Family Of Pore-forming Toxins Research Articles

Two pairs of back-to-back α-helices of Kingella kingae RtxA toxin are crucial for the formation of a membrane pore.

The RtxA cytotoxin, a member of the RTX (Repeats in ToXin) family of pore-forming toxins, is the primary virulence factor of the paediatric facultative pathogen Kingella kingae. Although structure-function studies of RTX toxins have defined their characteristic domains and features, the exact membrane topology of RTX toxins remains unknown. Here, we used labelling of cell-bound RtxA with a membrane-impermeable, lysine-reactive reagent and subsequent detection of the labelled lysine residues by mass spectrometry, which revealed that most of the membrane-bound toxin is localised extracellularly. A trypsin protection assay with cell-bound RtxA demonstrated that five of seven transmembrane α-helices, predicted by various algorithms within the N-terminal half of the molecule, are irreversibly embedded in the membrane. Structure-function analysis showed that these α-helices, four of which are arranged as two pairs of back-to-back helices, are essential for the formation of an ion-conducting membrane pore. In contrast, the C-terminal half of RtxA is required for the interaction with the cell surface and for the irreversible insertion of the toxin into the membrane via acyl chains covalently linked to the molecule. These findings advance our understanding of the structure-function relationships of RtxA and enable us to propose a membrane topology model of the toxin.

Distant relatives of a eukaryotic cell-specific toxin family evolved a complement-like mechanism to kill bacteria

Cholesterol-dependent cytolysins (CDCs) comprise a large family of pore-forming toxins produced by Gram-positive bacteria, which are used to attack eukaryotic cells. Here, we functionally characterize a family of 2-component CDC-like (CDCL) toxins produced by the Gram-negative Bacteroidota that form pores by a mechanism only described for the mammalian complement membrane attack complex (MAC). We further show that the Bacteroides CDCLs are not eukaryotic cell toxins like the CDCs, but instead bind to and are proteolytically activated on the surface of closely related species, resulting in pore formation and cell death. The CDCL-producing Bacteroides is protected from the effects of its own CDCL by the presence of a surface lipoprotein that blocks CDCL pore formation. These studies suggest a prevalent mode of bacterial antagonism by a family of two-component CDCLs that function like mammalian MAC and that are wide-spread in the gut microbiota of diverse human populations.

Unlatching of the stem domains in the Staphylococcus aureus pore-forming leukocidin LukAB influences toxin oligomerization

Staphylococcus aureus (S.aureus) is a serious global pathogen that causes a diverse range of invasive diseases. S.aureus utilizes a family of pore-forming toxins, known as bi-component leukocidins, to evade the host immune response and promote infection. Among these is LukAB (leukocidin A/leukocidin B), a toxin that assembles into an octameric β-barrel pore in the target cell membrane, resulting in host cell death. The established cellular receptor for LukAB is CD11b of the Mac-1 complex. Here, we show that hydrogen voltage-gated channel 1 is also required for the cytotoxicity of all major LukAB variants. We demonstrate that while each receptor is sufficient to recruit LukAB to the plasma membrane, both receptors are required for maximal lytic activity. Why LukAB requires two receptors, and how each of these receptors contributes to pore-formation remains unknown. To begin to resolve this, we performed an alanine scanning mutagenesis screen to identify mutations that allow LukAB to maintain cytotoxicity without CD11b. We discovered 30 mutations primarily localized in the stem domains of LukA and LukB that enable LukAB to exhibit full cytotoxicity in the absence of CD11b. Using crosslinking, electron microscopy, and hydroxyl radical protein footprinting, we show these mutations increase the solvent accessibility of the stem domain, priming LukAB for oligomerization. Together, our data support a model in which CD11b binding unlatches the membrane penetrating stem domains of LukAB, and this change in flexibility promotes toxin oligomerization.

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.

Recent insights into mechanisms of cellular toxicity and cell recognition associated with the ABC family of pore-forming toxins.

ABC toxins are pore-forming toxins characterised by the presence of three distinct components assembled into a hetero-oligomeric toxin complex ranging in size from 1.5-2.5 MDa. Most ABC toxins studied to date appear to be insecticidal toxins, although genes predicted to encode for homologous assemblies have also been found in human pathogens. In insects, they are delivered to the midgut either directly via the gastrointestinal tract, or via a nematode symbiont, where they attack the epithelial cells and rapidly trigger widespread cell death. At the molecular level, the homopentameric A subunit is responsible for binding to lipid bilayer membranes and introducing a protein translocation pore, through which a cytotoxic effector - encoded at the C-terminus of the C subunit - is delivered. The B subunit forms a protective cocoon that encapsulates the cytotoxic effector, part of which is contributed by the N-terminus of the C subunit. The latter also includes a protease motif that cleaves the cytotoxic effector, releasing it into the pore lumen. Here, we discuss and review recent studies that begin to explain how ABC toxins selectively target specific cells, establishing host tropism, and how different cytotoxic effectors trigger cell death. These findings allow for a more complete understanding of how ABC toxins function in an in vivo context, which in turn provides a stronger foundation for understanding how they cause disease in invertebrate (and potentially also vertebrate) hosts, and how they might be re-engineered for therapeutic or biotechnological purposes.

The Crystal Structure of Bacillus cereus HblL1.

The Hbl toxin is a three-component haemolytic complex produced by Bacillus cereus sensu lato strains and implicated as a cause of diarrhoea in B. cereus food poisoning. While the structure of the HblB component of this toxin is known, the structures of the other components are unresolved. Here, we describe the expression of the recombinant HblL1 component and the elucidation of its structure to 1.36 Å. Like HblB, it is a member of the alpha-helical pore-forming toxin family. In comparison to other members of this group, it has an extended hydrophobic beta tongue region that may be involved in pore formation. Molecular docking was used to predict possible interactions between HblL1 and HblB, and suggests a head to tail dimer might form, burying the HblL1 beta tongue region.

Structural and functional characterization of sticholysin III: A newly discovered actinoporin within the venom of the sea anemone Stichodactyla helianthus

Actinoporins are a family of pore-forming toxins produced by sea anemones as part of their venomous cocktail. These proteins remain soluble and stably folded in aqueous solution, but when interacting with sphingomyelin-containing lipid membranes, they become integral oligomeric membrane structures that form a pore permeable to cations, which leads to cell death by osmotic shock. Actinoporins appear as multigenic families within the genome of sea anemones: several genes encoding very similar actinoporins are detected within the same species. The Caribbean Sea anemone Stichodactyla helianthus produces three actinoporins (sticholysins I, II and III; StnI, StnII and StnIII) that differ in their toxic potency. For example, StnII is about four-fold more effective than StnI against sheep erythrocytes in causing hemolysis, and both show synergy. However, StnIII, recently discovered in the S. helianthus transcriptome, has not been characterized so far. Here we describe StnIII's spectroscopic and functional properties and show its potential to interact with the other Stns. StnIII seems to maintain the well-preserved fold of all actinoporins, characterized by a high content of β-sheet, but it is significantly less thermostable. Its functional characterization shows that the critical concentration needed to form active pores is higher than for either StnI or StnII, suggesting differences in behavior when oligomerizing on membrane surfaces. Our results show that StnIII is an interesting and unexpected piece in the puzzle of how this Caribbean Sea anemone species modulates its venomous activity.

Actinoporins: From the Structure and Function to the Generation of Biotechnological and Therapeutic Tools.

Actinoporins (APs) are a family of pore-forming toxins (PFTs) from sea anemones. These biomolecules exhibit the ability to exist as soluble monomers within an aqueous medium or as constitutively open oligomers in biological membranes. Through their conformational plasticity, actinoporins are considered good candidate molecules to be included for the rational design of molecular tools, such as immunotoxins directed against tumor cells and stochastic biosensors based on nanopores to analyze unique DNA or protein molecules. Additionally, the ability of these proteins to bind to sphingomyelin (SM) facilitates their use for the design of molecular probes to identify SM in the cells. The immunomodulatory activity of actinoporins in liposomal formulations for vaccine development has also been evaluated. In this review, we describe the potential of actinoporins for use in the development of molecular tools that could be used for possible medical and biotechnological applications.

Listeriolysin O: A phagosome-specific cytolysin revisited.

Listeriolysin O (LLO) is an essential determinant of Listeria monocytogenes pathogenesis that mediates the escape of L.monocytogenes from host cell vacuoles, thereby allowing replication in the cytosol without causing appreciable cell death. As a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins, LLO is unique in that it is secreted by a facultative intracellular pathogen, whereas all other CDCs are produced by pathogens that are largely extracellular. Replacement of LLO with other CDCs results in strains that are extremely cytotoxic and 10,000-fold less virulent in mice. LLO has structural and regulatory features that allow it to function intracellularly without causing cell death, most of which map to a unique N-terminal region of LLO referred to as the proline, glutamic acid, serine, threonine (PEST)-like sequence. Yet, while LLO has unique properties required for its intracellular site of action, extracellular LLO, like other CDCs, affects cells in a myriad of ways. Because all CDCs form pores in cholesterol-containing membranes that lead to rapid Ca2+ influx and K+ efflux, they consequently trigger a wide range of host cell responses, including mitogen-activated protein kinase activation, histone modification, and caspase-1 activation. There is no debate that extracellular LLO, like all other CDCs, can stimulate multiple cellular activities, but the primary question we wish to address in this perspective is whether these activities contribute to L.monocytogenes pathogenesis.

Enterotoxic Clostridia: Clostridium perfringens Enteric Diseases.

In humans and livestock, Clostridium perfringens is an important cause of intestinal infections that manifest as enteritis, enterocolitis, or enterotoxemia. This virulence is largely related to the toxin-producing ability of C. perfringens. This article primarily focuses on the C. perfringens type F strains that cause a very common type of human food poisoning and many cases of nonfoodborne human gastrointestinal diseases. The enteric virulence of type F strains is dependent on their ability to produce C. perfringens enterotoxin (CPE). CPE has a unique amino acid sequence but belongs structurally to the aerolysin pore-forming toxin family. The action of CPE begins with binding of the toxin to claudin receptors, followed by oligomerization of the bound toxin into a prepore on the host membrane surface. Each CPE molecule in the prepore then extends a beta-hairpin to form, collectively, a beta-barrel membrane pore that kills cells by increasing calcium influx. The cpe gene is typically encoded on the chromosome of type F food poisoning strains but is encoded by conjugative plasmids in nonfoodborne human gastrointestinal disease type F strains. During disease, CPE is produced when C. perfringens sporulates in the intestines. Beyond type F strains, C. perfringens type C strains producing beta-toxin and type A strains producing a toxin named CPILE or BEC have been associated with human intestinal infections. C. perfringens is also an important cause of enteritis, enterocolitis, and enterotoxemia in livestock and poultry due to intestinal growth and toxin production.

Multigene Family of Pore-Forming Toxins from Sea Anemone Heteractis crispa.

Sea anemones produce pore-forming toxins, actinoporins, which are interesting as tools for cytoplasmic membranes study, as well as being potential therapeutic agents for cancer therapy. This investigation is devoted to structural and functional study of the Heteractis crispa actinoporins diversity. Here, we described a multigene family consisting of 47 representatives expressed in the sea anemone tentacles as prepropeptide-coding transcripts. The phylogenetic analysis revealed that actinoporin clustering is consistent with the division of sea anemones into superfamilies and families. The transcriptomes of both H. crispa and Heteractis magnifica appear to contain a large repertoire of similar genes representing a rapid expansion of the actinoporin family due to gene duplication and sequence divergence. The presence of the most abundant specific group of actinoporins in H. crispa is the major difference between these species. The functional analysis of six recombinant actinoporins revealed that H. crispa actinoporin grouping was consistent with the different hemolytic activity of their representatives. According to molecular modeling data, we assume that the direction of the N-terminal dipole moment tightly reflects the actinoporins’ ability to possess hemolytic activity.

Pore-forming toxins impair pro-inflammatory macrophage responses

Abstract Necrotizing soft tissue infections (NSTIs) are lethal microbial infections. One virulence factor common to most bacteria responsible for NSTIs such as Streptococcus pyogenes is a pore-forming toxin (PFT). PFTs promote immune evasion through unknown mechanisms and lyse cells by forming pores. One mechanism by which cells resist these PFTs is intrinsic membrane repair which is sequestering and shedding toxin pores on microvesicles. It is possible that intrinsic membrane repair is hijacked by bacteria to promote immune evasion. We hypothesized that PFTs promote immune evasion by inducing immune receptor shedding during intrinsic membrane repair. We tested this hypothesis by challenging murine bone marrow derived macrophages with sublytic toxin doses prior to LPS or IFNγ challenge. We measured surface receptor expression and intracellular TNFα by flow cytometry in live cells. We found that toxins from one family of PFTs, cholesterol-dependent cytolysins (CDCs), suppressed LPS or IFNγ-stimulated TNFα production and CD69 and CD86 upregulation in a cell-intrinsic manner independently of cell death. CDCs further induced partial shedding of TLR4 and IFNγR, which might contribute to macrophage dysfunction. In contrast, we found that a toxin from a second PFT family, aerolysin, prevented TNFα production by inducing cell death in macrophages at equivalent hemolytic doses. Overall, these results suggest that different PFT families use different strategies to impair macrophages. CDCs may hijack intrinsic membrane repair to impair TNFα production, while aerolysin kills macrophages at similar doses. These findings might explain the immune suppression and lethality observed during NSTIs.

Using Molecular Dynamics Simulations to Compare the Stability of Lysenin Structures Obtained through X-ray Crystallography and Single-Particle Cryo-Electron Microscopy

Single-particle cryo-electron microscopy is being used more frequently to determine the atomic structure of macromolecules. Recent advances in technology have greatly improved the quality of structures obtained with this method, which is now comparable to x-ray crystallography in terms of resolution. Lysenin is a member of the aerolysin family of pore-forming toxins. Recently, both x-ray crystallography and cryo-EM structures of the lysenin pore have been published. We have used molecular dynamics simulations to analyze and compare both structures. In addition to the wild-type structures, we also analyzed two different experimentally studied lysenin mutants. Both mutants showed a reduction in the fixed negative charge on the pore wall, resulting in reduced pore size and functional activity compared to the wild-type pore. The simulation setup involved a nonameric lysenin pore, which was embedded in a membrane bilayer composed of POPC lipids. This protein-membrane complex was solvated in a water box and equilibrated to determine the stability and fluctuations of different regions of the protein. The two structures obtained from different techniques were initially modeled in their wild-type forms. The differential behavior of both structures, due to the mutation of functionally important residues, was then compared. This research sheds light on the structure-function relationship in lysenin and also investigates the relative reliability of the cryo-EM and crystal structures.

Environmental pH modulates inerolysin activity via post-binding blockade

The cholesterol dependent cytolysins (CDCs) are a family of pore-forming toxins produced by a wide range of bacteria. Some CDCs are important virulence factors for their cognate organisms, but their activity must be tightly regulated to ensure they operate at appropriate times and within the appropriate subcellular compartments. pH-dependent activity has been described for several CDCs, but the mechanism of such regulation has been studied in depth only for listeriolysin O (LLO), which senses environmental pH through a triad of acidic residues that mediate protein unfolding. Here we present data supporting a distinct mechanism for pH-dependence for inerolysin (INY), the CDC produced by Lactobacillus iners. Inerolysin (INY) has an acidic pH optimum with loss of activity at neutral pH. INY pH-dependence is characterized by reversible loss of pore formation with preservation of membrane binding. Fluorescent membrane probe assays indicated that INY insertion into host cell membranes, but not oligomerization, was defective at neutral pH. These data support the existence of a newly appreciated form of CDC pH-dependence functioning at a late stage of pore formation.

Interaction of Cholesterol with Perfringolysin O: What Have We Learned from Functional Analysis?

Cholesterol-dependent cytolysins (CDCs) constitute a family of pore-forming toxins secreted by Gram-positive bacteria. These toxins form transmembrane pores by inserting a large β-barrel into cholesterol-containing membranes. Cholesterol is absolutely required for pore-formation. For most CDCs, binding to cholesterol triggers conformational changes that lead to oligomerization and end in pore-formation. Perfringolysin O (PFO), secreted by Clostridium perfringens, is the prototype for the CDCs. The molecular mechanisms by which cholesterol regulates the cytolytic activity of the CDCs are not fully understood. In particular, the location of the binding site for cholesterol has remained elusive. We have summarized here the current body of knowledge on the CDCs-cholesterol interaction, with focus on PFO. We have employed sterols in aqueous solution to identify structural elements in the cholesterol molecule that are critical for its interaction with PFO. In the absence of high-resolution structural information, site-directed mutagenesis data combined with binding studies performed with different sterols, and molecular modeling are beginning to shed light on this interaction.

Structural, physicochemical and dynamic features conserved within the aerolysin pore-forming toxin family

Aerolysin is the founding member of a major class of β-pore-forming toxins (β-PFTs) found throughout all kingdoms of life. PFTs are cytotoxic proteins produced as soluble monomers, which oligomerize at the membrane of target host cells forming pores that may lead to osmotic lysis and cell death. Besides their role in microbial infection, they have become interesting for their potential as biotechnological sensors and delivery systems. Using an approach that integrates bioinformatics with molecular modeling and simulation, we looked for conserved features across this large toxin family. The cell surface-binding domains present high variability within the family to provide membrane receptor specificity. On the contrary, the novel concentric double β-barrel structure found in aerolysin is highly conserved in terms of sequence, structure and conformational dynamics, which likely contribute to preserve a common transition mechanism from the prepore to the mature pore within the family.Our results point to the key role of several amino acids in the conformational changes needed for oligomerization and further pore formation, such as Y221, W227, P248, Q263 and L277, which we propose are involved in the release of the stem loop and the two adjacent β-strands to form the transmembrane β-barrel.

Pseudomonas aeruginosa ExlA and Serratia marcescens ShlA trigger cadherin cleavage by promoting calcium influx and ADAM10 activation.

Pore-forming toxins are potent virulence factors secreted by a large array of bacteria. Here, we deciphered the action of ExlA from Pseudomonas aeruginosa and ShlA from Serratia marcescens on host cell-cell junctions. ExlA and ShlA are two members of a unique family of pore-forming toxins secreted by a two-component secretion system. Bacteria secreting either toxin induced an ExlA- or ShlA-dependent rapid cleavage of E-cadherin and VE-cadherin in epithelial and endothelial cells, respectively. Cadherin proteolysis was executed by ADAM10, a host cell transmembrane metalloprotease. ADAM10 activation is controlled in the host cell by cytosolic Ca2+ concentration. We show that Ca2+ influx, induced by ExlA or ShlA pore formation in the plasma membrane, triggered ADAM10 activation, thereby leading to cadherin cleavage. Our data suggest that ADAM10 is not a cellular receptor for ExlA and ShlA, further confirming that ADAM10 activation occurred via Ca2+ signalling. In conclusion, ExlA- and ShlA-secreting bacteria subvert a regulation mechanism of ADAM10 to activate cadherin shedding, inducing intercellular junction rupture, cell rounding and loss of tissue barrier integrity.

Structural Basis for Receptor Recognition by the Human CD59-Responsive Cholesterol-Dependent Cytolysins

Cholesterol-dependent cytolysins (CDCs) are a family of pore-forming toxins that punch holes in the outer membrane of eukaryotic cells. Cholesterol serves as the receptor, but a subclass of CDCs first binds to human CD59. Here we describe the crystal structures of vaginolysin and intermedilysin complexed to CD59. These studies, together with small-angle X-ray scattering, reveal that CD59 binds to each at different, though overlapping, sites, consistent with molecular dynamics simulations and binding studies. The CDC consensus undecapeptide motif, which for the CD59-responsive CDCs has a proline instead of a tryptophan in the motif, adopts a strikingly different conformation between the structures; our data suggest that the proline acts as a selectivity switch to ensure CD59-dependent CDCs bind their protein receptor first in preference to cholesterol. The structural data suggest a detailed model of how these water-soluble toxins assemble as prepores on the cell surface.

Proteomics Analysis Reveals Previously Uncharacterized Virulence Factors in Vibrio proteolyticus.

ABSTRACTMembers of the genus Vibrio include many pathogens of humans and marine animals that share genetic information via horizontal gene transfer. Hence, the Vibrio pan-genome carries the potential to establish new pathogenic strains by sharing virulence determinants, many of which have yet to be characterized. Here, we investigated the virulence properties of Vibrio proteolyticus, a Gram-negative marine bacterium previously identified as part of the Vibrio consortium isolated from diseased corals. We found that V. proteolyticus causes actin cytoskeleton rearrangements followed by cell lysis in HeLa cells in a contact-independent manner. In search of the responsible virulence factor involved, we determined the V. proteolyticus secretome. This proteomics approach revealed various putative virulence factors, including active type VI secretion systems and effectors with virulence toxin domains; however, these type VI secretion systems were not responsible for the observed cytotoxic effects. Further examination of the V. proteolyticus secretome led us to hypothesize and subsequently demonstrate that a secreted hemolysin, belonging to a previously uncharacterized clan of the leukocidin superfamily, was the toxin responsible for the V. proteolyticus-mediated cytotoxicity in both HeLa cells and macrophages. Clearly, there remains an armory of yet-to-be-discovered virulence factors in the Vibrio pan-genome that will undoubtedly provide a wealth of knowledge on how a pathogen can manipulate host cells.

Crystal structure of an invertebrate cytolysin pore reveals unique properties and mechanism of assembly.

The invertebrate cytolysin lysenin is a member of the aerolysin family of pore-forming toxins that includes many representatives from pathogenic bacteria. Here we report the crystal structure of the lysenin pore and provide insights into its assembly mechanism. The lysenin pore is assembled from nine monomers via dramatic reorganization of almost half of the monomeric subunit structure leading to a β-barrel pore ∼10 nm long and 1.6–2.5 nm wide. The lysenin pore is devoid of additional luminal compartments as commonly found in other toxin pores. Mutagenic analysis and atomic force microscopy imaging, together with these structural insights, suggest a mechanism for pore assembly for lysenin. These insights are relevant to the understanding of pore formation by other aerolysin-like pore-forming toxins, which often represent crucial virulence factors in bacteria.