Lipid interaction of Pseudomonas aeruginosa exotoxin A. Acid-triggered permeabilization and aggregation of lipid vesicles

Abstract

We have investigated the interaction of Pseudomonas exotoxin A with small unilamellar vesicles comprised of different phospholipids as a function of pH, toxin, and lipid concentration. We have found that this toxin induces vesicle permeabilization, as measured by the release of a fluorescent dye. Permeabilization is due to the formation of ion-conductive channels which we have directly observed in planar lipid bilayers. The toxin also produces vesicle aggregation, as indicated by an increase of the turbidity. Aggregation and permeabilization have completely different time course and extent upon toxin dose and lipid composition, thus suggesting that they are two independent events. Both time constants decrease by lowering the pH of the bulk phase or by introducing a negative lipid into the vesicles. Our results indicate that at least three steps are involved in the interaction of Pseudomonas exotoxin A with lipid vesicles. After protonation of one charged group the toxin becomes competent to bind to the surface of the vesicles. Binding is probably initiated by an electrostatic interaction because it is absolutely dependent on the presence of acidic phospholipids. Binding is a prerequisite for the subsequent insertion of the toxin into the lipid bilayer, with a special preference for phosphatidylglycerol-containing membranes, to form ionic channels. At high toxin and vesicle concentrations, bound toxin may also induce aggregation of the vesicles, particularly when phosphatidic acid is present in the lipid mixture. A quenching of the intrinsic tryptophan fluorescence of the protein, which is induced by lowering the pH of the solution, becomes more drastic in the presence of lipid vesicles. However, this further quenching takes so long that it cannot be a prerequisite to either vesicle permeabilization or aggregation. Pseudomonas exotoxin A shares many of these properties with other bacterial toxins like diphtheria and tetanus toxin.

Current models of the mode of action of Bacillus thuringiensis insecticidal crystal proteins: A critical review

“…Instead, although oligomers were shown to cause a much greater release of calcein than trypsin-activated toxin monomers, both forms of the toxin appeared to act instantaneously, at least within the time resolution of the instrument, upon addition of the toxin to the vesicle suspension Rausell et al, 2004a). This result is very surprising considering that although calcein release can be very rapid, it is nevertheless expected to take place at a measurable rate (Schwarz and Robert, 1990) as was reported for the Escherichia coli colicin E1 (Kayalar and Düzgüneçs, 1986) and hemolysin (Menestrina, 1988), the Clostridium tetani tetanus toxin (Menestrina et al, 1989), the Pseudomonas aeruginosa exotoxin A (Menestrina et al, 1991) and the mosquitocidal binary toxin from Bacillus sphaericus , to mention only a few examples of calcein-efflux experiments conducted with bacterial pore-forming toxins. Clearly, pore formation by Bt toxins is not expected to occur instantaneously, but requires some time for their insertion into the membrane.…”

Membrane interactions and surface hydrophobicity of Bacillus thuringiensis δ‐endotoxin CryIC

“…Indeed, the presence of a conformational transition, resulting in an increased exposure of hydrophobic sites on the surface of CryIC, is clearly indicated by the bis-ANS binding experiments. The behavior of CryIC under conditions of acidic pH shares certain characteristics common to a number of other bacterial toxins, such as diphtheria toxin [19], Pseudo-monas aemginosa exotoxin A [20,21], tetanus toxin 122,233 and colicins 124,251. All these proteins show a dramatic increase in the affinity for phospholipid membranes at acidic pH.…”

“…Another similarity between &endotoxin and some other membrane-active bacterial toxins is the preferential membrane interaction in the presence of acidic phospholipids [19,21,22,25]. In the case of CryIC, the effect of acidic lipids is clearly nonspecific and indicates that electrostatic interactions play a role in binding of the toxin to the membrane.…”

Discovery of a novel antimicrobial peptide using membrane binding-based approach

“…Ean der Goot, Gonzalez-Manas, Lakey, and Pattus (1991) observed that, depending on circumstances, the pH in the vicinity of the membrane might be 1-3 units lower than in the bulk solution. A decrease in pH has been linked to structural changes in many proteins, such as the Bt toxin Cry1C (Butko, Cournoyer, Pusztai-Carey, & Surewicz, 1994), P. aeruginosa exotoxin A (Menestrina, Pederzolli, Forti, & Gambale, 1991), tetanus toxin (Calappi, Masserini, Schiavo, Montecucco, & Tettamanti, 1992), and tumor necrosis factor a (Kagan, Baldwin, Munoz, & Wisnieski, 1992). All of these proteins increase their affinity to the membrane at acidic pH (Manceva, Pusztai-Carey, & Butko, 2004).…”