Effects of a Single Hit from the Alpha Hemolysin Produced by Escherichia coli on the Morphology of Sheep Erythrocytes

Abstract

Scanning electron micrographs of sheep erythrocytes showed that attachment of the alpha hemolysin produced by Escherichia coli resulted in the formation of spherocytes, with 10 to 20 small projections spaced relatively evenly over the surface of the erythrocyte membrane. This shape change was induced within 5 min after treatment. If the hemolysin concentration was reduced to a level which would lyse only a fraction of the total erythrocytes, the affected cells were easily identified against a background of normal, unaffected cells. Unlike sodium lauryl sulfate and other amphipathic agents which enter cell membranes and increase their flexibility, low concentrations of hemolysin did not provide protection against hypotonic hemolysis. These findings indicate that the surface projections were not the result of membrane expansion caused by incorporation of hemolysin into the outer portion of the lipid bilayer. The ability of a given amount of hemolysin to release a constant amount of hemoglobin in the presence of increasing concentrations of red cells confirmed that a single hit is sufficient for lysis. These results suggest that a single hemolysin molecule can bind to a sheep erythrocyte and trigger internal reactions which result in the derangement of membrane integrity at multiple sites on the surface. Confirmation of one-hit kinetics indicates that measurement of E. coli hemolysin activity should be carried out at low ratios of hemolysin to erythrocyte to decrease the possibility of multiple hits on a single cell.

Potent leukocidal action of Escherichia coli hemolysin mediated by permeabilization of target cell membranes.

“…In contrast to the large amount of molecular genetical data available on ECH, information on the mechanism of its cytolytic action and on its biological effects in a physiological environment has been scarce . Following earlier reports that together had suggested a membrane-perturbating effect ofECH on erythrocytes (21)(22)(23)(24), we recently showed that ECH forms discrete hydrophilic transmembrane pores of ti2 nm effective diameter both in erythrocyte membranes (25) and in planar lipid bilayers (26). These pores are probably generated by the insertion oftoxin monomers into the bilayer; in this respect, they differ from oligomerizing pore-formers including C5b-9 complement complexes (27), lymphocytolysins (28), and cytolysins of Grampositive organisms (29)(30)(31)(32)(33).…”

Characterization of Escherichia coli hemolysins conferring quantitative differences in virulence

“…Considering what is known about the lytic activity and kinetics of the E. coli hly, this appears to be a logical finding. Several groups have provided evidence that the E. coli hly exhibits a one-hit phenomenon that is easily saturable (5,11). This suggests a nonenzymatic, membrane insertional-type event similar to complement-mediated lysis.…”

Quantitative study of the binding and hemolytic efficiency of Escherichia coli hemolysin

“…We extended these studies by presenting evi-dence that the toxin forms fairly large, hydrophilic pores approximately 2 nm in diameter in both biological membranes (2) and protein-free, artificial lipid bilayers (27). In contrast to the oligomeric, structured pores formed by many cytolysins from gram-positive organisms (1, 3-5, 13) as well as by complement (3) and lymphocytolysins (16), the pores formed by E. coli hemolysin appeared to be generated by toxin monomers (2,19,26,27). The pores exhibited a marked selectivity for cations over anions, and pore opening was voltage dependent (27).…”