Pore formation in artificial membranes by the secreted hemolysins of Proteus vulgaris and Morganella morganii

Abstract

Lipid-bilayer experiments were performed with the related hemolysins from Proteus vulgaris and Morganella morganii (HlyA). The addition of the toxins to the aqueous phase bathing lipidbilayer membranes composed of different lipids resulted in the formation of transient ion-permeable channels. Membranes formed of pure lipids were rather inactive targets for the hemolysins as compared with lipid mixtures such as asolectin. The channels had several different substates. The major open state had single-channel conductances of 500 pS in 0.15 M KC1 at small transmembrane voltages. Experiments with different salts suggested that the hemolysin-induced channels of l? vulgaris and M . morganii were exclusively cation selective at neutral pH, caused by negative charges localized at the channel mouth. The mobility sequence of the cations within the channels was similar if not identical to their mobility sequence in the aqueous phase. The single-channel data were consistent with wide, water-filled channels with estimated minimal diameters of about 1 nm since the large organic cation Tris' can permeate the channels without any detectable interaction with its interior. Pore-forming properties of these hemolysins were compared with those of HlyA of Escherichia coli. All these toxins share common features, oligomerize probably to form pores in lipidbilayer membranes and form channels with similar properties which suggests that their structures are more or less identical. a-hemolysin (HlyA) of Escherichia coli is a member of a family of cytolysins from Gram-negative bacteria. Many of these toxins share common structural properties, especially a repeat domain in the C-terminal half of the toxin protein, and are therefore called RTX (repeats in toxin) proteins [l-31. The primary structure of HlyA has several different domains [4, 51. The C-terminus of HlyA is essential for its secretion out of the cell by the HlyB/HlyD system together with the minor outer membrane porin TolC [6-111 and its activation is catalysed by the cytoplasmic protein HlyC and involves acylation by an acyl-carrier protein [12, 131. The repeat domain binds Ca2+ and may be the essential part of the toxin for receptor recognition on the mammalian target cell but it is not essential for channel formation in lipid-bilayer membranes [14]. Further studies on the E. coli hemolysin have revealed that two other regions may also be involved in pore formation, the N-terminus and a hydrophobic domain at amino acid 177-411 [15, 161. Extensive similarity has been demonstrated between the four different hly genes of E. coli and those of the Proteacae [17, 181, and also to the hemolysins and leukotoxins of Actinobacillus Erythrocyte lysis by E. coli HlyA is characterised by three phases, an initial lag, an accelerated rate of hemolysis followed by a plateau, then a decline in rate [33]. An initial adsorption step (due to the repeat domain [l -31) may determine the lag step of erythrocyte lysis. Studies with erythrocytes and artificial membranes have demonstrated that a por...

Biochemical Identification and Biophysical Characterization of a Channel-Forming Protein from Rhodococcus erythropolis

“…A quantitative description of the effect of point charges on single-channel conductance may be given by the treatment proposed by Nelson and McQuarrie (25). It describes the effect of point charges on the conductance of a channel, which is dependent on ion concentration, on the channel diameter, and on the number of negative charges (5). A comparison of the crystal structure of the Rhodobacter capsulatus porin with the diameter derived from this theoretical treatment yields good agreement (30).…”

“…Accordingly, they influence both single-channel conductance and zero-current membrane potential, and the single-channel conductance is much larger than expected from the dimensions of the channel. A quantitative description of the effect of the point charges on the single-channel conductance may be given with the considerations of Nelson and McQuarrie (25), as previously described (5). A best fit of the data of Fig.…”

Low pH-induced Formation of Ion Channels by Clostridium difficile Toxin B in Target Cells

“…This result seems to represent a contradiction to the obvious receptor-mediated permeabilization of cells. However, it has to be kept in mind that other toxins, such as C2 toxin (20) or the repeat in toxin toxins (27)(28)(29), form channels in lipid bilayer membranes without the need for receptors, whereas they all need a receptor for biological activity. An important role of the toxin receptor in events finally resulting in channel formation is supported by the concentration effect studies at high concentration of toxin, which show that limitation of channel formation is most likely due to reaching the binding capacity of cells.…”

Clostridium perfringens Epsilon Toxin Induces a Rapid Change of Cell Membrane Permeability to Ions and Forms Channels in Artificial Lipid Bilayers

“…This means that they were mostly in the open configuration and did not close under our experimental conditions. This result indicated that the channel was formed by a defined structure, which does not show an associationdissociation equilibrium, such as the oligomers that form the hemolysin channels of Proteus vulgaris and Morganella morganii (25). A histogram of the channels formed by the activated C. perfringens epsilon toxin under the conditions of Fig.…”