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Expression and activity of recombinant proaerolysin derived from Aeromonas hydrophila cultured from diseased channel catfish

Zhang, Dunhua, Pridgeon, Julia W., Klesius, Phillip H.
Veterinary microbiology 2013 v.165 no.3-4 pp. 478
Aeromonas hydrophila, DNA, Ictalurus punctatus, acute toxicity, catfish, cytopathogenicity, farmed fish, gills, hemolysis, mechanism of action, molecular cloning, pathogens, recombinant proteins, toxins, trypsin, virulence
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.