Physicochemical and Biological Properties of an Extracellular Serine Protease of Aeromonas sobria

Abstract

Previously, we cloned a protease gene of Aeromonas sobria, determined its nucleotide sequence and established a method of purifying its product. In this study, we examined the properties of the purified protease. The protease was temperature‐labile and had an optimal pH of 7.5. Metallo‐protease inhibitors and a cysteine protease inhibitor did not block the proteolytic activity of the enzyme. The treatment with reagents to modify sulfhydryl group did not reduce the activity. But, serine protease inhibitors did, showing that it was a serine protease. Subsequently, we examined the ability of the protease to enhance vascular permeability in dorsal skin. The protease showed activity and the reaction was inhibited by a simultaneously injected antihistaminic agent. Histopathological examination showed that mast cells appeared around the site where the protease was injected. These findings show that the vascular permeability‐enhancing effect of the protease is due to histamine released at the site. Furthermore, we found that a soybean trypsin inhibitor (Kunitz) did not block the proteolytic action of the protease in vitro, but inhibited its vascular permeability‐enhancing activity in skin. This suggests that a trypsin‐like protease from skin mediates the activity of the protease to enhance its vascular permeability.

Case Reports: Fatal Necrotizing Fasciitis Caused by Aeromonas sobria in Two Diabetic Patients

“…Aeromonas sobria hemolysin and cytolytic enterotoxin possess enterotoxic activity and are the major virulence factors that cause diarrhea [11,23,30]. Yokoyama et al [30] reported Aeromonas sobria protease may enhance vascular permeability and bullous formation by stimulating the bradykinin release pathway in rat skin through the plasma kallikrein activation and histamine release.…”

“…Aeromonas species can produce many virulence factors, including hemolysin, cytotoxin, aerolysin, enterotoxin, endotoxin, protease, adhesins, leukocidin, and lipases [1,11,14,20,23,29,30]. Aeromonas hydrophila is the most commonly reported pathogen that causes Aeromonas necrotizing fasciitis and septicemia; these conditions often occur after soft tissue trauma with exposure to contaminated water or nonfecal organic materials and produce skin lesions similar to those observed in infections caused by Vibrio species [7,19,21,24,26,28].…”

“…Yokoyama et al [30] reported Aeromonas sobria protease may enhance vascular permeability and bullous formation by stimulating the bradykinin release pathway in rat skin through the plasma kallikrein activation and histamine release. Imamura et al [11] reported Aeromonas sobria can secret serine proteinase and may induce vascular leakage through prekallikrein activation and kinin release to cause lethal hypotension and septic shock.…”

Salt in Surroundings Influences the Production of Serine Protease into Milieu by Aeromonas sobria

“…The proteolytic activity of the cell free culture supernatant was determined by the lysis of casein (34). A portion (250 µl) of the sample was added to 250 µl of 10 mM Tris-HCl buffer (pH 7.5) containing azocasein at the concentration of 0.8% azocasein.…”

“…After cultivation at 37 C for 12 hr with shaking, their culture supernatants were prepared. Proteolytic activity of these samples was determined by azocasein assay (34). The amount of azocasein digested by the proteases was measured by absorbance at 450 nm, and the measured values were used as the value expressing the proteolytic activity of the corresponding sample.…”