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Virulence factors of fish pathogenic bacteria

鱼类病原菌毒力因子

基本信息

批准号：
06044063
负责人：
WAKABAYASHI Hisatsugu
金额：
$ 5.44万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

项目摘要

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.

1.用底物十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法分析了嗜铬细胞吞噬菌分离株的ECPs。114 kDa和152 kDa的酶对酪蛋白和明胶都有活性，32-86 kDa的酶对明胶有活性，但对酪蛋白没有活性。29个被研究的菌株根据蛋白酶形成了四个组。在对幼体鲑鱼的感染性实验中，表明了蛋白水解酶与毒力之间的某种联系。6株嗜冷链球菌产酶的最适温度为13.3SY+-1.ﾟC，最适生长温度为19.6SY+-0.5ﾟC。从鳗弧菌中克隆了编码溶血素的5kb DNA片段。溶血素基因(VAH1)的开放阅读框为2,253bp。利用VAH1在高要求条件下进行DNA杂交分析，结果表明VAH1与28株鳗弧菌中的25株发生杂交，而与其他弧菌不发生杂交。通过聚合酶链式反应-随机诱变技术，筛选到3株溶血活性阴性的嗜水气单胞菌AHH3-M1-3。比较AHH3和AHH3-M3的氨基酸序列，发现有9个氨基酸残基被替换。His355-Arg355的替换突变失去了溶血活性，His291-Gln291的突变降低了溶血活性。对EDDHA的最低抑菌浓度可区分迟缓爱德华氏菌的强毒株和无毒株。在含有EDDHA的HI发酵液中，强毒株产生类似铁载体的物质(S)，而无毒株则不产生。利用转座子(Tn5)获得了铁摄取能力较低的突变株。该突变株的致病力低于原始菌株。克隆了迟发性E.tarda(Eth)溶血素基因座，并对其进行了序列测定。该区域编码两个开放阅读框，分别命名为ethA和ethB，ethA是一个4782bp的溶血素基因，ethB是一个1677bp的激活/分泌蛋白基因。乙醇胺B蛋白是乙醇胺蛋白(溶血素)激活所必需的。EthB基因的转录受铁的调控。