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.

Epidemiology of Shiga toxin-producingEscherichia coliO157:H7 in Africa in review

“…This protein toxin damages cell membranes of erythrocytes and is used as a surrogate tool in detection of shiga toxin-producing E. coli. [68][69][70] Although enterohaemolysin activity can easily be visualised on blood agar cultures, confirmation is usually achieved by PCR amplification of the ehxA gene. 59,68 Some other E. coli strains such as O26, O103, O111, O118, O128, O121, O45 and O145 can produce disease syndromes and have been reported to be enterohaemolysin-positive and produce shiga toxins.…”

Engineering HlyA hypersecretion inEscherichia coli based on proteomic and microarray analyses

“…The liquid blood lysis assay was adapted from several previously reported hemolysis assays (Cortajarena et al, 2002;Jurgens et al, 2002;Vakharia et al, 2001). HlyAsecreting cells were grown to mid-logarithmic phase in tryptone water (1% tryptone, 0.5% NaCl) at 37jC with shaking at 250 rpm.…”

Serum high-density lipoprotein (HDL) inhibits in vitro enterohemolysin (EHly) activity produced by enteropathogenicEscherichia coli

“…Recently it has been suggested that EHly alters and disrupts cell membranes by a detergent‐like mechanism [12]. Detergents form a special group of lipids that are used to solubilize membranes [13], and act by forming thermodynamically stable colloidal aggregates or micelles [14] that may be asymmetrically distributed between the two leaflets of the membrane lipid bilayer.…”