:1) and AM (3.5:1) and the presence of palmitic, stearic, and tuberculostearic acids for a total of 7.8% lipid in LAM. Traces of phosphorus were found in the AMs, particularly LAM (0.05%). Nuclear magnetic resonance confirmed the presence of alpha-arabinosyl residues and the acylated nature of LAM. LAM exhibited lipid-dependent aggregation, as indicated by a Triton-induced decrease in molecular weight. By using bovine sera, LAM was found to be active in the complement fixation test, whereas AM was inactive and inhibited this activity. Thus, the presence of AM in crude polysaccharide could explain the variable complement fixation results. Triton-dissociated LAM exhibited a precipitin (Cl) in common with that of AM, confirming shared determinants. LAM in its lipid-dependent aggregated form, however, exhibited a second precipitin (C2), which may be due to the disparity in antigen size or a novel epitope. The lipid content of LAM rendered it 100 times more effective for coating plates in the enzyme immunoassay than lipid-free AM.

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Selected References

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  1. ANNAU E. A purified complement-fixing antigen from Mycobacterium johnei. Nature. 1958 Apr 26;181(4617):1206–1207. doi: 10.1038/1811206a0. [DOI] [PubMed] [Google Scholar]
  2. Abbas B., Riemann H. P., Lonnerdal B. Isolation of specific peptides from Mycobacterium paratuberculosis protoplasm and their use in an enzyme-linked immunosorbent assay for the detection of paratuberculosis (Johne's disease) in cattle. Am J Vet Res. 1983 Dec;44(12):2229–2236. [PubMed] [Google Scholar]
  3. Alpenfels W. F. A rapid and sensitive method for the determination of monosaccharides as their dansyl hydrazones by high-performance liquid chromatography. Anal Biochem. 1981 Jun;114(1):153–157. doi: 10.1016/0003-2697(81)90466-8. [DOI] [PubMed] [Google Scholar]
  4. Avigad G. Dansyl hydrazine as a fluorimetric reagent for thin-layer chromatographic analysis of reducing sugars. J Chromatogr. 1977 Sep 21;139(2):343–347. doi: 10.1016/s0021-9673(00)89330-9. [DOI] [PubMed] [Google Scholar]
  5. Azuma I., Kimura H., Niinaka T., Aoki T., Yamamura Y. Chemical and immunological studies on mycobacterial polysaccharides. 1. Purification and properties of polysaccharides from human tubercle bacilli. J Bacteriol. 1968 Feb;95(2):263–271. doi: 10.1128/jb.95.2.263-271.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Birkeland N. K., Hofstad T. Isolation by gel filtration and ion-exchange chromatography of a carbohydrate-rich LPS from phenol-water extracts of leptotrichia buccalis strain L11. Acta Pathol Microbiol Immunol Scand B. 1982 Dec;90(6):435–440. doi: 10.1111/j.1699-0463.1982.tb00142.x. [DOI] [PubMed] [Google Scholar]
  7. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  8. Caroff M., Bundle D. R., Perry M. B., Cherwonogrodzky J. W., Duncan J. R. Antigenic S-type lipopolysaccharide of Brucella abortus 1119-3. Infect Immun. 1984 Nov;46(2):384–388. doi: 10.1128/iai.46.2.384-388.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Collins F. M., Campbell S. G. Immunity to intracellular bacteria. Vet Immunol Immunopathol. 1982 Jan;3(1-2):5–66. doi: 10.1016/0165-2427(82)90031-9. [DOI] [PubMed] [Google Scholar]
  10. Daniel T. M., Misaki A. Carbohydrate analysis of concanavalin A-reactive and concanavalin A-nonreactive mycobacterial polysaccharides. Am Rev Respir Dis. 1976 May;113(5):705–706. doi: 10.1164/arrd.1976.113.5.705. [DOI] [PubMed] [Google Scholar]
  11. Daniel T. M. The purification of mycobacterial polysaccharides with concanavalin A. Am Rev Respir Dis. 1974 Nov;110(5):634–640. doi: 10.1164/arrd.1974.110.5.634. [DOI] [PubMed] [Google Scholar]
  12. Duncombe W. G. The colorimetric micro-determination of long-chain fatty acids. Biochem J. 1963 Jul;88(1):7–10. doi: 10.1042/bj0880007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. FOLCH J., LEES M., SLOANE STANLEY G. H. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957 May;226(1):497–509. [PubMed] [Google Scholar]
  14. Grushka E., Durst H. D., Kikta E. J., Jr Liquid chromatographic separation and detection of nanogram quantities of biologically important dicarboxylic acids. J Chromatogr. 1975 Oct 29;112:673–679. doi: 10.1016/s0021-9673(00)99996-5. [DOI] [PubMed] [Google Scholar]
  15. Keller J., Ballou C. E. The 6-O-methylglucose-containing lipopolysaccharide of Mycobacterium phlei. Identification of the lipid components. J Biol Chem. 1968 Jun 10;243(11):2905–2910. [PubMed] [Google Scholar]
  16. Lenzini L., Rottoli P., Rottoli L. The spectrum of human tuberculosis. Clin Exp Immunol. 1977 Feb;27(2):230–237. [PMC free article] [PubMed] [Google Scholar]
  17. Miller R. A., Dissanayake S., Buchanan T. M. Development of an enzyme-linked immunosorbent assay using arabinomannan from Mycobacterium smegmatis: a potentially useful screening test for the diagnosis of incubating leprosy. Am J Trop Med Hyg. 1983 May;32(3):555–564. doi: 10.4269/ajtmh.1983.32.555. [DOI] [PubMed] [Google Scholar]
  18. Misaki A., Azuma I., Yamamura Y. Structural and immunochemical studies on D-arabino-D-mannans and D-mannans of Mycobacterium tuberculosis and other Mycobacterium species. J Biochem. 1977 Dec;82(6):1759–1770. doi: 10.1093/oxfordjournals.jbchem.a131874. [DOI] [PubMed] [Google Scholar]
  19. Myrvang B., Feek C. M., Godal T. Antimycobacterial antibodies in sera from patients throughout the clinico-pathological disease spectrum of leprosy. Acta Pathol Microbiol Scand B Microbiol Immunol. 1974 Oct;82B(5):701–706. doi: 10.1111/j.1699-0463.1974.tb00238.x. [DOI] [PubMed] [Google Scholar]
  20. OHASHI M., TSUMITA T. THE FATTY ACID COMPOSITON OF THE LIPOPOLYSACCHARIDE OF MYCOBACTERIUM TUBERCULOSIS, THE HEMAGGLUTINATION ANTIGEN. Jpn J Exp Med. 1964 Dec;34:323–328. [PubMed] [Google Scholar]
  21. Oashi M. Studies on the chemical structure of serologically active arabinomannan from mycobacteria. Jpn J Exp Med. 1970 Feb;40(1):1–14. [PubMed] [Google Scholar]
  22. Pless D. D., Schmit A. S., Lennarz W. J. The characterization of mannan of Micrococcus lysodeikticus as an acidic lipopolysaccharide. J Biol Chem. 1975 Feb 25;250(4):1319–1327. [PubMed] [Google Scholar]
  23. Powell D. A., Duckworth M., Baddiley J. A membrane-associated lipomannan in micrococci. Biochem J. 1975 Nov;151(2):387–397. doi: 10.1042/bj1510387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rice C. E., Annau E., Konst H. Studies Of Johne's Disease In Canada: VII. Complement-Fixation Tests with A Polysaccharide Fraction Of Johne's Bacilli. Can J Comp Med Vet Sci. 1959 Sep;23(9):291–295. [PMC free article] [PubMed] [Google Scholar]
  25. Riemann H. P., Abbas B. Diagnosis and control of bovine paratuberculosis (Johne's disease). Adv Vet Sci Comp Med. 1983;27:481–506. [PubMed] [Google Scholar]
  26. TSUMITA T., MATSUMOTO R., MIZUNO D. Chemical and biological properties of the hemagglutination antigen, a lipopolysaccharide, of Mycobacterium tuberculosis, var. hominis. Jpn J Med Sci Biol. 1960 Jun;13:131–138. doi: 10.7883/yoken1952.13.131. [DOI] [PubMed] [Google Scholar]
  27. TSUMITA T., MATSUMOTO R., MIZUNO D. The nature of the Middlebrook-Dubos haemagglutination antigen of Mycobacterium tuberculosis. Jpn J Med Sci Biol. 1959 Jun;12:167–170. doi: 10.7883/yoken1952.12.167. [DOI] [PubMed] [Google Scholar]
  28. TSUMITA T., OHASHI M. A SYNTHETIC ACYL POLYSACCHARIDE AND THE HEMAGGLUTINATION ACTIVITY. J Exp Med. 1964 Jan 1;119:1017–1025. doi: 10.1084/jem.119.6.1017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Weber P. L., Gray G. R. Structural and immunochemical characterization of the acidic arabinomannan of Mycobacterium smegmatis. Carbohydr Res. 1979 Sep;74:259–278. doi: 10.1016/s0008-6215(00)84781-3. [DOI] [PubMed] [Google Scholar]
  30. Yokomizo Y., Merkal R. S., Lyle P. A. Enzyme-linked immunosorbent assay for detection of bovine immunoglobulin G1 antibody to a protoplasmic antigen of Mycobacterium paratuberculosis. Am J Vet Res. 1983 Nov;44(11):2205–2207. [PubMed] [Google Scholar]
  31. de Lisle G. W., Seguin P., Samagh B. S., Corner A. H., Duncan J. R. Bovine paratuberculosis I. A herd study using complement fixation and intradermal tests. Can J Comp Med. 1980 Apr;44(2):177–182. [PMC free article] [PubMed] [Google Scholar]

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