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Virulence factors of fish pathogenic bacteria

Research Project

Project/Area Number 06044063
Research Category

Grant-in-Aid for international Scientific Research

Allocation TypeSingle-year Grants
SectionJoint Research
Research InstitutionThe University of Tokyo

Principal Investigator

WAKABAYASHI Hisatsugu  Graduate School of Agricultural and Life Sciences, University of Tokyo, Professor, 大学院・農学生命科学研究科, 教授 (00011932)

Co-Investigator(Kenkyū-buntansha) HOLT Richard  Department of Microbiology, Oregon State University, Associate Professor, 微生物学科, 助教授
CROSA Jorge  Dept.of Microbiology and Immunology, Oregon Health Science University, Professor, 分子生物免疫学科, 教授
FRYER John  Center for Salmon Disease Research, Oregon State University, Director, Professor, 魚病研究センター, 所長
HIRONO Ikuo  Department of Aquatic Biosciences, Tokyo University of Fisheries, Assistnat Prof, 資源育成学科, 助手 (00270926)
IIDA Takaji  Faculty of Agriculture, Miyazaki University, Associate Professor, 農学部, 助教授 (70159557)
AOKI Takashi  Department of Aquatic Biosciences, Tokyo University of Fisheries, Professor, 資源育成学科, 教授 (00051805)
Project Period (FY) 1994 – 1996
Project Status Completed (Fiscal Year 1996)
Budget Amount *help
¥8,500,000 (Direct Cost: ¥8,500,000)
Fiscal Year 1996: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 1995: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 1994: ¥3,100,000 (Direct Cost: ¥3,100,000)
KeywordsCytophaga psychrophila / Aeromonas hydrophila / Vibrio anguillarum / Edwardsiella tarda / fish-pathogenicity / protease / siderophore / hemolysin gene / virulence factor / トランスポゾン / 鉄代謝 / Flavobacterium branchiophilum / Aeromonas salmonicida / スーパーオキシドジスムターゼ / Cytophage psychrophila / 線毛
Research Abstract

1. The ECPs of Cytophaga phychrophila isolates were analyzed by substrate SDS-PAGE.Proteases of 114 and 152 kDa had activity against both casein and gelatin, and proteases from 32 to 86 kDa were active against gelatin but not casein. The 29 isolates studied formed four groups based on the proteases. In infectivity experiments with juvenile salmonids indicated some association between protease group and virulence.
2. The optimum temperatures for protease production of six C.psychrophila isolates was 13.3(]SY+-[)1.゚C,while 19.6(]SY+-[)0.5゚C for growth.
3. A 5 kb DNA fragment encoding a hemolysin was cloned from Vibrio anguillarum. An open reading frame of the hemolysin gene (VAH1) was 2,253 bp. DNA hybridization analysis under high-stringent conditions using VAH1, demonstrated that VAH1 hybridized with 25 out of 28 strains of V.anguillarum, but did not with other species of Vibrio.
4. Three hemolytic activity negative mutants of Aeromonas hydrophila AHH3 were isolated by the PCR-random mutagenesis (AHH3-m1-3). When compared the amino acid sequences of AHH3 with those of AHH3-m3, nine amino acid residues replaced. The replacement mutant of His355-Arg355 lost the hemolytic activity, and mutant of His291-Gln291 decreased the hemolytic activity.
5. MICs against EDDHA may distinguish virulent strains from avirulent ones of Edwardsiella tarda. In HI broth containing EDDHA,virulent strains produced siderophore-like substance (s), while avirulent ones did not. Mutant strains with lower iron acquisition ability were produced by using transposon (Tn5). This mutants had less virulence than the original strain.
6. A hemolysin gene locus from E.tarda (ETH) was cloned and sequenced. This region coded two open reading frames, designated ethA and ethB.The ethA is a hemolysin gene consisting of 4782bp and ethB is an activation/secretion protein gene of 1677 bp. The EthB protein was necessary for activation of EthA protein (hemolysin). Transcription of the ethB gene was regulated by iron.

Report

(4 results)
  • 1996 Annual Research Report   Final Research Report Summary
  • 1995 Annual Research Report
  • 1994 Annual Research Report
  • Research Products

    (11 results)

All Other

All Publications (11 results)

  • [Publications] M.Bertolini,H.Wakabayashi,V.G.Watral,M.J.Whipple and J.S.Rohovec: "Electrophoretic Detection of Proteases from Selected Strains of Flexibacter psychrophilus and Assessment of Their Variability." Journal of Aquatic Animal Health. 6. 224-233 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] I.Hirono and T.Aoki: "Randam and Site-Directed Mutagenesis of the AHH3 Hemolysin Gene of Aeromonas hydrophila." Proceedings of the International Symposium on Biotecnology in Aquaculture,Asian Fisheries Society Special Publication. No.10. 153-159 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] I.Hirono,T.Masuda and T.Aoki: "Cloning and Detection of the Hemolysin gene of Vibrio anguillarum." Microbial Pathogenesis. 21. 173-182 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] J.M.Bertolini, H.Wakabayashi, V.G.Watral, M.J.Whipple and J.S.Rohovec: "Electrophoretic Detection of Proteases from Selected Strains of Flexibecter phychrophilus and Assessment of Their Variability." Journal of Aquatic Animal Health. 6. 224-233 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] I.Hirono and T.Aoki: "Randam and Site-Directed Mutagenesis of the AHH3 Hemolysin Gene of Aeromonas hydrophila." Proceedings of the International Symposium on Biotecnology in Aquaculture, Asian Fisheries Society Special Publication. 10. 153-159 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] I.Hirono, T.Masuda and T.Aoki: "Cloning and Detection of the Hemolysin gene of Vibrio anguillarum." Microbial Pathogenesis. 21. 173-182 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Hirono,I他1名: "Cloning and detection of the hemolysin gene of Vibrio anguillarum" Microbial Pathogenesis. 21. 173-183 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Hirono,I.他1名: "Random and site-directed mutagenesis of the AHH3 hemolysin gene of Aeromonas hydrophila" Proceedings of International Sympsium on Biotechnology Applications in Aquaculture(ed. C-M.,Kou,et al.). Asian Fisheries Society Special Publication. No.10. 153-159 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Toyama,M.他2名: "Identification of Flexibacter maritimus,Flavobacterium branchiophilum and Cytophaga columnaris by PCR targeted 16S ribosomal DNA." Fish Pathology. 31. 25-31 (1996)

    • Related Report
      1995 Annual Research Report
  • [Publications] Bertolini,J.M.他4名: "Electrophoretic detection of proteases from selected strains of Flexibacter psychrophilus and assessment of their variability." Journal of Aquatic Animal Health. 6. 224-233 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] 遠山峰司 他2名: "Identification of Cytophaga psychrophila by PCR targeted 16S ribosomal RNA." 魚病研究. 25. 271-275 (1994)

    • Related Report
      1994 Annual Research Report

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Published: 1994-04-01   Modified: 2016-04-21  

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