We examined the ability of Aeromonas hydrophila to lyse elastin. Eight of 13 strains showed elastolytic activity on agar medium containing elastin and 5 strains did not. In order to examine the involvement of the metalloprotease of A. hydrophila (AMP) in elastolytic activity, we made the amp-deletion mutant strain from an elastolytic strain. The elastolytic activity of the strain decreased with this deletion. The analysis of AMP released into the culture supernatant showed that AMP appeared outside of the cell as the intermediate consisting of a mature domain and carboxy terminal (C-terminal) propeptide domain. Further analysis showed that the intermediate has the ability to lyse elastin and that loss of the C-terminal domain causes loss of the elastolytic activity of the intermediate. We then determined the nucleotide sequence of the amps of all strains used in this study. Phylogenetic analysis revealed that these AMPs were divided into three groups. The AMPs from elastolytic strains belong to group I or group II, and AMPs from non-elastolytic strains belong to group III. The distance between group I and group II is small, but group III is located separately from groups I and II. Comparison of the amino acid residues of the C-terminal domain revealed that there are 13 amino acid residues specific to the C-terminal domain of group III. This indicates that the conformation of the C-terminal propeptide domain formed by these specific amino acid residues is important for AMP to express elastolytic activity.
Key words Aeromonas hydrophila; metalloprotease; elastolytic activityThe mesophilic Aeromonas species are ubiquitous in aquatic environments.1-4) The mesophilic species cause sporadic diarrhea in both adults and children, 5,6) and it has been reported that A. hydrophila and A. sobria occasionally cause extraintestinal infections such as sepsis and necrotizing softtissue infection in patients. Such extraintestinal infections often occur in patients suffering from hepatic dysfunction and diabetes mellitus. [7][8][9] As pathogenic factors of Aeromonas, various extracellular proteins containing hemolysin, proteases, lipases and so on have been reported. 10,11) In addition, a virulent type III secretion system has been proposed.12) The hemolysin produced by A. hydrophila and A. sobria is thought to be a major etiological agent of diarrhea because it induces fluid accumulation in the intestinal loop test and the antiserum against hemolysin neutralizes the enterotoxic activity of these bacteria 13) ; however, it is unclear how A. hydrophila and A. sobria, which infect the intestine, cause exointestinal infection.Cascón et al. have reported that elastase of A. hydrophila plays an important role in the pathogenesis of the bacteria. They determined the LD 50 of two isogenic strains of A. hydrophila, wild-type strain and the elastase gene-deleted mutant strain of the wild type by intraperitoneal challenge of rainbow trout. The LD 50 of the mutant strain is about 100 times higher than that of the wild type, indicating that ...
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