Emerging enterococcus pore-forming toxins with MHC/HLA-I as receptors.

Enterococci are a part of human microbiota and a leading cause of multidrug resistant infections. Here, we identify a family of Enterococcus pore-forming toxins (Epxs) in E. faecalis, E. faecium, and E. hirae strains isolated across the globe. Structural studies reveal that Epxs form a branch of ß-barrel pore-forming toxins with a ß-barrel protrusion (designated the top domain) sitting atop the cap domain. Through a genome-wide CRISPR-Cas9 screen, we identify human leukocyte antigen class I (HLA-I) complex as a receptor for two members (Epx2 and Epx3), which preferentially recognize human HLA-I and homologous MHC-I of equine, bovine, and porcine, but not murine, origin. Interferon exposure, which stimulates MHC-I expression, sensitizes human cells and intestinal organoids to Epx2 and Epx3 toxicity. Co-culture with Epx2-harboring E. faecium damages human peripheral blood mononuclear cells and intestinal organoids, and this toxicity is neutralized by an Epx2 antibody, demonstrating the toxin-mediated virulence of Epx-carrying Enterococcus.

The septin cytoskeleton is required for plasma membrane repair.

Plasma membrane repair is a fundamental homeostatic process of eukaryotic cells. Here, we report a new function for the conserved cytoskeletal proteins known as septins in the repair of cells perforated by pore-forming toxins or mechanical disruption. Using a silencing RNA screen, we identified known repair factors (e.g. annexin A2, ANXA2) and novel factors such as septin 7 (SEPT7) that is essential for septin assembly. Upon plasma membrane injury, the septin cytoskeleton is extensively redistributed to form submembranous domains arranged as knob and loop structures containing F-actin, myosin IIA, S100A11, and ANXA2. Formation of these domains is Ca2+-dependent and correlates with plasma membrane repair efficiency. Super-resolution microscopy revealed that septins and F-actin form intertwined filaments associated with ANXA2. Depletion of SEPT7 prevented ANXA2 recruitment and formation of submembranous actomyosin domains. However, ANXA2 depletion had no effect on domain formation. Collectively, our data support a novel septin-based mechanism for resealing damaged cells, in which the septin cytoskeleton plays a key structural role in remodeling the plasma membrane by promoting the formation of SEPT/F-actin/myosin IIA/ANXA2/S100A11 repair domains.

The mechanistic basis of the membrane-permeabilizing activities of the virulence-associated protein A (VapA) from Rhodococcus equi.

Pathogenic Rhodococcus equi release the virulence-associated protein A (VapA) within macrophage phagosomes. VapA permeabilizes phagosome and lysosome membranes and reduces acidification of both compartments. Using biophysical techniques, we found that VapA interacts with model membranes in four steps: (i) binding, change of mechanical properties, (ii) formation of specific membrane domains, (iii) permeabilization within the domains, and (iv) pH-specific transformation of domains. Biosensor data revealed that VapA binds to membranes in one step at pH 6.5 and in two steps at pH 4.5 and decreases membrane fluidity. The integration of VapA into lipid monolayers was only significant at lateral pressures <20 mN m-1 indicating preferential incorporation into membrane regions with reduced integrity. Atomic force microscopy of lipid mono- and bilayers showed that VapA increased the surface heterogeneity of liquid disordered domains. Furthermore, VapA led to the formation of a new microstructured domain type and, at pH 4.5, to the formation of 5 nm high domains. VapA binding, its integration and lipid domain formation depended on lipid composition, pH, protein concentration and lateral membrane pressure. VapA-mediated permeabilization is clearly distinct from that caused by classical microbial pore formers and is a key contribution to the multiplication of Rhodococcus equi in phagosomes.

Patch repair protects cells from the small pore-forming toxin aerolysin.

Aerolysin family pore-forming toxins damage the membrane, but membrane repair responses used to resist them, if any, remain controversial. Four proposed membrane repair mechanisms include toxin removal by caveolar endocytosis, clogging by annexins, microvesicle shedding catalyzed by MEK, and patch repair. Which repair mechanism aerolysin triggers is unknown. Membrane repair requires Ca2+, but it is controversial if Ca2+ flux is triggered by aerolysin. Here, we determined Ca2+ influx and repair mechanisms activated by aerolysin. In contrast to what is seen with cholesterol-dependent cytolysins (CDCs), removal of extracellular Ca2+ protected cells from aerolysin. Aerolysin triggered sustained Ca2+ influx. Intracellular Ca2+ chelation increased cell death, indicating that Ca2+-dependent repair pathways were triggered. Caveolar endocytosis failed to protect cells from aerolysin or CDCs. MEK-dependent repair did not protect against aerolysin. Aerolysin triggered slower annexin A6 membrane recruitment compared to CDCs. In contrast to what is seen with CDCs, expression of the patch repair protein dysferlin protected cells from aerolysin. We propose aerolysin triggers a Ca2+-dependent death mechanism that obscures repair, and the primary repair mechanism used to resist aerolysin is patch repair. We conclude that different classes of bacterial toxins trigger distinct repair mechanisms.

Entangling roles of cholesterol-dependent interaction and cholesterol-mediated lipid phase heterogeneity in regulating listeriolysin O pore-formation.

Cholesterol-dependent cytolysins (CDCs) are the distinct class of ß-barrel pore-forming toxins (ß-PFTs) that attack eukaryotic cell membranes, and form large, oligomeric, transmembrane ß-barrel pores. Listeriolysin O (LLO) is a prominent member in the CDC family. As documented for the other CDCs, membrane cholesterol is essential for the pore-forming functionality of LLO. However, it remains obscure how exactly cholesterol facilitates its pore formation. Here, we show that cholesterol promotes both membrane-binding and oligomerization of LLO. We demonstrate cholesterol not only facilitates membrane-binding, it also enhances the saturation threshold of LLO-membrane association, and alteration of the cholesterol-recognition motif in the LLO mutant (LLOT515G-L516G) compromises its pore-forming efficacy. Interestingly, such defect of LLOT515G-L516G could be rescued in the presence of higher membrane cholesterol levels, suggesting cholesterol can augment the pore-forming efficacy of LLO even in the absence of a direct toxin-cholesterol interaction. Furthermore, we find the membrane-binding and pore-forming abilities of LLOT515G-L516G, but not those of LLO, correlate with the cholesterol-dependent rigidity/ordering of the membrane lipid bilayer. Our data further suggest that the line tension derived from the lipid phase heterogeneity of the cholesterol-containing membranes could play a pivotal role in LLO function, particularly in the absence of cholesterol binding. Therefore, in addition to its receptor-like role, we conclude cholesterol can further facilitate the pore-forming, membrane-damaging functionality of LLO by asserting the optimal physicochemical environment in membranes. To the best of our knowledge, this aspect of the cholesterol-mediated regulation of the CDC mode of action has not been appreciated thus far.

The sphingolipids ceramide and inositol phosphorylceramide protect the Leishmania major membrane from sterol-specific toxins.

The accessibility of sterols in mammalian cells to exogenous sterol-binding agents has been well-described previously, but sterol accessibility in distantly related protozoa is unclear. The human pathogen Leishmania major uses sterols and sphingolipids distinct from those used in mammals. Sterols in mammalian cells can be sheltered from sterol-binding agents by membrane components, including sphingolipids, but the surface exposure of ergosterol in Leishmania remains unknown. Here, we used flow cytometry to test the ability of the L. major sphingolipids inositol phosphorylceramide (IPC) and ceramide to shelter ergosterol by preventing binding of the sterol-specific toxins streptolysin O and perfringolysin O and subsequent cytotoxicity. In contrast to mammalian systems, we found that Leishmania sphingolipids did not preclude toxin binding to sterols in the membrane. However, we show that IPC reduced cytotoxicity and that ceramide reduced perfringolysin O- but not streptolysin O-mediated cytotoxicity in cells. Furthermore, we demonstrate ceramide sensing was controlled by the toxin L3 loop, and that ceramide was sufficient to protect L. major promastigotes from the anti-leishmaniasis drug amphotericin B. Based on these results, we propose a mechanism whereby pore-forming toxins engage additional lipids like ceramide to determine the optimal environment to sustain pore formation. Thus, L. major could serve as a genetically tractable protozoan model organism for understanding toxin-membrane interactions.

Vibrio parahaemolyticus thermostable direct haemolysin induces non-classical programmed cell death despite caspase activation.

Thermostable direct haemolysin (TDH) is the key virulence factor secreted by the human gastroenteric bacterial pathogen Vibrio parahaemolyticus. TDH is a membrane-damaging pore-forming toxin. It evokes potent cytotoxicity, the mechanism of which still remains under-explored. Here, we have elucidated the mechanistic details of cell death response elicited by TDH. Employing Caco-2 intestinal epithelial cells and THP-1 monocytic cells, we show that TDH induces some of the hallmark features of apoptosis-like programmed cell death. TDH triggers caspase-3 and 7 activations in the THP-1 cells, while caspase-7 activation is observed in the Caco-2 cells. Interestingly, TDH appears to induce caspase-independent cell death. Higher XIAP level and lower Smac/Diablo level upon TDH intoxication provide plausible explanation for the functional inability of caspases in the THP-1 cells, in particular. Further exploration reveals that mitochondria play a central role in the TDH-induced cell death. TDH triggers mitochondrial damage, resulting in the release of AIF and endonuclease G, responsible for the execution of caspase-independent cell death. Among the other critical mediators of cell death, ROS is found to play an important role in the THP-1 cells, while PARP-1 appears to play a critical role in the Caco-2 cells. Altogether, our work provides critical new insights into the mechanism of cell death induction by TDH, showing a common central theme of non-classical programmed cell death. Our study also unravels the interplay of crucial molecules in the underlying signalling processes. Our findings add valuable insights into the role of TDH in the context of the host-pathogen interaction processes.

Caenorhabditis elegans LIN-24, a homolog of bacterial pore-forming toxin, protects the host from microbial infection.

Aerolysin-like pore-forming protein (af-PFP) superfamily members are double-edge swords that assist the bacterial infection but shied bacteria from the host by various mechanisms in some species including the toad Bombina maxima and zebrafish. While members of this family are widely expressed in all kingdoms, especially non-bacteria species, it remains unclear whether their anti-bacterial function is conserved. LIN-24 is an af-PFP that is constitutively expressed throughout the Caenorhabditis elegans lifespan. Here, we observed that LIN-24 knockdown reduced the maximum lifespan of worms. RNA-seq analysis identified 323 differentially expressed genes (DEGs) post-LIN-24 knockdown that were enriched in "immune response" and "lysosome pathway," suggesting a possible role for LIN-24 in resisting microbial infection. In line with this, we found that Pseudomonas aeruginosa 14 (PA14) infection induced LIN-24 expression, and that survival after PA14 infection was significantly reduced by LIN-24 knockdown. In contrast, LIN-24 overexpression (LIN-24-OE) conferred protection against PA14 infection, with worms showing longer survival time and reduced bacterial load. Weighted gene co-expression network analysis of LIN-24-OE worms showed that the highest correlation module was enriched in factors related to immunity and the defense response. Finally, by predicting transcription factors from RNA-seq data and knocking down candidate transcription factors in LIN-24-OE worms, we revealed that LIN-24 may protect worms against bacterial infection by stimulating DAF-16-mediated immune responses. These findings agree with our previous studies showing an anti-microbial role for the amphibian-derived af-PFP complex ßγ-CAT, suggesting that af-PFPs may play a conserved role in combatting microbial infections. Further research is needed to determine the roles this protein family plays in other physio-pathological processes, such as metabolism, longevity, and aging.

Cryo-EM structure of the octameric pore of Clostridium perfringens ß-toxin.

Clostridium perfringens is one of the most widely distributed and successful pathogens producing an impressive arsenal of toxins. One of the most potent toxins produced is the C. perfringens ß-toxin (CPB). This toxin is the main virulence factor of type C strains. We describe the cryo-electron microscopy (EM) structure of CPB oligomer. We show that CPB forms homo-octameric pores like the hetero-oligomeric pores of the bi-component leukocidins, with important differences in the receptor binding region and the N-terminal latch domain. Intriguingly, the octameric CPB pore complex contains a second 16-stranded ß-barrel protrusion atop of the cap domain that is formed by the N-termini of the eight protomers. We propose that CPB, together with the newly identified Epx toxins, is a member a new subclass of the hemolysin-like family. In addition, we show that the ß-barrel protrusion domain can be modified without affecting the pore-forming ability, thus making the pore particularly attractive for macromolecule sensing and nanotechnology. The cryo-EM structure of the octameric pore of CPB will facilitate future developments in both nanotechnology and basic research.

Glu289 residue in the pore-forming motif of Vibrio cholerae cytolysin is important for efficient ß-barrel pore formation.

Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging ß-barrel pore-forming toxin. Upon binding to the target membranes, VCC monomers first assemble into oligomeric prepore intermediates and subsequently transform into transmembrane ß-barrel pores. VCC harbors a designated pore-forming motif, which, during oligomeric pore formation, inserts into the membrane and generates a transmembrane ß-barrel scaffold. It remains an enigma how the molecular architecture of the pore-forming motif regulates the VCC pore-formation mechanism. Here, we show that a specific pore-forming motif residue, E289, plays crucial regulatory roles in the pore-formation mechanism of VCC. We find that the mutation of E289A drastically compromises pore-forming activity, without affecting the structural integrity and membrane-binding potential of the toxin monomers. Although our single-particle cryo-EM analysis reveals WT-like oligomeric ß-barrel pore formation by E289A-VCC in the membrane, we demonstrate that the mutant shows severely delayed kinetics in terms of pore-forming ability that can be rescued with elevated temperature conditions. We find that the pore-formation efficacy of E289A-VCC appears to be more profoundly dependent on temperature than that of the WT toxin. Our results suggest that the E289A mutation traps membrane-bound toxin molecules in the prepore-like intermediate state that is hindered from converting into the functional ß-barrel pores by a large energy barrier, thus highlighting the importance of this residue for the pore-formation mechanism of VCC.

Structural insights into thermostable direct hemolysin of Vibrio parahaemolyticus using single-particle cryo-EM.

Thermostable direct hemolysin (TDH) is a ~19 kDa, hemolytic pore-forming toxin from the gram-negative marine bacterium Vibrio parahaemolyticus, one of the causative agents of seafood-borne acute gastroenteritis and septicemia. Previous studies have established that TDH exists as a tetrameric assembly in physiological state; however, there is limited knowledge regarding the molecular arrangement of its disordered N-terminal region (NTR)-the absence of which has been shown to compromise TDH's hemolytic and cytotoxic abilities. In our current study, we have employed single-particle cryo-electron microscopy to resolve the solution-state structures of wild-type TDH and a TDH construct with deletion of the NTR (NTD), in order to investigate structural aspects of NTR on the overall tetrameric architecture. We observed that both TDH and NTD electron density maps, resolved at global resolutions of 4.5 and 4.2 Å, respectively, showed good correlation in their respective oligomeric architecture. Additionally, we were able to locate extra densities near the pore opening of TDH which might correspond to the disordered NTR. Surprisingly, under cryogenic conditions, we were also able to observe novel supramolecular assemblies of TDH tetramers, which we were able to resolve to 4.3 Å. We further investigated the tetrameric and inter-tetrameric interaction interfaces to elaborate upon the key residues involved in both TDH tetramers and TDH super assemblies. Our current structural study will aid in understanding the mechanistic aspects of this pore-forming toxin and the role of its disordered NTR in membrane interaction.

Wheat Pore-Forming Toxin-Like Protein Confers Broad-Spectrum Resistance to Fungal Pathogens in Arabidopsis.

Fusarium head blight (FHB), caused by the hemibiotrophic fungus Fusarium graminearum, is one of the major threats to global wheat productivity. A wheat pore-forming toxin-like (PFT) protein was previously reported to underlie Fhb1, the most widely used quantitative trait locus in FHB breeding programs worldwide. In the present work, wheat PFT was ectopically expressed in the model dicot plant Arabidopsis. Heterologous expression of wheat PFT in Arabidopsis provided a broad-spectrum quantitative resistance to fungal pathogens including F. graminearum, Colletotrichum higginsianum, Sclerotinia sclerotiorum, and Botrytis cinerea. However, there was no resistance to bacterial or oomycete pathogens Pseudomonas syringae and Phytophthora capsici, respectively in the transgenic Arabidopsis plants. To explore the reason for the resistance response to, exclusively, the fungal pathogens, purified PFT protein was hybridized to a glycan microarray having 300 different types of carbohydrate monomers and oligomers. It was found that PFT specifically hybridized with chitin monomer, N-acetyl glucosamine (GlcNAc), which is present in fungal cell walls but not in bacteria or oomycete species. This exclusive recognition of chitin may be responsible for the specificity of PFT-mediated resistance to fungal pathogens. Transfer of the atypical quantitative resistance of wheat PFT to a dicot system highlights its potential utility in designing broad-spectrum resistance in diverse host plants. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Characterization of tigurilysin, a novel human CD59-specific cholesterol-dependent cytolysin, reveals a role for host specificity in augmenting toxin activity.

Cholesterol-dependent cytolysins (CDCs) are a large family of pore-forming toxins, produced by numerous Gram-positive pathogens. CDCs depend on host membrane cholesterol for pore formation; some CDCs also require surface-associated human CD59 (hCD59) for binding, conferring specificity for human cells. We purified a recombinant version of a putative CDC encoded in the genome of Streptococcus oralis subsp. tigurinus, tigurilysin (TGY), and used CRISPR/Cas9 to construct hCD59 knockout (KO) HeLa and JEG-3 cell lines. Cell viability assays with TGY on wild-type and hCD59 KO cells showed that TGY is a hCD59-dependent CDC. Two variants of TGY exist among S. oralis subsp. tigurinus genomes, only one of which is functional. We discovered that a single amino acid change between these two TGY variants determines its activity. Flow cytometry and oligomerization Western blots revealed that the single amino acid difference between the two TGY isoforms disrupts host cell binding and oligomerization. Furthermore, experiments with hCD59 KO cells and cholesterol-depleted cells demonstrated that TGY is fully dependent on both hCD59 and cholesterol for activity, unlike other known hCD59-dependent CDCs. Using full-length CDCs and toxin constructs differing only in the binding domain, we determined that having hCD59 dependence leads to increased lysis efficiency, conferring a potential advantage to organisms producing hCD59-dependent CDCs.

Pore formation-independent cell death induced by a ß-barrel pore-forming toxin.

Vibrio cholerae cytolysin (VCC) is a ß-barrel pore-forming toxin (ß-PFT). It exhibits potent hemolytic activity against erythrocytes that appears to be a direct outcome of its pore-forming functionality. However, VCC-mediated cell-killing mechanism is more complicated in the case of nucleated mammalian cells. It induces apoptosis in the target nucleated cells, mechanistic details of which are still unclear. Furthermore, it has never been explored whether the ability of VCC to trigger programmed cell death is stringently dependent on its pore-forming activity. Here, we show that VCC can evoke hallmark features of the caspase-dependent apoptotic cell death even in the absence of the pore-forming ability. Our study demonstrates that VCC mutants with abortive pore-forming hemolytic activity can trigger apoptotic cell death responses and cytotoxicity, similar to those elicited by the wild-type toxin. VCC as well as its pore formation-deficient mutants display prominent propensity to translocate to the target cell mitochondria and cause mitochondrial membrane damage. Therefore, our results for the first time reveal that VCC, despite being an archetypical ß-PFT, can kill target nucleated cells independent of its pore-forming functionality. These findings are intriguing for a ß-PFT, whose destination is generally expected to remain limited on the target cell membranes, and whose mode of action is commonly attributed to the membrane-damaging pore-forming ability. Taken together, our study provides critical new insights regarding distinct implications of the two important virulence functionalities of VCC for the V. cholerae pathogenesis process: hemolytic activity for iron acquisition and cytotoxicity for tissue damage by the bacteria.

Utilizing Extraepitopic Amino Acid Substitutions to Define Changes in the Accessibility of Conformational Epitopes of the Bacillus cereus HlyII C-Terminal Domain.

Hemolysin II (HlyII)-one of the pathogenic factors of Bacillus cereus, a pore-forming ß-barrel toxin-possesses a C-terminal extension of 94 amino acid residues, designated as the C-terminal domain of HlyII (HlyIICTD), which plays an important role in the functioning of the toxin. Our previous work described a monoclonal antibody (HlyIIC-20), capable of strain-specific inhibition of hemolysis caused by HlyII, and demonstrated the dependence of the efficiency of hemolysis on the presence of proline at position 324 in HlyII outside the conformational antigenic determinant. In this work, we studied 16 mutant forms of HlyIICTD. Each of the mutations, obtained via multiple site-directed mutagenesis leading to the replacement of amino acid residues lying on the surface of the 3D structure of HlyIICTD, led to a decrease in the interaction of HlyIIC-20 with the mutant form of the protein. Changes in epitope structure confirm the high conformational mobility of HlyIICTD required for the functioning of HlyII. Comparison of the effect of the introduced mutations on the effectiveness of interactions between HlyIICTD and HlyIIC-20 and a control antibody recognizing a non-overlapping epitope enabled the identification of the amino acid residues N339 and K340, included in the conformational antigenic determinant recognized by HlyIIC-20.

Region Met225 to Ile412 of Bacillus cereus Hemolysin II Is Capable to Agglutinate Red Blood Cells.

Hemolysin II (HlyII) is one of the virulence factors of the opportunistic bacterium Bacillus cereus belonging to the group of ß-pore-forming toxins. This work created a genetic construct encoding a large C-terminal fragment of the toxin (HlyIILCTD, M225-I412 according to the numbering of amino acid residues in HlyII). A soluble form of HlyIILCTD was obtained using the SlyD chaperone protein. HlyIILCTD was first shown to be capable of agglutinating rabbit erythrocytes. Monoclonal antibodies against HlyIILCTD were obtained by hybridoma technology. We also proposed a mode of rabbit erythrocyte agglutination by HlyIILCTD and selected three anti-HlyIILCTD monoclonal antibodies that inhibited the agglutination.

Tyrosine in the hinge region of the pore-forming motif regulates oligomeric ß-barrel pore formation by Vibrio cholerae cytolysin.

ß-barrel pore-forming toxins perforate cell membranes by forming oligomeric ß-barrel pores. The most crucial step is the membrane-insertion of the pore-forming motifs that create the transmembrane ß-barrel scaffold. Molecular mechanism that regulates structural reorganization of these pore-forming motifs during ß-barrel pore-formation still remains elusive. Using Vibrio cholerae cytolysin as an archetypical example of the ß-barrel pore-forming toxin, we show that a key tyrosine residue (Y321) in the hinge region of the pore-forming motif plays crucial role in this process. Mutation of Y321 abrogates oligomerization of the membrane-bound toxin protomers, and blocks subsequent steps of pore-formation. Our study suggests that the presence of Y321 in the hinge region of the pore-forming motif is crucial for the toxin molecule to sense membrane-binding, and to trigger essential structural rearrangements required for the subsequent oligomerization and pore-formation process. Such a regulatory mechanism of pore-formation by V. cholerae cytolysin has not been documented earlier in the structurally related ß-barrel pore-forming toxins.

Toxin-Enabled "On-Demand" Liposomes for Enhanced Phototherapy to Treat and Protect against Methicillin-Resistant Staphylococcus aureus Infection.

An effective therapeutic strategy against methicillin-resistant Staphylococcus aureus (MRSA) that does not promote further drug resistance is highly desirable. While phototherapies have demonstrated considerable promise, their application toward bacterial infections can be limited by negative off-target effects to healthy cells. Here, a smart targeted nanoformulation consisting of a liquid perfluorocarbon core stabilized by a lipid membrane coating is developed. Using vancomycin as a targeting agent, the platform is capable of specifically delivering an encapsulated photosensitizer along with oxygen to sites of MRSA infection, where high concentrations of pore-forming toxins trigger on-demand payload release. Upon subsequent near-infrared irradiation, local increases in temperature and reactive oxygen species effectively kill the bacteria. Additionally, the secreted toxins that are captured by the nanoformulation can be processed by resident immune cells to promote multiantigenic immunity that protects against secondary MRSA infections. Overall, the reported approach for the on-demand release of phototherapeutic agents into sites of infection could be applied against a wide range of high-priority pathogens.

Membrane Dynamics and Remodelling in Response to the Action of the Membrane-Damaging Pore-Forming Toxins.

Pore-forming protein toxins (PFTs) represent a diverse class of membrane-damaging proteins that are produced by a wide variety of organisms. PFT-mediated membrane perforation is largely governed by the chemical composition and the physical properties of the plasma membranes. The interaction between the PFTs with the target membranes is critical for the initiation of the pore-formation process, and can lead to discrete membrane reorganization events that further aids in the process of pore-formation. Punching holes on the plasma membranes by the PFTs interferes with the cellular homeostasis by disrupting the ion-balance inside the cells that in turn can turn on multiple signalling cascades required to restore membrane integrity and cellular homeostasis. In this review, we discuss the physicochemical attributes of the plasma membranes associated with the pore-formation processes by the PFTs, and the subsequent membrane remodelling events that may start off the membrane-repair mechanisms.

Cholesterol-dependent cytolysins: The outstanding questions.

The cholesterol-dependent cytolysins (CDCs) are a major family of bacterial pore-forming proteins secreted as virulence factors by Gram-positive bacterial species. CDCs are produced as soluble, monomeric proteins that bind specifically to cholesterol-rich membranes, where they oligomerize into ring-shaped pores of more than 30 monomers. Understanding the details of the steps the toxin undergoes in converting from monomer to a membrane-spanning pore is a continuing challenge. In this review we summarize what we know about CDCs and highlight the remaining outstanding questions that require answers to obtain a complete picture of how these toxins kill cells.