The Cloning Express Andp Rathogenicity Analysis Of A Novel Viru Lence-Associated Metalloprotease From Aeromonas Hydrophila

Aeromonas hydrophila is a Gram-negative bacterium that belongs to the family Aeromonadaceae. It is not only an important disease-causing pathogen of fish, but also it is the etiological agent responsible for a variety of infectious complications in humans. Especially this pathogen has brought a serious harm in the aquaculture industry.Metalloprotease is a kind of extracellular products secreted by bacteria, which might be different in primary structure but has a conservative active center. Metal ion is usually found in the active center, such as Zn2+and Co2+, Ni2+. It depends on the metal ion to catalyze peptide chain.According to the gene sequence of metalloprotease of A. hydrophila ATCC7966(accession number:AHA-0616), we designed a pairs of primers and amplified the gene fragment from A. Hydrophila J-1using PCR. The gene was named ahMP. The sequence analysis showed, the ahMP gene with the length of1281bp, encodes a421-amino acid enzyme, including41basic amino acids,51acidic amino acids,129hydrophobic amino acids and134polar amino acids. The gene sequence has98%similarity with that of A. hydrophila ATCC7966, and97%with those of A. hydrophila SSU and A. aquariorum AAK1. The ahMP gene contains Zn2+consensus domain sequence in151to161aa, and the amino acid sequence of the domain is HEXXHNMGLNH, in which X stands for any amino acids and Glu is an actor. It belongs to ZnMc superfamily. The ahMP gene was detected in155stains of Aeromonas by PCR, and the result showed that the distribution in A. hydrophila strains was wide and up to87.88%.To investigate the relationship between metalloprotease and its pathogenicity in A. hydrophila, the protease gene from A. Hydrophila J-1was cloned and expressed in Escherichia coli. A64kD recombinant protease was obtained. Biological characteristics analysis showed that it was a thermostable metalloprotease with the high activity between42℃and56℃. The proteolytic activity of recombinant ahMP showed that the ahMP was inhibited by metal ion chelators, such as EDTA and1,10-phenanthroline, and also it was inhibited by SDS. In contrast, serine protease inhibitors PMSF did not affect the ahMP. The ahMP was most likey a zinc-dependant metalloprotease as it was still inhibited by zinc specific metal. These results indicated that ahMP could be regarded as a metalloprotease, with Zn2+as its active center.To further confirm the role of this protease in the pathogenicity of A. hydrophila, experimental inoculation was performed in mice and fish. The protease could cause death in fish, but not in mice. The histopathological analysis showed that clear pathological changes, including hemorrhage, hyperemia or necrosis, in the heart, lung, kidney and spleen in ahMP injected group. Meanwhile, the blood analysis and cytotoxicity assay showed the ahMP could make the decrease the number of white blood cells (WBC), and damage the epithelial cells. This study provides a basis for the exploration of A. Hydrophila pathogenesis.