Targeted small molecule inhibitors blocking the cytolytic effects of pneumolysin and homologous toxins.

Pneumolysin (PLY) is a cholesterol-dependent cytolysin (CDC) from Streptococcus pneumoniae, the main cause for bacterial pneumonia. Liberation of PLY during infection leads to compromised immune system and cytolytic cell death. Here, we report discovery, development, and validation of targeted small molecule inhibitors of PLY (pore-blockers, PB). PB-1 is a virtual screening hit inhibiting PLY-mediated hemolysis. Structural optimization provides PB-2 with improved efficacy. Cryo-electron tomography reveals that PB-2 blocks PLY-binding to cholesterol-containing membranes and subsequent pore formation. Scaffold-hopping delivers PB-3 with superior chemical stability and solubility. PB-3, formed in a protein-templated reaction, binds to Cys428 adjacent to the cholesterol recognition domain of PLY with a KD of 256 nM and a residence time of 2000 s. It acts as anti-virulence factor preventing human lung epithelial cells from PLY-mediated cytolysis and cell death during infection with Streptococcus pneumoniae and is active against the homologous Cys-containing CDC perfringolysin (PFO) as well.

Delivery of membrane impermeable molecules to primary mouse T lymphocytes.

The pore-forming toxin streptolysin-O (SLO) enables intracellular delivery of molecules up to 100 kDa and has been used for short-term delivery of membrane-impermeable substances to assess their effects on cellular activities. A limitation of this technique is the loss of intracellular components and the potential unpredicted alterations of cellular metabolism and signaling. This protocol, optimized for primary mouse T lymphocytes, describes steps for SLO-mediated cell membrane permeabilization and substance supplementation, followed by immunoblotting and immunofluorescent microscopy for assessing cellular effects. For complete details on the use and execution of this protocol, please refer to Xu et al., 2021a, Xu et al., 2021b.

Mapping of Recognition Sites of Monoclonal Antibodies Responsible for the Inhibition of Pneumolysin Functional Activity.

The pathogenicity of many bacteria, including Streptococcus pneumoniae, depends on pore-forming toxins (PFTs) that cause host cell lysis by forming large pores in cholesterol-containing cell membranes. Therefore, PFTs-neutralising antibodies may provide useful tools for reducing S. pneumoniae pathogenic effects. This study aimed at the development and characterisation of monoclonal antibodies (MAbs) with neutralising activity to S. pneumoniae PFT pneumolysin (PLY). Five out of 10 produced MAbs were able to neutralise the cytolytic activity of PLY on a lung epithelial cell line. Epitope mapping with a series of recombinant overlapping PLY fragments revealed that neutralising MAbs are directed against PLY loops L1 and L3 within domain 4. The epitopes of MAbs 3A9, 6E5 and 12F11 located at L1 loop (aa 454-471) were crucial for PLY binding to the immobilised cholesterol. In contrast, the MAb 12D10 recognising L3 (aa 403-423) and the MAb 3F3 against the conformational epitope did not interfere with PLY-cholesterol interaction. Due to conformation-dependent binding, the approach to use overlapping peptides for fine epitope mapping of the neutralising MAbs was unsuccessful. Therefore, the epitopes recognised by the MAbs were analysed using computational methods. This study provides new data on PLY sites involved in functional activity.

Domain 4 of pneumolysin from Streptococcus pneumoniae is a multifunctional domain contributing TLR4 activating and hemolytic activity.

The pneumolysin (Ply) protein of Streptococcus pneumoniae is composed of four domains and possesses several different but related activities. In this study, recombinant Ply and two truncated forms, Ply domain 1-3 and Ply domain 4 (rPly4), were expressed and characterized regarding their participation in apoptosis, the stimulation of cytokine production, hemolytic activity and virulence. rPly4 activated murine bone marrow-derived dendritic cells in a Toll-like receptor (TLR) 4-dependent manner. The rPly4 alone was able to produce hemolytic activity at high concertation and penetrate the lipid bilayer. We further demonstrated that domain 4 of Ply involved in the virulence of the bacteria in mouse model. In the absence of apoptotic activity, the virulence level caused by rPly4 was similar to that of full length Ply. Our data suggested that domain 4 of Ply alone with TLR4 agonist and hemolytic activity may play roles in virulence of Streptococcus pneumoniae.

Intrinsic repair protects cells from pore-forming toxins by microvesicle shedding.

Pore-forming toxins (PFTs) are used by both the immune system and by pathogens to disrupt cell membranes. Cells attempt to repair this disruption in various ways, but the exact mechanism(s) that cells use are not fully understood, nor agreed upon. Current models for membrane repair include (1) patch formation (e.g., fusion of internal vesicles with plasma membrane defects), (2) endocytosis of the pores, and (3) shedding of the pores by blebbing from the cell membrane. In this study, we sought to determine the specific mechanism(s) that cells use to resist three different cholesterol-dependent PFTs: Streptolysin O, Perfringolysin O, and Intermedilysin. We found that all three toxins were shed from cells by blebbing from the cell membrane on extracellular microvesicles (MVs). Unique among the cells studied, we found that macrophages were 10 times more resistant to the toxins, yet they shed significantly smaller vesicles than the other cells. To examine the mechanism of shedding, we tested whether toxins with engineered defects in pore formation or oligomerization were shed. We found that oligomerization was necessary and sufficient for membrane shedding, suggesting that calcium influx and patch formation were not required for shedding. However, pore formation enhanced shedding, suggesting that calcium influx and patch formation enhance repair. In contrast, monomeric toxins were endocytosed. These data indicate that cells use two interrelated mechanisms of membrane repair: lipid-dependent MV shedding, which we term 'intrinsic repair', and patch formation by intracellular organelles. Endocytosis may act after membrane repair is complete by removing inactivated and monomeric toxins from the cell surface.

In vivo efficacy and molecular docking of designed peptide that exhibits potent antipneumococcal activity and synergises in combination with penicillin.

We have previously designed a series of antimicrobial peptides (AMPs) and in the current study, the in vivo therapeutic efficacy and toxicity were investigated. Among all the peptides, DM3 conferred protection to a substantial proportion of the lethally infected mice caused by a strain of penicillin-resistant Streptococcus pneumoniae. Synergism was reported and therapeutic efficacy was significantly enhanced when DM3 was formulated in combination with penicillin (PEN). No toxicity was observed in mice receiving these treatments. The in silico molecular docking study results showed that, DM3 has a strong affinity towards three protein targets; autolysin and pneumococcal surface protein A (pspA). Thus AMPs could serve as supporting therapeutics in combination with conventional antibiotics to enhance treatment outcome.

The cholesterol-dependent cytolysins pneumolysin and streptolysin O require binding to red blood cell glycans for hemolytic activity.

The cholesterol-dependent cytolysin (CDC) pneumolysin (Ply) is a key virulence factor of Streptococcus pneumoniae. Membrane cholesterol is required for the cytolytic activity of this toxin, but it is not clear whether cholesterol is the only cellular receptor. Analysis of Ply binding to a glycan microarray revealed that Ply has lectin activity and binds glycans, including the Lewis histo-blood group antigens. Surface plasmon resonance analysis showed that Ply has the highest affinity for the sialyl LewisX (sLeX) structure, with a K(d) of 1.88 × 10(-5) M. Ply hemolytic activity against human RBCs showed dose-dependent inhibition by sLeX. Flow cytometric analysis and Western blots showed that blocking binding of Ply to the sLeX glycolipid on RBCs prevents deposition of the toxin in the membrane. The lectin domain responsible for sLeX binding is in domain 4 of Ply, which contains candidate carbohydrate-binding sites. Mutagenesis of these predicted carbohydrate-binding residues of Ply resulted in a decrease in hemolytic activity and a reduced affinity for sLeX. This study reveals that this archetypal CDC requires interaction with the sLeX glycolipid cellular receptor as an essential step before membrane insertion. A similar analysis conducted on streptolysin O from Streptococcus pyogenes revealed that this CDC also has glycan-binding properties and that hemolytic activity against RBCs can be blocked with the glycan lacto-N-neotetraose by inhibiting binding to the cell surface. Together, these data support the emerging paradigm shift that pore-forming toxins, including CDCs, have cellular receptors other than cholesterol that define target cell tropism.

Molecular analysis of Streptococcus anginosus-derived SagA peptides.

BACKGROUND: SagA1 and SagA2 molecules produced from beta-hemolytic Streptococcus anginosus subsp. anginosus are composed of a leader peptide and a propeptide, and their mature form has hemolytic activity as a well-known Streptococcal peptide toxin, streptolysin. The function of these SagA molecules is thought to be dependent on intra-molecular heterocycle formation. In this study, we examined the heterocycle-involved molecular features of SagA1, SagA2, and S. pyogenes SagA (SPySagA), focusing on their heterocycle formation. MATERIALS AND METHODS: Molecular models of SagA1, SagA2, and SPySagA were constructed using a molecular modeling technique. Molecular dynamics and molecular mechanic analyses of the modeled SagA molecules were performed to obtain their energy profiles. RESULTS: Total energy of the modeled SagA1, SagA2, and SPySagA decreased with heterocycle formation, and the border between the leader peptide and propeptide was clearly observed after heterocycle formation. CONCLUSION: The flexibility of SagA molecules was changed by intramolecular heterocycle formation, and their function (e.g. hemolytic activity) seems to be regulated by structural transition with heterocycle formation.

Visualization of bacterial toxin induced responses using live cell fluorescence microscopy.

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type(1). In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines(2). Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms. Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism(3,4). This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation(4). In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines(5,6,7). A link between IL-1ß release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes(8). To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Neutralizing antibodies elicited by a novel detoxified pneumolysin derivative, PlyD1, provide protection against both pneumococcal infection and lung injury.

Streptococcus pneumoniae pneumolysin (PLY) is a virulence factor that causes toxic effects contributing to pneumococcal pneumonia. To date, deriving a PLY candidate vaccine with the appropriate detoxification and immune profile has been challenging. A pneumolysin protein that is appropriately detoxified and that retains its immunogenicity is a desirable vaccine candidate. In this study, we assessed the protective efficacy of our novel PlyD1 detoxified PLY variant and investigated its underlying mechanism of protection. Results have shown that PlyD1 immunization protected mice against lethal intranasal (i.n.) challenge with pneumococci and lung injury mediated by PLY challenge. Protection was associated with PlyD1-specific IgG titers and in vitro neutralization titers. Pretreatment of PLY with PlyD1-specific rat polyclonal antiserum prior to i.n. delivery of toxin reduced PLY-mediated lung lesions, interleukin-6 (IL-6) production, and neutrophil infiltration into lungs, indicating that protection from lung lesions induced by PLY is antibody mediated. Preincubation of PLY with a neutralizing monoclonal PLY antibody also specifically reduced the cytotoxic effects of PLY after i.n. inoculation in comparison to nonneutralizing monoclonal antibodies. These results indicate that the induction of neutralizing antibodies against PLY can contribute to protection against bacterial pneumonia by preventing the development of PLY-induced lung lesions and inflammation. Our detoxified PlyD1 antigen elicits such PLY neutralizing antibodies, thus serving as a candidate vaccine antigen for the prevention of pneumococcal pneumonia.

Time course of virulence factors produced by group A streptococcus during a food-borne epidemic.

We studied the protein amount and activity of the major virulence factors hemolysin, cysteine protease streptococcal pyrogenic exotoxin B (SpeB), and NAD glycohydrolase (NADase), which are produced by Streptococcus pyogenes type T-25, with a food poisoning outbreak. The three virulence factors were analyzed by activity and amount of protein using supernatants at 2-30 h of culture. All these virulence factors were confirmed by their activity. Streptolysin O (SLO), SpeB, and NADase were immunochemically confirmed at protein level by Western blot analysis. Two hemolytic forms (70 and 60 kDa) of SLO were identified. SpeB was detected as a 44-kDa precursor form and a 30-kDa mature form. NADase was 50 kDa. SLO protein peaked at 8 h of culture, which corresponded with the hemolytic activity peak. Conversion from precursor to SpeB protein peaked at 14 h of culture. The conversion peak corresponded to the activity expression time. Also, mature SpeB protein peaked at 24 h of culture and corresponded to SpeB activity peak. Electrophoretic analysis clarified the relationship between SLO protein and SpeB protein, although amounts of SLO and SpeB have been reported to be inversely proportional to activity. NADase protein peaked at 12 h of culture, but protein level did not correspond to the peak. Because the NADase protein peak was closer to SpeB activity than SLO protein, our results suggested NADase protein was degraded at 12 h of culture. The time course production of these virulence factors is discussed.

Properties of metabolic substances produced by group A streptococcus from a food-borne epidemic.

Here we report a large food poisoning outbreak by Streptococcus pyogenes that occurred in Kanagawa, Japan, in July 2005. To compare cases of type T-B3264 (Chiba) and type T-28 (Tokyo) reported to date, we studied the properties and activity of the major virulence factors produced by Streptococcus pyogenes type T-25 (Kanagawa): hemolysin, cysteine protease streptococcal pyrogenic exotoxin B (SpeB), and NAD glycohydrolase (NADase). These virulence factors were also analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The titer of hemolysin was 9 50% hemolytic dose (HD(50)) per milliliter (HD(50)/ml) for T-25, 173 HD(50)/ml for T-28, and 147 HD(50)/ml for T-B3264. The hemolytic titer of T-25 was very low compared with those of T-28 and T-B3264. Each hemolysin produced by the three strains was dependent on its reductant, and its properties differed among strains. The major hemolysin of T-25 was identified as streptolysin O (SLO), because cholesterol or γ-globulin, but not phospholipids, inhibited its hemolysis. In contrast, the major hemolysin of T-28 and T-B3264 was streptolysin S (SLS). Although the SpeB activity of T-25 (4.8 U/ml) was lower than that of T-B3264, its NADase activity (19.1 U) was the largest of the three strains. The conversion from the SpeB precursor to mature SpeB was confirmed by SDS-PAGE analysis of T-25 at 6 h of culture; no conversion was identified for T-28 and T-B3264 at 6 h. SpeB of T-25 was converted quickly, most likely because of the degradation of SLO by SpeB, thereby resulting in the very low hemolytic titer of T-25. These results suggest that the three strains have diverse properties and activities of major virulence factors. The specific interactions of these virulence factors are thought to be involved in the pathosis of these strains.

Streptococcus pyogenes cytolysin-mediated translocation does not require pore formation by streptolysin O.

Bacterial toxin injection into the host cell is required for the virulence of numerous pathogenic bacteria. Cytolysin-mediated translocation (CMT) of Streptococcus pyogenes uses streptolysin O (SLO) to translocate the S. pyogenes nicotinamide adenine dinucleotide-glycohydrolase (SPN) into the host cell cytosol, resulting in the death of the host cell. Although SLO is a pore-forming protein, previous studies have shown that pore formation alone is not sufficient for CMT to occur. Thus, the role and requirement of the SLO pore remains unclear. In this study, we constructed various S. pyogenes strains expressing altered forms of SLO to assess the importance of pore formation. We observed that SLO mutants that are unable to form pores retain the ability to translocate SPN. In addition, SPN translocation occurs after inhibition of actin polymerization, suggesting that CMT occurs independently of clathrin-mediated endocytosis. Moreover, despite the ability of mutants to translocate SPN, their cytotoxic effect requires SLO pore formation.

Oriented immobilization of anti-pneumolysin tagged recombinant antibody fragments.

Recombinant antibodies such as Fab and scFv are monovalent and small in size, although their functional affinity can be improved through tag-specific immobilization. In order to find the optimum candidate for oriented immobilization, we generated Fab and scFv fragments derived from an anti-pneumolysin monoclonal antibody PLY-7, with histidine and cysteine residues added in diverse arrangements. Tagged antibody fragments scFv-Cys7-His6, His6-scFv-Cys7, and Fab-Cys7 lost considerable affinity for the antigen; however, Fab-His6, Fab-Cys1, and scFv-His6-Cys1 were able to detect immobilized antigen, revealing that the position and number of histidine and cysteine residues are involved differently in the reactivity of antibody fragments. Random and orientated immobilizations were carried out using conventional polystyrene and commercial surface-pretreated ELISA plates. The best orientation performance was obtained with Fab-Cys1-biotin on streptavidin-coated plates with increased signal levels of 62%, while oriented immobilization of Fab-His6 and scFv-His6-Cys1 on nickel- and maleimide-coated plates failed to improve the ELISA sensitivity.

Epithelial cells are sensitive detectors of bacterial pore-forming toxins.

Epithelial cells act as an interface between human mucosal surfaces and the surrounding environment. As a result, they are responsible for the initiation of local immune responses, which may be crucial for prevention of invasive infection. Here we show that epithelial cells detect the presence of bacterial pore-forming toxins (including pneumolysin from Streptococcus pneumoniae, alpha-hemolysin from Staphylococcus aureus, streptolysin O from Streptococcus pyogenes, and anthrolysin O from Bacillus anthracis) at nanomolar concentrations, far below those required to cause cytolysis. Phosphorylation of p38 MAPK appears to be a conserved response of epithelial cells to subcytolytic concentrations of bacterial poreforming toxins, and this activity is inhibited by the addition of high molecular weight osmolytes to the extracellular medium. By sensing osmotic stress caused by the insertion of a sublethal number of pores into their membranes, epithelial cells may act as an early warning system to commence an immune response, while the local density of toxin-producing bacteria remains low. Osmosensing may thus represent a novel innate immune response to a common bacterial virulence strategy.

Molecular dynamics of human-specific cytolysin: analysis of membrane binding motif for therapeutic application.

BACKGROUND: Intermedilysin (ILY) is a human-specific cytolysin secreted from Streptococcus intermedius. In this study, the dynamic structure of ILY, StreptolysinO (SLO) and their 12mer substituted mutants for 500 ps was analyzed. Several parameters, such as dipole moment and electrostatic potential, were determined to elucidate the molecular mechanism of membrane binding. MATERIALS AND METHODS: Molecular models of lLY, SLO and their mutants were constructed using Insightll-Discover with the Homology module. Their molecular dynamics were simulated with the Discover3 (Insight module), and z-matrix data of the membrane-binding 12mer region were extracted to calculate the molecular orbital (MO) parameters (i.e., dipole moment, solvation free energy (dGW)). RESULTS: Cytolysins vibrated like a bow, and the dipole moment direction of ILY 12mer region was different from that of SLO. Certain ILY mutants indicated the SLO-like dipole properties, which had an SLO-type limer cysteine motif amino acid sequence. The ILY 11mer region was more hydrophobic than that of SLO, and seemed to interact easily with the cell membrane without cholesterol. The electrostatic potential field distribution of ILY differed from that of SLO, especially in the 11mer region. CONCLUSION: In the 11mer region, the dipole moment directions of these cytolysins were constant during molecular movement, and ready to interact with membrane components (i.e., cholesterol, phospholipid).

Inhibition of the membrane fusion machinery prevents exit from the TGN and proteolytic processing by furin.

The Semliki Forest virus (SFV) glycoprotein precursor p62 is processed to the E2 and E3 during the transport from the trans-Golgi network (TGN) to the cell surface. We have studied the regulation of the membrane fusion machinery (Rab/N-ethylmaleimide (NEM)-sensitive fusion protein (NSF)/soluble NSF attachment protein (SNAP)-SNAP receptor) in this processing. Activation of the disassembly of this complex with recombinant NSF stimulated the cleavage of p62 in permeabilized cells. Inactivation of NSF with a mutant alpha-SNAP(L294A) or NEM treatment inhibited processing of p62. Rab GDP dissociation inhibitor inhibited the cleavage. Inactivation of NSF blocks the transport of SFV glycoproteins and vesicular stomatitis virus G-glycoprotein from the TGN membranes to the cell surface. The results support the conclusion that inhibition of membrane fusion arrests p62 in the TGN and prevents its processing by furin.

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

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

Protective immunity and gene expression related to pneumococcal glycoconjugate.

The immunogenicity of most polysaccharides (PSs) contained in the pneumococcal vaccine is low in children less than 2 years of age. Enhancement of immune response in early life can be induced by immunization of pneumococcal glycoconjugate as well as plasma DNA coding for cell-surface protein antigen. Pneumococcal type 19F PS conjugated with inactivated pneumolysin (Ply) induced in mice remarkable antibody responses to both type 19F PS and the protein carrier. In addition, the conjugate was administered to pregnant mice during gestation and/or lactation, and to their offspring during early infancy. When the young mice were challenged with type 19F pneumococci, the bacteria were cleared more rapidly from the blood of immunized mice than from that of the control group. The mortality rate of young mice from immunized mothers was also significantly lower than the control group. These results indicate that the effective protective immunity against pneumococcal infection can be induced in young mice by the maternal immunization with the glycoconjugate during gestation and at early infancy. Studies have been conducted to express type 19A pneumolysin gene (ply) in mammalian cells. Ply DNA was inserted into the cloning site of a vector containing CMV promoter. The recombinant plasmid DNA containing ply was transfected in human rhabdomyosarcoma cells and the gene expression was confirmed by immunoblot. Injection of mice three times 50-100 ug per dose ply DNA produced high serum levels of Ply IgG and IgM antibodies and showed rapid bacterial clearance from the blood.

Streptolysin O: inhibition of the conformational change during membrane binding of the monomer prevents oligomerization and pore formation.

Streptolysin O is a four-domain protein toxin that permeabilizes animal cell membranes. The toxin first binds as a monomer to membrane cholesterol and subsequently assembles into oligomeric transmembrane pores. Binding is mediated by a C-terminally located tryptophan-rich motif. In a previous study, conformational effects of membrane binding were characterized by introducing single mutant cysteine residues that were then thiol-specifically derivatized with the environmentally sensitive fluorophoracrylodan. Membrane binding of the labeled proteins was accompanied by spectral shifts of the probe fluorescence, suggesting that the toxin molecule had undergone a conformational change. Here we provide evidence that this change corresponds to an allosteric transition of the toxin monomer that is required for the subsequent oligomerization and pore formation. The conformational change is reversible with reversal of binding, and it is related to temperature in a fashion that closely parallels the temperature-dependency of oligomerization. Furthermore, we describe a point mutation (N402E) that, while compatible with membrane binding, abrogates the accompanying conformational change. At the same time, the N402E mutation also abolishes oligomerization. These findings corroborate the contention that the target membrane acts as an allosteric effector to activate the oligomerizing and pore-forming capability of streptolysin O.