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Bacillus cereus hemolysin II, a pore-forming β-barrel toxin (HlyII), has a C-terminal extension of 94 amino acid residues, designated as the C-terminal domain of HlyII (HlyIICTD). HlyIICTD is capable of forming oligomers in aqueous solutions. Oligomerization of HlyIICTD significantly increased in the presence of erythrocytes and liposomes. Its affinity for erythrocytes of various origins differed insignificantly but was noticeably higher for T-cells. HlyIICTD destroyed THP-1 monocytes and J774 macrophages, acted most effectively on Jurkat T-lymphocytes and had virtually no impact on B-cell lines. HlyIICTD was able to form ion-conducting channels on an artificial bilayer membrane. • The features of HlyIICTD (C-terminal domain of HlyII B. cereus) were studied. • The parameters of the binding of HlyIICTD to various cells were determined. • HlyIICTD oligomerized in the presence of erythrocytes and liposomes. • HlyIICTD destroyed monocytes, macrophages and T-lymphocytes. • HlyIICTD formed ion conducting channels on an artificial bilayer membrane. [ABSTRACT FROM AUTHOR]

Bacterial vaginosis (BV) is a common type of vaginal inflammation caused by a proliferation of pathogenic bacteria, among which Mobiluncus curtisii. In our previous studies on M. curtisii genome, we identified the presence of a genomic fragment encoding a 25 kDa pore-forming toxin, the CAMP factor, which is known to be involved in the synergistic lysis of erythrocytes namely CAMP reaction. However, whether this hypothetical gene product has hemolytic activity is unknown. Moreover, its relative structure and function are not yet solved. Here we found that the M. curtisii CAMP factor is a monomer at pH 4.4 and oligomer at pH > 4.6. Hemolysis assays showed that M. curtisii CAMP factor could lyse sheep red blood cells efficiently in pH 5.4–7.4. Negative staining electron microscope analysis of the CAMP factor revealed ring-like structures at pH above 4.6. Additionally, the crystal structure of M. curtisii CAMP factor, determineded at 1.85 Å resolution, reveals a 5 + 3 helix motif. Further functional analysis suggested that the structural rearrangement of the N-terminal domain might be required for protein function. In conclusion, this structure-function relationship study of CAMP factor provides a new perspective of the M. curtisii role in BV development. [ABSTRACT FROM AUTHOR]

Bacillus thuringiensis crystal (Cry) proteins, used for decades as insecticidal toxins, are well known to be toxic to certain insects, but not to mammals. A novel group of Cry toxins called parasporins possess a strong cytocidal activity against some human cancer cells. Cry41Aa, or parasporin3, closely resembles commercially used insecticidal toxins and yet is toxic to the human hepatic cancer cell line HepG2, disrupting membranes of susceptible cells, similar to its insecticidal counterparts. In this study, we explore the protective effect that the common divalent metal chelator EGTA exerts on Cry41Aa's activity on HepG2 cells. Our results indicate that rather than interfering with a signalling pathway as a result of chelating cations in the medium, the chelator prevented the toxin's interaction with the membrane, and thus the subsequent steps of membrane damage and p38 phosphorylation, by removing cations bound to plasma membrane components. BAPTA and DTPA also inhibited Cry41Aa toxicity but at higher concentrations. We also show for the first time that Cry41Aa induces pore formation in planar lipid bilayers. This activity is not altered by EGTA, consistent with a biological context of chelation. Salt supplementation assays identified Ca2+, Mn2+ and Zn2+ as being able to reinstate Cry41Aa activity. Our data suggest the existence of one or more metal cation-dependent receptors in the Cry41Aa mechanism of action. • EGTA, EDTA, BAPTA and DTPA chelators protect HepG2 cells from Cry41Aa cytotoxicity. • EGTA removes membrane bound cations preventing toxin binding and membrane damage. • Cry41Aa induces pore formation in planar lipid bilayers, also in EGTA's presence. • Ca2+, Mn2+ and Zn2+ reinstated cytotoxicity in salt supplementation assays. • Toxin's action is dependent on interaction with metal cation-dependent receptor(s). [ABSTRACT FROM AUTHOR]

Abstract Sticholysin I and II (Sts: St I and St II) are proteins of biomedical interest that form pores upon the insertion of their N-terminus in the plasma membrane. Peptides spanning the N-terminal residues of StI (StI 1-31) or StII (StII 1-30) can mimic the permeabilizing ability of these toxins, emerging as candidates to rationalize their potential biomedical applications. These peptides have different activities that correlate with their hydrophobicity. However, it is not clear how this property contributes to peptide folding in solution or upon binding to membranes. Here we compared the conformational properties of these peptides and shorter versions lacking the hydrophobic segment 1–11 of StI (StI 12-31) or 1–10 of StII (StII 11-30). Folding of peptides was assessed in solution and in membrane mimetic systems and related with their ability to bind to membranes and to permeabilize lipid vesicles. Our results suggest that the differences in activity among peptides could be ascribed to their different folding propensity and different membrane binding properties. In solution, StII 1-30 tends to acquire α-helical conformation coexisting with self-associated structures, while StI 1-31 remains structureless. Both peptides fold as α-helix in membrane; but StII 1-30 also self-associates in the lipid environment, a process that is favored by its higher affinity for membrane. We stress the contribution of the non-polar/polar balance of the 1–10 amino acid sequence of the peptides as a determining factor for different self-association capabilities. Such difference in hydrophobicity seems to determine the molecular path of peptides folding upon binding to membranes, with an impact in their permeabilizing activity. This study contributes to a better understanding of the molecular mechanisms underlying the permeabilizing activity of Sts N-terminal derived peptides, with connotation for the exploitation of these small molecules as alternative of the full-length toxins in clinical settings. Graphical abstract Image 1 Highlights • Hydrophobicity of peptides StI 1-31 and StII 1-30 determines their self-association capabilities. • StII 1-30 but not StI 1-31 self-associates in solution and in the lipid environment. • Folding of StI 1-31 and StII 1-30 peptides is strongly conditioned by the membrane environment. • The higher hydrophobicity of StII 1-30 facilitates its partition into the membrane. • The permeabilizing activity of StII 1-30 could be favored by pre-association in solution. [ABSTRACT FROM AUTHOR]

A bstract Actinoporins constitute a unique class of pore-forming toxins found in sea anemones that being secreted as soluble monomers are able to bind and permeabilize membranes leading to cell death. The interest in these proteins has risen due to their high cytotoxicity that can be properly used to design immunotoxins against tumor cells and antigen-releasing systems to cell cytosol. In this work we describe a novel actinoporin produced by Anthopleura nigrescens, an anemone found in the Central American Pacific Ocean. Here we report the amino acid sequence of an actinoporin as deduced from cDNA obtained from total body RNA. The synthetic DNA sequence encoding for one cytolysin variant was expressed in BL21 Star (DE3) Escherichia coli and the protein purified by chromatography on CM Sephadex C-25 with more than 97% homogeneity as verified by MS-MS and HPLC analyses. This actinoporin comprises 179 amino acid residues, consistent with its observed isotope-averaged molecular mass of 19 661 Da. The toxin lacks Cys and readily permeabilizes erythrocytes, as well as L1210 cells. CD spectroscopy revealed that its secondary structure is dominated by beta structure (58.5%) with 5.5% of α-helix, and 35% of random structure. Moreover, binding experiments to lipidic monolayers and to liposomes, as well as permeabilization studies in vesicles, revealed that the affinity of this toxin for sphingomyelin-containing membranes is quite similar to sticholysin II (StII). Comparison by spectroscopic techniques and modeling the three-dimensional structure of nigrelysin (Ng) showed a high homology with StII but several differences were also detectable. Taken together, these results reinforce the notion that Ng is a novel member of the actinoporin pore-forming toxin (PFT) family with a HA as high as that of StII, the most potent actinoporin so far described, but with peculiar structural characteristics contributing to expand the understanding of the structure-function relationship in this protein family. Highlights • Nigrelysin (Ng) is a novel 20 kDa actinoporin produced by sea anemone A. nigrescens. • Ng efficiently permeabilizes cells and model membranes containing sphingomyelin. • Ng´s high permeabilizing activity is similar to that of StII with C 50 values ∼2 nM. [ABSTRACT FROM AUTHOR]

Sticholysin II (StII) is a pore-forming toxin of biomedical interest that belongs to the actinoporin protein family. Sticholysins are currently under examination as an active immunomodulating component of a vaccinal platform against tumoral cells and as a key element of a nucleic acids delivery system to cell cytosol. These proteins form pores in the plasma membrane leading to ion imbalance and cell lysis. However, the intracellular mechanisms triggered by actinoporins upon binding to membranes and its consequences for cell death are barely understood. Here, we have examined the cytotoxicity and intracellular responses induced by StII upon binding to human B-cell lymphoma Raji in vitro . StII cytotoxicity involves a functional actin cytoskeleton, induces cellular swelling, lysis and the concomitant release of cytosol content. In addition, StII induces calcium release mainly from the Endoplasmic Reticulum, activates Mitogen-Activated Protein Kinase ERK and impairs mitochondrial membrane potential. Furthermore, StII stimulates the expression of receptor interacting protein kinase 1 (RIP1), normally related to different forms of regulated cell death such as apoptosis and necroptosis. In correspondence, necrostatin-1, an inhibitor of this kinase, reduces StII cytotoxicity. However, the mechanism of cell death activated by StII does not involve caspases activation, typical molecular features of apoptosis and pyroptosis. Our results suggest that, beyond pore-formation and cell lysis, StII-induced cytotoxicity could involve other regulated intracellular mechanisms connected to RIP1-MEK1/2 –ERK1/2- pathways. This opens new perspectives and challenges the general point of view that these toxins induce a completely unregulated mechanism of necrotic cell death. This study contributes to a better understanding of the molecular mechanisms involved in toxin-cell interaction and the implications for cell functioning, with connotation for the exploitations of these toxins in clinical settings. [ABSTRACT FROM AUTHOR]

The pore-forming S. aureus α-toxin (Hla) contributes to virulence and disease pathogenesis. While high concentrations of toxin induce cell death, neutrophils exhibit relative resistance to lysis, suggesting that the action of Hla may not be solely conferred by lytic susceptibility. Using intravital microscopy, we observed that Hla disrupts neutrophil localization and clustering early in infection. Hla forms a narrow, ion-selective pore, suggesting that Hla may dysregulate calcium or other ions to impair neutrophil function. We found that sub-lytic Hla did not permit calcium influx but caused rapid membrane depolarization. Depolarization decreases the electrogenic driving force for calcium, and concordantly, Hla suppressed calcium signaling in vitro and in vivo and calcium-dependent leukotriene B4 (LTB4) production, a key mediator of neutrophil clustering. Thus, Hla disrupts the early patterning of the neutrophil response to infection, in part through direct impairment of neutrophil calcium signaling. This early mis-localization of neutrophils may contribute to establishment of infection. [Display omitted] • S. aureus α-toxin disrupts the cell membrane potential to impair neutrophil SOCE • S. aureus α-toxin impairs neutrophil localization and clustering at sites of infection • Neutrophil mis-localization correlates with impaired bacterial control Yang et al. find that the pore-forming S. aureus α-toxin (Hla) disrupts the neutrophil response to infection by impairing calcium signaling. Acting as an ion-selective channel, Hla permits sodium influx leading to membrane depolarization and decreased electrogenic driving force for calcium entry. Mis-localization of neutrophils correlates with impaired bacterial control. [ABSTRACT FROM AUTHOR]

Clostridium perfringens delta-toxin is a β-barrel-pore-forming toxin (β-PFT) and a presumptive virulence factor of type B and C strains, which are causative organisms of fatal intestinal diseases in animals. We showed previously that delta-toxin causes cytotoxicity via necrosis in sensitive cells. Here, we examined the effect of delta-toxin on intestinal membrane integrity. Delta-toxin led to a reduction in transepithelial electrical resistance (TEER) and increased the permeability of fluorescence isothiocyanate-conjugated dextran in human intestinal epithelial Caco-2 cells without changing the tight junction proteins, such as zonula occludens-1 (ZO-1), occludin, and claudin-1. On the other hand, delta-toxin reduced the cellular levels of adherence junction protein E-cadherin before cell injury. A disintegrin and metalloprotease (ADAM) 10 facilitates E-cadherin cleavage and was identified as the cellular receptor for alpha-toxin, a β-PFT produced by Staphylococcus aureus . ADAM10 inhibitor (GI254023X) blocked the toxin-induced decrease in TEER and cleavage of E-cadherin. Delta-toxin enhanced ADAM10 activity in a dose- and time-dependent manner. Furthermore, delta-toxin colocalized with ADAM10. These results indicated that ADAM10 plays a key role in delta-toxin-induced intestinal injury. [ABSTRACT FROM AUTHOR]

Background Beta-toxin produced by Clostridium perfringens is a key virulence factor of fatal hemorrhagic enterocolitis and enterotoxemia. This toxin belongs to a family of β-pore-forming toxins (PFTs). We reported recently that the ATP-gated P2X 7 receptor interacts with beta-toxin. The ATP-release channel pannexin 1 (Panx1) is an important contributor to P2X 7 receptor signaling. Hence, we investigated the involvement of Panx1 in beta-toxin-caused cell death. Methods We examined the effect of Panx1 in beta-toxin-induced cell death utilizing selective antagonists, knockdown of Panx1, and binding using dot-blot analysis. Localization of Panx1 and the P2X 7 receptor after toxin treatment was determined by immunofluorescence staining. Results Selective Panx1 antagonists (carbenoxolone [CBX], probenecid, and Panx1 inhibitory peptide) prevented beta-toxin-caused cell death in THP-1 cells. CBX did not block the binding of the toxin to cells. Small interfering knockdown of Panx1 blocked beta-toxin-mediated cell death through inhibiting the oligomer formation of the toxin. Beta-toxin triggered a transient ATP release from THP-1 cells, but this early ATP release was blocked by CBX. ATP scavengers (apyrase and hexokinase) inhibited beta-toxin-induced cytotoxicity. Furthermore, co-administration of ATP with beta-toxin enhanced the binding and cytotoxicity of the toxin. Conclusions Based on our results, Panx1 activation is achieved through the interaction of beta-toxin with the P2X 7 receptor. Then, ATP released by the Panx1 channel opening promotes oligomer formation of the toxin, leading to cell death. General significance Pannexin 1 is a novel candidate therapeutic target for beta-toxin-mediated disease. [ABSTRACT FROM AUTHOR]

Helicobacter pylori is associated with severe and chronic diseases of the stomach and duodenum such as peptic ulcer, non-cardial adenocarcinoma and gastric lymphoma, making Helicobacter pylori the only bacterial pathogen which is known to cause cancer. The worldwide rate of incidence for these diseases is extremely high and it is estimated that about half of the world's population is infected with H. pylori . Among the bacterial virulence factors is the vacuolating cytotoxin A (VacA), which represents an important determinant of pathogenicity. Intensive characterization of VacA over the past years has provided insight into an ample variety of mechanisms contributing to host-pathogen interactions. The toxin is considered as an important target for ongoing research for several reasons: i) VacA displays unique features and structural properties and its mechanism of action is unrelated to any other known bacterial toxin; ii) the toxin is involved in disease progress and colonization by H. pylori of the stomach; iii) VacA is a potential and promising candidate for the inclusion as antigen in a vaccine directed against H. pylori and iv) the vacA gene is characterized by a high allelic diversity, and allelic variants contribute differently to the pathogenicity of H. pylori . Despite the accumulation of substantial data related to VacA over the past years, several aspects of VacA-related activity have been characterized only to a limited extent. The biologically most significant effect of VacA activity on host cells is the formation of membrane pores and the induction of vacuole formation. This review discusses recent findings and advances on structure-function relations of the H. pylori VacA toxin, in particular with a view to membrane channel formation, oligomerization, receptor binding and apoptosis. [ABSTRACT FROM AUTHOR]

Staphylococcal α-hemolysin (α-HL) is a β-barrel pore-forming toxin (PFT) expressed by Staphylococcus aureus . α-HL is secreted as a water-soluble monomeric protein, which binds to target membranes and forms membrane-inserted heptameric pores. To explore the pore-forming mechanism of α-HL in detail, we determined the crystal structure of the α-HL monomer and prepore using H35A mutant and W179A/R200A mutant, respectively. Although the overall structure of the monomer was similar to that of other staphylococcal PFTs, a marked difference was observed in the N-terminal amino latch, which bent toward the prestem. Moreover, the prestem was fastened by the cap domain with a key hydrogen bond between Asp45 and Tyr118. Prepore structure showed that the transmembrane region is roughly formed with flexibility, although the upper half of the β-barrel is formed appropriately. Structure comparison among monomer, prepore and pore revealed a series of motions, in which the N-terminal amino latch released upon oligomerization destroys its own key hydrogen bond between Asp45–Tyr118. This action initiated the protrusion of the prestem. Y118F mutant and the N-terminal truncated mutant markedly decreased in the hemolytic activity, indicating the importance of the key hydrogen bond and the N-terminal amino latch on the pore formation. Based on these observations, we proposed a dynamic molecular mechanism of pore formation for α-HL. [ABSTRACT FROM AUTHOR]

Background Clostridium perfringens beta-toxin is a pore-forming toxin (PFT) and an important agent of necrotic enteritis and enterotoxemia. We recently reported that beta-toxin strongly induced cell death in THP-1 cells via the formation of oligomers. We here describe that the P2X 7 receptor, which is an ATP receptor, interacts with beta-toxin. Methods We tested the role of P2X 7 receptor in beta-toxin-induced toxicity using specific inhibitors, knockdown of receptor, expression of the receptor and interaction by dot-blot assay. The potency of P2X 7 receptor was further determined using an in vivo mouse model. Results Selective P2X 7 receptor antagonists (oxidized ATP (o-ATP), oxidized ADP, and Brilliant Blue G (BBG)) inhibited beta-toxin-induced cytotoxicity in THP-1 cells. o-ATP also blocked the binding of beta-toxin to cells. The P2X 7 receptor and beta-toxin oligomer were localized in the lipid rafts of THP-1 cells. siRNA for the P2X 7 receptor inhibited toxin-induced cytotoxicity and binding of the toxin. In contrast, the siRNA knockdown of P2Y 2 or P2Y 6 had no effect on beta-toxin-induced cytotoxicity. The addition of beta-toxin to P2X 7 -transfected HEK-293 cells resulted in binding of beta-toxin oligomer. Moreover, beta-toxin specifically bound to immobilized P2X 7 receptors in vitro and colocalized with the P2X 7 receptor on the THP-1 cell surface. Furthermore, beta-toxin-induced lethality in mice was blocked by the preadministration of BBG. Conclusions The results of this study indicate that the P2X 7 receptor plays a role in beta-toxin-mediated cellular injury. General significance P2X 7 receptor is a potential target for the treatment of C. perfringens type C infection. [ABSTRACT FROM AUTHOR]

In this study, the antibacterial activities of Clove oil and liposome-encapsulated Clove oil were investigated. First, the antibacterial activity of Clove oil demonstrated that the essential oil exhibited favorable antimicrobial activity for both Escherichia coli and Staphylococcus aureus . However, a setback of using Clove oil as a disinfectant is its low chemical stability. Then Clove oil was incorporated into a liposome formulation to increase its stability. The optimal polydispersity index (PDI) (0.196), Zeta potential (−24.5 mV) and entrapment efficiency (20.41%) of liposome were obtained at the concentration of Clove oil to 5.0 mg/mL. In addition, selective antimicrobial activity for S. aureus by utilizing pore-forming toxins (PFTs) to activate Clove oil release from liposome was observed. By contrast, liposome-encapsulated Clove oil has no effect on E. coli that doesn't secrete PFTs because antimicrobial component can't reach bacteria. Gas chromatography (GC) assay found that when liposome met S. aureus that secrete PFTs, PFTs would insert into the liposome membranes and form pores, through which the encapsulated Clove oil was released. Besides, liposome-encapsulated Clove oil exhibited efficient antimicrobial activity for S. aureus in tofu. [ABSTRACT FROM AUTHOR]

Actinoporins are basic pore-forming proteins produced by sea anemones, with molecular weight around 20 kDa showing high affinity for sphingomyelin-containing membranes. Most sea anemones produce more than one actinoporin isoform differing in isoelectric point, molecular weigth and cytolytic activity. Examples of sea anemones with actinoporin isoforms are: Actinia equina with at least five isoform genes; Actinia tenebrosa , three isoforms; Actinia fragacea , five isoforms; Actineria villosa , Phyllodiscus semoni , Stichodactyla helianthus and Oulactis orientalis , with two isoforms each one, and Heteractis crispa with twenty-four isoforms. Additionally, thirty-four different amino acid sequences were deduced from fifty-two nucleotide sequences of Heteractis magnifica toxins suggesting the presence of a large number of isoforms or allelic variants. Many amino acidic changes in the isoforms are located in important regions for pore formation. The genetic structure of actinoporins comprises a pre-propeptide and a mature toxin region; therefore, actinoporins could be synthetized in the Golgi apparatus as precursor forms. The subsequent maturation of the toxins involves a proteolytic processing during secretion. Here we hypothesize that sea anemones could have suffered duplication, conversion and mutation of genes that produced multigene families as an efficient response to evolutionary pressure, leading to successful strategies of predatory and defensive function. [ABSTRACT FROM AUTHOR]

Pore-forming toxins (PFTs) rupture plasma membranes and kill target cells. PFTs are secreted as soluble monomers that undergo drastic structural rearrangements upon interacting with the target membrane and generate transmembrane oligomeric pores. A detailed understanding of the molecular mechanisms of the pore-formation process remains unclear due to limited structural insights regarding the transmembrane oligomeric pore states of the PFTs. However, recent advances in the field of cryo-electron microscopy (cryo-EM) have led to the high-resolution structure determination of the oligomeric pore forms of diverse PFTs. Here, we discuss the pore-forming mechanisms of various PFTs, specifically the mechanistic details contributed by the cryo-EM-based structural studies. [Display omitted] • Pore-forming toxins (PFTs) are membrane-damaging toxins. • Soluble monomers of PFTs form oligomeric pores in the membranes. • Pore-formation mechanism highlights prominent structural and assembly changes. • Cryo-EM elucidates structural and mechanistic details of the PFT pore-formation. [ABSTRACT FROM AUTHOR]

This study demonstrates that the polyketide toxin karlotoxin 2 (KmTx 2) produced by Karlodinium veneficum , a dinoflagellate associated with fish kills in temperate estuaries world-wide, alters vertebrate cell membrane permeability. Microfluorimetric and electrophysiological measurements were used to determine that vertebrate cellular toxicity occurs through non-selective permeabilization of plasma membranes, leading to osmotic cell lysis. Previous studies showed that KmTx 2 is lethal to fish at naturally-occurring concentrations measured during fish kills, while sub-lethal doses severely damage gill epithelia. This study provides a mechanistic explanation for the association between K. veneficum blooms and fish kills that has long been observed in temperate estuaries worldwide. [ABSTRACT FROM AUTHOR]

Abstract: α-Hemolysin (HlyA) is a protein toxin, a member of the pore-forming Repeat in Toxin (RTX) family, secreted by some pathogenic strands of Escherichia coli. The mechanism of action of this toxin seems to involve three stages that ultimately lead to cell lysis: binding, insertion, and oligomerization of the toxin within the membrane. Since the influence of phase segregation on HlyA binding and insertion in lipid membranes is not clearly understood, we explored at the meso- and nanoscale—both in situ and in real-time—the interaction of HlyA with lipid monolayers and bilayers. Our results demonstrate that HlyA could insert into monolayers of dioleoylphosphatidylcholine/sphingomyelin/cholesterol (DOPC/16:0SM/Cho) and DOPC/24:1SM/Cho. The time course for HlyA insertion was similar in both lipidic mixtures. HlyA insertion into DOPC/16:0SM/Cho monolayers, visualized by Brewster-angle microscopy (BAM), suggest an integration of the toxin into both the liquid-ordered and liquid-expanded phases. Atomic-force-microscopy imaging reported that phase boundaries favor the initial binding of the toxin, whereas after a longer time period the HlyA becomes localized into the liquid-disordered (Ld) phases of supported planar bilayers composed of DOPC/16:0SM/Cho. Our AFM images, however, showed that the HlyA interaction does not appear to match the general strategy described for other invasive proteins. We discuss these results in terms of the mechanism of action of HlyA. [Copyright &y& Elsevier]

Abstract: Pore-forming toxins are an important group of natural molecules that damage cellular membranes by forming transmembrane pores. They are used by many organisms for attack or defense and similar proteins are employed in the immune system of vertebrates. Various biophysical approaches have been used to understand how these proteins act at the molecular level. One of the most useful, in terms of monitoring pore formation in real time, is a method that employs planar lipid membranes and involves ionic current measurements. Here we highlight the advantages and possibilities that this approach offers and show how it can advance understanding of the pore-forming mechanism and pore properties for one of the most important families of natural toxins, the cholesterol-dependent cytolysins. [Copyright &y& Elsevier]

Abstract: Epsilon toxin (ET), produced by Clostridium perfringens types B and D, ranks among the four most potent poisonous substances known so far. ET-intoxication is responsible for enterotoxaemia in animals, mainly sheep and goats. This disease comprises several manifestations indicating the attack of the nervous system. This review aims to summarize the effects of ET on central nervous system. ET binds to endothelial cells of brain capillary vessels before passing through the blood–brain barrier. Therefore, it induces perivascular oedema and accumulates into brain. ET binding to different brain structures and to different component in the brain indicates regional susceptibility to the toxin. Histological examination has revealed nerve tissue and cellular lesions, which may be directly or indirectly caused by ET. The naturally occurring disease caused by ET-intoxication can be reproduced experimentally in rodents. In mice and rats, ET recognizes receptor at the surface of different neural cell types, including certain neurons (e.g. the granule cells in cerebellum) as well as oligodendrocytes, which are the glial cells responsible for the axons myelination. Moreover, ET induces release of glutamate and other transmitters, leading to firing of neural network. The precise mode of action of ET on neural cells remains to be determined. [Copyright &y& Elsevier]

Abstract: Sticholysins (Sts) I and II (StI/II) are pore-forming toxins (PFTs) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin family, a unique class of eukaryotic PFTs exclusively found in sea anemones. The role of lipid phase co-existence in the mechanism of the action of membranolytic proteins and peptides is not clearly understood. As for actinoporins, it has been proposed that phase separation promotes pore forming activity. However little is known about the effect of sticholysins on the phase separation of lipids in membranes. To gain insight into the mechanism of action of sticholysins, we evaluated the effect of these proteins on lipid segregation using differential scanning calorimetry (DSC) and atomic force microscopy (AFM). New evidence was obtained reflecting that these proteins reduce line tension in the membrane by promoting lipid mixing. In terms of the relevance for the mechanism of action of actinoporins, we hypothesize that expanding lipid disordered phases into lipid ordered phases decreases the lipid packing at the borders of the lipid raft, turning it into a more suitable environment for N-terminal insertion and pore formation. [Copyright &y& Elsevier]

Highlights: [•] NetB from Clostridium perfringens is the major virulence factor in avian necrotic enteritis. [•] Vaccination with a NetB genetic or formaldehyde toxoid protects chicken in an in vivo disease model. [•] NetB toxoids could form the bases of an efficient vaccine against necrotic enteritis. [ABSTRACT FROM AUTHOR]

Abstract: Background: CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata that shows Ca2+-dependent Gal/GalNAc-binding specificity. This lectin is composed of two carbohydrate-recognition domains (domains 1 and 2) and an oligomerization domain (domain 3) that facilitates CEL-III assembly in the target cell membrane to form ion-permeable pores. Methods: Several amino acid residues in domain 3 were replaced by alanine, and hemolytic activity of the mutants was examined. Results: K344A, K351A, K405A, K420A and K425A showed marked increases in activity. In particular, K405A had activity that was 360-fold higher than the wild-type recombinant CEL-III and 3.6-fold higher than the native protein purified from sea cucumber. Since these residues appear to play roles in the stabilization of domain 3 through ionic and hydrogen bonding interactions with other residues, the mutations of these residues presumably lead to destabilization of domain 3, which consequently induces the oligomerization of the protein through association of domain 3 in the membrane. In contrast, K338A, R378A and R408A mutants exhibited a marked decrease in hemolytic activity. Since these residues are located on the surface of domain 3 without significant interactions with other residue, they may be involved in the interaction with components of the target cell membrane. Conclusions: Several amino acid residues, especially basic residues, are found to be involved in the hemolytic activity as well as the oligomerization ability of CEL-III. General significance: The results provide important clues to the membrane pore-forming mechanism of CEL-III, which is also related to that of bacterial pore-forming toxins. [Copyright &y& Elsevier]

Highlights: [•] Genetically attenuated Plasmodium berghei parasites for vaccination trials have been generated. [•] Double attenuation increases safety of vaccination. [•] This new approach of genetic attenuation allows parasite development to the schizont stage. [•] Vaccination with the newly generated double-attenuated parasite strain induces sterile protection. [Copyright &y& Elsevier]

Listeriolysin O (LLO) is a toxin produced by Listeria monocytogenes, an opportunistic bacterial pathogen responsible for the disease listeriosis. This disease starts with the ingestion of contaminated foods and mainly affects immunocompromised individuals, newborns, and pregnant women. In the laboratory, L. monocytogenes is used as a model organism to study processes such as cell invasion, intracellular survival, and cell-to-cell spreading, as this Gram-positive bacterium has evolved elaborate molecular strategies to subvert host cell functions. LLO is a major virulence factor originally shown to be crucial for bacterial escape from the internalization vacuole after entry into cells. However, recent studies are revisiting the role of LLO during infection and are revealing new insights into the action of LLO, in particular before bacterial entry. These latest findings along with their impact on the infectious process will be discussed. [ABSTRACT FROM AUTHOR]

Abstract: Experimental evidence shows that the mechanism of pore formation by actinoporins is a multistep process, involving binding of the water-soluble monomer to the membrane and subsequent oligomerization on the membrane surface, leading to the formation of a functional pore. However, as for other eukaryotic pore-forming toxins, the molecular details of the mechanism of membrane insertion and oligomerization are not clear. In order to obtain further insight with regard to the structure-function relationship in sticholysins, we designed and produced three cysteine mutants of recombinant sticholysin I (rStI) in relevant functional regions for membrane interaction: StI E2C and StI F15C (in the N-terminal region) and StI R52C (in the membrane binding site). The conformational characterization derived from fluorescence and CD spectroscopic studies of StI E2C, StI F15C and StI R52C suggests that replacement of these residues by Cys in rStI did not noticeably change the conformation of the protein. The substitution by Cys of Arg52 in the phosphocholine-binding site, provoked noticeable changes in rStI permeabilizing activity; however, the substitutions in the N-terminal region (Glu2, Phe15) did not modify the toxin’s permeabilizing ability. The presence of a dimerized population stabilized by a disulfide bond in the StI E2C mutant showed higher pore-forming activity than when the protein is in the monomeric state, suggesting that sticholysins pre-ensembled at the N-terminal region could facilitate pore formation. [Copyright &y& Elsevier]

Abstract: Adenylate cyclase-haemolysin toxin (CyaA) is a virulence factor secreted from the etiologic agent of whooping cough, Bordetella pertussis. Previously, the haemolysin or pore-forming domain (CyaA-PF) has been shown to cause cell lysis of sheep erythrocytes independently, and the predicted helix 3(570−593) within the PF-hydrophobic stretch could be a pore-lining constituent. Here, a plausible involvement in haemolytic activity of polar or charged residues (Glu570, Gln574, Glu581, Ser584 and Ser585) lining the hydrophilic side of CyaA-PF helix 3 was investigated via single-alanine substitutions. All the 126-kDa mutant proteins over-expressed in Escherichia coli were verified for toxin acylation as the results are corresponding to the wild-type toxin. When haemolytic activity of E. coli lysates containing soluble mutant proteins was tested against sheep erythrocytes, the importance of Glu570, which is highly conserved among the pore-forming RTX cytotoxin family, was revealed for pore formation, conceivably for a general pore-lining residue involved in ion conduction. [Copyright &y& Elsevier]

Abstract: A novel two-component hemolysin, erylysin A and B (EryA and EryB), was isolated from an edible mushroom, Pleurotus eryngii. Hemolytic activity was exhibited only by the EryA and EryB mixture. EryA showed one band at 15 kDa on SDS-PAGE while EryB showed two bands at 15 kDa (EryB1) and 37 kDa (EryB2). MALDI-TOF MS showed that the molecular masses of EryA, EryB1 and EryB2 were 14,945 Da, 14,593 Da and 37,417 Da, respectively. EryA and EryB were very similar to pleurotolysin A and B in terms of molecular mass, and the N-terminus and inner sequences. At pH 7.2, EryA exists as a homodimer whereas EryB exists as a heterodimer of B1 and B2. CD spectrum analysis showed Tm values of 47 °C and 37 °C for EryA and EryB, respectively. EryB was particularly unstable. [ABSTRACT FROM AUTHOR]

Abstract: Lysenin is a self-assembling, pore-forming toxin which specifically recognizes sphingomyelin. Mutation of tryptophan 20 abolishes lysenin oligomerization and cytolytic activity. We studied the interaction of lysenin WT and W20A with sphingomyelin in membranes of various lipid compositions which, according to atomic force microscopy studies, generated either homo- or heterogeneous sphingomyelin distribution. Liposomes composed of SM/DOPC, SM/DOPC/cholesterol and SM/DPPC/cholesterol could bind the highest amounts of GST-lysenin WT, as shown by surface plasmon resonance analysis. These lipid compositions enhanced the release of carboxyfluorescein from liposomes induced by lysenin WT, pointing to the importance of heterogeneous sphingomyelin distribution for lysenin WT binding and oligomerization. Lysenin W20A bound more weakly to sphingomyelin-containing liposomes than did lysenin WT. The same amounts of lysenin W20A bound to sphingomyelin mixed with either DOPC or DPPC, indicating that the binding was not affected by sphingomyelin distribution in the membranes. The mutant lysenin had a limited ability to penetrate hydrophobic region of the membrane as indicated by measurements of surface pressure changes. When applied to detect sphingomyelin on the cell surface, lysenin W20A formed large conglomerates on the membrane, different from small and regular clusters of lysenin WT. Only lysenin WT recognized sphingomyelin pool affected by formation of raft-based signaling platforms. During fractionation of Triton X-100 cell lysates, SDS-resistant oligomers of lysenin WT associated with membrane fragments insoluble in Triton X-100 while monomers of lysenin W20A partitioned to Triton X-100-soluble membrane fractions. Altogether, the data suggest that oligomerization of lysenin WT is a prerequisite for its docking in raft-related domains. [Copyright &y& Elsevier]

Abstract: The use of membrane active toxins as toxic moieties in the construction of immunotoxins (ITs) is an attractive alternative to overcome some of the problems of classical ITs since these new conjugates are based in the use of a different mechanism of killing undesired cells. Pore-forming cytolysins from sea anemones were used in the construction of ITs targeted to different cell types including tumour cell lines and the parasite Giardia duodenalis. The results obtained support the feasibility of directing these cytolysins to the surface of the cancer cells or the parasite through their conjugation to monoclonal antibodies recognizing tumour-associated or parasite antigens, respectively. However the main problem with the IT constructed in this fashion is the lack of specificity associated with the toxin moiety. An approach designed to overcome this limitation was the construction of inactive cytolysin with built-in biological “trigger” that renders the toxin active in the presence of tumour-specific proteinases. This construction is considered as a proof of concept to demonstrate the feasibility of such activation systems in the construction of ITs based on pore-forming cytolysins from sea anemones with reduced unspecific activity. The future prospects of the use of the N-terminal region of actinoporins for construction of IT is also described. [Copyright &y& Elsevier]

Abstract: Sticholysins (Sts) I and II (StI/II) are pore-forming toxins (PFTs) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin family, a unique class of eukaryotic PFTs exclusively found in sea anemones. As for the rest of the members of this family, Sts are cysteine-less proteins, with molecular weights around 20kDa, high isoelectric points (>9.5), and a preference for sphingomyelin-containing membranes. A three-dimensional structure of StII, solved by X-ray crystallography, showed that it is composed of a hydrophobic β-sandwich core flanked on the opposite sides by two α helices comprising residues 14–23 and 128–135. A variety of experimental results indicate that the first thirty N-terminal residues, which include one of the helices, are directly involved in pore formation. This region contains an amphipathic stretch, well conserved in all actinoporins, which is the only portion of the molecule that can change conformation without perturbing the general protein fold; in fact, binding to model membranes only produces a slight increase in the regular secondary structure content of Sts. Sts are produced in soluble form but they readily bind to different cell and model membrane systems such as lipidic monolayers, micelles, and lipid vesicles. Remarkably, both the binding and pore-formation steps are critically dependent on the physico-chemical nature of the membrane. In fact, a large population of toxin irreversibly binds with high affinity in membranes containing sphingomyelin whereas binding in membranes lacking this sphingolipid is relatively low and reversible. The joint presence of SM and cholesterol largely promotes binding and pore formation. Minor amounts of lipids favoring a non-lamellar organization also augment the efficiency of pore formation. The functional pore formed in cellular and model membranes has a diameter of ∼2.0nm and is presumably formed by the N-terminal α helices of four monomers tilted 31° in relation to the bilayer normal. Experimental evidence supports the hypothesis that sticholysins, as well as equinatoxin II, another actinoporin, form a toroidal pore in membranes in which the polypeptide chains as well as the polar head groups of phospholipids are involved. [Copyright &y& Elsevier]

Abstract: Hemolysin E (HlyE), a pore-forming protein-toxin and a potential virulence factor of Escherichia coli, exhibits cytotoxic activity to mammalian cells. However, very little is known about how the different individual segments contribute in the toxic activity of the protein. Toward this end, the role of a 33-residue segment comprising the amino acid region 88 to 120, which contains the putative transmembrane domain in the tail region of HlyE has been addressed in the toxic activity of the protein-toxin by characterizing the related wild type and mutant peptides and the whole protein. Along with the 33-residue wild type peptide, H-88, two mutants of the same size were synthesized; in one mutant a conserved valine at 89th position was replaced by aspartic acid and in the other both glycine and valine at the 88th and 89th positions were substituted by aspartic acid residues. These mutations were also incorporated in the whole toxin HlyE. Results showed that only H-88 but not its mutants permeabilized both lipid vesicles and human red blood cells (hRBCs). Interestingly, while H-88 exhibited a moderate lytic activity to human red blood cells, the mutants were not active. Drastic reduction in the depolarization of hRBCs and hemolytic activity of the whole toxin HlyE was also observed as a result of the same double and single amino acid substitution in it. The results indicate an important role of the amino acid segment 88–120, containing the putative transmembrane domain of the tail region of the toxin in the toxic activity of hemolysin E. [Copyright &y& Elsevier]

Abstract: Functional homologs of the Escherichia coli cytolysin A (clyA, hlyE, sheA) gene have recently been detected in Salmonella enterica serovars Typhi (S. Typhi) and Paratyphi A (S. Paratyphi A). In this study, analysis of a collection of Salmonella strains showed that all S. Typhi and S. Paratyphi A strains tested harbor an intact copy of the corresponding clyA variant, i.e. clyA STy and clyA SPaA, respectively. On the other hand, clyA proved to be absent in the S. enterica serovar Paratyphi B and serovar Paratyphi C strains, in various non-typhoid S. enterica subsp. enterica serovars (Typhimurium, Enteritidis, Choleraesuis, Dublin, and Gallinarum), and in S. enterica subsp. arizonae and Salmonella bongori strains. When grown under normal laboratory conditions, the S. Typhi and S. Paratyphi A strains produced only basal amounts of ClyA protein and did not exhibit a clyA-dependent hemolytic phenotype. RT-PCR and immunoblot analyses as well as phenotypic data revealed, however, that the expression of clyA STy and clyA SPaA can be activated by the Salmonella transcription factor SlyA. In addition, osmotic protection assays and lipid bilayer experiments demonstrated that the hemolytic ClyASTy and ClyASPaA proteins are effective pore-forming toxins which, similar to E. coli ClyA, generate large, stable, moderately cation-selective channels in target membranes. Taken together with our recent serological findings which have indicated that S. Typhi and S. Paratyphi A strains produce substantial amounts of ClyA during human infection, these data suggest that ClyA may play a role in S. Typhi and S. Paratyphi A pathogenesis. [Copyright &y& Elsevier]

Abstract: Bacteria, as well as higher organisms such as sea anemones or earthworms, have developed sophisticated virulence factors such as the pore-forming toxins (PFTs) to mount their attack against the host. One of the most fascinating aspects of PFTs is that they can adopt a water-soluble form at the beginning of their lifetime and become an integral transmembrane protein in the membrane of the target cells. There is a growing understanding of the sequence of events and the various conformational changes undergone by these toxins in order to bind to the host cell surface, to penetrate the cell membranes and to achieve pore formation. These points will be addressed in this review. [Copyright &y& Elsevier]

Abstract: Pneumolysin is a cytolytic toxin of Streptococcus pneumoniae, a causative agent of pneumonia and meningitis. The prepore and pore states of pneumolysin have recently been investigated by cryo-electron microscopy and atomic force microscopy, confirming the existence of arc-shaped as well as ring-form oligomers. Here we provide further insights into the pneumolysin oligomer by studying the interaction of pneumolysin with cholesterol crystals, comparing the results to those obtained for polyene antibiotics, which also bind cholesterol. [Copyright &y& Elsevier]

Abstract: 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°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. [Copyright &y& Elsevier]

Abstract: Vibrio cholerae cytolysin (VCC) forms oligomeric transmembrane pores in cholesterol-rich membranes. To better understand this process, we used planar bilayer membranes. In symmetric membranes, the rate of the channel formation by VCC has a superlinear dependency on the cholesterol membrane fraction. Thus, more than one cholesterol molecule can facilitate VCC-pore formation. In asymmetric membranes, the rate of pore formation is limited by the leaflet with the lower cholesterol content. Methyl-β-cyclodextrin, which removes cholesterol from membranes, rapidly inhibits VCC pore formation, even when it is added to the side opposite that of VCC addition. The results suggest that cholesterol in both membrane leaflets aid VCC-pore formation and that either leaflet can function as a kinetic bottleneck with respect to the rate of pore-formation. [Copyright &y& Elsevier]

Abstract: Hemolysin II (HlyII), one of several cytolytic proteins encoded by the opportunistic human pathogen Bacillus cereus, is a member of the family of oligomeric β-barrel pore-forming toxins. This work has studied the pore-forming properties of HlyII using a number of biochemical and biophysical approaches. According to electron microscopy, HlyII protein interacts with liposomes to form ordered heptamer-like macromolecular assemblies with an inner pore diameter of 1.5–2 nm and an outer diameter of 6–8 nm. This is consistent with inner pore diameter obtained from osmotic protection assay. According to the 3D model obtained, seven HlyII monomers might form a pore, the outer size of which has been estimated to be slightly larger than by the other method, with an inner diameter changing from 1 to 4 nm along the channel length. The hemolysis rate has been found to be temperature-dependent, with an explicit lag at lower temperatures. Temperature jump experiments have indicated the pore structures formed at 37 °C and 4 °C to be different. The channels formed by HlyII are anion-selective in lipid bilayers and show a rising conductance as the salt concentration increases. The results presented show for the first time that at high salt concentration HlyII pores demonstrate voltage-induced gating observed at low negative potentials. Taken together we have found that the membrane-binding properties of hemolysin II as well as the properties of its pores strongly depend on environmental conditions. The study of the properties together with structural modeling allows a better understanding of channel functioning. [Copyright &y& Elsevier]

Abstract: Sticholysin II (St II) is a cytolysin produced by the sea anemone Stichodactyla helianthus, characterized by forming oligomeric pores in natural and artificial membranes. In the present work the influence of the membrane lipidic components sphingomyelin (SM) and cholesterol (Cho) on binding and functional activity of St II, was evaluated using ELISA, lipid monolayers and liposomes. The aim of this work was to establish the promoting role of Cho and SM, both in St II binding and pore formation efficiency. In general the association (evaluated by ELISA and incorporation to phospholipid monolayers) of St II to lipids mixtures was better than to any one of the single components. Regarding the unique role of SM, it was found that, albeit inefficiently, St II binds to phosphatidylcholine (PC):Cho monolayers and liposomes, and is able to form active pores in these bilayers. The results in monolayers and liposomes show that the presence of SM and large amounts of Cho leads to the highest values of critical pressure and rate of association to monolayers, the most favorable interaction with liposomes, and the fastest rate of pore formation, in spite of the rigidity of the layers as suggested by the high generalized polarization (GP) of Laurdan incorporated to liposomes and FTIR data. Taken together, the present results show that the joint presence of SM and Cho, both in binary and ternary (PC containing) mixtures provide conditions particularly suitable for St II binding and function. We suggest that microdomains present in the bilayers could be important for toxin-membrane association. [Copyright &y& Elsevier]

Sticholysin I (StI), a potent cytolysin isolated from the sea anemone Stichodactyla helianthus, was linked to the monoclonal antibody (mAb) ior C5. StI acts by forming hydrophilic pores in the membrane of the attacked cells leading to osmotic lysis. ior C5 is a murine IgG1, which recognizes the tumor associated antigen (TAA) ior C2. The cytolysin and the mAb were coupled by using the heterobifunctional cross-linking reagent sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC). Two hybrid molecules composed by one ior C5 and one or two StI molecules were obtained (named conjugated I and II, respectively). The purified conjugates were evaluated by a binding affinity assay against an ior C2-positive colon cancer cell line (SW948). Both molecules were able to recognize the antigen (Ag) in the same way that unconjugated ior C5 does. The activity of both conjugates against human erythrocytes and SW948 cells was assessed. They lost most of their hemolytic activity but their residual activity was very similar. Nevertheless, when their cytotoxicity was studied on the SW948 cell line, only conjugate II killed efficiently the cells, indicating a specific mAb-Ag interaction. In this chimeric molecule the ratio between the cytotoxic and the hemolytic activity was larger than that of the free cytolysin. This fact indicates an increase of the specificity of the toxic effect toward the SW948 cell line and consequently an increase of the difference between its hemolytic and cytotoxic doses. The results herein support the feasibility of directing StI to the surface of cancer cells expressing ior C2 Ag via the mAb ior C5. [Copyright &y& Elsevier]

Sticholysin II (St II) a potent cytolysin from the sea anemone Stichodactyla helianthus was obtained by recombinant procedures exhibiting six histidine residues in its N-terminus (St IIn6H) (). The functional comparison between St II and St IIn6H showed a lesser pore-forming ability for the recombinant than for the native in human or rat red blood cells (RBC) and in large unilamellar vesicles (LUV) of different phospholipid composition. However, binding of St IIn6H to small unilamellar vesicles (SUV) was higher with regard to St II. The explanation to the different permeabilizing capacity of both protein variants is not clear, but a different anchoring of St IIn6H to the lipid bilayer could delay the organization of the competent pore into membrane. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

Listeria monocytogenes is an intracellular food-borne pathogen that causes listeriosis, a severe and potentially life-threatening disease. Listeria uses a number of virulence factors to proliferate and spread to various cells and tissues. In this process, three bacterial virulence factors, the pore-forming protein listeriolysin O and phospholipases PlcA and PlcB, play a crucial role. Listeriolysin O belongs to a family of cholesterol-dependent cytolysins that are mostly expressed by gram-positive bacteria. Its unique structural features in an otherwise conserved three-dimensional fold, such as the acidic triad and proline-glutamate-serine-threonine-like sequence, enable the regulation of its intracellular activity as well as distinct extracellular functions. The stability of listeriolysin O is pH- and temperature-dependent, and this provides another layer of control of its activity in cells. Moreover, many recent studies have demonstrated a unique mechanism of pore formation by listeriolysin O, i.e., the formation of arc-shaped oligomers that can subsequently fuse to form membrane defects of various shapes and sizes. During listerial invasion of host cells, these membrane defects can disrupt phagosome membranes, allowing bacteria to escape into the cytosol and rapidly multiply. The activity of listeriolysin O is profoundly dependent on the amount and accessibility of cholesterol in the lipid membrane, which can be modulated by the phospholipase PlcB. All these prominent features of listeriolysin O play a role during different stages of the L. monocytogenes life cycle by promoting the proliferation of the pathogen while mitigating excessive damage to its replicative niche in the cytosol of the host cell. [Display omitted] • Listeriolysin O is the most important virulence factor of Listeria monocytogenes. • The stability of listeriolysin O is pH- and temperature-dependent. • Listeriolysin O acts via the formation of transmembrane oligomers in lipid membranes. • Arcs represent the predominant functional oligomeric form of listeriolysin O. • The pathogenic action of listeriolysin O is supported by two listerial phospholipases. [ABSTRACT FROM AUTHOR]

The membrane pore-forming α-toxin is an important virulence factor of Staphylococcus aureus. Target cells can remove pores from their surface, but recent work shows that α-toxin may undermine this self-defense by clinging to epithelial cell junctions. The findings could lead to the development of novel remedies against S. aureus infections. [ABSTRACT FROM AUTHOR]