Summary␣-Haemolysin is an extracellular protein toxin (Ϸ107 kDa) secreted by Escherichia coli that acts at the level of the plasma membranes of target eukaryotic cells. The nature of the toxin interaction with the membrane is not known at present, although it has been established that receptor-mediated binding is not essential. In this work, we have studied the perturbation produced by purified ␣-haemolysin on pure phosphatidylcholine bilayers in the form of large unilamellar vesicles, under conditions in which the toxin has been shown to induce vesicle leakage. The bilayer systems containing bound protein have been examined by differential scanning calorimetry, fluorescence spectroscopy, differential solubilization by Triton X-114, and freeze-fracture electron microscopy. All the data concur in indicating that ␣-haemolysin, under conditions leading to cell lysis, becomes inserted in the target membrane in the way of intrinsic or integral proteins. In addition, the experimental results support the idea that inserted ␣-haemolysin occupies only one of the membrane phospholipid monolayers, i.e. it is not a transmembrane protein. The experimental data are complemented by structure prediction studies according to which as many as ten amphipathic ␣-helices, appropriate for protein-lipid interaction, but no hydrophobic transmembrane helices are predicted in ␣-haemolysin. These observations and predictions have important consequences for the mechanism of cell lysis by ␣-haemolysin; in particular, a non-transmembrane arrangement of the toxin in the target membrane is not compatible with the concept of ␣-haemolysin as a pore-forming toxin.
Previous studies from this laboratory had shown that calcium ions were essential for the membrane lytic activity of E. coli alpha-haemolysin (HlyA), while zinc ions did not sustain such a lytic activity. The present data indicate that calcium-binding does not lead to major changes in the secondary structure, judging from circular dichroism spectra. However binding to Ca2+ exposes new hydrophobic residues at the protein surface, as indicated by the increased binding of the fluorescent probe aniline naphtholsulphonate (ANS), and by the increased tendency of the Ca2+-bound protein to self-aggregate. In addition zinc ions are seen to decrease the thermal stability of HlyA which, according to intrinsic fluorescence and differential scanning calorimetry data, is stable below 95 degrees C when bound to calcium, while it undergoes irreversible denaturation above 60 degrees C in the zinc-bound form. Binding to phosphatidylcholine bilayers is quantitatively similar in the presence of both cations, but about one-third of the zinc-bound HlyA is released in the presence of 2 M NaCl. Differential scanning calorimetry of dimyristoylglycerophosphocholine large unilamellar vesicles reveals that Zn2+-HlyA interaction with the lipid bilayer has a strong polar component, while Ca2+-HlyA appears to interact mainly through hydrophobic forces. Experiments in which HIyA transfer is measured from phospholipid vesicles to red blood cells demonstrate that Ca2+ ions promote the irreversible binding of the toxin to bilayers. All these data can be interpreted in terms of a specific Ca2+ effect that increases the surface hydrophobicity of the protein, thus facilitating its irreversible bilayer insertion in the fashion of intrinsic membrane proteins.
a-haemolysin, an extracellular protein toxin of Escherichia coli, is known to disrupt eukaryotic cell membranes. In spite of genetic evidence of Ca2+-binding motifs in its sequence, conflicting results are found in the literature on the requirement of divalent cations for the membranolytic activity of the toxin. Moreover, Ca2+-binding sites have not been characterized to date in the native protein. The results in this paper show that when Cazf levels are kept sufficiently low during bacterial growth and toxin purification, membrane lysis does not occur in the absence of added divalent cations. CaZ+ and, at higher concentrations, Sr2+ and Ba2+, support the lytic activity, but Mg", Mn2+, Zn2+ and Cd2+ appear to be inactive in this respect. Binding of metal ions can be followed by changes in the intrinsic fluorescence of a-haemolysin; ions supporting lytic activity produce changes in the intrinsic fluorescence that are not caused by the inactive ones. Scatchard analysis of "Ca*+ binding reveals three equivalent, independent sites, with Kd = 0.11 mM. No 45Ca2' binding is observed when the protein is incubated with Zn"; conversely, incubation with Ca2+ prevents subsequent binding of 65Zn2+. In the light of three-dimensional data available for a structurally related protein, alkaline protease of Pseudomonas aeruginosn [Baumann, U., Wu, S., Flaherty, K. M. & McKay, D. B. (1993) EMBO J. 12, 3357-33641 it is suggested that a-haemolysin may bind a larger number of Ca2+ than the three that are more easily exchangeable and are thus detected in the 45Ca2+-binding experiments. In addition, structural similarities and conservation of ion-binding motifs support the hypothesis that His 859 is involved in the mutually exclusive binding of Zn2+ and Ca2+.Keywords. a-haemolysin ; bacterial toxins ; RTX toxins ; Ca2+-binding proteins ; fluorescence.Escherichia coli a-haemolysin is an extracellular protein toxin whose lytic activities on eukaryotic cells, including red blood cells, are well known [l]. Early efforts in the elucidation of the mechanism of a-haemolysin-induced erythrocyte lysis [2] pointed out that a rapid K+ efflux and Ca" influx took place preceding cell lysis; entry of Caz+ was thought to be the trigger for a hypothetical enzyme cascade leading to haemolysis. Later workers suggested that the primary cause of a-haemolysin-induced haemolysis is the formation of discrete transmembrane toxin pores [3, 41. The toxin also formed pores in model membranes [5-71. However, results from this laboratory [8] cast doubts on the hypothesis of pore formation as the main haemolytic mechanism, pointing instead to a detergent-like action of the toxin. 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 ...
Pro-haemolysin (= 1 1 0 kDa), the inactive precursor of the membrane-lytic toxin a-haemolysin, has been purified from an overproducing strain of Escheuichia coli. Pro-haemolysin forms aggregates in aqueous media, like the mature protein, suggesting an amphipathic structure. Direct measurements of protein binding to liposomal membranes, following a novel procedure, show that pro-haemolysin can bind the lipid bilayers to a similar extent as a-haemolysin. This is confirmed by the observed changes in the intrinsic fluorescence emission of the protein upon binding the bilayers. However, pro-haemolysin is totally unable to induce liposoinal membrane lysis. Binding of Ca2+, that is essential for the lytic activity of rx-haemolysin, is greatly diminished in the precursor protein, as shown both by direct measurements of 45Ca'+-binding and by fluorescence measurements. The results suggest that binding of a fatty acyl residue in the activation step brings about an important conformational change in the protein that involves the Ca"-binding domain.Keyword.p: a-haemolysin ; pro-haemolysin ; amphipathic protein ; bacterial toxin ; calcium-binding protein.a-Haemolysin is an extracellular protein toxin (=I 10 kDa) secreted by some strains of Escherichia coli, that is held responsible for the pathogenicity of those strains. The protein is also interesting because it is secreted through a peculiar mechanism, in which the secretion signal lies in the C-terminus of the protein [I]. a-Haemolysin is synthesized as a precursor, called pro-haemolysin. The synthesis, maturation and export of u-haemolysin depend on the hly operon; the product of the structural gene hlyA is pro-haemolysin, and this is converted, by acylation, into mature a-haemolysin in a process catalyzed by HlyC 121. Immediately after, the products of genes hlyB and h1yD produce the export of a-haemolysin to the external medium.Previous work from this laboratory has dealt with the purification, to a high specific activity, of a-haemolysin from a plasmidic determinant [3] and with thc lytic effect of the toxin on large unilamellar vesicles (LUV) of defined phospholipid composition (41. More recently, we have characterized the binding of divalent cations to a-haemolysin [S], and the binding of a-haemolysin to lipid bilayers [6]. The present paper describes a procedure for the purification of pro-haemolysin, that has not been available up to now, and a parallel study of the interaction of the mature and precursor toxins with lipid bilayers and divalent cations. It was known that pro-haemolysin could be exported in a manner very similar to that of the mature toxin, but it could not produce red blood cell lysis 121. We have found that the precursor does bind lipid bilayers, although its calciumbinding capacity is severely reduced with respect to that of the mature protein. promoter. Plasmid pSU2781 [cat, rep(pl5A), h l y B f , hlyD] is compatible with pSU2726 and was used as a helper for the secretion of HlyA. The construction of both plasmids was described by BartolomC [7]....
alpha-haemolysin, an extracellular protein toxin of Escherichia coli, is known to disrupt eukaryotic cell membranes. In spite of genetic evidence of Ca(2+)-binding motifs in its sequence, conflicting results are found in the literature on the requirement of divalent cations for the membranolytic activity of the toxin. Moreover, Ca(2+)-binding sites have not been characterized to date in the native protein. The results in this paper show that when Ca2+ levels are kept sufficiently low during bacterial growth and toxin purification, membrane lysis does not occur in the absence of added divalent cations. Ca2+ and, at higher concentrations, Sr2+ and Ba2+, support the lytic activity, but Mg2+, Mn2+, Zn2+ and Cd2+ appear to be inactive in this respect. Binding of metal ions can be followed by changes in the intrinsic fluorescence of alpha-haemolysin; ions supporting lytic activity produce changes in the intrinsic fluorescence that are not caused by the inactive ones. Scatchard analysis of 45Ca2+ binding reveals three equivalent, independent sites, with Kd approximately 0.11 mM. No 45Ca2+ binding is observed when the protein is incubated with Zn2+; conversely, incubation with Ca2+ prevents subsequent binding of 65Zn2+. In the light of three-dimensional data available for a structurally related protein, alkaline protease of Pseudomonas aeruginosa [Baumann, U., Wu, S., Flaherty, K. M. & McKay, D. B. (1993) EMBO J. 12, 3357-3364] it is suggested that alpha-haemolysin may bind a larger number of Ca2+ than the three that are more easily exchangeable and are thus detected in the 45Ca(2+)-binding experiments. In addition, structural similarities and conservation of ion-binding motifs support the hypothesis that His 859 is involved in the mutually exclusive binding of Zn2+ and Ca2+.
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