1887

Abstract

Two neurotoxic alkaloids, anatoxin-a and its homologue homoanatoxin-a, were purified from the filamentous cyanobacteria sp. strain 193 (PCC 9240) and NIVA CYA-92 (PCC 10111), respectively, and characterized by mass spectrometry. Biological activity was determined by examining the capacity of the toxins to competitively inhibit the binding of radiolabelled bungarotoxin to acetylcholine receptors, using post-synaptic membrane fractions of electric tissue. Inhibition was concentration dependent, with a of 5·4±1·1×10 M for anatoxin-a and 7·4±0·9×10 M for homoanatoxin-a. Their high affinities for the nicotinic cholinergic receptors were exploited to adapt the radioligand-binding assay for routine detection of this class of neurotoxins directly in low-molecular-mass cell extracts of cyanobacteria. Confirmation of the results and toxin identification were achieved by coupled gas chromatography-mass spectrometry (GC/MS). Seventy-six axenic strains, representative of 13 genera, were analysed. Five strains of the genus , hitherto unknown for their toxicity, inhibited bungarotoxin binding. GC/MS revealed that sp. strains PCC 6407, PCC 6412 and PCC 9107 synthesized exclusively anatoxin-a, whereas both anatoxin-a and homoanatoxin-a were produced by strain PCC 9029. sp. strain PCC 6506, an isolate co-identic with strain PCC 9029, also produced both neurotoxins, but their respective presence depended upon growth conditions. The latter results suggest that regulatory differences in at least some of the cyanobacterial strains may account for the preferential synthesis of only one of the two neurotoxins or for their simultaneous occurrence.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.27660-0
2005-04-01
2019-11-22
Loading full text...

Full text loading...

/deliver/fulltext/micro/151/4/mic1511263.html?itemId=/content/journal/micro/10.1099/mic.0.27660-0&mimeType=html&fmt=ahah

References

  1. Adeyemo, O. M. & Sirén, A. L. ( 1992; ). Cardio-respiratory changes and mortality in the conscious rat induced by (+)- and (±)-anatoxin-a. Toxicon 30, 899–905.[CrossRef]
    [Google Scholar]
  2. Amar, M., Thomas, P., Johnson, C., Lunt, G. G. & Wonnacott, S. ( 1993; ). Agonist pharmacology of the neuronal α7 nicotinic receptor expressed in Xenopus oocytes. FEBS Lett 327, 284–288.[CrossRef]
    [Google Scholar]
  3. Aronstam, R. S. & Witkop, B. ( 1981; ). Anatoxin-a interactions with cholinergic synaptic molecules. Proc Natl Acad Sci U S A 78, 4639–4643.[CrossRef]
    [Google Scholar]
  4. Booker, M. J. & Walsby, A. E. ( 1979; ). The relative form resistance of straight and helical blue-green algal filaments. Br Phycol J 14, 141–150.[CrossRef]
    [Google Scholar]
  5. Bruno, M., Barbini, D. A., Pierdominici, E., Serse, A. P. & Ioppolo, A. ( 1994; ). Anatoxin-a and a previously unknown toxin in Anabaena planctonica from blooms found in Lake Mulargia (Italy). Toxicon 32, 369–373.[CrossRef]
    [Google Scholar]
  6. Burger-Wiersma, T., Veenhuis, M., Korthals, H. J., Van de Wiel, C. C. M. & Mur, L. R. ( 1986; ). A new prokaryote containing chlorophylls a and b. Nature 320, 262–264.[CrossRef]
    [Google Scholar]
  7. Carmichael, W. W. ( 1994; ). The toxins of cyanobacteria. Sci Am 270, 78–86.[CrossRef]
    [Google Scholar]
  8. Carmichael, W. W. & Gorham, P. R. ( 1978; ). Anatoxins from clones of Anabaena flos-aquae isolated from lakes of western Canada. Mitt Int Ver Limnol 21, 285–295.
    [Google Scholar]
  9. Castenholz, R. W., Rippka, R., Herdman, M. & Wilmotte, A. ( 2001; ). Subsection III (Formerly Oscillatoriales Elenkin 1934). In Bergey's Manual of Systematic Bacteriology, 2nd edn, vol. 1, the Archaea and the Deeply Branching and Phototrophic Bacteria, pp. 539–562. Edited by D. R. Boone, R. W. Castenholz & G. M. Garrity. New York, Berlin, Heidelberg: Springer.
  10. Cheng, Y. & Prusoff, W. H. ( 1973; ). Relationship between the inhibition constant (K i) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 22, 3099–3108.[CrossRef]
    [Google Scholar]
  11. Devlin, J. P., Edwards, O. E., Gorham, P. R., Hunter, N. R., Pike, R. K. & Stavric, B. ( 1977; ). Anatoxin-a, a toxic alkaloid from Anabaena flos-aquae NRC-44h. Can J Chem 55, 1367–1371.[CrossRef]
    [Google Scholar]
  12. Edwards, C., Beattie, K. A., Scrimgeour, C. M. & Codd, G. A. ( 1992; ). Identification of anatoxin-a in benthic cyanobacteria (blue-green algae) and in associated dog poisonings at Loch Insh, Scotland. Toxicon 30, 1165–1175.[CrossRef]
    [Google Scholar]
  13. Fawell, J. K., Michell, R. E., Hill, R. E. & Everett, D. J. ( 1999; ). The toxicity of cyanobacterial toxins in the mouse. II. anatoxin-a. Hum Exp Toxicol 18, 168–173.[CrossRef]
    [Google Scholar]
  14. Furey, A., Crowley, J., Lehane, M. & James, K. J. ( 2003a; ). Liquid chromatography with electrospray ion-trap mass spectrometry for the determination of anatoxins in cyanobacteria and drinking water. Rapid Commun Mass Spectrom 17, 583–588.[CrossRef]
    [Google Scholar]
  15. Furey, A., Crowley, J., Shuilleabhain, A. N., Skulberg, O. M. & James, K. J. ( 2003b; ). The first identification of the rare cyanobacterial toxin, homoanatoxin-a, in Ireland. Toxicon 41, 297–303.[CrossRef]
    [Google Scholar]
  16. Garcia-Pichel, F., López-Cortés, A. & Nübel, U. ( 2001; ). Phylogenetic and morphological diversity of cyanobacteria in soil desert crusts from the Colorado plateau. Appl Environ Microbiol 67, 1902–1910.[CrossRef]
    [Google Scholar]
  17. Harada, K.-I., Kimura, Y., Ogawa, K., Suzuki, M., Dahlem, A. M., Beasley, V. R. & Carmichael, W. W. ( 1989; ). A new procedure for the analysis and purification of naturally occurring anatoxin-a from the blue-green alga Anabaena flos-aquae. Toxicon 27, 1289–1296.[CrossRef]
    [Google Scholar]
  18. Harada, K.-I., Nagai, H., Kimura, Y., Suzuki, M., Park, H.-D., Watanabe, M. F., Luukkainen, R., Sivonen, K. & Carmichael, W. W. ( 1993; ). Liquid chromatography/mass spectrometric detection of anatoxin-a, a neurotoxin from cyanobacteria. Tetrahedron 49, 9251–9260.[CrossRef]
    [Google Scholar]
  19. Hemscheidt, T., Rapala, J., Sivonen, K. & Skulberg, O. M. ( 1995; ). Biosynthesis of anatoxin-a in Anabaena flos-aquae and homoanatoxin-a in Oscillatoria formosa. J Chem Soc Commun 13, 1361–1362.
    [Google Scholar]
  20. Hill, J. A., Nghiêm, H. O. & Changeux, J.-P. ( 1991; ). Serine-specific phosphorylation of nicotinic receptor associated 43K protein. Biochemistry 30, 5579–5585.[CrossRef]
    [Google Scholar]
  21. Himberg, K. ( 1989; ). Determination of anatoxin-a, the neurotoxin of Anabaena flos-aquae cyanobacterium, in algae and water by gas chromatography-mass spectrometry. J Chromatogr 481, 358–362.[CrossRef]
    [Google Scholar]
  22. Mahmood, N. A. & Carmichael, W. W. ( 1986; ). The pharmacology of anatoxin-a(s), a neurotoxin produced by the freshwater cyanobacterium Anabaena flos-aquae NRC 525-17. Toxicon 24, 425–434.[CrossRef]
    [Google Scholar]
  23. Namikoshi, M., Murakami, T., Watanabe, M. F., Oda, T., Yamada, J., Tsujimura, S., Nagai, H. & Oishi, S. ( 2003; ). Simultaneous production of homoanatoxin-a, anatoxin-a, and a new non-toxic 4-hydroxyhomoanatoxin-a by the cyanobacterium Raphidiopsis mediterranea Skuja. Toxicon 42, 533–538.[CrossRef]
    [Google Scholar]
  24. Rippka, R. ( 1988; ). Isolation and purification of cyanobacteria. Methods Enzymol 167, 3–27.
    [Google Scholar]
  25. Rippka, R. & Herdman, M. ( 1992; ). Pasteur Culture Collection of Cyanobacterial Strains in Axenic Culture: Catalogue & Taxonomic Handbook, vol. I, Catalogue of Strains. Paris: Institut Pasteur.
  26. Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M. & Stanier, R. Y. ( 1979; ). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111, 1–61.[CrossRef]
    [Google Scholar]
  27. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  28. Schlösser, U. G. ( 1994; ). SAG – Sammlung von Algenkulturen at the University of Göttingen, Catalogue of Strains 1994. Bot Acta 107, 113–186.[CrossRef]
    [Google Scholar]
  29. Sivonen, K. & Jones, G. ( 1999; ). Cyanobacterial toxins. In Toxic Cyanobacteria in Water: a Guide to their Public Health Consequences, Monitoring and Management, pp. 41–111. Edited by I. Chorus & J. Bartram. London: E. & F. N. Spon.
  30. Sivonen, K., Himberg, K., Luukkainen, R., Niemelä, S. I., Poon, G. K. & Codd, G. A. ( 1989; ). Preliminary characterization of neurotoxic cyanobacteria blooms and strains from Finland. Toxic Assess 4, 339–352.[CrossRef]
    [Google Scholar]
  31. Sivonen, K., Niemelä, S. I., Niemi, R. M., Lepistö, L., Luoma, T. H. & Räsänen, L. A. ( 1990; ). Toxic cyanobacteria (blue-green algae) in Finnish fresh and coastal waters. Hydrobiologia 190, 267–275.[CrossRef]
    [Google Scholar]
  32. Skulberg, O. M., Carmichael, W. W., Andersen, R. A., Matsunaga, S., Moore, R. E. & Skulberg, R. ( 1992; ). Investigations of a neurotoxic oscillatorialean strain (Cyanophyceae) and its toxin. Isolation and characterization of homoanatoxin-a. Environ Toxicol Chem 11, 321–329.[CrossRef]
    [Google Scholar]
  33. Starr, R. C. & Zeikus, J. A. ( 1993; ). UTEX – the culture collection of algae at the University of Texas at Austin. J Phycol 29 (supplement), 1–106.[CrossRef]
    [Google Scholar]
  34. Stevens, D. K. & Krieger, R. I. ( 1991; ). Stability studies on the cyanobacterial nicotinic alkaloid anatoxin-a. Toxicon 29, 167–179.[CrossRef]
    [Google Scholar]
  35. Sugiyama, J. ( 1998; ). Microalgae. In IAM Catalogue of Strains, 2nd edn, pp. 237–264. Edited by the Editorial Board of the IAM Catalogue of Strains. Center for Cellular and Molecular Research, Institute of Molecular and Cellular Biosciences, University of Tokyo. Tokyo: Microbiology Research Foundation.
  36. Swanson, K. L., Allen, C. N., Aronstam, R. S., Rapoport, H. & Albuquerque, E. X. ( 1985; ). Molecular mechanisms of the potent and stereospecific nicotinic receptor agonist (+)-anatoxin-a. Mol Pharmacol 29, 250–257.
    [Google Scholar]
  37. Swanson, K. L., Aronstam, R. S., Wonnacott, S., Rapoport, H. & Albuquerque, E. X. ( 1991; ). Nicotinic pharmacology of anatoxin analogs. I. Side chain structure-activity relationships at peripheral agonist and noncompetitive antagonist sites. J Pharmacol Exp Ther 259, 377–386.
    [Google Scholar]
  38. Takino, M., Daishima, S. & Yamaguchi, K. ( 1999; ). Analysis of anatoxin-a in freshwaters by automated on-line derivatization-liquid chromatography-electrospray mass spectrometry. J Chromatogr A 862, 191–197.[CrossRef]
    [Google Scholar]
  39. Thomas, P., Stephens, M., Wilkie, G. & 8 other authors ( 1993; ). (+)-Anatoxin-a is a potent agonist at neuronal nicotinic acetylcholine receptors. J Neurochem 60, 2308–2311.[CrossRef]
    [Google Scholar]
  40. Tomaselli, L., Giovannetti, L. & Torzillo, G. ( 1993; ). Physiology of stress response in Spirulina spp. Bull Inst Océanogr (Monaco) no. spécial 12, 65–75.
    [Google Scholar]
  41. Viti, C., Ventura, S., Lotti, F., Capolino, E., Tomaselli, L. & Giovannetti, L. ( 1997; ). Genotypic diversity and typing of cyanobacterial strains of the genus Arthrospira by very sensitive total DNA restriction profile analysis. Res Microbiol 148, 605–611.[CrossRef]
    [Google Scholar]
  42. Wonnacott, S., Jackman, S., Swanson, K. L., Rapoport, H. & Albuquerque, E. X. ( 1991; ). Nicotinic pharmacology of anatoxin analogs. II. Side chain structure-activity relationships at neuronal nicotinic ligand binding sites. J Pharmacol Exp Ther 259, 387–391.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.27660-0
Loading
/content/journal/micro/10.1099/mic.0.27660-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error