pH-dependent Cytotoxicity of a Peptide Toxin, Tolaasin

펩티드 독소 Tolaasin에 의한 세포독성의 pH 의존성

  • Kim, Sung-Tae (Department of Agricultural Chemistry, Chungbuk National University) ;
  • Choi, Tae-Keun (Department of Agricultural Chemistry, Chungbuk National University) ;
  • Kim, Young-Kee (Department of Agricultural Chemistry, Chungbuk National University)
  • 김성태 (충북대학교 농업생명환경대학 농화학과) ;
  • 최태근 (충북대학교 농업생명환경대학 농화학과) ;
  • 김영기 (충북대학교 농업생명환경대학 농화학과)
  • Published : 2007.12.31


Tolaasin, a peptide toxin produced by Pseudomonas tolaasii, causes a serious disease on the cultivated mushrooms, known as brown blotch disease. Hemolysis using red blood cells was designed to measure the cytotoxicity of tolaasin molecules. Since tolaasin has two amine groups near the C-terminus, its membrane binding will be dependent on the ionic states of the amine groups. When the tolaasin peptide was titrated, its titration curve indicated the presence of titratable amine(s) at pH ranges from 7.0 to 9.6. When the pH-dependence of tolaasin-induced hemolysis was measured at various pHs, hemolysis was more efficient at alkaline pHs. In order to measure the membrane binding activity of tolaasin at different pHs, RBCs were incubated with tolaasin molecules for short time periods and washed out with fresh buffer. Because of the tolaasin binding during the preincubation period, fast hemolyses were observed at pH 8 or higher. These results imply that non-charged or less positively charged states of tolaasin molecules easily bind to membrane and show high hemolytic activity.

Pseudomonas tolaasii에 의해서 분비되는 펩티드 독소인 tolaasin은 재배버섯에 세균성 갈반병을 유발한다. Tolaasin의 독성은 적혈구를 파괴하는 용혈활성으로 평가된다. Tolaasin 펩티드는 C-말단부위에 두 개의 amine기를 갖고있어, 이 펩티드의 세포막 결합은 amine기의 전하상태에 따라 다를 수 있다. 이것을 확인하기 위하여 tolaasin을 적정하였을 때, 적정곡선은 pH 7.0에서 9.6 사이에서 적정이 되는 amine기가 있음을 보였다. Tolaasin에 의한 용혈활성의 pH 의존성을 조사하였을 때, 용혈활성은 알칼리 조건에서 증가함을 확인하였다. 따라서, pH 변화에 따른 tolaasin의 막결합 특성을 조사하기 위하여 적혈구를 tolaasin과 사전배양한 후, tolaasin을 포함하지 않은 완충액으로 씻어내고, 사전배양중 적혈구에 결합한 tolaasin의 용혈활성을 측정하였을 때, 활성은 pH 8 이상에서 크게 증가하였다. 이러한 결과는 tolaasin이 전하가 없거나 양전하량이 적은 상태에서 세포막에 잘 결합하여 세포독성이 커진다는 것을 의미한다.


  1. Kim, J. W., Kim, K. H. and Kang, H. J. (1994) Studies on the pathogenic Pseudomonas causing bacterial disease of cultivated mushroom in Korea: 1. On the causal organisms of the rots of Agaricus bisporus, Pleurotus ostreatus, and Lentinus edodes. Kor. J. Plant Pathol. 10, 197-210
  2. Jin, K. S., Kim, J. J., Jeon, C. S. and Lee, E. J. (1994) Characteristical differentiation on isolation and identification of mushroom bacterial brown blotch pathogens Pseudomonas tolaasii and Pseudomonas agarici. RDA. J. Agri. Sci. 36, 343-348
  3. Kim, J. W., Kwon, S. I. and Kang, H. J. (1995) Studies on the pathogenic Pseudomonas causing bacterial diseases of cultivated mushrooms in Korea: 2. Bacteriological characteristics of P. tolaasii causing mushroom brown blotch and white line reacting organism. Kor. J. Plant Pathol. 11, 353-360
  4. Wong, W. C. and Preece, T. F. (1985) Pseudomonas tolaasii in cultivated mushroom (Agaricus bisporus) crops effect of sodium hypochloride on the bacterium and on blotch disease severity. J. Appl. Bacteriol. 58, 259-267
  5. Geels, F. P., van Griensven, L. D. and Rutjens, A. J. (1991) Chlorine dioxide and the control of bacterial blotch on mushrooms, caused by Pseudomonas tolaasii, In Science and Cultivation of Edible Fungi (Maher, ed.) Vol. 1, p. 437-442, Balkema Pub., Rotterdam, Netherlands
  6. Guillaumes, J., Houdeau, G., Germain, R. and Oliver, J. M. (1998) Amelioration de la lutte biologique contre Pseudomonas tolaasii utilisation de bacteriophages. Bull. OEPP/EPPO 18, 77-82
  7. Munsch, P., Oliver, J. M. and Houdeau, G. (1991) Experimental control of bacterial blotch by bacteriophages. In Science and Cultivation of Edible Fungi, (Maher ed.,) Vol. 1., p. 389-396, Balkema Pub., Rotterdam, Netherlands
  8. Geels, F. P. (1995) Pseudomonas tolaasii control by kasugamycin cultivated mushrooms (Agaricus bisporus). J. Appl. Bacteriol. 79, 38-42
  9. Tolaas, A. G. (1915) A bacterial disease of cultivated mushrooms. Phytopathol. 5, 51-54
  10. Nutkins, J. C., Mortishire-Smith, R. J., Packman, L. C., Brodey, C. L., Rainey, P. B., Johnstone, K. and Williams, D. H. (1991) Structure determination of tolaasin, an extracellular lipodepsipeptide produced by the mushroom pathogen Pseudomonas tolaasii Paine. J. Am. Chem. Soc. 113, 2621-2627
  11. Brodey, C. L., Rainey, P. B., Tester, M. and Johnstone, K. (1991) Bacterial blotch disease of the cultivated mushroom is caused by an ion channel forming lipodepsipeptide toxin. Mol. Plant-Microbe Interact. 4, 407-411
  12. Cho, K. H. and Kim, Y. K. (2003) Two types of ion channel formation of tolaasin, a Pseudomonas peptide toxin. FEMS Microbiol. Lett. 221, 221-226
  13. Cho, K. H., Park, K. S. and Kim, Y. K. (2000) Hemolytic properties of tolaasin causing the brown blotch disease on oyster mushroom. J. Kor. Soc. Agricul. Chem. Biotechnol. 43, 190-195
  14. Lee, H. I., Lee, S. D., Park, K. S., Kim, Y. K. and Cha, J. S. (1997) Pathogenicity of bacterial isolates from brown blotchdiseased oyster mushrooms in Chungcheungbuk-do. J. Agr. Sci., Chungbuk Nat'l Univ. 14, 121-132
  15. Tsuneda, A., Suyama, K., Murakami, S. and Ohira, I. (1995) Occurrence of Pseudomonas tolaasii on fruiting bodies of Lentinula edodes formed on Quercus logs. Mycosci. 36, 283-288
  16. Cho, K. H., Kim, S. T. and Kim, Y. K. (2007) Purification of a pore-forming toxin, tolaasin, produced by Pseudomonas tolaasii 6264. J. Biochem. Mol. Biol. 40, 113-118
  17. Rainey, P. B., Brodey, C. L. and Johnstone, K. (1991) Biological properties and spectrum of activity of tolaasin, a lipodepsipeptide toxin produced by the mushroom pathogen Pseudomonas tolaasii. Physiol. Mol. Plant Pathol. 39, 57-70
  18. Bernheimer, A. W. and Rudy, B. (1986) Interactions between membranes and cytolytic peptides. Biochim. Biophys. Acta 864, 123-141
  19. Jacobs, M. H. and Stewart, D. R. (1947) Osmotic properties of the erythrocyte. XII. ionic and osmotic equilibria with a complex external solution. J. Cell. Comp. Physiol. 30, 79-103
  20. Freedman, J. C. and Hoffman, J. F. (1979) Ionic and osmotic equilibria of human red blood cells treated with nystatin. J. Gen. Physiol. 74, 157-185
  21. Glaser, R. and Donath, J. (1984) Stationary ionic states in human red blood cells. Bioelectrochem. Bioenerg. 13, 71-83
  22. Shai, Y., Hadari, Y. R. and Finkels, A. (1991) pH-dependent pore formation properties of pardaxin analogues. J. Biol. Chem. 266, 22346-22354