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Site-Directed Mutagenesis of HlyA of Vibrio Cholerae at Common Domain Found in Vibroio Vulnificus Cytolysin.

Research Project

Project/Area Number 01570236
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field 細菌学
Research InstitutionResearch Institute for Microbial Diseases, Osaka University

Principal Investigator

YAMAMOTO Koichiro  Biken, Osaka Univ., Research Instructor, 微生物病研究所, 助手 (30158274)

Co-Investigator(Kenkyū-buntansha) YOH Myonsun  Biken, Osaka Univ., Research Instructor, 微生物病研究所, 助手 (70093482)
Project Period (FY) 1989 – 1990
Project Status Completed (Fiscal Year 1990)
Budget Amount *help
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1990: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1989: ¥1,200,000 (Direct Cost: ¥1,200,000)
Keywordsmutation / Amino Acid Sequence / hemolysin / Pathogenic Factor / cloning / domain / vibrio / Nucleotide Sequence / 毒素 / コレラ菌 / サイトディレクティド変異 / バルニフィカス菌 / タンパク毒素
Research Abstract

Site-directed mutagenesis of hlyA of Vibrio cholerae at common domain found in Vibrio vulnificus cytolysin was carried out.
1. Oligonucleotide primers were synthesized as one of the amino acids of the domain in substituted to another amino acid.
The mutations were designed to substitute to another amino acid of which
(A) hydropathy and
(B) charge was greatly changed and
(C) the third nucleotide of the codons was changed
where it gives amino acid substitution.
2. Site-directed mutations were carried out using these oligonucleotide primers and the mutation was confirmed by DNA sequencing and ten mutants were obtained.
3. The mutant precursors were over-produced in E. Coli. The mutant cytolysins were extracted and partially purified. The quantities of the cytolysin antigens and the protein contents were determined.
4. The specific activities of the mutant cytolysins were 1/10 - 1/100 of the native cytolysin. This result suggest that the common domain found in V. Cholerae and V. Vulnificus play an important role(s) in cytolytic activities of the cytolysins.

Report

(3 results)
  • 1990 Annual Research Report   Final Research Report Summary
  • 1989 Annual Research Report
  • Research Products

    (45 results)

All Other

All Publications (45 results)

  • [Publications] Yamamoto,K.,et al.: "Processing for activation of Vibrio cholerae El Tor cytolysin." Jpn.J.Med.Sci.Biol.42. 178- (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Honda,T.,et al.: "Production of monoclonal antibodies against thermostable direct hemolysin of Vibrio parahaemolyticus and applicatio of the antibodies for enzymeーlinked immunosorbent assay." Med.Microbiol.Immunol.178. 245-253 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Yoh,M.,et al.: "Homology of a hemolysin (VhーrTDH) produced by clinical and environmental isoltes of Vibrio hollisae." FEMS Microbiol.Lett.61. 171-176 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Honda,T.,et al.: "Evidence of immunologic crossーreactivity between hemolysins of Vibrio hollisae and Vibrio parahaemolyticus demonstrated by monoーclonal antibodies." J.Infectious Diseases. 160. 1089-1090 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Yoh,M.,et al.: "Comparative amino acid sequence analysis of hemolysins produced by Vibrio hollisae and Vibrio parahaemolyticus." J.Bacteriol.171. 6859-6861 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Yamamoto,K.,et al.: "The cytolysin gene of Vibrio vulnificus :Sequence and relationーships to the Vibrio cholerae El Tor hemolysin gene." Infect.Immun.58. 2706-2709 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Iida,T.,et al.: "Cloning and expression of two genes encoding higly homologous hemolysins from a Kanagawa phenomenonーpositive Vibrio parahaemolyticus T4750 strain." Gene. 93. 9-5 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Yamsmoto,K.,et al.: "Twoーstep processing for activation of the cytolysin/hemolysin of Vibrio cholerae Ol biotype El Tor:Nucleotide sequence of the structtural gene (hlyA)and characterization of the processed products." Infect.Immun.58. 4109-4116 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Yoh,M.,et al.: "Characterizatrion of thermostable direct hemolysin encoded by four representative tdh genes of Vibrio parahaemolyticus." Microbial Pathogenesis,.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Honda,T.,et al.: "Characterization of a new thermostable direct haemolysin produced by a Kanagawaーphenomenonーnegative clinical isolate of Vibrio parahaemolyticus." J.General Microbiol.,.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Honda,T.,et al.: "Characterization of TDHーrelated hemolysins (VpーTRH) produced by Kanagawa phenomenonーnegative clinical isolates of Vibrio parahaeーmolyticus" Advances in Research on Cholera and Related Diarrheas. 7. 277-286 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Yoh,M.,et al: "Characterization of four thermostable direct hemolysins encoded by four representative tdh genes of Vibrio parahaemolyticus." Jpn.J.Med.Sci.Biol.,.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Naka,A.,et al.: "Characterization of Vibrio cholerae nonーOl hemagglulutinin/protese." Jpn.Med.Sci.Biol.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Yoh,M.,et al.: "Characterization and amino and sequence of Vibrio hollisae hemolysin that relates to the thermostable direct hemolysin of Vibrio parahaemolyticus." Advances in Reserch on Cholera and Related Diarrheas,.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Honda,T.,et al.: "Characterization of TDHーrelated hemolysins (VpーTRH) produced Kanagawa phenomenonーnegative clinical isolates of Vibrio parahaeーmolyticus" Advances in Research on Chlolera and Related Diarrheas,.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] 本田 武司 他: "Vibrio cholerae nonーOIの産生する多様な病原毒素 (医学細菌学第5巻、三輪谷俊夫監修)" 菜根出版(東京), 31 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Yamamoto, K., Honda, T., Miwatani, T., and Ichinose, Y.: "Two-step processing in a Vibro cholerae cytolysin." Jpn. J. Med. Sci. Biol.42. 178 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Honda, T., Ni, Y., Yoh, M., and Miwatani, T: "Production of monoclonal antibodies against thermostable direct hemolysin of Vibrio parahaemolyticus and applicatio of the antibodies for enzyme-linked immunosorbent assay." Med. Microbiol. Immunol.178. 245-253 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Yoh, M., Honda, T., and Miwatani, T.: "Homology of a hemolysin (Vh-rTDH) produced by clinical and environmental isoltes of Vibrio hollisae." FEMS Microbiol. Lett.61. 171-176 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Honda, T., Ni, Y., Yoh, M., and Miwatani, T.: "Evidence of immunologic cross-reactivity between hemolysins of Vibrio hollisae and Vibrio parahaemolyticus demonstrated by monoclonal antibodies." J. Infectious Diseases. 160. 1089-1090 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Yoh, M., Honda, T., Miwatani, T, Tsunasawa, S., and Sakiyama, F.: "Comparative amino acid sequence analysis of hemolysins produced by Vibrio hollisae and Vibrio parahaemolyticus." J. Bacteriol. 171. 6859-6861 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Yamamoto, K., Wright, A. C., Kaper, J. B. and Morris, J. G. Jr.: "The cytolysin gene of Vibrio vulnificus : Sequence and relationships to the Vibrio cholerae El Tor hemolysin gene." Infection and Immunity. 58. 2706-2709 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Iida, T. and Yamamoto, K.: "Cloning and expression of two genes encoding highly homologous hemolysins from a Kanagawa-phenomenon-positive Vibrio parahaemolyticus T4750 strain." Gene. 93. 9-5 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Yamamoto, K., Ichinose, Y., Shinagawa, H., Makino, K., Nakata, A., Iwanaga, M., Honda, T. and Miwatani, T.: "Two-step processing for activation of the cytolysin/hemolysin of Vibrio cholerae O1 biotype E1 Tor : Nucleotide sequence of the structural gene (hlyA) and characterization of the processed products." Infection and Immunity. 58. 4106-4116 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Yoh, M., Honda, T., Miwatani, T., and Nishibuchi, M.: "Characterizatrion of thermostable direct hemolysin encoded by four representative tdh genes of Vibrio Parahaemolyticus." Microbial Pathogenesis. (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Honda, T., Abad-Lapuebla, M. A., Ni, Y., Yamamoto, K. and Miwatani, T.: "Characterization of a new thermostable direct haemolysin produced by a Kanagawa-phenomenon-negative clinical isolate of Vibrio parahaemolyticus." J. General Microbiol.(1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Honda, T., Ni, Y., Yoh, M., Nishibuchi, M., Miwatani, T., Ishibashi, M., Kinoshita, Y., Honda, S., Goto, I., Minematsu, I., Shimoirisa, K., Ikeda, N., and Asano, N.: "Characterization of a TDH-related hemolysin produced by Kanagawa phenomenon-negative Vibrio parahaemolyticus which caused an outbreak of traveller's diarrhea." Jpn. J. Med. Sci. Biol.,.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Yoh, M., Honda, T., Miwatani, T., and Nishibuchi, M.: "Characterization of four thermostable direct hemolysins encoded by four representative tdh genes of Vibrio parahaemolyticus." Advances in Research on Cholera and Related Diarrheas, 7, (Eds., R. B. Sack and Y. Zinnaka). KTK Scientific Publishers, Tokyo. 277-286 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Naka, A., Lertpocasombat, K., Yamamoto, K., Honda, T., and Miwatani, T.,: "Characterization of Vibrio cholerae non-O1 hemagglulutinin/protease." Jpn. Med. Sci. Biol.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Yoh, M., Honda, T., Miwatani, T., Tsunasawa, S. and Sakiyama, F.: "Characterization and amino acid sequence of vibrio hollisae hemolysin that relates to the thermostable direct hemolysin of Vibrio parahaemolyticus." Advances in Research on Cholera and Related Diarrheas.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Honda, T., Ni, Y., Iida, T., Yoshida, H., Yoh, M., Yamamoto, K., Miwatani, T., Okamoto, T., Goshima, K., Takakura, H., Tsunasawa, S. and Sakiyama, F.: "Characterization of TDH-related hemolysins (Vp-TRH) produced by Kanagawa phenomenon-negative clinical isolates of Vibrio parahaemolyticus" Advances in Research on Cholera and Related Diarrheas.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Yamamoto, K., Ichinose, Y., Honda, T., and Miwatani, T.: "Processing for activation of Vibrio cholerae El Tor cytolysin." Advances in Research on Cholera and Related Diarrheas.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Yamamoto,K.: "The cytolysin gene of Vibrio vulnificus i:Sequence and relationship to the Vibrio cholerae hemolysin gene" Infection and Immunity. 58. 2706-2709 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Iida,T.: "Cloning and expression of two genes encoding highly homologous hemolysins from a Kanagawaーphenomenonーpositive Vibrio parahaemolyticus T4750 strain." Gene. 93. 9-15 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Yamamoto,K.: "Twoーstep procssing for activation of the cytolysin/hemolysin of Vibrio cholerae 01 biotype El Tor:nucleotide sequence of the structural gene (hlyA) and characterization of the processed products." Infection and Immunity. 58. 4106-4116 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Honda,T.: "Characterization of a new thermostable direct hemolysin produced by a kanagawaーnegative clinical isolate of Vibrio parahaemolyticus." Journal of General Microbiology. (1991)

    • Related Report
      1990 Annual Research Report
  • [Publications] Tamatsukuri,S.: "Detection of heatーlabile enterotoxi gene in enterotoxigenic Escherichia coli by densitometric evaluation using highly specific enzymeーlinked oligoーnucleotide probes."

    • Related Report
      1990 Annual Research Report
  • [Publications] Yamamoto,K.: "Processing and activation of Vibrio cholerae hemolysin precusor by the V.cholerae soluble hemagglutinin/protease and trypsin."

    • Related Report
      1990 Annual Research Report
  • [Publications] 本田 武司: "Vibrio cholerae nonー01の産生する多様な病原毒素 (医学細菌学5巻,三輪谷 俊夫監修)" 菜根出版(東京), 31 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Yamamoto K.: "The cytolysin gene of Vibrio vulnificus:Sequence and relationship to Vibrio cholerae El Tor hemolysin."

    • Related Report
      1989 Annual Research Report
  • [Publications] Iida,T.: "Cloning and expression of two genes encoding highly homologous hemolysins from a Kanagawa phenomenonpositive Vibrio parahaemolyticus T4750 strain." Gene. (1990)

    • Related Report
      1989 Annual Research Report
  • [Publications] Yamamoto K.: "Twoーstep processing for activation of cytolysin/hemolysin of Vibrio cholerae 01,E1 Tor,N86:Nucleotide sequence of the structure gene(hlyA)and characterization of the processed products"

    • Related Report
      1989 Annual Research Report
  • [Publications] Yamamoto K.: "Activation of procytolysin of Vibrio cholerae 01,E1 Tor by soluble hemagglutinin/protease of Vibrio choleae and trypsin."

    • Related Report
      1989 Annual Research Report
  • [Publications] Yoh,M.: "Homogeneity of hemolysin(VhーrTDH)produced by clinical and environmental isolates of Vibrio hollisae." FEMS Microbiol.Lett.61. 171-176 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Yoh,M.: "Comparative amino acid sequence analysis of hemolysins produced by Vibrio hollisae and Vibeio parahaemolyticus." J.Bacteriol.171. 6859-6861 (1989)

    • Related Report
      1989 Annual Research Report

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Published: 1989-04-01   Modified: 2016-04-21  

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