Lysenin: a sphingomyelin specific pore-forming toxin

Sphingomyelin is a major sphingolipid in mammalian cells. Recent results indicate that sphingomyelin is a reservoir of lipid second messengers, ceramide and sphingosine-1-phosphate. Sphingomyelin is also a major component of sphingolipid and cholesterol-rich membrane domains (lipid rafts). Lysenin is a pore-forming toxin that specifically binds sphingomyelin. The binding of lysenin to sphingomyelin is dependent on the membrane distribution of the lipid, i.e. the toxin selectively binds sphingomyelin clusters. Development of a non-toxic lysenin mutant revealed the spatial and functional heterogeneity of sphingolipid-rich membrane domains.     

Epsilon toxin: a fascinating pore-forming toxin

Epsilon toxin (ETX) is produced by strains of Clostridium perfringens classified as type B or type D. ETX belongs to the heptameric β-pore-forming toxins including aerolysin and Clostridium septicum alpha toxin, which are characterized by the formation of a pore through the plasma membrane of eukaryotic cells consisting in a β-barrel of 14 amphipatic β strands. By contrast to aerolysin and C. septicum alpha toxin, ETX is a much more potent toxin and is responsible for enterotoxemia in animals, mainly sheep. ETX induces perivascular edema in various tissues and accumulates in particular in the kidneys and brain, where it causes edema and necrotic lesions. ETX is able to pass through the blood-brain barrier and stimulate the release of glutamate, which accounts for the symptoms of nervous excitation observed in animal enterotoxemia. At the cellular level, ETX causes rapid swelling followed by cell death involving necrosis. The precise mode of action of ETX remains to be determined. ETX is a powerful toxin, however, it also represents a unique tool with which to vehicle drugs into the central nervous system or target glutamatergic neurons.     

Effects of the eukaryotic pore-forming cytolysin Equinatoxin II on lipid membranes and the role of sphingomyelin

Equinatoxin II (EqtII), a protein toxin from the sea anemone Actinia equina, readily creates pores in sphingomyelin-containing lipid membranes. The perturbation by EqtII of model lipid membranes composed of dimyristoylphosphatidycholine and sphingomyelin (10 mol %) was investigated using wideline phosphorus-31 and deuterium NMR. The preferential interaction between EqtII (0.1 and 0.4 mol %) and the individual bilayer lipids was studied by (31)P magic angle spinning NMR, and toxin-induced changes in bilayer morphology were examined by freeze-fracture electron microscopy. Both NMR and EM showed the formation of an additional lipid phase in sphingomyelin-containing mixed lipid multilamellar suspensions with 0.4 mol % EqtII. The new toxin-induced phase consisted of small unilamellar vesicles 20-40 nm in diameter. Deuterium NMR showed that the new lipid phase contains both dimyristoylphosphatidycholine and sphingomyelin. Solid-state (31)P NMR showed an increase in spin-lattice and a decrease in spin-spin relaxation times in mixed-lipid model membranes in the presence of EqtII, consistent with an increase in the intensity of low frequency motions. The (2)H and (31)P spectral intensity distributions confirmed a change in lipid mobility and showed the creation of an isotropic lipid phase, which was identified as the small vesicle structures visible by electron microscopy in the EqtII-lipid suspensions. The toxin appears to enhance slow motions in the membrane lipids and destabilize the membrane. This effect was greatly enhanced in sphingomyelin-containing mixed lipid membranes compared with pure phosphatidylcholine bilayers, suggesting a preferential interaction between the toxin and bilayer sphingomyelin.     

Molecular determinants of protein half-lives in eukaryotic cells

Multiple pathways of intracellular protein breakdown operate within cells, and the activities of different pathways can be regulated under different physiological conditions. Recent studies suggest that molecular determinants within proteins target them for different pathways of proteolysis. Proteins that are partially unfolded and have an unblocked amino-terminal amino acid with a bulky side chain appear to be good substrates for cytosolic, ubiquitin-mediated pathways of proteolysis. Certain modifications of internal residues such as oxidation of methionines also increase the susceptibility of certain proteins to ubiquitin-mediated proteolysis. Rapidly degraded normal proteins contain peptide regions rich in proline, glutamate, serine, and threonine (PEST regions). The pathway of degradation for these proteins has not been established, but they may be good substrates for calcium-activated proteases. In addition, a lysosomal pathway of protein degradation is activated when serum is withdrawn from cultured cells and is selective for cytosolic proteins containing peptide regions similar to Lys-Phe-Glu-Arg-Gln (KFERQ). This short review summarizes our current understanding of mechanisms of protein breakdown in eukaryotes and evaluates potential molecular determinants of protein half-lives.     

Pore formation by equinatoxin, a eukaryotic pore-forming toxin, requires a flexible N-terminal region and a stable beta-sandwich

Actinoporins are eukaryotic pore-forming proteins that create 2-nm pores in natural and model lipid membranes by the self-association of four monomers. The regions that undergo conformational change and form part of the transmembrane pore are currently being defined. It was shown recently that the N-terminal region (residues 10-28) of equinatoxin, an actinoporin from Actinia equina, participates in building of the final pore wall. Assuming that the pore is formed solely by a polypeptide chain, other parts of the toxin should constitute the conductive channel and here we searched for these regions by disulfide scanning mutagenesis. Only double cysteine mutants where the N-terminal segment 1-30 was attached to the beta-sandwich exhibited reduced hemolytic activity upon disulfide formation, showing that other parts of equinatoxin, particularly the beta-sandwich and importantly the C-terminal alpha-helix, do not undergo large conformational rearrangements during the pore formation. The role of the beta-sandwich stability was independently assessed via destabilization of a part of its hydrophobic core by mutations of the buried Trp117. These mutants were considerably less stable than the wild-type but exhibited similar or slightly lower permeabilizing activity. Collectively these results show that a flexible N-terminal region and stable beta-sandwich are pre-requisite for proper pore formation by the actinoporin family.     

Cytotoxic activity of a tumor protease-activated pore-forming toxin

Equinatoxin II is a pore forming toxin produced by the sea anemone Actinia equina. It is able to kill very unspecifically most cell types by the membrane-perturbing action of an amphiphilic alpha-helix located at its N-terminal. A normally active N-terminal mutant, containing one single cys in the amphiphilic alpha-helix, becomes totally inactive when it is bound to avidin via a biotinylated linker. By choosing, as a linker, a peptide containing a tumor protease cleavage site, we were able to construct an enzymatically activable conjugate which should be selective for tumor cells. The introduced cleavage site was designed in order to be digested by both cathepsin B and matrix metalloproteases (MMPs). We confirmed that this conjugate could be activated in vitro by cathepsin B and MMPs. After having measured the enzymatic activity of fibrosarcoma and breast carcinoma cells, we analyzed the cytotoxic effect of the conjugate on the same lines and on human red blood cells (HRBC) as controls. We found that the conjugate was activated, at least in part, by the tumor cell lines used, whereas it was inactive on HRBC. That the activation process was dependent on the enzymatic action of cathepsin B and MMPs, was indicated by three lines of evidence: (1) binding occurred normally on all type of cells including HRBC which however were insensitive being devoid of enzymes; (2) the cytotoxic effect correlated with the amount of cathepsin B activity expressed by the cells; (3) conjugate activation was reduced by specific inhibitors of cathepsin B and MMPs. These results demonstrate the possibility of tumor cell killing by a pore-forming toxin conjugate specifically activated by tumor proteases.     

Properties of nonfused liposomes immobilized on an L1 Biacore chip and their permeabilization by a eukaryotic pore-forming toxin

The L1 chip is used intensively for protein-membrane interaction studies in Biacore surface plasmon resonance systems. The exact form of captured lipid membranes on the chip is, however, not precisely known. Evidence exists that the vesicles both remain intact after the binding to the chip and fuse to form a large single-bilayer membrane. In this study, we were able to bind up to approximately 11,500 resonance units of zwitterionic liposomes (100 nm in diameter) at a low flow rate. We show by fluorescence microscopy that the entire surface of the flow cell is covered homogeneously by liposomes. Negatively charged vesicles (i.e., those composed of phosphatidylcholine/phosphatidylglycerol [1:1]) always deposited less densely, but we were able to increase the density slightly with the use of calcium chloride that promotes fusion of the vesicles. Finally, we used zwitterionic liposomes loaded with fluorescent probe calcein to show that they remain intact after the capture on the L1 chip. The fluorescence was lost only after we used equinatoxin, a well-studied pore-forming toxin, to perform on-chip permeabilization of vesicles. The characteristics of permeabilization process for chip-immobilized liposomes are similar to those of liposomes free in solution. All results collectively suggest that liposomes do not fuse to form a single bilayer on the surface of the chip.     

Cysteine-scanning mutagenesis of an eukaryotic pore-forming toxin from sea anemone: topology in lipid membranes.

Equinatoxin II is a cysteineless pore-forming protein from the sea anemone Actinia equina. It readily creates pores in membranes containing sphingomyelin. Its topology when bound in lipid membranes has been studied using cysteine-scanning mutagenesis. At approximately every tenth residue, a cysteine was introduced. Nineteen single cysteine mutants were produced in Escherichia coli and purified. The accessibility of the thiol groups in lipid-embedded cysteine mutants was studied by reaction with biotin maleimide. Most of the mutants were modified, except those with cysteines at positions 105 and 114. Mutants R144C and S160C were modified only at high concentrations of the probe. Similar results were obtained if membrane-bound biotinylated mutants were tested for avidin binding, but in this case three more mutants gave a negative result: S1C, S13C and K43C. Furthermore, mutants S1C, S13C, K20C, K43C and S95C reacted with biotin only after insertion into the lipid, suggesting that they were involved in major conformational changes occurring upon membrane binding. These results were further confirmed by labeling the mutants with acrylodan, a polarity-sensitive fluorescent probe. When labeled mutants were combined with vesicles, the following mutants exhibited blue-shifts, indicating the transfer of acrylodan into a hydrophobic environment: S13C, K20C, S105C, S114C, R120C, R144C and S160C. The overall results suggest that at least two regions are embedded within the lipid membrane: the N-terminal 13-20 region, probably forming an amphiphilic helix, and the tryptophan-rich 105-120 region. Arg144, Ser160 and residues nearby could be involved in making contacts with lipid headgroups. The association with the membrane appears to be unique and different from that of bacterial pore-forming proteins and therefore equinatoxin II may serve as a model for eukaryotic channel-forming toxins.     

Adventures of a pore-forming toxin at the target cell surface

The past three years have shed light on how the pore-forming toxin aerolysin binds to its target cell and then hijacks cellular devices to promote its own polymerization and pore formation. This selective permeabilization of the plasma membrane has unexpected intracellular consequences that might explain the importance of aerolysin in Aeromonas pathogenicity.     

Characterization of Streptococcus agalactiae CAMP factor as a pore-forming toxin

A recombinant form of CAMP factor of Streptococcus agalactiae has been expressed as glutathione S-transferase-CAMP fusion protein in Escherichia coli. After thrombin cleavage of the fusion protein, the recombinant CAMP factor exhibited hemolytic activity comparable with that of the native form. Osmotic protection experiments with polyethylene glycols show that CAMP factor forms discrete transmembrane pores with a diameter upward of 1.6 nm on susceptible membranes; electron microscopy reveals circular membrane lesions of heterogeneous size, up to 12-15 nm in diameter. Liposome permeabilization studies show that pore formation is a highly cooperative process, which suggests that it involves the oligomerization of CAMP factor. Chemical cross-linking experiments also support an oligomeric mode of action.     

Molecular determinants of substrate specificity in plant 5'-methylthioadenosine nucleosidases

5′-Methylthioadenosine (MTA)/S-adenosylhomocysteine (SAH) nucleosidase (MTAN) is essential for cellular metabolism and development in many bacterial species. While the enzyme is found in plants, plant MTANs appear to select for MTA preferentially, with little or no affinity for SAH. To understand what determines substrate specificity in this enzyme, MTAN homologues from Arabidopsis thaliana (AtMTAN1 and AtMTAN2, which are referred to as AtMTN1 and AtMTN2 in the plant literature) have been characterized kinetically. While both homologues hydrolyze MTA with comparable kinetic parameters, only AtMTAN2 shows activity towards SAH. AtMTAN2 also has higher catalytic activity towards other substrate analogues with longer 5′-substituents. The structures of apo AtMTAN1 and its complexes with the substrate- and transition-state-analogues, 5′-methylthiotubercidin and formycin A, respectively, have been determined at 2.0-1.8 Å resolution. A homology model of AtMTAN2 was generated using the AtMTAN1 structures. Comparison of the AtMTAN1 and AtMTAN2 structures reveals that only three residues in the active site differ between the two enzymes. Our analysis suggests that two of these residues, Leu181/Met168 and Phe148/Leu135 in AtMTAN1/AtMTAN2, likely account for the divergence in specificity of the enzymes. Comparison of the AtMTAN1 and available Escherichia coli MTAN (EcMTAN) structures suggests that a combination of differences in the 5′-alkylthio binding region and reduced conformational flexibility in the AtMTAN1 active site likely contribute to its reduced efficiency in binding substrate analogues with longer 5′-substituents. In addition, in contrast to EcMTAN, the active site of AtMTAN1 remains solvated in its ligand-bound forms. As the apparent pK_a of an amino acid depends on its local environment, the putative catalytic acid Asp225 in AtMTAN1 may not be protonated at physiological pH and this suggests the transition state of AtMTAN1, like human MTA phosphorylase and Streptococcus pneumoniae MTAN, may be different from that found in EcMTAN.     

K1 killer toxin, a pore-forming protein from yeast

K1 killer toxin is a secreted, pore-forming protein that kills sensitive yeast cells. The heterodimeric toxin is processed from a precursor in the Golgi, and has allowed identification of the KEX2- and KEX1-encoded proteases. The toxin binds to a glucan receptor on the cell wall of target yeast, and mutational analysis implicates both the alpha- and beta-toxin subunits in receptor binding. Toxin-resistant mutants with altered cell-wall glucans have helped to outline a pathway of assembly of these polysaccharides. Patch-clamp technology has demonstrated the nature of the lethal channel in toxin-treated plasma membranes. The hydrophobic alpha-subunit-encoding region is the site of all mutations affecting channel formation. Immunity to the toxin is conferred by the toxin precursor, and immunity mutations map to the region encoding the alpha subunit. The precursor probably competes with the toxin to prevent channel formation in toxin-producing cells, but the basis of this remains unknown. This toxin/immunity system has a domain structure that differs from that of other characterized toxins and has no known homologues.     

Mimicry of a host anion channel by a Helicobacter pylori pore-forming toxin

Bacterial pore-forming toxins have traditionally been thought to function either by causing an essentially unrestricted flux of ions and molecules across a membrane or by effecting the transmembrane transport of an enzymatically active bacterial peptide. However, the Helicobacter pylori pore-forming toxin, VacA, does not appear to function by either of these mechanisms, even though at least some of its effects in cells are dependent on its pore-forming ability. Here we show that the VacA channel exhibits two of the most characteristic electrophysiological properties of a specific family of cellular channels, the ClC channels: an open probability dependent on the molar ratio of permeable ions and single channel events resolvable as two independent, voltage-dependent transitions. The sharing of such peculiar properties by VacA and host ClC channels, together with their similar magnitudes of conductance, ion selectivities, and localization within eukaryotic cells, suggests a novel mechanism of toxin action in which the VacA pore largely mimics the electrophysiological behavior of a host channel, differing only in the membrane potential at which it closes. As a result, VacA can perturb, but not necessarily abolish, the homeostatic ionic imbalance across a membrane and so change cellular physiology without necessarily jeopardizing vitality.     

Elimination of a bacterial pore-forming toxin by sequential endocytosis and exocytosis

Staphylococcus aureus α-toxin is the archetype of bacterial pore forming toxins and a key virulence factor secreted by the majority of clinical isolates of S. aureus. Toxin monomers bind to target cells and oligomerize to form small β-barrel pores in the plasma membrane. Many nucleated cells are able to repair a limited number of lesions by unknown, calcium-independent mechanisms. Here we show that cells can internalize α-toxin, that uptake is essential for cellular survival, and that pore-complexes are not proteolytically degraded, but returned to the extracellular milieu in the context of exosome-like structures, which we term toxosomes.     

Molecular determinants of substrate specificity for Semliki Forest virus nonstructural protease

The C-terminal cysteine protease domain of Semliki Forest virus nonstructural protein 2 (nsP2) regulates the virus life cycle by sequentially cleaving at three specific sites within the virus-encoded replicase polyprotein P1234. The site between nsP3 and nsP4 (the 3/4 site) is cleaved most efficiently. Analysis of Semliki Forest virus-specific cleavage sites with shuffled N-terminal and C-terminal half-sites showed that the main determinants of cleavage efficiency are located in the region preceding the cleavage site. Random mutagenesis analysis revealed that amino acid residues in positions P4, P3, P2, and P1 of the 3/4 cleavage site cannot tolerate much variation, whereas in the P5 position most residues were permitted. When mutations affecting cleavage efficiency were introduced into the 2/3 and 3/4 cleavage sites, the resulting viruses remained viable but had similar defects in P1234 processing as observed in the in vitro assay. Complete blockage of the 3/4 cleavage was found to be lethal. The amino acid in position P1' had a significant effect on cleavage efficiency, and in this regard the protease markedly preferred a glycine residue over the tyrosine natively present in the 3/4 site. Therefore, the cleavage sites represent a compromise between protease recognition and other requirements of the virus life cycle. The protease recognizes at least residues P4 to P1', and the P4 arginine residue plays an important role in the fast cleavage of the 3/4 site.     

Molecular determinants of the cofactor specificity of ribitol dehydrogenase, a short-chain dehydrogenase/reductase

Ribitol dehydrogenase from Zymomonas mobilis (ZmRDH) catalyzes the conversion of ribitol to d-ribulose and concomitantly reduces NAD(P)(+) to NAD(P)H. A systematic approach involving an initial sequence alignment-based residue screening, followed by a homology model-based screening and site-directed mutagenesis of the screened residues, was used to study the molecular determinants of the cofactor specificity of ZmRDH. A homologous conserved amino acid, Ser156, in the substrate-binding pocket of the wild-type ZmRDH was identified as an important residue affecting the cofactor specificity of ZmRDH. Further insights into the function of the Ser156 residue were obtained by substituting it with other hydrophobic nonpolar or polar amino acids. Substituting Ser156 with the negatively charged amino acids (Asp and Glu) altered the cofactor specificity of ZmRDH toward NAD(+) (S156D, [k(cat)/K(m)(,NAD)]/[k(cat)/K(m)(,NADP)] = 10.9, where K(m)(,NAD) is the K(m) for NAD(+) and K(m)(,NADP) is the K(m) for NADP(+)). In contrast, the mutants containing positively charged amino acids (His, Lys, or Arg) at position 156 showed a higher efficiency with NADP(+) as the cofactor (S156H, [k(cat)/K(m)(,NAD)]/[k(cat)/K(m)(,NADP)] = 0.11). These data, in addition to those of molecular dynamics and isothermal titration calorimetry studies, suggest that the cofactor specificity of ZmRDH can be modulated by manipulating the amino acid residue at position 156.     

Membrane interaction of Pasteurella multocida toxin involves sphingomyelin

Pasteurella multocida toxin (PMT) is an AB toxin that causes pleiotropic effects in targeted host cells. The N-terminus of PMT (PMT-N) is considered to harbor the membrane receptor binding and translocation domains responsible for mediating cellular entry and delivery of the C-terminal catalytic domain into the host cytosol. Previous studies have implicated gangliosides as the host receptors for PMT binding. To gain further insight into the binding interactions involved in PMT binding to cell membranes, we explored the role of various membrane components in PMT binding, utilizing four different approaches: (a) TLC-overlay binding experiments with (125) I-labeled PMT, PMT-N or the C-terminus of PMT; (b) pull-down experiments using reconstituted membrane liposomes with full-length PMT; (c) surface plasmon resonance analysis of PMT-N binding to reconstituted membrane liposomes; (d) and surface plasmon resonance analysis of PMT-N binding to HEK-293T cell membranes without or with sphingomyelinase, phospholipase D or trypsin treatment. The results obtained revealed that, in our experimental system, full-length PMT and PMT-N did not bind to gangliosides, including monoasialogangliosides GM(1) , GM(2) or GM(3) , but instead bound to membrane phospholipids, primarily the abundant sphingophospholipid sphingomyelin or phosphatidylcholine with other lipid components. Collectively, these studies demonstrate the importance of sphingomyelin for PMT binding to membranes and suggest the involvement of a protein co-receptor.     

The cytotoxic fimbrial structural subunit of Xenorhabdus nematophila is a pore-forming toxin

We have purified a fimbrial shaft protein (MrxA) of Xenorhabdus nematophila. The soluble monomeric protein lysed larval hemocytes of Helicoverpa armigera. Osmotic protection of the cells with polyethylene glycol suggested that the 17-kDa MrxA subunit makes pores in the target cell membrane. The internal diameter of the pores was estimated to be >2.9 nm. Electron microscopy confirmed the formation of pores by the fimbrial subunit. MrxA protein oligomerized in the presence of liposomes. Electrophysiological studies demonstrated that MrxA formed large, voltage-gated passive-diffusion channels in lipid bilayers.     

The thermostable direct hemolysin of Vibrio parahaemolyticus is a pore-forming toxin.

The hemolytic mechanism of thermostable direct hemolysin (TDH), a possible virulence factor of Vibrio parahaemolyticus, was studied. We demonstrated that TDH acts as a "pore-forming toxin" in temperature-dependent and -independent steps. The first temperature-dependent step requires only about 1-2 min incubation at 37 degrees C and makes a "pore" with a functional diameter of approximately 2 nm. The pore size was deduced from the molecular diameter of the colloidal inhibitory polysaccharides. The formation of the pores on TDH-treated erythrocyte membranes was also demonstrated by electron microscopic examination. The second step, which is a temperature-independent lytic step, causes the erythrocytes to swell owing to a colloidal osmotic influx of water via the "pores" into cells, resulting in erythrocyte lysis (or rupture) owing to increased intracellular pressure.