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Staphylococcus aureus bi-component pore-forming leukocidins are secreted toxins that directly target and lyse immune cells. Intriguingly, one of the leukocidins, Leukocidin AB (LukAB), is found associated with the bacterial cell envelope in addition to secreted into the extracellular milieu. Here, we report that retention of LukAB on the bacterial cells provides S. aureus with a pre-synthesized active toxin that kills immune cells. On the bacteria, LukAB is distributed as discrete foci in two distinct compartments: membrane-proximal and surface-exposed. Through genetic screens, we show that a membrane lipid, lysyl-phosphatidylglycerol (LPG), and lipoteichoic acid (LTA) contribute to LukAB deposition and release. Furthermore, by studying non-covalently surface-bound proteins we discovered that the sorting of additional exoproteins, such as IsaB, Hel, ScaH, and Geh, are also controlled by LPG and LTA. Collectively, our study reveals a multistep secretion system that controls exoprotein storage and protein translocation across the S. aureus cell wall., (© 2021. The Author(s).)

Mast cells are potent immune sensors of the tissue microenvironment. Within seconds of activation, they release various preformed biologically active products and initiate the process of de novo synthesis of cytokines, chemokines, and other inflammatory mediators. This process is regulated at multiple levels. Besides the extensively studied IgE and IgG receptors, toll-like receptors, MRGPR, and other protein receptor signaling pathways, there is a critical activation pathway based on cholesterol-dependent, pore-forming cytolytic exotoxins produced by Gram-positive bacterial pathogens. This pathway is initiated by binding the exotoxins to the cholesterol-rich membrane, followed by their dimerization, multimerization, pre-pore formation, and pore formation. At low sublytic concentrations, the exotoxins induce mast cell activation, including degranulation, intracellular calcium concentration changes, and transcriptional activation, resulting in production of cytokines and other inflammatory mediators. Higher toxin concentrations lead to cell death. Similar activation events are observed when mast cells are exposed to sublytic concentrations of saponins or some other compounds interfering with the membrane integrity. We review the molecular mechanisms of mast cell activation by pore-forming bacterial exotoxins, and other compounds inducing cholesterol-dependent plasma membrane perturbations. We discuss the importance of these signaling pathways in innate and acquired immunity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Draberova, Tumova and Draber.)

Cholesterol-dependent cytolysins (CDCs), of which intermedilysin (ILY) is an archetypal member, are a group of pore-forming toxins secreted by a large variety of pathogenic bacteria. These toxins, secreted as soluble monomers, oligomerize upon interaction with cholesterol in the target membrane and transect it as pores of diameters of up to 100 to 300 Å. These pores disrupt cell membranes and result in cell lysis. The immune receptor CD59 is a well-established cellular factor required for intermedilysin pore formation. In this study, we applied genome-wide CRISPR-Cas9 knock-out screening to reveal additional cellular co-factors essential for ILY-mediated cell lysis. We discovered a plethora of genes previously not associated with ILY, many of which are important for membrane constitution. We show that heparan sulfates facilitate ILY activity, which can be inhibited by heparin. Furthermore, we identified hits in both protein and lipid glycosylation pathways and show a role for glucosylceramide, demonstrating that membrane organization is important for ILY activity. We also cross-validated identified genes with vaginolysin and pneumolysin and found that pneumolysin's cytolytic activity strongly depends on the asymmetric distribution of membrane phospholipids. This study shows that membrane-targeting toxins combined with genetic screening can identify genes involved in biological membrane composition and metabolism., Competing Interests: The authors have declared that no competing interests exist.

Cholesterol-dependent cytolysins (CDCs) are pore-forming proteins that serve as major virulence factors for pathogenic bacteria. They target eukaryotic cells using different mechanisms, but all require the presence of cholesterol to pierce lipid bilayers. How CDCs use cholesterol to selectively lyse cells is essential for understanding virulence strategies of several pathogenic bacteria, and for repurposing CDCs to kill new cellular targets. Here we address that question by trapping an early state of pore formation for the CDC intermedilysin, bound to the human immune receptor CD59 in a nanodisc model membrane. Our cryo electron microscopy map reveals structural transitions required for oligomerization, which include the lateral movement of a key amphipathic helix. We demonstrate that the charge of this helix is crucial for tuning lytic activity of CDCs. Furthermore, we discover modifications that overcome the requirement of cholesterol for membrane rupture, which may facilitate engineering the target-cell specificity of pore-forming proteins.

We investigated whether the modification of the negatively charged carboxyl groups with semicarbazide could confer membrane-disrupting and cytotoxic properties to bovine α-lactalbumin (LA). MALDI-TOF analysis revealed that eighteen of the twenty-one carboxyl groups in LA were coupled with semicarbazide molecules. Measurement of circular dichroism spectra and Trp fluorescence quenching studies showed that semicarbazide-modified LA (SEM-LA) had a molten globule-like conformation that retained the α-helix secondary structure but lost the tertiary structure of LA. Compared to LA, SEM-LA had a higher structural flexibility in response to trifluoroethanol- and temperature-induced structural transitions. In sharp contrast to LA, SEM-LA exhibited membrane-damaging activity and cytotoxicity. Furthermore, SEM-LA-induced membrane permeability promoted the uptake of daunorubicin and thereby its cytotoxicity. The microenvironment surrounding the Trp residues of SEM-LA was enriched in positive charges, as revealed by iodide quenching studies. The binding of SEM-LA with lipid vesicles altered the positively charged cluster around Trp residues. Although LA and SEM-LA displayed similar lipid-binding affinities, the membrane interaction modes of SEM-LA and LA differed. Collectively, these results suggest that blocking of negatively charged residues enables the formation of a molten-globule conformation of LA with structural flexibility and increased positive charge, thereby generating functional LA with membrane-disrupting activity and cytotoxicity., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Vibrio cholerae cytolysin (VCC) is a membrane-damaging protein toxin with potent cytolytic/cytotoxic activity against wide range of eukaryotic cells. VCC is a β-barrel pore-forming toxin (β-PFT), and it inflicts damage to the target cell membranes by forming transmembrane heptameric β-barrel pores. To exert pore-forming activity, VCC must bind to the cell membranes in an efficient manner. Efficient interaction with the cell membranes is an essential pre-requisite to trigger subsequent structural/conformational and organizational changes in the toxin molecules leading toward formation of the transmembrane oligomeric β-barrel pores. Based on the large numbers of studies investigating the mode of action of VCC, it is now evident that VCC is capable of using multiple distinct mechanisms to recognize and bind to the membrane components and cell surface molecules. In this review article, we present an overview of our current understanding regarding the membrane interaction mechanisms of VCC, and their functional implications for the pore-forming activity of the toxin. © 2018 IUBMB Life, 70(4):260-266, 2018., (© 2018 International Union of Biochemistry and Molecular Biology.)

Actinoporins are pore-forming toxins from sea anemones. Upon interaction with sphingomyelin-containing bilayers, they become integral oligomeric membrane structures that form a pore. Sticholysin II from Stichodactyla helianthus contains five tryptophans located at strategic positions; its role has now been studied using different mutants. Results show that W43 and W115 play a determinant role in maintaining the high thermostability of the protein, while W146 provides specific interactions for protomer-protomer assembly. W110 and W114 sustain the hydrophobic effect, which is one of the major driving forces for membrane binding in the presence of Chol. However, in its absence, additional interactions with sphingomyelin are required. These conclusions were confirmed with two sphingomyelin analogues, one of which had impaired hydrogen bonding properties. The results obtained support actinoporins' Trp residues playing a major role in membrane recognition and binding, but their residues have an only minor influence on the diffusion and oligomerization steps needed to assemble a functional pore.

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., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Pediatric septic arthritis in patients under age of four is frequently caused by the oral Gram-negative bacterium Kingella kingae. This organism may be responsible for a severe form of infective endocarditis in otherwise healthy children and adults. A major virulence factor of K. kingae is RtxA, a toxin that belongs to the RTX (Repeats-in-ToXin) group of secreted pore forming toxins. To understand the RtxA effects on host cell membranes, the toxin activity was studied using planar lipid bilayers. K. kingae strain PYKK081 and its isogenic RtxA-deficient strain, KKNB100, were tested for their ability to form pores in artificial membranes of asolectin/n-decane. RtxA, purified from PYKK081, was able to rapidly form pores with an apparent diameter of 1.9nm as measured by the partition of nonelectrolytes in the pores. The RtxA channels are cation-selective and showed strong voltage-dependent gating. In contrast to supernatants of PYKK081, those of KKNB100 did not show any pore forming activity. We concluded that RtxA toxin is the only secreted protein from K. kingae forming large channels in host cell membranes where it induces cation flux leading to programmed cell death. Furthermore, our findings suggested that the planar lipid bilayer technique can effectively be used to test possible inhibitors of RTX toxin activity and to investigate the mechanism of the toxin binding to the membrane., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Islet amyloid polypeptide (IAPP) is a peptide hormone whose pathological self-assembly is a hallmark of the progression of type II diabetes. IAPP-membrane interactions catalyze its higher-order self-assembly and also underlie its toxic effects toward cells. While there is great interest in developing small molecule reagents capable of altering the structure and behavior of oligomeric, membrane-bound IAPP, the dynamic and heterogeneous nature of this ensemble makes it recalcitrant to traditional approaches. Here, we build on recent insights into the nature of membrane-bound states and develop a combined computational and experimental strategy to address this problem. The generalized structural approach efficiently identified diverse compounds from large commercial libraries with previously unrecognized activities toward the gain-of-function behaviors of IAPP. The use of appropriate computational prescreening reduced the experimental burden by orders of magnitude relative to unbiased high-throughput screening. We found that rationally targeting experimentally derived models of membrane-bound dimers identified several compounds that demonstrate the remarkable ability to enhance IAPP-membrane binding and one compound that enhances IAPP-mediated cytotoxicity. Taken together, these findings imply that membrane binding per se is insufficient to generate cytotoxicity; instead, enhanced sampling of rare states within the membrane-bound ensemble may potentiate IAPP's toxic effects.

The cell membrane is crucial for protection of the cell from its environment. MACPF/CDC proteins are a large superfamily known to be essential for bacterial pathogenesis and proper functioning of the immune system. The three most studied groups of MACPF/CDC proteins are cholesterol-dependent cytolysins from bacteria, the membrane attack complex of complement and human perforin. Their primary function is to form transmembrane pores in target cell membranes. The common mechanism of action comprises water-soluble monomeric proteins binding to the host cell membrane, oligomerization, and formation of a functional pore. This causes a disturbance in gradients of ions and other molecules across the membrane and can lead to cell death. Cells react to this form of attack in a complex manner. Responses can be general, like removing the perforated part of the membrane, or more specific, in many cases depending on binding of proteins to specific receptors to trigger various signalling cascades.

Globoid cell leukodystrophy (GLD) is a neurological disease caused by deficiency of the lysosomal enzyme galactosylceramidase (GALC). In the absence of GALC, the cytotoxic glycosphingolipid, psychosine (psy), accumulates in the nervous system. Psychosine accumulation preferentially affects oligodendrocytes, leading to progressive demyelination and infiltration of activated monocytes/macrophages into the CNS. GLD is characterized by motor defects, cognitive deficits, seizures, and death by 2-5 years of age. It has been hypothesized that psychosine accumulation, primarily within lipid rafts, results in the pathogenic cascade in GLD. However, the mechanism of psychosine toxicity has yet to be elucidated. Therefore, we synthesized the enantiomer of psychosine (ent-psy) to use as a probe to distinguish between protein-psy (stereo-specific enantioselective) or membrane-psy (stereo-insensitive nonenantioselective) interactions. The enantiomer of psychosine has equal or greater toxicity compared with psy, suggesting that psy exerts its toxicity through a nonenantioselective mechanism. Finally, in this study we demonstrate that psy and ent-psy localize to lipid rafts, perturb natural and artificial membrane integrity, and inhibit protein Kinase C translocation to the plasma membrane. Although other mechanisms may play a role in disease, these data strongly suggest that psy exerts its effects primarily through membrane perturbation rather than through specific protein-psy interactions.

Recent work on the MACPF/CDC superfamily of pore-forming proteins has focused on the structural analysis of monomers and pore-forming oligomeric complexes. We set the family of proteins in context and highlight aspects of their function which the direct and exclusive equation of oligomers with pores fails to explain. Starting with a description of the distribution of MACPF/CDC proteins across the domains of life, we proceed to show how their evolutionary relationships can be understood on the basis of their structural homology and re-evaluate models for pore formation by perforin, in particular. We furthermore highlight data showing the role of incomplete oligomeric rings (arcs) in pore formation and how this can explain small pores generated by oligomers of proteins belonging to the family. We set this in the context of cell biological and biophysical data on the proteins' function and discuss how this helps in the development of an understanding of how they act in processes such as apicomplexan parasites gliding through cells and exiting from cells.

Targeted disruption of the plasma membrane is a ubiquitous form of attack used in all three domains of life. Many bacteria secrete pore-forming proteins during infection with broad implications for pathogenesis. The cholesterol-dependent cytolysins (CDC) are a family of pore-forming toxins expressed predominately by Gram-positive bacterial pathogens. The structure and assembly of some of these oligomeric toxins on the host membrane have been described, but how the targeted cell responds to intoxication by the CDCs is not as clearly understood. Many CDCs induce lysis of their target cell and can activate apoptotic cascades to promote cell death. However, the extent to which intoxication causes cell death is both CDC- and host cell-dependent, and at lower concentrations of toxin, survival of intoxicated host cells is well documented. Additionally, the effect of CDCs can be seen beyond the plasma membrane, and it is becoming increasingly clear that these toxins are potent regulators of signaling and immunity, beyond their role in intoxication. In this review, we discuss the cellular response to CDC intoxication with emphasis on the effects of pore formation on the host cell plasma membrane and subcellular organelles and whether subsequent cellular responses contribute to the survival of the affected cell.

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.

Vibrio vulnificus secretes a hemolysin/cytolysin (VVH) that induces cytolysis in target cells. A detergent resistant membrane domain (DRM) fraction of the cells after sucrose gradient centrifugation includes cholesterol-rich membrane microdomains which have been called "lipid rafts". It was reported that some pore-forming toxins require association with DRM and/or lipid rafts to exert their cytotoxicity. It has also been thought that cellular cholesterol is involved in VVH cytotoxicity because VVH cytotoxicity was inhibited by pre-incubation with cholesterol. However, both cellular localization and mode of action of VVH cytotoxicity remain unclear. In this study, we investigated the relationship between VVH localization on the cellular membrane and its cytotoxicity. Oligomers of VVH were detected from DRM fractions by sucrose gradient ultracentrifugation but all of these oligomers shifted from DRM fractions to non-DRM fractions after treatment with methyl-beta-cyclodextrin (MβCD), a cholesterol sequestering agent. On the other hand, immunofluorescence analysis showed that VVH did not co-localize with major lipid raft markers on cellular membrane of CHO cells. These data suggested that VVH localized at membrane regions which are relatively abundant in cholesterol but which are not identical with lipid rafts. To determine the linkage between localization and cytotoxicity of VVH, cytotoxicity was evaluated in MβCD-treated CHO cells. The cytotoxicity of VVH was not decreased by the MβCD treatment. In addition, the amount of VVH oligomer did not decrease in MβCD-treated CHO cells. Thus, we found that the amount of oligomer on cellular membrane is important for induction of cytotoxicity, whereas localization to lipid rafts on the cellular membrane was not essential to cytotoxicity.

The cholesterol-dependent cytolysins (CDCs) are a family of beta-barrel pore-forming toxins secreted by Gram-positive bacteria. These toxins are produced as water-soluble monomeric proteins that after binding to the target cell oligomerize on the membrane surface forming a ring-like pre-pore complex, and finally insert a large beta-barrel into the membrane (about 250 A in diameter). Formation of such a large transmembrane structure requires multiple and coordinated conformational changes. The presence of cholesterol in the target membrane is absolutely required for pore-formation, and therefore it was long thought that cholesterol was the cellular receptor for these toxins. However, not all the CDCs require cholesterol for binding. Intermedilysin, secreted by Streptoccocus intermedius only binds to membranes containing a protein receptor, but forms pores only if the membrane contains sufficient cholesterol. In contrast, perfringolysin O, secreted by Clostridium perfringens, only binds to membranes containing substantial amounts of cholesterol. The mechanisms by which cholesterol regulates the cytolytic activity of the CDCs are not understood at the molecular level. The C-terminus of perfringolysin O is involved in cholesterol recognition, and changes in the conformation of the loops located at the distal tip of this domain affect the toxin-membrane interactions. At the same time, the distribution of cholesterol in the membrane can modulate toxin binding. Recent studies support the concept that there is a dynamic interplay between the cholesterol-binding domain of the CDCs and the excess of cholesterol molecules in the target membrane.

Ammodytin L (AtnL) is a Ser-49 secretory phospholipase A2 (sPLA2) homologue with myotoxic activity. By analogy to the Lys-49 sPLA2 myotoxins, AtnL has been predicted to be enzymatically inactive due to the absence of the conserved Asp-49 that participates in coordination of the Ca2+ cofactor. By substituting Ser-49 and three other residues in the Ca2+-binding loop of AtnL, we obtained the first two enzymatically active mutants of Lys-49/Ser-49 sPLA2 homologues. The mutants LW and LV, which differed only by the presence of Trp and Val at position 31, respectively, efficiently hydrolyzed phospholipid vesicles, while recombinant AtnL displayed no activity. In contrast to AtnL but similarly to ammodytoxin A (AtxA), a homologous neurotoxic sPLA2, both mutants exhibited catalysis-dependent membrane-damaging ability, involving vesicle contents leakage and fusion. However, LW and LV also exhibited the potent, Ca2+-independent disruption of vesicle integrity characteristic of AtnL, but not of AtxA, in which leakage of the contents is not associated with membrane fusion. Although LV and, especially, LW have the advantage over AtnL of being able to act in both Ca2+-independent and Ca2+-dependent modes, and display higher cytotoxicity and higher lethal potency, they have a lower Ca2+-independent membrane-damaging potency and display reduced specificity in targeting muscle fibers in vitro. Our results indicate that, in evolution, Lys-49 and Ser-49 sPLA2 myotoxins have lost their Ca2+-binding ability and enzymatic activity through subtle changes in the Ca2+-binding network without affecting the rest of the catalytic machinery, thereby optimizing their Ca2+-independent membrane-damaging ability and myotoxic activity.

Pseudomonas aeruginosa ExoS (453 amino acids) is a bifunctional type III cytotoxin, comprising a Rho GTPase-activating protein domain (RhoGAP), and a 14-3-3 dependent ADP-ribosyltransferase domain. In addition, ExoS contains a membrane localization domain (termed MLD, residues 51-77) which localizes and traffics ExoS within intoxicated host cells. While membrane localization has been shown to be essential for ExoS to ADP-ribosylate Ras, the relationship between intracellular localization and expression of RhoGAP activity has not been addressed. In this study, loss of MLD function was observed to abolish expression of ExoS RhoGAP activity in HeLa cells. One mutation within the MLD (R56, R63, D70 mutated to N, RRD-->N) diminished plasma membrane localization and altered the cell rounding phenotype elicited by ExoS RhoGAP. In addition, cell rounding caused by ExoS-MLD(RRD-->N) was reversed by dominant active Rac1, but not dominant active Cdc42, indicating a switch in ExoS RhoGAP substrate specificity. Mutation of the C-terminal polybasic region abolished the ability of dominant active Rac1 to protect HeLa cells from expression of the RhoGAP activity of ExoS-MLD(RRD-->N). This study shows the importance of membrane localization in the targeting of Rho GTPases by ExoS RhoGAP.

The main pathological features in the Alzheimer's brain are progressive depositions of amyloid protein plaques among nerve cells, and neurofibrillary tangles within the nerve cells. The major components of plaques are Abeta peptides. Numerous reports have provided evidence that Abeta peptides are cytotoxic and may play a role in the pathogenesis of AD. An increasing number of research reports support the concept that the Abeta-membrane interaction event may be followed by the insertion of Abeta into the membrane in a structural configuration which forms an ion channel. This review summarizes experimental procedures which have been designed to test the hypothesis that the interaction of Abeta with a variety of membranes, both artificial and natural, results in the subsequent formation of Abeta ion channels We describe experiments, by ourselves and others, that support the view that Abeta is cytotoxic largely due to the action of Abeta channels in the cell membrane. The interaction of Abeta with the surface of the cell membrane may results in the activation of a chain of processes that, when large enough, become cytotoxic and induce cell death by apoptosis. Remarkably, the blockage of Abeta ion channels at the surface of the cell absolutely prevents the activation of these processes at different intracellular levels, thereby preserving the life of the cells. As a prospect for therapy for Alzheimer's disease, our findings at cellular level may be testable on AD animal models to elucidate the potential role and the magnitude of the contribution of the Abeta channels for induction of the disease.

Increasing evidence suggests that the misfolding and deposition of IAPP plays an important role in the pathogenesis of type II, or non-insulin-dependent diabetes mellitus (T2DM). Membranes have been implicated in IAPP-dependent toxicity in several ways: Lipid membranes have been shown to promote the misfolding and aggregation of IAPP. Thus, potentially toxic forms of IAPP can be generated when IAPP interacts with cellular membranes. In addition, membranes have been implicated as the target of IAPP toxicity. IAPP has been shown to disrupt membrane integrity and to permeabilize membranes. Since disruption of cellular membranes is highly toxic, such a mechanism has been suggested to explain the observed IAPP toxicity. Here, we review IAPP-membrane interaction in the context of (1) catalyzing IAPP misfolding and (2) being a potential origin of IAPP toxicity.

Listeriolysin O (LLO), a member of the cholesterol-dependent cytolysin (CDC) family, is a major virulence factor of Listeria monocytogenes and contributes to bacterial escape from intracellular killing of macrophages. LLO is activated under weakly acidic conditions; however, the molecular mechanism of this pH-dependent expression of cytolytic activity of LLO is poorly understood. In this study, CDCs including LLO, ivanolysin O (ILO), seeligeriolysin O (LSO), pneumolysin (PLY), streptolysin O (SLO) and perfringolysin O (PFO) were prepared as recombinant proteins and examined for their functional changes after treatment under various pH conditions. Haemolytic and membrane cholesterol-binding activities were not affected in PLY, SLO and PFO at any pH examined. By contrast, all the Listeria-derived cytolysins, LLO, ILO and LSO, were active only at an acidic pH and rapidly inactivated under neutral or alkaline conditions. Once inactivated, LLO could not be reactivated even by a downward pH shift. The hydrophobicity of LLO treated at neutral or alkaline pH was increased. These data suggested that the pH-dependent loss of cytolytic activity appeared to be due to irreversible structural changes of domain 4 that resulted in the loss of target membrane cholesterol binding.

Bacillus anthracis has recently been shown to secrete a potently hemolytic/cytolytic protein that has been designated anthrolysin O (ALO). In this work, we initiated a study of this potential anthrax virulence factor in an effort to understand the membrane-binding properties of this protein. Recombinant anthrolysin O (rALO35-512) and two N-terminally truncated versions of ALO (rALO390-512 and rALO403-512) from B. anthracis were overproduced in Escherichia coli and purified to homogeneity. The role of cholesterol in the cytolytic activity of ALO was probed in cellular cholesterol depletion assays using mouse and human macrophage-like lines, and also Drosophila Schneider 2 cells. Challenging the macrophage cells with rALO35-512, but not rALO390-512 or rALO403-512, resulted in cell death by lysis, with this cytolysis being abolished by depletion of the membrane cholesterol. Drosophila cells, which contain ergosterol as their major membrane sterol, were resistant to rALO-mediated cytolysis. In order to determine the molecular mechanism of this resistance, the interaction of rALO with model membranes comprised of POPC alone, or with a variety of structurally similar sterols including ergosterol, was probed using Biacore. Both rALO35-512 and rALO403-512 demonstrated robust binding to model membranes composed of POPC and cholesterol, with amount of protein bound proportional to the cholesterol content. Ergosterol supported greatly reduced binding of both rALO35-512 and rALO403-512, whereas other sterols tested did not support binding. The rALO403-512--membrane interaction demonstrated an equilibrium dissociation constant (KD) in the low nanomolar range, whereas rALO35-512 exhibited complex kinetics likely due to the multiple events involved in pore formation. These results establish the pivotal role of cholesterol in the action of rALO. The biosensor method developed to measure ALO recognition of cholesterol in a membrane environment could be extended to provide a platform for the screening of inhibitors of other membrane-binding proteins and peptides., (Copyright 2006 John Wiley & Sons, Ltd.)

FRET measurements were used to determine the domain-specific topography of perfringolysin O, a pore-forming toxin, on a membrane surface at different stages of pore formation. The data reveal that the elongated toxin monomer binds stably to the membrane in an "end-on" orientation, with its long axis approximately perpendicular to the plane of the membrane bilayer. This orientation is largely retained even after monomer association to form an oligomeric prepore complex. The domain 3 (D3) polypeptide segments that ultimately form transmembrane beta-hairpins remain far above the membrane surface in both the membrane-bound monomer and prepore oligomer. Upon pore formation, these segments enter the bilayer, whereas D1 moves to a position that is substantially closer to the membrane. Therefore, the extended D2 beta-structure that connects D1 to membrane-bound D4 appears to bend or otherwise reconfigure during the prepore-to-pore transition of the perfringolysin O oligomer.

In a show piece for electron microscopy (EM), Tilley at al. used single-particle cryo-EM to visualize the structural rearrangements in the bacterial toxin pneumolysin that occur when it assembles into a membrane-associated prepore and when the prepore subsequently transitions into a fully membrane-inserted pore.

Sea anemones produce a family of 18-20 kDa proteins, the actinoporins, which lyse cells by forming pores in cell membranes. Sphingomyelin plays an important role in their lytic activity, with membranes lacking this lipid being largely refractory to these toxins. As a means of characterising membrane binding by the actinoporin equinatoxin II (EqTII), we have used 19F NMR to probe the environment of Trp residues in the presence of micelles and bicelles. Trp was chosen as previous data from mutational studies and truncated analogues had identified the N-terminal helix of EqTII and the surface aromatic cluster including tryptophan residues 112 and 116 as being important for membrane interactions. The five tryptophan residues were replaced with 5-fluorotryptophan and assigned by site-directed mutagenesis. The 19F resonance of W112 was most affected in the presence of phospholipid micelles or bicelles, followed by W116, with further change induced by the addition of sphingomyelin. Although binding to phosphatidylcholine is not sufficient to enable pore formation in bilayer membranes, this interaction had a greater effect on the tryptophan residues in our studies than the subsequent interaction with sphingomyelin. Furthermore, sphingomyelin had a direct effect on EqTII in both model membranes, so its role in EqTII pore formation involves more than simply an indirect effect mediated via bulk lipid properties. The lack of change in chemical shift for W149 even in the presence of sphingomyelin indicates that, at least in the model membranes studied here, interaction with sphingomyelin was not sufficient to trigger dissociation of the N-terminal helix from the beta-sandwich, which forms the bulk of the protein.

Perfringolysin O (PFO) is a prototype of the large family of pore-forming cholesterol-dependent cytolysins (CDCs). A central enigma of the cytolytic mechanism of the CDCs is that their membrane-spanning beta-hairpins (the transmembrane amphipathic beta-hairpins (TMHs)) appear to be approximately 40 A too far above the membrane surface to cross the bilayer and form the pore. We now present evidence, using atomic force microscopy (AFM), of a significant difference in the height by which the prepore and pore protrude from the membrane surface: 113+/-5 A for the prepore but only 73+/-5 A for the pore. Time-lapse AFM micrographs show this change in height in real time. Moreover, the monomers in both complexes exhibit nearly identical surface features and these results in combination with those of spectrofluorimetric analyses indicate that the monomers remain in a perpendicular orientation to the bilayer plane during this transition. Therefore, the PFO undergoes a vertical collapse that brings its TMHs to the membrane surface so that they can extend across the bilayer to form the beta-barrel pore.

Pore-formation in cell membranes is used by many toxins to kill cells. It is usually a process involving multiple steps that are difficult to analyse at the molecular level. The use of surface plasmon resonance (SPR) has only recently been introduced into the study of pore-forming toxins (PFT). It can give useful data mostly on the first steps of the pore-forming process; the binding to the lipid membranes. In particular, it can make unique contributions to our knowledge of ligand specificity and the kinetics of binding. This mini-review summarizes some recent SPR studies of PFT.

Cytosolic internalization is a requirement for the toxicity of secretory ribonucleases. Here, we investigate the mechanism of internalization of Onconase (ONC), a toxic protein, and ribonuclease A (RNase A), a nontoxic homolog. Microscopy studies indicate that both ribonucleases readily bind to the cell surface and are internalized via acidic vesicles. Blocking dynamin-dependent endocytosis prevents transferrin internalization but does not hinder RNase A internalization. ONC and G88R RNase A, which is a toxic variant, demonstrate enhanced cytotoxicity in the absence of clathrin- and dynamin-mediated endocytosis. The cytosolic entry of ribonucleases does not require an acidic environment or transport to the ER and probably occurs from endosomes. Thus, common proteins - secretory ribonucleases - enter the cytosol by a pathway that is distinct from that of other known toxins.

The repertoire of the cytolytic pore-forming protein toxins (PFT) comprises 81 identified members. The essential feature of these cytolysins is their capacity to provoke the formation of hydrophilic pores in the cytoplasmic membranes of target eukaryotic cells. This process results from the binding of the proteins on the cell surface, followed by their oligomerization which leads to the insertion of the oligomers into the membrane and formation of protein-lined channels. It impairs the osmotic balance of the cell and causes cytolysis. In this review the molecular aspects of a number of important PFT and their respective encoding structural genes will be briefly described.

Listeriolysin O (LLO) is a major virulence factor secreted by the pathogenic Listeria monocytogenes and acts as pore-forming cytolysin. Based on sequence similarities between LLO and perfringolysin (PFO), the cytolysin from Clostridium perfringens of known crystallographic structure, two truncated LLO proteins were produced: LLO-d123, comprising the first three predicted domains, and LLO-d4, the last C-terminal domain. The two proteins were efficiently secreted into the culture supernatant of L. monocytogenes and were able to bind to cell membranes. Strikingly, when expressed simultaneously, the two secreted domains LLO-d123 and LLO-d4 reassembled into a haemolytically active form. Two in-frame linker insertions were generated in the hinge region between the d123 and d4 domains. In both cases, the insertion created a major cleavage site for proteolytic degradation and abolished cytolytic activity, which might suggest that the region connecting d123 and d4 participates in the interaction between the two portions of the monomer.

In view of the recent studies on the CDCs, a reasonable schematic of the stages leading to membrane insertion of the CDCs can be assembled. As shown in Fig. 3, we propose that the CDC first binds to the membrane as a monomer. These monomers then diffuse laterally on the membrane surface to encounter other monomers or incomplete oligomeric complexes. Presumably, once the requisite oligomer size is reached, the prepore complex is converted into the pore complex and a large membrane channel is formed. During the conversion of the prepore complex to the pore complex, we predict that the TMHs of the subunits in the prepore complex insert into the bilayer in a concerted fashion to form the large transmembrane beta-barrel, although this still remains to be confirmed experimentally. Many intriguing problems concerning the cytolytic mechanism of the CDCs remain unsolved. The nature of the initial interaction of the CDC monomer with the membrane is currently one of the most controversial questions concerning the CDC mechanism. Is cholesterol involved in this interaction, as previously assumed, or do specific receptors exist for these toxins that remain to be discovered? Also, the trigger for membrane insertion and the regions of these toxins that facilitate the [figure: see text] interaction of the monomers during prepore complex formation are unknown. In addition, the temporal sequence of the multiple structural changes that accompany the conversion of the soluble CDC monomer into a membrane-inserted oligomer have yet to be defined or characterized kinetically.

Thiol-activated cytolysins share a conserved hydrophobic, Trp-rich undecapeptide that is suggested to be involved in membrane binding and intercalation. The neutralizing monoclonal antibody PLY-5 recognizes all members of this toxin family and peptide mapping assigned its epitope to the undecapeptide motif. This antibody inhibited binding of the toxins to host cell membranes and the epitope was no longer available for binding when a preformed toxin/membrane complex was tested. These results confirm the model of cytolysin binding suggested by structural data.

RTX toxins are important virulence factors produced by a wide range of Gram-negative bacteria. They fall into two categories: the hemolysins, which affect a variety of cell types, and the leukotoxins, which are cell-type- and species-specific. These toxins offer interesting models for targeting, insertion and translocation of aqueous proteins into lipid membranes.

The mechanisms by which proteins gain entry into membranes is a fundamental problem in biology. Here, we present the first crystal structure of a thiol-activated cytolysin, perfringolysin O, a member of a large family of toxins that kill eukaryotic cells by punching holes in their membranes. The molecule adopts an unusually elongated shape rich in beta sheet. We have used electron microscopy data to construct a detailed model of the membrane channel form of the toxin. The structures reveal a novel mechanism for membrane insertion. Surprisingly, the toxin receptor, cholesterol, appears to play multiple roles: targeting, promotion of oligomerization, triggering a membrane insertion competent form, and stabilizing the membrane pore.