Reversible adsorption and nonreversible insertion of Escherichia coli alpha-hemolysin into lipid bilayers

Abstract

Alpha-Hemolysin is an extracellular protein toxin (107 kDa) produced by some pathogenic strains of Escherichia coli. Although stable in aqueous medium, it can bind to lipid bilayers and produce membrane disruption in model and cell membranes. Previous studies had shown that toxin binding to the bilayer did not always lead to membrane lysis. In this paper, we find that alpha-hemolysin may bind the membranes in at least two ways, a reversible adsorption and an irreversible insertion. Reversibility is detected by the ability of liposome-bound toxin to induce hemolysis of added horse erythrocytes; insertion is accompanied by an increase in the protein intrinsic fluorescence. Toxin insertion does not necessarily lead to membrane lysis. Studies of alpha-hemolysin insertion into bilayers formed from a variety of single phospholipids, or binary mixtures of phospholipids, or of phospholipid and cholesterol, reveal that irreversible insertion is favored by fluid over gel states, by low over high cholesterol concentrations, by disordered liquid phases over gel or ordered liquid phases, and by gel over ordered liquid phases. These results are relevant to the mechanism of action of alpha-hemolysin and provide new insights into the membrane insertion of large proteins.

Aggregatibacter actinomycetemcomitans Leukotoxin Utilizes a Cholesterol Recognition/Amino Acid Consensus Site for Membrane Association

“…The RTX toxins are known to be secreted into the aqueous solution but also to be membrane-interacting proteins (11). Neither the water-soluble nor the membrane-embedded structures of these toxins (and LtxA in particular) have been solved; however, it has been proposed that this family of toxins undergoes a conformational change upon membrane association (12).…”

The Calcium-binding C-terminal Domain of Escherichia coli α-Hemolysin Is a Major Determinant in the Surface-active Properties of the Protein

“…HlyA interaction with the target membrane is a complex process, of which many aspects are still poorly understood. The process includes two main steps, namely reversible binding (adsorption) and irreversible insertion (7). Binding occurs probably via specific receptors (8 -10), although the latter are not essential, since the toxin has also a lytic effect, at higher concentrations, on pure lipid liposomes (9).…”

E. coli <FONT FACE=Symbol>a</FONT>-hemolysin: a membrane-active protein toxin

“…However, only when the protein had been preincubated with this cation did the lytic effect follow toxin binding. In a further study, Bakás et al (66) applied to α-hemolysin a procedure developed by Tomita et al (67) that allowed the distinction between reversibly and irreversibly bound HlyA (Figure 4). The method consists essentially of incubating liposomes in the presence of toxin, then washing to remove the non-bound protein.…”

“…Using this procedure, Bakás et al (66) were able to show that α-hemolysin may bind the membranes in at least two ways, a reversible adsorption and an irreversible insertion. Comparison of irreversible binding and cell lysis results demonstrated that not even irreversible insertion leads necessarily to membrane damage.…”

“…Measurements of HlyA insertion into bilayers formed by a variety of single phospholipids, or binary mixtures of phospholipids, or of phospholipid and cholesterol, allowed Bakás et al (66) to reach the conclusion that irreversible insertion of the toxin was favored by fluid over gel states, by low over high cholesterol concentrations, by disordered liquid phases over gel or ordered liquid phases, and by gel over ordered liquid phases. These results are relevant to the mechanism of action of α-hemolysin, and provide new insights into the membrane insertion of large proteins.…”