Virulence factors of fish pathogenic bacteria

1. 采用底物SDS-PAGE分析嗜绿细胞噬菌分离株的ECPs。114和152 kDa的蛋白酶对酪蛋白和明胶均有活性，32 ~ 86 kDa的蛋白酶对明胶无活性。29株分离菌根据蛋白酶组成4个类群。对鲑鱼幼鱼的感染性实验表明，蛋白酶组与毒力有一定的相关性。6株嗜冷杆菌产蛋白酶的最适温度为13.3(]SY+-[)1。而19.6(]SY+-[)0.5 (C)用于生长。从鳗弧菌中克隆了一个编码溶血素的5kb DNA片段。溶血素基因（VAH1）的开放阅读框为2253 bp。在高严格条件下使用VAH1进行DNA杂交分析，结果表明VAH1与鳗弧菌28株中的25株杂交，但与其他弧菌没有杂交。采用pcr随机诱变法分离出3株嗜水气单胞菌AHH3溶血活性阴性突变体（AHH3-m1-3）。AHH3与AHH3-m3的氨基酸序列比较，有9个氨基酸残基被替换。替换突变体His355-Arg355丧失溶血活性，而His291-Gln291突变体溶血活性降低。针对EDDHA的mic可以区分迟发爱德华菌的强毒株和无毒株。在含有EDDHA的HI肉汤中，毒性菌株产生了类似铁载体的物质，而无毒菌株则没有。利用转座子（Tn5）产生铁获取能力较低的突变株。这个突变株的毒力比原菌株小。克隆并测定了迟孢霉（e.c arda， ETH）溶血素基因位点。该区域编码两个开放阅读帧，分别为ethA和ethB。其中ethA是溶血素基因，全长4782bp； ethB是激活/分泌蛋白基因，全长1677bp。EthB蛋白是溶血素（EthA）蛋白活化所必需的。ethB基因的转录受铁的调控。

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.