Structures of perfringolysin O suggest a pathway for activation of cholesterol-dependent cytolysins

Cholesterol-dependent cytolysins (CDCs), a large family of bacterial toxins, are secreted as water-soluble monomers and yet are capable of generating oligomeric pores in membranes. Previous work has demonstrated that large scale structural rearrangements occur during this transition but the detailed mechanism by which these changes take place remains a puzzle. Despite evidence of structural and functional couplings between domains 3 and 4, the crystal structure of the CDC, perfringolysin O (PFO), shows the two domains do not make direct contact. Here, we present crystal structures of PFO that demonstrate movements of domain 4 are sufficient to trigger conformational changes that are transmitted through the molecule to the distant domain 3. These coupled movements result in a loss of many contacts between domain 3 and rest of the molecule that would eventually lead to the exposure of transmembrane regions in preparation for membrane insertion. The structures reveal a detailed molecular pathway that may be the basis for the allosteric transition that occurs on initial membrane binding leading to the exposure of membrane-spanning regions in a domain distant from the initial site of interaction.     

Crystal structure of cytotoxin protein suilysin from Streptococcus suis

Cholesterol-dependent cytolysins (CDC) are pore forming toxins. A prototype of the CDC family members is perfringolysin O (PFO), which directly binds to cholesterol rich cell membrane and lyses the cell. However, as an exception of this general observation, Streptococcus intermedius intermedilysin (ILY) requires human CD59 as its receptor in addition to cholesterol when exhibiting hemolytic activity. It was attempted to explain this functional difference based on a conformational variation in the C-terminal domain of the two toxin proteins, particularly a highly conserved undecapeptide termed tryptophan rich motif. Here, we present the crystal structure of suilysin, a CDC toxin from the swine infectious pathogen Streptococcus suis. Like PFO, suilysin does not require a host receptor for hemolytic activity; yet in the suilysin crystal it shares a similar conformation in the tryptophan rich motif with ILY. This observation suggests that current views of structure-function relationship of CDC proteins in membrane association are still far from complete.     

The Cholesterol-Dependent Cytolysin Signature Motif: A Critical Element in the Allosteric Pathway that Couples Membrane Binding to Pore Assembly

The cholesterol-dependent cytolysins (CDCs) constitute a family of pore-forming toxins that contribute to the pathogenesis of a large number of Gram-positive bacterial pathogens.The most highly conserved region in the primary structure of the CDCs is the signature undecapeptide sequence (ECTGLAWEWWR). The CDC pore forming mechanism is highly sensitive to changes in its structure, yet its contribution to the molecular mechanism of the CDCs has remained enigmatic. Using a combination of fluorescence spectroscopic methods we provide evidence that shows the undecapeptide motif of the archetype CDC, perfringolysin O (PFO), is a key structural element in the allosteric coupling of the cholesterol-mediated membrane binding in domain 4 (D4) to distal structural changes in domain 3 (D3) that are required for the formation of the oligomeric pore complex. Loss of the undecapeptide function prevents all measurable D3 structural transitions, the intermolecular interaction of membrane bound monomers and the assembly of the oligomeric pore complex. We further show that this pathway does not exist in intermedilysin (ILY), a CDC that exhibits a divergent undecapeptide and that has evolved to use human CD59 rather than cholesterol as its receptor. These studies show for the first time that the undecapeptide of the cholesterol-binding CDCs forms a critical element of the allosteric pathway that controls the assembly of the pore complex.     

The mechanism of pore assembly for a cholesterol-dependent cytolysin: formation of a large prepore complex precedes the insertion of the transmembrane beta-hairpins

Perfringolysin O (PFO) is a member of the cholesterol-dependent cytolysin (CDC) family of membrane-penetrating toxins. The CDCs form large homooligomers (estimated to be comprised of up to 50 CDC monomers) that are responsible for generating a large pore in cholesterol-containing membranes of eukaryotic cells. The assembly of the PFO cytolytic complex was examined to determine whether it forms an oligomeric prepore complex on the membrane prior to the insertion of its membrane-spanning beta-sheet. A PFO oligomeric complex was formed on liposomes at both 4 degrees C and 37 degrees C and shown by SDS-agarose gel electrophoresis to be comprised of a large, comparatively homogeneous complex instead of a distribution of oligomer sizes. At low temperature, the processes of oligomerization and membrane insertion could be resolved, and PFO was found to form an oligomer without significant membrane insertion of its beta-hairpins. Furthermore, PFO was found to increase the ion conductivity through a planar bilayer by large and discrete stepwise changes in conductance that are consistent with the insertion of a preassembled pore complex into the bilayer. The combined results of these analyses strongly support the hypothesis that PFO forms a large oligomeric prepore complex on the membrane surface prior to the insertion of its transmembrane beta-sheet.     

Specificity of streptolysin O in cytolysin-mediated translocation

Cytolysin-mediated translocation (CMT) is a recently described process in the Gram-positive pathogen Streptococcus pyogenes that translocates an effector protein of streptococcal origin into the cytoplasm of a host cell. At least two proteins participate in CMT, the pore-forming molecule streptolysin O (SLO) and an effector protein with the characteristics of a signal transduction protein, the Streptococcus pyogenes NAD-glycohydrolase (SPN). In order to begin to elucidate the molecular details of the translocation process, we examined whether perfringolysin O (PFO), a pore-forming protein related to SLO, could substitute for SLO in the translocation of SPN. When expressed by S. pyogenes, PFO, like SLO, had the ability to form functional pores in keratinocyte membranes. However, unlike SLO, PFO was not competent for translocation of SPN across the host cell membrane. Thus, pore formation by itself was not sufficient to promote CMT, suggesting that an additional feature of SLO was required. This conclusion was supported by the construction of a series of mutations in SLO that uncoupled pore formation and competence for CMT. These mutations defined a domain in SLO that was dispensable for pore formation, but was essential for CMT. However, introduction of this domain into PFO did not render PFO competent for CMT, implying that an additional domain of SLO is also critical for translocation. Taken together, these data indicate that SLO plays an active role in the translocation process that extends beyond that of a passive pore.     

The mechanism of membrane insertion for a cholesterol-dependent cytolysin: a novel paradigm for pore-forming toxins.

Perfringolysin O (PFO), a water-soluble monomeric cytolysin secreted by pathogenic Clostridium perfringens, oligomerizes and forms large pores upon encountering cholesterol-containing membranes. Whereas all pore-forming bacterial toxins examined previously have been shown to penetrate the membrane using a single amphipathic beta hairpin per polypeptide, cysteine-scanning mutagenesis and multiple independent fluorescence techniques here reveal that each PFO monomer contains a second domain involved in pore formation, and that each of the two amphipathic beta hairpins completely spans the membrane. In the soluble monomer, these transmembrane segments are folded into six alpha helices. The insertion of two transmembrane hairpins per toxin monomer and the major change in secondary structure are striking and define a novel paradigm for the mechanism of membrane insertion by a cytolytic toxin.     

A novel cholesterol‐insensitive mode of membrane binding promotes cytolysin‐mediated translocation by Streptolysin O

Cytolysin‐mediated translocation (CMT), performed by Streptococcus pyogenes, utilizes the cholesterol‐dependent cytolysin Streptolysin O (SLO) to translocate the NAD⁺‐glycohydrolase (SPN) into the host cell during infection. SLO is required for CMT and can accomplish this activity without pore formation, but the details of SLO's interaction with the membrane preceding SPN translocation are unknown. Analysis of binding domain mutants of SLO and binding domain swaps between SLO and homologous cholesterol‐dependent cytolysins revealed that membrane binding by SLO is necessary but not sufficient for CMT, demonstrating a specific requirement for SLO in this process. Despite being the only known receptor for SLO, this membrane interaction does not require cholesterol. Depletion of cholesterol from host membranes and mutation of SLO's cholesterol recognition motif abolished pore formation but did not inhibit membrane binding or CMT. Surprisingly, SLO requires the coexpression and membrane localization of SPN to achieve cholesterol‐insensitive membrane binding; in the absence of SPN, SLO's binding is characteristically cholesterol‐dependent. SPN's membrane localization also requires SLO, suggesting a co‐dependent, cholesterol‐insensitive mechanism of membrane binding occurs, resulting in SPN translocation.     

The Cholesterol-dependent Cytolysin Membrane-binding Interface Discriminates Lipid Environments of Cholesterol to Support β-Barrel Pore Insertion

The majority of cholesterol-dependent cytolysins (CDCs) utilize cholesterol as a membrane receptor, whereas a small number are restricted to the GPI-anchored protein CD59 for initial membrane recognition. Two cholesterol-binding CDCs, perfringolysin O (PFO) and streptolysin O (SLO), were found to exhibit strikingly different binding properties to cholesterol-rich natural and synthetic membranes. The structural basis for this difference was mapped to one of the loops (L3) in the membrane binding interface that help anchor the toxin monomers to the membrane after receptor (cholesterol) binding by the membrane insertion of its amino acid side chains. A single point mutation in this loop conferred the binding properties of SLO to PFO and vice versa. Our studies strongly suggest that changing the side chain structure of this loop alters its equilibrium between membrane-inserted and uninserted states, thereby affecting the overall binding affinity and total bound toxin. Previous studies have shown that the lipid environment of cholesterol has a dramatic effect on binding and activity. Combining this data with the results of our current studies on L3 suggests that the structure of this loop has evolved in the different CDCs to preferentially direct binding to cholesterol in different lipid environments. Finally, the efficiency of β-barrel pore formation was inversely correlated with the increased binding and affinity of the PFO L3 mutant, suggesting that selection of a compatible lipid environment impacts the efficiency of membrane insertion of the β-barrel pore.     

Arresting pore formation of a cholesterol-dependent cytolysin by disulfide trapping synchronizes the insertion of the transmembrane beta-sheet from a prepore intermediate

Perfringolysin O (PFO), a member of the cholesterol-dependent cytolysin family of pore-forming toxins, forms large oligomeric complexes comprising up to 50 monomers. In the present study, a disulfide bridge was introduced between cysteine-substituted serine 190 of transmembrane hairpin 1 (TMH1) and cysteine-substituted glycine 57 of domain 2 of PFO. The resulting disulfide-trapped mutant (PFO(C190-C57)) was devoid of hemolytic activity and could not insert either of its transmembrane beta-hairpins (TMHs) into the membrane unless the disulfide was reduced. Both the size of the oligomer formed on the membrane and its rate of formation were unaffected by the oxidation state of the Cys(190)-Cys(57) disulfide bond; thus, the disulfide-trapped PFO was assembled into a prepore complex on the membrane. The conversion of this prepore to the pore complex was achieved by reducing the C190-C57 disulfide bond. PFO(C190-C57) that was allowed to form the prepore prior to the reduction of the disulfide exhibited a dramatic increase in the rate of PFO-dependent hemolysis and the membrane insertion of its TMHs when compared with toxin that had the disulfide reduced prior mixing the toxin with membranes. Therefore, the rate-limiting step in pore formation is prepore assembly, not TMH insertion. These data demonstrate that the prepore is a legitimate intermediate during the insertion of the large transmembrane beta-sheet of the PFO oligomer. Finally, the PFO TMHs do not appear to insert independently, but instead their insertion is coupled.     

Phospholipid hydrolysis caused by Clostridium perfringens α-toxin facilitates the targeting of perfringolysin O to membrane bilayers

Clostridium perfringens causes gas gangrene and gastrointestinal disease in humans. These pathologies are mediated by potent extracellular protein toxins, particularly α-toxin and perfringolysin O (PFO). While α-toxin hydrolyzes phosphatidylcholine and sphingomyelin, PFO forms large transmembrane pores on cholesterol-containing membranes. It has been suggested that the ability of PFO to perforate the membrane of target cells is dictated by how much free cholesterol molecules are present. Given that C. perfringens α-toxin cleaves the phosphocholine headgroup of phosphatidylcholine, we reasoned that α-toxin may increase the number of free cholesterol molecules in the membrane. Our present studies reveal that α-toxin action on membrane bilayers facilitates the PFO?cholesterol interaction as evidenced by a reduction in the amount of cholesterol required in the membrane for PFO binding and pore formation. These studies suggest a mechanism for the concerted action of α-toxin and PFO during C. perfringens pathogenesis.     

Helical crystallization on nickel-lipid nanotubes: perfringolysin O as a model protein

To facilitate purification and subsequent structural studies of recombinant proteins the most widely used genetically encoded tag is the histidine tag (His-tag) which specifically binds to N-nitrilotriacetic-acid-chelated nickel ions. Lipids derivatized with a nickel-chelating head group can be mixed with galactosylceramide glycolipids to prepare lipid nanotubes that bind His-tagged proteins. In this study, we use His-tagged perfringolysin O (PFO), a soluble toxin secreted by the bacterial pathogen Clostridium perfringens, as a model protein to test the utility of nickel-lipid nanotubes as a tool for structural studies of His-tagged proteins. PFO is a member of the cholesterol dependent cytolysin family (CDC) of oligomerizing, pore-forming toxins found in a variety of Gram-positive bacterial pathogens. CDC pores have been difficult to study by X-ray crystallography because they are membrane associated and vary in size. We demonstrate that both a wild-type and a mutant form of PFO form helical arrays on nickel-lipid containing nanotubes. Cryo-electron microscopy and image analysis of the helical arrays were used to reconstruct a 3D density map of wild-type PFO. This study suggests that the use of nickel-lipid nanotubes may offer a general approach for structural studies of recombinant proteins and may provide insights into the molecular interactions of proteins that have a natural affinity for a membrane surface.     

Conformational changes that effect oligomerization and initiate pore formation are triggered throughout perfringolysin O upon binding to cholesterol

Pore formation by the cholesterol-dependent cytolysins (CDCs) requires the presence of cholesterol in the target membrane. Cholesterol was long thought to be the cellular receptor for these toxins, but not all CDCs require cholesterol for binding. Intermedilysin, secreted by Streptococcus intermedius, only binds to membranes containing the human protein CD59 but forms pores only if the membrane contains sufficient cholesterol. In contrast, perfringolysin O (PFO), secreted by Clostridium perfringens, only binds to membranes containing substantial amounts of cholesterol. Given that different steps in the assembly of various CDC pores require cholesterol, here we have analyzed to what extent cholesterol molecules, by themselves, can modulate the conformational changes associated with PFO oligomerization and pore formation. PFO binds to cholesterol when dispersed in aqueous solution, and this binding triggers the distant rearrangement of a beta-strand that exposes an oligomerization interface. Moreover, upon binding to cholesterol, PFO forms a prepore complex, unfolds two amphipathic transmembrane beta-hairpins, and positions their nonpolar surfaces so they associate with the hydrophobic cholesterol surface. The interaction of PFO with cholesterol is therefore sufficient to initiate an irreversible sequence of coupled conformational changes that extend throughout the toxin molecule.     

How interaction of perfringolysin O with membranes is controlled by sterol structure, lipid structure, and physiological low pH: insights into the origin of perfringolysin O-lipid raft interaction

Perfringolysin O (PFO) is a sterol-dependent, pore-forming cytolysin. To understand the molecular basis of PFO membrane interaction, we studied its dependence upon sterol and lipid structure and aqueous environment. PFO interacted with diverse sterols, although binding was affected by double bond location in the sterol rings, sterol side chain structure, and sterol polar group structure. Importantly, a sterol structure promoting formation of ordered membrane domains (lipid rafts) was not critical for interaction. PFO membrane interaction was also affected by phospholipid acyl chain structure, being inversely related to tight acyl chain packing with cholesterol. Experiments using the pre-pore Y181A mutant demonstrated that sterol binding strength and specificity was not affected by whether PFO forms a transmembrane beta-barrel. Combined, these observations are consistent with a model in which the strength and specificity of sterol interaction arises from both sterol interactions with domain 4 and sterol chemical activity within membranes. The lipid raft-binding portions of sterol bound to PFO may remain largely exposed to the lipid bilayer. These results place important constraints upon the origin of PFO raft affinity. Additional experiments demonstrated that the structure of membrane-inserted PFO at low and neutral pH was similar as judged by the effect of phospholipid and sterol structure upon PFO properties and membrane interaction. However, low pH enhanced PFO membrane binding, oligomerization, and pore formation. In lipid vesicles mimicking the exofacial (outer) membrane leaflet, PFO-membrane binding was maximal at pH 5.5-6. This is consistent with the hypothesis that PFO function involves acidic vacuoles.     

Ultrastructural analysis of the membrane insertion of domain 3 of streptolysin O

Streptolysin O (SLO) is a membrane-damaging toxic protein produced by group A streptococci. We performed an ultrastructural analysis of pore formation and the mechanism of hemolysis by SLO, using a mutant form of SLO [SLO(C/A)-SS] and native SLO. SLO(C/A)-SS was unable to penetrate the erythrocyte membrane as a consequence of immobilization that was due to a disulfide bond between domains. The SLO(C/A)-SS molecules that bound to membranes formed numerous single-layered ring-shaped structures that did not result in pores on the membranes. These structures were similar to the structures formed by native SLO at 0 degrees C. After treatment with dithiothreitol, SLO(C/A)-SS that had bound to membranes formed double-layered rings with pores on the membranes, as does native SLO at room temperature. Our morphological evidence demonstrates that an increase in temperature is necessary for the occurrence of conformational changes and for the formation of double-layered rings after the insertion of domain 3 into the host cell membrane. On the basis of a model of the oligomeric structure of SLO, we propose some new details of the mechanism of hemolysis by SLO.     

A basic model for the association of ligands with membrane cholesterol: application to cytolysin binding

Almost all the cholesterol in cellular membranes is associated with phospholipids in simple stoichiometric complexes. This limits the binding of sterol ligands such as filipin and perfringolysin O (PFO) to a small fraction of the total. We offer a simple mathematical model that characterizes this complexity. It posits that the cholesterol accessible to ligands has two forms: active cholesterol, which is that not complexed with phospholipids; and extractable cholesterol, that which ligands can capture competitively from the phospholipid complexes. Simulations based on the model match published data for the association of PFO oligomers with liposomes, plasma membranes, and the isolated endoplasmic reticulum. The model shows how the binding of a probe greatly underestimates cholesterol abundance when its affinity for the sterol is so weak that it competes poorly with the membrane phospholipids. Two examples are the understaining of plasma membranes by filipin and the failure of domain D4 of PFO to label their cytoplasmic leaflets. Conversely, the exaggerated staining of endolysosomes suggests that their cholesterol, being uncomplexed, is readily available. The model is also applicable to the association of cholesterol with intrinsic membrane proteins. For example, it supports the hypothesis that the sharp threshold in the regulation of homeostatic endoplasmic reticulum proteins by cholesterol derives from the cooperativity of their binding to the sterol weakly held by the phospholipids. Thus, the model explicates the complexity inherent in the binding of ligands like PFO and filipin to the small accessible fraction of membrane cholesterol.     

Change in membrane potential induced by streptolysin O,a pore-forming toxin: flow cytometric analysis using a voltage-sensitive fluorescent probe and rat thymic lymphocytes

Streptolysin O (SLO) is a bacterial pore-forming toxin that is employed to permeabilize cell membranes in some biological experiments. SLO forms various types of pores with different shapes, increasing membrane ion permeability and subsequently inducing changes in membrane potential. To characterize the pores formed by SLO, the changes in membrane potential induced by SLO in rat lymphocytes were considered using flow cytometry with a voltage-sensitive fluorescent probe, bis-(1,3-dibutylbarbituric acid)trimethine oxonol (Oxonol). SLO caused three types of membrane potential responses accessed with Oxonol. One type induces a great decrease in Oxonol fluorescence (large hyperpolarization) that may be elicited via the increase of Ca²⁺-dependent K⁺ permeability by SLO-induced influx of external Ca²⁺. A second type is an increase in Oxonol fluorescence (depolarization) that may be caused by a nonspecific increase in membrane cation permeability. The third type is a small decrease in Oxonol fluorescence (small hyperpolarization), probably via an increase in Cl^– permeability. That SLO transitionally changes membrane ion permeability may have implications in the pathology of pyogenic group streptococci infections in which SLO is thought to be one of the key virulence factors.     

Differential interaction of the two cholesterol-dependent, membrane-damaging toxins, streptolysin O and Vibrio cholerae cytolysin, with enantiomeric cholesterol

Membrane cholesterol is essential to the activity of at least two structurally unrelated families of bacterial pore-forming toxins, represented by streptolysin O (SLO) and Vibrio cholerae cytolysin (VCC), respectively. Here, we report that SLO and VCC differ sharply in their interaction with liposome membranes containing enantiomeric cholesterol (ent-cholesterol). VCC had very low activity with ent-cholesterol, which is in line with a stereospecific mode of interaction of this toxin with cholesterol. In contrast, SLO was only slightly less active with ent-cholesterol than with cholesterol, suggesting a rather limited degree of structural specificity in the toxin-cholesterol interaction.     

P2X7 receptors mediate resistance to toxin-induced cell lysis

In the majority of cells, the integrity of the plasmalemma is recurrently compromised by mechanical or chemical stress. Serum complement or bacterial pore-forming toxins can perforate the plasma membrane provoking uncontrolled Ca^2 + influx, loss of cytoplasmic constituents and cell lysis. Plasmalemmal blebbing has previously been shown to protect cells against bacterial pore-forming toxins. The activation of the P2X7 receptor (P2X7R), an ATP-gated trimeric membrane cation channel, triggers Ca^2 + influx and induces blebbing. We have investigated the role of the P2X7R as a regulator of plasmalemmal protection after toxin-induced membrane perforation caused by bacterial streptolysin O (SLO).     

Unusual Thermal Disassembly of the SPFH Domain Oligomer from Pyrococcus horikoshii

Stomatin, prohibitin, flotillin, and HflK/C (SPFH) domain proteins are membrane proteins that are widely conserved from bacteria to mammals. The molecular functions of these proteins have not been established. In mammals, the domain is often found in raft-associated proteins such as flotillin and podocin. We determined the structure of the SPFH domain of PH0470 derived from Pyrococcus horikoshii using NMR. The structure closely resembles that of the SPFH domain of the paralog PH1511, except for two C-terminal helices. The results show that the SPFH domain forms stable dimers, trimers, tetramers, and multimers, although it lacks the coiled-coil region for oligomerization, which is a highly conserved region in this protein family. The oligomers exhibited unusual thermodynamic behavior, as determined by circular dichroism, NMR, gel filtration, chemical cross-linking, and analytical ultracentrifugation. The oligomers were converted into monomers when they were heated once and then cooled. This transition was one-way and irreversible. We propose a mechanism of domain swapping for forming dimers as well as successive oligomers. The results of this study provide what to our knowledge are new insights into the common molecular function of the SPFH domain, which may act as a membrane skeleton through oligomerization by domain swapping.     

Streptolysin O: the C-terminal, tryptophan-rich domain carries functional sites for both membrane binding and self-interaction but not for stable oligomerization

Streptolysin O belongs to the class of thiol-activated toxins, which are single chain, four-domain proteins that bind to membranes containing cholesterol and then assemble to form large oligomeric pores. Membrane binding involves a conserved tryptophan-rich sequence motif located within the C-terminally located domain 4. In contrast, sites involved in oligomerization and pore formation have been assigned to domains 1 and 3, respectively. We here examined the functional properties of domain 4, which was recombinantly expressed with an N-terminal histidine tag for purification and an additional cysteine residue for covalent labeling. The fluorescently labeled fragment readily bound to membranes, but it did not form oligomers nor lyse cell membranes. Moreover, the labeled fragment did not detectably become incorporated into hybrid oligomers when combined with lytically active full-length toxin. However, when present in large excess over the active toxin, the domain 4 fragment effected reduction of hemolytic activity and of functional pore size, which indicates interference with oligomerization of the lytically active species. Our findings support the notion that domain 4 of the streptolysin O molecule may fold autonomously, is essential for membrane binding and is capable not of irreversible but of reversible association with the entire toxin molecule.