Location: Aquatic Animal Health Research
Title: Expression and activity of recombinant proaerolysin derived from Aeromonas hydrophila cultured from diseased channel catfish Authors
Submitted to: Veterinary Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 22, 2013
Publication Date: May 1, 2013
Repository URL: http://handle.nal.usda.gov/10113/56873
Citation: Zhang, D., Wei Pridgeon, Y., Klesius, P.H. 2013. Expression and activity of recombinant proaerolysin derived from Aeromonas hydrophila cultured from diseased channel catfish. Veterinary Microbiology. 165:478-482. Interpretive Summary: Bacterial pathogen, Aeromonas hydrophila, has been implicated many fish diseases, mostly known as motile aeromonas septicemia (MAS). The bacterium was involved in some MAS outbreaks in fish farms, causing high mortality rates and severe economic losses. The MAS pathogenesis was attributed to multiple virulence factors present in extra-cellular proteins (ECP) of the bacterium. Aerolysin was recognized as one of the virulence-related ECPs. The aerolysin gene has hence been used as one of virulence markers to identify potentially pathogenic Aeromonas strains. In this study, the aerolysin gene was cloned from a high virulent A. hydrophila and over-expressed in Escherichia coli. The recombinant aerolysin produced in E. coli was inactive but functional; it could be processed and activated on cell surface as well as by furin, trypsin and ECPs of A. hydrophila. The activated recombinant aerolysin was highly hemolytic and cytotoxic. The findings revealed in this study provided in vitro confirmation that proaerolysin was a protoxin, which may be an important death-causing factor during A. hydrophila infection. The recombinant aerolysin would be applicable for further research on virulence, pathogenicity and antigenicity associated with A. hydrophila infection.
Technical Abstract: Proaerolysin-coding gene was cloned from the genomic DNA of A. hydrophila and heterologously expressed in E. coli. The purified recombinant proaerolysin was inactive and could be activated by treatment with proteases, furin and trypsin, and extra-cellular proteins (ECPs, the cell-free supernatant of A. hydrophila liquid culture) although different treatments produced different cleavage profiles shown on SDS-PAGE and resulted in differential hemolytic and cytotoxic activities. The highest activity was observed from aerolysin processed by furin, a proprotein convertase, while treatment of proaerolysin with trypsin and ECPs 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, suggesting cell surface activation mechanism.