The channel-forming toxin aerolysin

Abstract Aeromonas sp. secrete a precursor of the cytolytic protein aerolysin into the culture medium, where it is activated by proteolytic removal of a C-terminal fragment. Activation can be achieved by a variety of mammalian proteases as well as by proteases released by the bacteria itself. Activated toxin binds with high affinity to the transmembrane protein glycophorin on the surface of eucaryotic cells. Binding is followed by oligomerization and the formation of transmembrane channels, leading to cell death. Using chemical modification and site-directed mutagenesis, we have identified regions of the molecule which are important in transfer across the outer membrane of the bacteria, and in proteolytic activation, binding, and oligomerization. A preliminary electron density map of proaerolysin crystals nidicates that the protein is organized into three domains. Analysis of two-dimensional crystals of aerolysin suggests that the oligomeric form of the protein is heptameric.     

Dimer dissociation of the pore-forming toxin aerolysin precedes receptor binding

The pore-forming toxin aerolysin is secreted by Aeromonas hydrophila as an inactive precursor. Based on chemical cross-linking and gel filtration, we show here that proaerolysin exists as a monomer at low concentrations but is dimeric above 0.1 mg/ml. At intermediate concentrations, monomers and dimers appeared to be in rapid equilibrium. All together our data indicate that, at low concentrations, the toxin is a monomer and that this species is competent for receptor binding. In contrast, a mutant toxin that forms a covalent dimer was unable to bind to target cells.     

Purification of cloned proaerolysin released by a low protease mutant of Aeromonas salmonicida

The precursor to the hole-forming toxin aerolysin has been purified in high yield from culture supernatants of a mutant of Aeromonas salmonicida containing the cloned structural gene. The mutant strain was generated by Tn5 mutagenesis. It released little or no protease or other extracellular proteins, including phospholipase, suggesting that it is a regulatory mutant. The absence of protease allowed the isolation of protoxin free from contaminating aerolysin. Typically, more than 50 mg of pure proaerolysin was obtained from 2 L of culture supernatant. The purified protein was completely unable to lyse human erythrocytes without prior activation with trypsin.     

Clostridium perfringens epsilon-toxin forms a heptameric pore within the detergent-insoluble microdomains of Madin-Darby canine kidney cells and rat synaptosomes

Clostridium perfringens epsilon-toxin, which is responsible for enterotoxaemia in ungulates, forms a heptamer in rat synaptosomal and Madin-Darby canine kidney (MDCK) cell membranes, leading to membrane permealization. Thus, the toxin may target the detergent-resistant membrane domains (DRMs) of these membranes, in analogy to aerolysin, a heptameric pore-forming toxin that associates with DRMs. To test this idea, we examined the distribution of radiolabeled epsilon-toxin in DRM and detergent-soluble membrane fractions of MDCK cells and rat synaptosomal membranes. When MDCK cells and synaptosomal membranes were incubated with the toxin and then fractionated by cold Triton X-100 extraction and flotation on sucrose gradients, the heptameric toxin was detected almost exclusively in DRMs. The results of a toxin overlay assay revealed that the toxin preferentially bound to and heptamerized in the isolated DRMs. Furthermore, cholesterol depletion by methyl-beta-cyclodextrin abrogated their association and lowered the cytotoxicity of the toxin toward MDCK cells. When epsilon-protoxin, an inactive precursor able to bind to but unable to heptamerize in the membrane, was incubated with MDCK cell membranes, it was detected mainly in their DRMs. These results suggest that the toxin is concentrated and induced to heptamerize on binding to a putative receptor located preferentially in DRMs, with all steps from initial binding through pore formation completed within the same DRMs.     

Macromolecular crowding extended to a heptameric system: the Co-chaperonin protein 10

Experiments on monomeric proteins have shown that macromolecular crowding can stabilize toward heat perturbation and also modulate native-state structure. To assess the effects of macromolecular crowding on unfolding of an oligomeric protein, we here tested the effects of the synthetic crowding agent Ficoll 70 on human cpn10 (GroES in E. coli), a heptameric protein consisting of seven identical β-barrel subunits assembling into a ring. Using far-UV circular dichroism (CD), tyrosine fluorescence, nuclear magnetic resonance (NMR), and cross-linking experiments, we investigated thermal and chemical stability, as well as the heptamer-monomer dissociation constant, without and with crowding agent. We find that crowding shifts the heptamer-monomer equilibrium constant in the direction of the heptamer. The cpn10 heptamer is both thermally and thermodynamically stabilized in 300 mg/mL Ficoll 70 as compared to regular buffer conditions. Kinetic unfolding experiments show that the increased stability in crowded conditions, in part, is explained by slower unfolding rates. A thermodynamic cycle reveals that in presence of 300 mg/mL Ficoll the thermodynamic stability of each cpn10 monomer increases by over 30%, whereas the interfaces are stabilized by less than 10%. We also introduce a new approach to analyze the spectroscopic data that makes use of multiple wavelengths: this provides robust error estimates of thermodynamic parameters.     

Membrane insertion of the heptameric staphylococcal alpha-toxin pore. A domino-like structural transition that is allosterically modulated by the target cell membrane

Staphylococcal alpha-toxin forms heptameric pores on eukaryotic cells. After binding to the cell membrane in its monomeric form, the toxin first assembles into a heptameric pre-pore. Subsequently, the pre-pore transforms into the final pore by membrane insertion of an amphipathic beta-barrel, which comprises the "central loop" domains of all heptamer subunits. The process of membrane insertion was analyzed here using a set of functionally altered toxin mutants. The results show that insertion may be initiated within an individual protomer when its NH2 terminus activates its central loop. The activated state is then shared with the central loops of the residual heptamer subunits, which results in cooperative membrane penetration. This cooperation of the central loops commences while these are still remote from the lipid bilayer. Nevertheless, it is subject to modulation by the target membrane, which therefore acts across a distance much like an allosteric effector. However, while allosteric transitions usually are reversible, membrane insertion of alpha-toxin is an irreversible event, and we show here that it can proceed to completion in a domino-like fashion when triggered by as little as a single foreign atom within the entire heptamer.     

Site-directed mutagenesis of a single tryptophan near the middle of the channel-forming toxin aerolysin inhibits its transfer across the outer membrane of Aeromonas salmonicida

The channel-forming protein aerolysin must cross both the inner and outer bacterial membranes during its secretion from Aeromonas hydrophila or from Aeromonas salmonicida containing the cloned structural gene. We examined the fate of three mutant proteins in which Trp-227, near the middle of the amino acid chain, was replaced with glycine, leucine, or phenylalanine by site-directed mutagenesis. All three proteins crossed the inner membrane and entered the periplasm in the same way as wild-type, and in each case the signal sequence was removed correctly. Little or none of the proaerolysin substituted with glycine or leucine was released into the culture supernatant. Instead, significant amounts became associated with the outer membrane. The Phe-227 protoxin was secreted by the bacteria but at a reduced rate. The leucine and phenylalanine mutant proteins were purified and compared with native proaerolysin. They were processed correctly to the mature forms by treatment with trypsin, and like native aerolysin, both were resistant to further proteolysis. In each case, processing was followed by the formation of oligomers similar to those produced by native toxin. The hemolytic activity of the processed Phe-227 mutant was one-quarter that of wild-type toxin whereas Leu-227 aerolysin had less than one-hundredth the wild-type activity. These results are further evidence that aerolysin is secreted in at least two steps. As well, they show that the last step, crossing the outer membrane, can be blocked by an apparently small change in the structure of the protein.     

Crossing three membranes. Channel formation by aerolysin.

Aerolysin is a channel-forming toxin responsible for the pathogenicity of Aeromonas hydrophila. It crosses the inner and outer membranes of the bacteria in separate steps and is released as a 52-kDa inactive protoxin which is activated by proteolytic removal of approximately 40 amino acids from the C terminus. The toxin binds to the erythrocyte transmembrane protein glycophorin and oligomerizes before inserting into the membrane, producing a voltage gated, anion selective channel about 1 nm in diameter. Remarkably, proaerolysin appears to be dimeric, whereas the oligomer is a heptamer. Using chemical modification and site-directed mutagenesis, we have identified some of the regions of the molecule which appear to be involved in secretion and in channel formation.     

Expression and properties of an aerolysin--Clostridium septicum alpha toxin hybrid protein

Aerolysin is a bilobal channel-forming toxin secreted by Aeromonas hydrophila. The alpha toxin produced by Clostridium septicum is homologous to the large lobe of aerolysin. However, it does not contain a region corresponding to the small lobe of the Aeromonas toxin, leading us to ask what the function of the small lobe is. We fused the small lobe of aerolysin to alpha toxin, producing a hybrid protein that should structurally resemble aerolysin. Unlike aerolysin, the hybrid was not secreted when expressed in Aeromonas salmonicida. The purified hybrid was activated by proteolytic processing in the same way as both parent proteins and, after activation, it formed oligomers that corresponded to the aerolysin heptamer. Like aerolysin, the hybrid was far more active than alpha toxin against human erythrocytes and mouse T lymphocytes. Both aerolysin and the hybrid bound to human glycophorin, and both were inhibited by preincubation with this erythrocyte glycoprotein, whereas alpha toxin was unaffected. We conclude that aerolysin contains two receptor binding sites, one for glycosyl-phosphatidylinositol-anchored proteins that is located in the large lobe and is also found in alpha toxin, and a second site, located in the small lobe, that binds a surface carbohydrate determinant.     

Structural stability of covalently linked GroES heptamer: advantages in the formation of oligomeric structure

In order to understand how inter-subunit association stabilizes oligomeric proteins, a single polypeptide chain variant of heptameric co-chaperonin GroES (tandem GroES) was constructed from Escherichia coli heptameric GroES by linking consecutively the C-terminal of one subunit to the N-terminal of the adjacent subunit with a small linker peptide. The tandem GroES (ESC7) showed properties similar to wild-type GroES in structural aspects and co-chaperonin activity. In unfolding and refolding equilibrium experiments using guanidine hydrochloride (Gdn-HCl) as a denaturant at a low protein concentration (50 microg ml(-1)), ESC7 showed a two-state transition with a greater resistance toward Gdn-HCl denaturation (Cm=1.95 M) compared to wild-type GroES (Cm=1.1 M). ESC7 was found to be about 10 kcal mol(-1) more stable than the wild-type GroES heptamer at 50 microg ml(-1). Kinetic unfolding and refolding experiments of ESC7 revealed that the increased stability was mainly attributed to a slower unfolding rate. Also a transient intermediate was detected in the refolding reaction. Interestingly, at the physiological GroES concentration (>1 mg ml(-1)), the free energy of unfolding for GroES heptamer exceeded that for ESC7. These results showed that at low protein concentrations (<1 mg ml(-1)), the covalent linking of subunits contributes to the stability but also complicates the refolding kinetics. At physiological concentrations of GroES, however, the oligomeric state is energetically preferred and the advantages of covalent linkage are lost. This finding highlights a possible advantage in transitioning from multi-domain proteins to oligomeric proteins with small subunits in order to improve structural and kinetic stabilities.     

Structural stability of oligomeric chaperonin 10: the role of two beta-strands at the N and C termini in structural stabilization

Chaperonin 10 (cpn10) is a well-conserved subgroup of the molecular chaperone family. GroES, the cpn10 from Escherichia coli, is composed of seven 10kDa subunits, which form a dome-like oligomeric ring structure. From our previous studies, it was found that GroES unfolded completely through a three-state unfolding mechanism involving a partly folded monomer and that this reaction was reversible. In order to study whether these unfolding-refolding characteristics were conserved in other cpn10 proteins, we have examined the structural stabilities of cpn10s from rat mitochondria (RatES) and from hyperthermophilic eubacteria Thermotoga maritima (TmaES), and compared the values to those of GroES. From size-exclusion chromatography experiments in the presence of various concentrations of Gdn-HCl at 25 degrees C, both cpn10s showed unfolding-refolding characteristics similar to those of GroES, i.e. two-stage unfolding reactions that include formation of a partially folded monomer. Although the partially folded monomer of TmaES was considerably more stable compared to GroES and RatES, it was found that the overall stabilities of all three cpn10s were achieved significantly by inter-subunit interactions. We studied this contribution of inter-subunit interactions to overall stability in the GroES heptamer by introducing a mutation that perturbed subunit association, specifically the interaction between the two anti-parallel beta-strands at the N and C termini of this protein. From analyses of the mutants' stabilities, it was revealed that the anti-parallel beta-strands at the subunit interface are crucial for subunit association and stabilization of the heptameric GroES protein.     

Protein export by a gram-negative bacterium: production of aerolysin by Aeromonas hydrophila.

The synthesis and export of aerolysin, an extracellular protein toxin released by the gram-negative bacterium Aeromonas hydrophila, was studied by pulse-labeling with [35S]methionine. The toxin was synthesized as a higher-molecular-weight precursor. This was processed cotranslationally, resulting in the appearance within the cell of the mature protein, which was then exported to the supernatant. Precursor aerolysin accumulated in cells incubated in the presence of carbonyl cyanide m-chlorophenyl hydrazone, a substance which also inhibited the export of mature aerolysin from the cell. The entrapped mature toxin could not be shocked from the cells, although it could be digested by protease applied to shocked cells. The toxin was processed and translocated across the inner membrane of pleiotropic export mutants and accumulated in the periplasm. The results indicate that more than one step is required for the export of the protein and that aerolysin does not cross the inner and outer membranes simultaneously.     

Conversion of a transmembrane to a water-soluble protein complex by a single point mutation

Proteins exist in one of two generally incompatible states: either membrane associated or soluble. Pore-forming proteins are exceptional because they are synthesized as a water-soluble molecule but end up being located in the membrane -- that is, they are nonconstitutive membrane proteins. Here we report the pronounced effect of the single point mutation Y221G of the pore-forming toxin aerolysin. This mutation blocks the hemolytic activity of the toxin but does not affect its initial structure, its ability to bind to cell-surface receptors or its capacity to form heptamers, which constitute the channel-forming unit. The overall structure of the Y221G protein as analyzed by cryo-negative staining EM and three-dimensional reconstruction is remarkably similar to that of the wild type heptamer. The mutant protein forms a mushroom-shaped complex whose stem domain is thought to be within the membrane in the wild type toxin. In contrast to the wild type heptamer, which is a hydrophobic complex, the Y221G heptamer is fully hydrophilic. This point mutation has, therefore, converted a normally membrane-embedded toxin into a soluble complex.     

Unfolding and refolding of Escherichia coli chaperonin GroES is expressed by a three-state model.

The guanidine-hydrochloride (Gdn-HCl) induced unfolding and refolding characteristics of the co-chaperonin GroES from Escherichia coli, a homoheptamer of subunit molecular mass 10,000 Da, were studied by using intrinsic fluorescence, 1-anilino-8-naphthalene sulfonate (ANS) binding, and size-exclusion HPLC. When monitored by tyrosine fluorescence, the unfolding reaction of GroES consisted of a single transition, with a transition midpoint at around 1.0 M Gdn-HCl. Interestingly, however, ANS binding and size-exclusion HPLC experiments strongly suggested the existence of an intermediate state in the transition. In order to confirm the existence of an intermediate state between the native heptameric and unfolded monomeric states, a tryptophan residue was introduced into the interface of GroES subunits as a fluorescent probe. The unfolding reaction of GroES I48W as monitored by tryptophyl fluorescence showed a single transition curve with a transition midpoint at 0.5 M Gdn-HCl. This unfolding transition curve as well as the refolding kinetics were dependent on the concentration of GroES protein. CD spectrum and size-exclusion HPLC experiments demonstrated that the intermediates assumed a partially folded conformation at around 0.5 M Gdn-HCl. The refolding of GroES protein from 3 M Gdn-HCl was probed functionally by measuring the extent of inhibition of GroEL ATPase activity and the enhancement of lactate dehydrogenase refolding yields in the presence of GroEL and ADP. These results clearly demonstrated that the GroES heptamer first dissociated to monomers and then unfolded completely upon increasing the concentration of Gdn-HCl, and that both transitions were reversible. From the thermodynamic analysis of the dissociation reaction, it was found that the partially folded monomer was only marginally stable and that the stability of GroES protein is governed mostly by the association of the subunits.     

Cloned aerolysin of Aeromonas hydrophila is exported by a wild-type marine Vibrio strain but remains periplasmic in pleiotropic export mutants.

With a wide host range vector, the structural gene aerA for the hole-forming extracellular protein aerolysin of Aeromonas hydrophila was cloned into the marine Vibrio sp. strain 60 and into three pleiotropic export mutants (epr mutants). The parent strain and all of the mutants were able to express the protein with the aerA promoter in the plasmid. The parent strain exported proaerolysin into the medium, while all of the mutants accumulated the protoxin in their periplasms. Two of the mutants also accumulated protease; however, as we have found earlier with A. hydrophila, the periplasmic form of proaerolysin in the Vibrio sp. must somehow be protected from proteolysis because it was not converted to active toxin until the cells were shocked. Conversion could be prevented by adding o-phenanthroline to the solutions used in shocking. These results show that the export pathway in the marine Vibrio sp. is very similar to the pathway in A. hydrophila.     

The aerolysin membrane channel is formed by heptamerization of the monomer.

The cytolytic toxin aerolysin has been found to form heptameric oligomers by SDS-PAGE electrophoresis, STEM mass measurements of single oligomers and image analysis of two-dimensional membrane crystals. Two types of crystal, flat sheets and long regular tubes, have been obtained by reconstitution of purified protein and Escherichia coli phospholipids. A noise-filtered image of the best crystalline sheets reveals a structure with 7-fold symmetry containing a central strongly stain-excluding ring that encircles a dark stain-filled channel 17 A in diameter. The ring is surrounded by seven arms each made up of two unequal sized domains. By combining projected views and side-views, a simplified model of the aerolysin channel complex has been constructed. The relevance of this structure to the mode of action of aerolysin is discussed.     

Reversible denaturation of oligomeric human chaperonin 10: denatured state depends on chemical denaturant

Chaperonins cpn60/cpn10 (GroEL/GroES in Escherichia coli) assist folding of nonnative polypeptides. Folding of the chaperonins themselves is distinct in that it entails assembly of a sevenfold symmetrical structure. We have characterized denaturation and renaturation of the recombinant human chaperonin 10 (cpn10), which forms a heptamer. Denaturation induced by chemical denaturants urea and guanidine hydrochloride (GuHCl) as well as by heat was monitored by tyrosine fluorescence, far-ultraviolet circular dichroism, and cross-linking; all denaturation reactions were reversible. GuHCl-induced denaturation was found to be cpn10 concentration dependent, in accord with a native heptamer to denatured monomer transition. In contrast, urea-induced denaturation was not cpn10 concentration dependent, suggesting that under these conditions cpn10 heptamers denature without dissociation. There were no indications of equilibrium intermediates, such as folded monomers, in either denaturant. The different cpn10 denatured states observed in high [GuHCl] and high [urea] were supported by cross-linking experiments. Thermal denaturation revealed that monomer and heptamer reactions display the same enthalpy change (per monomer), whereas the entropy-increase is significantly larger for the heptamer. A thermodynamic cycle for oligomeric cpn10, combining chemical denaturation with the dissociation constant in absence of denaturant, shows that dissociated monomers are only marginally stable (3 kJ/mol). The thermodynamics for co-chaperonin stability appears conserved; therefore, instability of the monomer could be necessary to specify the native heptameric structure.     

Beta-barrel membrane protein folding and structure viewed through the lens of alpha-hemolysin

The beta-barrel is a transmembrane structural motif commonly encountered in bacterial outer membrane proteins and pore-forming toxins (PFTs). Alpha-hemolysin (alphaHL) is a cytotoxin secreted by Staphylococcus aureus that assembles from a water-soluble monomer to form a membrane-bound heptameric beta-barrel on the surface of susceptible cells, perforating the cell membranes, leading to cell death and lysis. The mechanism of heptamer assembly, which has been studied extensively, occurs in a stepwise manner, and the structures of the initial, monomeric form and final, membrane-embedded pore are known. The toxin's ability to assemble from an aqueous, hydrophilic species to a membrane-inserted oligomer is of interest in understanding the assembly of PFTs in particular and the folding and structure of beta-barrel membrane proteins in general. Here we review the structures of the monomeric and heptamer states of LukF and alphaHL, respectively, the mechanism of toxin assembly, and the relationships between alphaHL and nontoxin beta-barrel membrane proteins.     

Spectroscopic study of the activation and oligomerization of the channel-forming toxin aerolysin: identification of the site of proteolytic activation.

The channel-forming protein aerolysin is secreted as a protoxin which can be activated by proteolytic removal of a C-terminal peptide. The activation and subsequent oligomerization of aerolysin were studied using a variety of spectroscopic techniques. Mass spectrometric determination of the molecular weights of proaerolysin and aerolysin permitted identification of the sites at which the protoxin is processed by trypsin and chymotrypsin. The results of far- and near-UV circular dichroism measurements indicated that processing with trypsin does not lead to major changes in secondary or tertiary structure of the protein. An increase in tryptophan fluorescence intensity and a small red shift in the maximum emission wavelength of tryptophans could be observed, suggesting that there is a change in the environment of some of the tryptophans. There was also a dramatic increase in the binding of the hydrophobic fluorescent probe 1-anilino-8-naphthalenesulfonate during activation, leading us to conclude that a hydrophobic region in the protein is exposed by trypsin treatment. Using measurements of light scattering, various parameters influencing oligomerisation of trypsin-activated aerolysin were determined. Oligomerization rates were found to increase with the concentration of aerolysin, whereas they decreased with increasing ionic strength.     

Unfolding of Vibrio cholerae hemolysin induces oligomerization of the toxin monomer

Vibrio cholerae hemolysin (HlyA) is a pore-forming toxin that exists in two stable forms: a hemolytically active water-soluble monomer with a native molecular weight of 65,000 and a hemolytically inactive SDS-stable heptamer with the configuration of a transmembrane diffusion channel. Transformation of the monomer into the oligomer is spontaneous but very slow in the absence of interaction with specific membrane components like cholesterol and sphingolipids. In this report, we show that mild disruption of the native tertiary structure of HlyA by 1.75 M urea triggered rapid and quantitative conversion of the monomer to an oligomer. Furthermore, the HlyA monomer when unfolded in 8 M urea refolded and reconstituted on renaturation into the oligomer biochemically and functionally similar to the heptamer formed in target lipid bilayer, suggesting that the HlyA polypeptide had a strong propensity to adopt the oligomer as the stable native state in preference to the monomer. On the basis of our results, we propose that (a) the hemolytically active HlyA monomer represents a quasi-stable conformation corresponding to a local free energy minimum and the transmembrane heptameric pore represents a stable conformation corresponding to an absolute free energy minimum and (b) any perturbation of the native tertiary structure of the HlyA monomer causing relaxation of conformational constraints tends to promote self-assembly to the oligomer with membrane components playing at most an accessory role.