Evaluation of the vaccine potential of a cytotoxic protease and a protective immunogen from a pathogenic Vibrio harveyi strain.
Vibrio harveyi is an important aquaculture pathogen that can infect a number of fish species and marine invertebrates. A putative protease, Vhp1, was identified from a pathogenic V. harveyi strain isolated from diseased fish as a protein with secretion capacity. Vhp1 is 530 amino acids in length and shares high sequence identities with several extracellular serine proteases of the Vibrio species. In silico analysis identified a protease domain in Vhp1, which is preceded by a subtilisin-N domain and followed by a bacterial pre-peptidase C-terminal domain. Purified recombinant protein corresponding to the protease domain of Vhp1 exhibited apparent proteolytic activity that was relatively heat-stable and reached maximum at pH 8.0 and 50 degrees C. The activity of purified recombinant Vhp1 protease was enhanced by Ca(2+) and inhibited by Mn(2+) and ethylenedinitrilotetraacetic acid. Cytotoxicity analyses indicated that recombinant Vhp1 protease was toxic to cultured Japanese flounder cells and could cause complete cell lysis. Immunoprotective analysis using Japanese flounder as an animal model showed that purified recombinant Vhp1 in the form of a denatured and proteolytically inactive protein was an effective subunit vaccine. To improve the vaccine potential of Vhp1, an Escherichia coli strain that expresses and secrets a cytotoxically impaired Vhp1 was constructed, which, when used as a live vaccine, afforded a high level of protection upon the vaccinated fish against lethal V. harveyi challenge. Taken together, these results demonstrate that Vhp1 is a cytotoxic protease and an effective vaccine candidate against V. harveyi infection.
Cheng
Shuang
S
Institute of Oceanology, Chinese Academy of Sciences, Qingdao, PR China.
Zhang
Wei-wei
WW
Zhang
Min
M
Sun
Li
L
D000818:Animals; D000942:Antigens, Bacterial / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D001427:Bacterial Toxins / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D001428:Bacterial Vaccines / Q000276:immunology*; D002118:Calcium / Q000494:pharmacology; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D020536:Enzyme Activators / Q000494:pharmacology; D022361:Escherichia coli Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology; D005393:Fish Diseases / Q000276:immunology / Q000517:prevention & control*; D005432:Flounder / Q000276:immunology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D055550:Protein Stability; D017422:Sequence Analysis, DNA; D016019:Survival Analysis; D022223:Vaccines, Subunit / Q000276:immunology; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D014735:Vibrio Infections / Q000276:immunology / Q000517:prevention & control / Q000662:veterinary*
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D001428:Bacterial Vaccines; D004269:DNA, Bacterial; D020536:Enzyme Activators; D022361:Escherichia coli Vaccines; D022223:Vaccines, Subunit; D008345:Manganese; D002118:Calcium