HYPERPRODUCTION, PURIFICATION, AND MECHANISM OF ACTION OF THE CYTOTOXIC ENTEROTOXIN PRODUCED BY AEROMONAS-HYDROPHILA

A gene encoding the cytotoxic enterotoxin (Act) from Aeromonas hydroph ila was hyperexpressed with the pET, pTRX, and pGEX vector systems. Ma ximum toxin yield was obtained with the pTRX vector. Approximately 40 to 60% of Act was in a soluble form with the pTRX and pET vector syste ms. The toxin protein was purified to homogeneity by a combination of ammonium sulfate precipitation and fast protein liquid chromatography- based column chromatographies, including hydrophobic, anion-exchange, sizing, and hydroxylapatite chromatographies. Purified mature toxin mi grated as a 52-kDa polypeptide on a sodium dodecyl sulfate (SDS)polyac rylamide gel that reacted with Act-specific antibodies in immunoblots. The minimal amount of toxin needed to cause fluid secretion in rat il eal loops was 200 ng, and the 50% lethal dose for mice was 27.5 ng whe n injected intravenously. Binding of the toxin to erythrocytes was tem perature dependent, with no binding occurring at 4 degrees C. However, at 37 degrees C the toxin bound to erythrocytes within 1 to 2 min. It was determined that the mechanism of action of the toxin involved the formation of pores in erythrocyte membranes, and the diameter of the pores was estimated to be 1.14 to 2.8 nm, as determined by the use of saccharides of different sizes and by electron microscopy. Calcium chl oride prevented lysis of erythrocytes by the toxin; however, it did no t affect the binding and pore-forming capabilities of the toxin, A dos e-dependent reduction in hemoglobin release from erythrocytes was obse rved when Act was preincubated with cholesterol, but not with myristyl ated cholesterol. With C-14-labeled cholesterol and gel filtration, th e binding of cholesterol to Act was demonstrated. None of the other ph ospholipids and glycolipids tested reduced the hemolytic activity of A ct. The toxin also appeared to undergo aggregation when preincubated w ith cholesterol, as determined by SDS-polyacrylamide gel electorphores is. As a result of this aggregation, Act's capacity to form pores in t he erythrocyte membrane was inhibited.