1996 Fiscal Year Final Research Report Summary
Virulence factors of fish pathogenic bacteria
| Project/Area Number |
06044063
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Institution | The University of Tokyo |
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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)
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| 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)
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| Project Period (FY) |
1994 – 1996
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| Keywords | Cytophaga psychrophila / Aeromonas hydrophila / Vibrio anguillarum / Edwardsiella tarda / fish-pathogenicity / protease / siderophore / hemolysin gene / virulence factor |
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| 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.
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