Ultrastructural Analysis of Mechanism of Protein-Domain-Assembly on Cell Membrane of Specific Lipid-Binding Toxin.

• Project/Area Number: 15590396
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Bacteriology (including Mycology)
• Research Institution: Kitasato University
• Principal Investigator: SEKIYA Kachiko Kitasato Univ., Sch. of Pharm., Assistant Prof., 薬学部, 講師 (30050579)
• Co-Investigator(Kenkyū-buntansha): ABE Akio Kitasato Univ., Kitasato Institute for Life Sciences, Prof., 北里生命科学研究所, 教授 (50184205)
• Project Period (FY): 2003 – 2005
• Project Status: Completed (Fiscal Year 2005)
• Budget Amount: ¥3,500,000 (Direct Cost: ¥3,500,000); Fiscal Year 2005: ¥1,000,000 (Direct Cost: ¥1,000,000); Fiscal Year 2004: ¥1,100,000 (Direct Cost: ¥1,100,000); Fiscal Year 2003: ¥1,400,000 (Direct Cost: ¥1,400,000)
• Keywords: Streptolysin O / Hemolysis / Electron Microscopy / Negative staining / SLO / cholesterol / Protein-domain / Pore-forming Toxin / ストレプトリジンO / 超微形態 / 分子会合モデル / SLO変異体 / ネガティブ染色 / 分子会合 / リポソーム

Research Abstract

Streptolysin O (SLO) is a membrane-damaging toxic protein produced by group A streptococci. An ultrastructural analysis of pore formation and the mechanism of hemolysis by SLO, using a mutant form of SLO [SLO(C/A)-SS and native SLO. Electron micrographs of negatively stained preparations revealed that SLO(C/A)-SS was unable to penetrate the erythrocyte membrane as a consequence of immobilization that was due to a disulfide bond between domains. The SLO(C/A)-SS molecules that bound to membranes formed numerous single-layered ring-shaped structures that did not result in pores on the membranes. These structures were similar to the structures formed by native SLO at 0℃. After treatment with dithiothreitol (DTT), SLO(C/A)-SS that had bound to membranes formed double-layered rings with pores on the membranes, as does native SLO at room temperature. There was a correlation between the number of double-layered ring structure with pores that formed and the duration of treatment with DTT. From these results, the following two points were concluded ; (1) Each inner and outer layer of the previously proposed ring model of SLO (J.Bacteriol. 1993) was composed of single SLO molecules associated in a circle. (2) Hemolysis by SLO toxin involves two defined steps; a temperature-independent step and a temperature-dependent step. An increase in temperature is necessary for the occurrence of conformational changes and the formation of the double-layered ring after the insertion of domain 3 into the host cell membrane.

Research Products

• [Journal Article] Assembly of the type III secretion apparatus of enteropathogenic Escherichia coli. (2006)
  • Author(s): Ogino, T., Ohno, R., Sekiya, K., Kuwae, A., Matsuzawa, T., Abe, A.et al.
  • Journal Title: J. Baceriol. 188(8) Pages: 2801-2811
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Assembly of the type III secretion apparatus of enteropathogenic Escherichia coll. (2006)
  • Author(s): Ogino, T., Ohno, R., Sekiya, K., Kuwae, A., Matsuzawa, T., Nonaka, T., Fukuda, H., Imajoh-Ohmi, S., Abe, A.
  • Journal Title: J.Baceriol. 188(8) Pages: 2801-2811
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Assembly of the typeIII secretion apparatus of enteropathogenic Escherichia coli. (2006)
  • Author(s): Tomoaki Ogino, Kachiko Sekiya, Asaomi Kuwae, Akio Abe, et al.
  • Journal Title: J.Bacteriol. (In press)
• [Journal Article] Nocardioides aromaticivorans sp. nov., a dibenzofuran-degrading bacterium isolated from dioxin-polluted environments. (2005)
  • Author(s): Kubota, M., Kawahara, K., Sekiya, K., Hiraishi, A.et al.
  • Journal Title: Systematic Applied Microbiol. 28 Pages: 165-174
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Azithromycin inhibits the formation of flagellar filaments without suppressing flagellin synthesis in Salmonella enterica serovar Typhimurium. (2005)
  • Author(s): Matsui, H., Eguchi, M., Ohsumi, K., Isshiki, Y., Sekiya, K., Kikuchi, Y., et al.
  • Journal Title: Antimicrob. Agents Chemother. 49(8) Pages: 3396-3403
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] 病原細菌由来蛋白質の構造解析 -ネガティブ染色法- (2005)
  • Author(s): 関矢加智子
  • Journal Title: 医生電顕技術誌 19(2) Pages: 132-134
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Nocardioides aromaticivorans sp.nov., a dibenzofuran-degrading bacterium isolated from dioxin-polluted environments. (2005)
  • Author(s): Kubota, M., Kawahara, K., Sekiya, K., Uchida, T., Hattori, Y., Futamata, H., Hiraishi, A.
  • Journal Title: Systematic Applied Microbiol. 28 Pages: 165-174
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] A zithromycin inhibits the formation of flagellar filaments without suppressing flagellin synthesis in Salmonella enterica serovar Typhimurium. (2005)
  • Author(s): Matsui, H., Eguchi, M., Ohsumi, K., Nakamura, A., Isshiki, Y., Sekiya, K., Kikuchi, Y., Nagamitsu, T., Masuma, R., Sunazuka, T., Omura, S.
  • Journal Title: Antimicrob.Agents Chemother. 49(8) Pages: 3396-3403
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Analysis of Morphological Structure of Proteins Produced by Pathogenic Bacteria. (Negative Staining) (2005)
  • Author(s): Sekiya, K.
  • Journal Title: J.Electr.Microsc.Technol.Med.Biol. 19(2) Pages: 132-134
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] 病原細菌由来蛋白質の構造解析 -ネガティブ染色法- (2005)
  • Author(s): 関矢 加智子
  • Journal Title: 医生電顕技術誌 19(2) Pages: 132-134
• [Journal Article] Nocardioides aromaticivorans sp.nov., a dibenzofuran-degrading bacterium isolated from dioxin-polluted environments. (2005)
  • Author(s): Masahiro Kubota, Kazuyoshi kawahara, Kachiko Sekiya, Akira Hiraishi et al.
  • Journal Title: Systematic and Applied Microbiology
• [Book] 基礎病原微生物学 (2005)
  • Author(s): 檀原宏文, 田口文章, 関矢加智子ら20名
  • Total Pages: 835
  • Publisher: 廣川書店
  • Description: 「研究成果報告書概要(和文)」より