title: Production and characterization of Escherichia coli enterohemolysin and its effects on the structure of erythrocyte membranes.
abstract: Hemolysins are cell-damaging protein toxins produced by pathogenic bacteria, which are usually released into the extracellular medium. Escherichia coli enterohemolysin is an intracellular toxin produced during the log phase of growth, with a maximal intracellular accumulation in the late log phase. In the present study, we have employed electron microscopy and SDS-PAGE to assess the effects of enterohemolysin on erythocyte membranes from different species. The erythrocyte cell damage began immediately after exposure to enterohemolysin with chemically detectable changes in cell membrane permeability, and the formation of surface lesions which increased rapidly in size. This process resulted in complete cell destruction. Ring-shaped structures with a diameter of 10nm were observed by electron microscopy after treatment of horse erythrocyte membranes with enterohemolysin. The ring structures were found clustered and irregularly distributed on the surface of the membranes. Following incubation of the toxin with horse erythrocyte ghosts and detergent-solubilization, the enterohemolysin was isolated from the cytoplasm in its membrane-bound form by sucrose density gradient. SDS-PAGE and silver staining of deoxycholate-solubilized target membranes revealed heterogeneous forms of the toxin. By using SDS-PAGE and gel filtration, the molecular weight of the toxin was estimated to be 35 kDa. With respect to species specificity, horse erythrocytes showed the highest sensitivity to the enterohemolysin, followed by human and guinea pig erythrocytes. The hemolytic sensitivity correlated with the toxin binding capacity of erythrocyte membranes of different animal species. The degree of hemolysis was unaffected by temperature in the range of 4 degrees C-37 degrees C and was optimal at pH 9.0. In contrast to pore-forming cytolysins, the hemolytic activity of enterohemolysin was enhanced continuously in the presence of increasing concentrations of dextran 4 and dextran 8 within the range of 5 to 30 mM. Trypsin sensitivity of membrane-bound enterohemolysin indicates that the cell surface is the most likely target site for this toxin. Additionally, the fact that proteinase and phosphatase inhibitors failed to inhibit lysis suggests that enterohemolysin alters and disrupts cell membranes by a detergent-like mechanism.
authors.affiliation: Robert Koch-Institut, Berlin, Germany.
authors.forename: Dagmar
authors.initials: D
authors.lastname: Jürgens
authors.forename: Muhsin
authors.initials: M
authors.lastname: Ozel
authors.forename: Ntondo B
authors.initials: NB
authors.lastname: Takaisi-Kikuni
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D003593:Cytoplasm / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D003911:Dextrans / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism* / Q000648:ultrastructure; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006168:Guinea Pigs; D006460:Hemolysin Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D013696:Temperature; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
chemical_list: D003902:Detergents; D003911:Dextrans; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C066219:enterohemolysin; D000255:Adenosine Triphosphate; D014357:Trypsin