title: Hyperproduction, purification, and mechanism of action of the cytotoxic enterotoxin produced by Aeromonas hydrophila.
abstract: A gene encoding the cytotoxic enterotoxin (Act) from Aeromonas hydrophila was hyperexpressed with the pET, pTRX, and pGEX vector systems. Maximum 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 systems. 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 migrated as a 52-kDa polypeptide on a sodium dodecyl sulfate (SDS)polyacrylamide gel that reacted with Act-specific antibodies in immunoblots. The minimal amount of toxin needed to cause fluid secretion in rat ileal loops was 200 ng, and the 50% lethal dose for mice was 27.5 ng when injected intravenously. Binding of the toxin to erythrocytes was temperature 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 chloride prevented lysis of erythrocytes by the toxin; however, it did not affect the binding and pore-forming capabilities of the toxin. A dose-dependent reduction in hemoglobin release from erythrocytes was observed when Act was preincubated with cholesterol, but not with myristylated cholesterol. With 14C-labeled cholesterol and gel filtration, the binding of cholesterol to Act was demonstrated. None of the other phospholipids and glycolipids tested reduced the hemolytic activity of Act. The toxin also appeared to undergo aggregation when preincubated with cholesterol, as determined by SDS-polyacrylamide gel electorphoresis. As a result of this aggregation, Act's capacity to form pores in the erythrocyte membrane was inhibited.
authors.affiliation: Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston 77555-1070, USA.
authors.forename: M R
authors.initials: MR
authors.lastname: Ferguson
authors.forename: X J
authors.initials: XJ
authors.lastname: Xu
authors.forename: C W
authors.initials: CW
authors.lastname: Houston
authors.forename: J W
authors.initials: JW
authors.lastname: Peterson
authors.forename: D H
authors.initials: DH
authors.lastname: Coppenhaver
authors.forename: V L
authors.initials: VL
authors.lastname: Popov
authors.forename: A K
authors.initials: AK
authors.lastname: Chopra
mesh_terms: D016980:Aeromonas hydrophila; D000818:Animals; D001426:Bacterial Proteins; D001681:Biological Assay; D002118:Calcium / Q000494:pharmacology; D002241:Carbohydrates / Q000494:pharmacology; D003603:Cytotoxins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004768:Enterotoxins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000648:ultrastructure; D004912:Erythrocytes / Q000187:drug effects*; D004926:Escherichia coli / Q000235:genetics; D006162:Guanylate Cyclase / Q000737:chemistry; D006461:Hemolysis / Q000187:drug effects; D051379:Mice; D011092:Polyethylene Glycols / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011817:Rabbits; D051381:Rats; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D012995:Solubility; D018675:Toxicity Tests
chemical_list: D001426:Bacterial Proteins; D002241:Carbohydrates; D003603:Cytotoxins; D004768:Enterotoxins; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins; C082174:cytolytic enterotoxin protein, Aeromonas; D011092:Polyethylene Glycols; D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D002118:Calcium