Aquatic Animal Health Research Unit, USDA-ARS, 990 Wire Road, Auburn, AL 36832, USA.
Vet Microbiol. 2013 Aug 30;165(3-4):478-82. doi: 10.1016/j.vetmic.2013.04.023. Epub 2013 May 1.
Aerolysin is one of the putative toxins in extracellular products (ECP) produced by Aeromonas hydrophila, an important pathogen of catfish. To better understand the molecular mechanism and mode of action of this toxin, proaerolysin-coding gene was cloned from the genomic DNA of an A. hydrophila strain, cultured from diseased channel catfish, and heterologously expressed in E. coli. Functional recombinant proaerolysin was obtained, revealing some unique properties. The purified recombinant proaerolysin was inactive but could be activated by treatment with furin, trypsin, and ECP although different treatments produced different cleavage profiles and resulted in differential hemolytic and cytotoxic activities. The highest activity was observed from aerolysin processed by furin while treatment of proaerolysin with trypsin and ECP resulted in reduced activities. The unprocessed proaerolysin, though not hemolytic in vitro, had the same cytopathic effect on cultured walking catfish gill cells as the furin-processed had. In in vivo assay, the recombinant proaerolysin was found to be lethal to catfish when injected via intraperitoneal (IP) route. The lethal toxicity was acute and dose-dependent, as observed in IP injection of live A. hydrophila. This is the first recombinant proaerolysin confirmed to be a virulence factor; the recombinant protein could be used to further evaluate virulence, pathogenicity and antigenicity associated with A. hydrophila infection.
气溶素是嗜水气单胞菌（一种鲶鱼的重要病原体）产生的细胞外产物（ECP）中的一种假定毒素。为了更好地了解这种毒素的分子机制和作用模式，从患病鲶鱼分离的嗜水气单胞菌菌株的基因组 DNA 中克隆了原气溶素编码基因，并在大肠杆菌中异源表达。获得了功能性重组原气溶素，揭示了一些独特的性质。纯化的重组原气溶素没有活性，但可以通过弗林蛋白酶、胰蛋白酶和 ECP 的处理而被激活，尽管不同的处理产生了不同的切割图谱，并导致不同的溶血和细胞毒性活性。用弗林蛋白酶处理的气溶素表现出最高的活性，而用胰蛋白酶和 ECP 处理原气溶素则导致活性降低。未经处理的原气溶素虽然在体外没有溶血活性，但对培养的鲶鱼鳃细胞具有与弗林蛋白酶处理的气溶素相同的细胞病变效应。在体内试验中，重组原气溶素经腹腔（IP）注射对鲶鱼具有致死毒性。致死毒性是急性和剂量依赖性的，与活体嗜水气单胞菌的 IP 注射观察结果一致。这是第一个被证实为毒力因子的重组原气溶素；重组蛋白可用于进一步评估与嗜水气单胞菌感染相关的毒力、致病性和抗原性。
