Pore formation by the Escherichia coli hemolysin: evidence for an association-dissociation equilibrium of the pore-forming aggregates

Abstract

Lipid bilayer experiments were performed in the presence of hemolysin of Escherichia coli. The toxin had a rather low activity in membranes formed of pure lipids, such as phosphatidylcholine or phosphatidylserine. In membranes from asolectin, a crude lipid mixture from soybean, hemolysin was able to increase the conductance by many orders of magnitude in a steep concentration-dependent fashion, which suggested that several hemolysin molecules could be involved in the conductive unit. Furthermore, the much higher toxin activity in asolectin membranes would be consistent with the assumption that this lipid contains a receptor needed for membrane activity of the toxin. The results of single-channel records showed that the membrane activity of hemolysin is due to the formation of ion-permeable channels with a single-channel conductance of about 500 pS in 0.15 M KCl. The hemolysin channel seemed to be formed by a toxin oligomer which showed an association-dissociation reaction and had a mean lifetime of about 2 s at small transmembrane voltages. The conductance of the hemolysin channels was only moderately dependent on the salt concentration in the aqueous phase. Zero-current membrane potential experiments showed that the hemolysin channel is cation selective. The mobility sequence of the cations in the channel was similar to their mobility sequence in the aqueous phase, which was consistent with the assumption that the hemolysin channel is wide and that the interior field strength is not very high. From the single-channel conductance, a lower limit of about 1.0 nm for the effective channel diameter could be estimated.

Interaction of Serratia marcescens hemolysin (ShlA) with artificial and erythrocyte membranes

“…This latency could be caused by a slow aqueous diffusion of the toxin or a slow diffusion rate into the membrane. The conductance of membranes made of different lipids varied, but ShlA did not show a lipid specificity as did HlyA of E. coli [25]. Membrane conductance was increased with higher toxin concentrations.…”

“…In the case of the E. coli hemolysin pores, an effective diameter of 2-3 nm was derived from osmotic protection data [30] while significantly smaller diameters (1 -1.3 nm) were estimated from results obtained by measuring the channel conductivity in artificial lipid bilayers [25,311. To obtain a more reliable estimation of the ShlA pore diameter, we compared it with the well-characterised channel formed by a-toxin of S. a u r a s [8].…”

“…Thi\ means that ShlA forms channels that are not voltage gated. Other toxins, such as the hemolysin of E. coli [25] and the aerolysin of A. sobria [28], form voltagedependent channels that close at high voltages starting at approximately 30 mV (HlyA) and 50 mV (aerolysin). Fig.…”

Pore‐Forming Properties of the Plasmid‐Encoded Hemolysin of Enterohemorrhagic Escherichia Coli 0157: H7

“…were prepared as described above. Extracellular a-hemolysin (HlyA) of Escherichia coli was isolated from the supernatants of an E. coli 5 WpANN202-812 culture (100 ml) grown at 37°C in double-concentrated yeast tryptone broth (2-fold YT) to a density of 5x1O8 cells/ml as previously described [17] then precipitated with poly(ethy1ene glycol) as described above.…”

The Binding of Divalent Cations to Escherichia coli alpha-Haemolysin

“…a-haemolysin is synthesized as an inactive precursor, whose activation is believed to include binding of fatty acyl groups [9]. The so-called pro-haemolysin is inactive with either red blood cells or model membranes [7]. The toxin belongs to a diverse group of proteins secreted by Gram-negative organisms, and characterized by the presence of a domain with a variable number of repeats of a nonapeptide of consensus sequence GGXGXDXUX, where X is an arbitrary amino acid residue, and Correspondence to F. M. Gofii, Department of Biochemistry, Univer-Fa: +34 4 4648500.…”