Bacterial cholesterol-dependent cytolysins and their interaction with the human immune response.

PURPOSE OF REVIEW: Many cholesterol-dependent cytolysin (CDC)-producing pathogens pose a significant threat to human health. Herein, we review the pore-dependent and -independent properties CDCs possess to assist pathogens in evading the host immune response. RECENT FINDINGS: Within the last 5 years, exciting new research suggests CDCs can act to inhibit important immune functions, disrupt critical cell signaling pathways, and have tissue-specific effects. Additionally, recent studies have identified a key region of CDCs that generates robust immunity, providing resources for the development of CDC-based vaccines. SUMMARY: This review provides new information on how CDCs alter host immune responses to aid bacteria in pathogenesis. These studies can assist in the design of more efficient vaccines and therapeutics against CDCs that will enhance the immune response to CDC-producing pathogens while mitigating the dampening effects CDCs have on the host immune response.

Pore-forming activity of S. pneumoniae pneumolysin disrupts the paracellular localization of the epithelial adherens junction protein E-cadherin.

Streptococcus pneumoniae, a common cause of community-acquired bacterial pneumonia, can cross the respiratory epithelial barrier to cause lethal septicemia and meningitis. S. pneumoniae pore-forming toxin pneumolysin (PLY) triggers robust neutrophil (PMN) infiltration that promotes bacterial transepithelial migration in vitro and disseminated disease in mice. Apical infection of polarized respiratory epithelial monolayers by S. pneumoniae at a multiplicity of infection (MOI) of 20 resulted in recruitment of PMNs, loss of 50% of the monolayer, and PMN-dependent bacterial translocation. Reducing the MOI to 2 decreased PMN recruitment two-fold and preserved the monolayer, but apical-to-basolateral translocation of S. pneumoniae remained relatively efficient. At both MOI of 2 and 20, PLY was required for maximal PMN recruitment and bacterial translocation. Co-infection by wild-type S. pneumoniae restored translocation by a PLY-deficient mutant, indicating that PLY can act in trans. Investigating the contribution of S. pneumoniae infection on apical junction complexes in the absence of PMN transmigration, we found that S. pneumoniae infection triggered the cleavage and mislocalization of the adherens junction (AJ) protein E-cadherin. This disruption was PLY-dependent at MOI of 2 and was recapitulated by purified PLY, requiring its pore-forming activity. In contrast, at MOI of 20, E-cadherin disruption was independent of PLY, indicating that S. pneumoniae encodes multiple means to disrupt epithelial integrity. This disruption was insufficient to promote bacterial translocation in the absence of PMNs. Thus, S. pneumoniae triggers cleavage and mislocalization of E-cadherin through PLY-dependent and -independent mechanisms, but maximal bacterial translocation across epithelial monolayers requires PLY-dependent neutrophil transmigration.

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.

Pneumolysin Induces 12-Lipoxygenase-Dependent Neutrophil Migration during Streptococcus pneumoniae Infection.

Streptococcus pneumoniae is a major cause of pneumonia, wherein infection of respiratory mucosa drives a robust influx of neutrophils. We have previously shown that S. pneumoniae infection of the respiratory epithelium induces the production of the 12-lipoxygenase (12-LOX)-dependent lipid inflammatory mediator hepoxilin A3, which promotes recruitment of neutrophils into the airways, tissue damage, and lethal septicemia. Pneumolysin (PLY), a member of the cholesterol-dependent cytolysin (CDC) family, is a major S. pneumoniae virulence factor that generates ∼25-nm diameter pores in eukaryotic membranes and promotes acute inflammation, tissue damage, and bacteremia. We show that a PLY-deficient S. pneumoniae mutant was impaired in triggering human neutrophil transepithelial migration in vitro. Ectopic production of PLY endowed the nonpathogenic Bacillus subtilis with the ability to trigger neutrophil recruitment across human-cultured monolayers. Purified PLY, several other CDC family members, and the α-toxin of Clostridium septicum, which generates pores with cross-sectional areas nearly 300 times smaller than CDCs, reproduced this robust neutrophil transmigration. PLY non-pore-forming point mutants that are trapped at various stages of pore assembly did not recruit neutrophils. PLY triggered neutrophil recruitment in a 12-LOX-dependent manner in vitro. Instillation of wild-type PLY but not inactive derivatives into the lungs of mice induced robust 12-LOX-dependent neutrophil migration into the airways, although residual inflammation induced by PLY in 12-LOX-deficient mice indicates that 12-LOX-independent pathways also contribute to PLY-triggered pulmonary inflammation. These data indicate that PLY is an important factor in promoting hepoxilin A3-dependent neutrophil recruitment across pulmonary epithelium in a pore-dependent fashion.

The Unique Molecular Choreography of Giant Pore Formation by the Cholesterol-Dependent Cytolysins of Gram-Positive Bacteria.

The mechanism by which the cholesterol-dependent cytolysins (CDCs) assemble their giant ß-barrel pore in cholesterol-rich membranes has been the subject of intense study in the past two decades. A combination of structural, biophysical, and biochemical analyses has revealed deep insights into the series of complex and highly choreographed secondary and tertiary structural transitions that the CDCs undergo to assemble their ß-barrel pore in eukaryotic membranes. Our knowledge of the molecular details of these dramatic structural changes in CDCs has transformed our understanding of how giant pore complexes are assembled and has been critical to our understanding of the mechanisms of other important classes of pore-forming toxins and proteins across the kingdoms of life. Finally, there are tantalizing hints that the CDC pore-forming mechanism is more sophisticated than previously imagined and that some CDCs are employed in pore-independent processes.

Conformational changes during pore formation by the perforin-related protein pleurotolysin.

Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ∼70° opening of the bent and distorted central ß-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane ß-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of ß-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into ß-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted ß-barrel. The intermediate structures of the MACPF domain during refolding into the ß-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters, suggesting why this region is targeted by endogenous inhibitors of MACPF function.

An intermolecular electrostatic interaction controls the prepore-to-pore transition in a cholesterol-dependent cytolysin.

ß-Barrel pore-forming toxins (ßPFTs) form an obligatory oligomeric prepore intermediate before the formation of the ß-barrel pore. The molecular components that control the critical prepore-to-pore transition remain unknown for ßPFTs. Using the archetype ßPFT perfringolysin O, we show that E183 of each monomer within the prepore complex forms an intermolecular electrostatic interaction with K336 of the adjacent monomer on completion of the prepore complex. The signal generated throughout the prepore complex by this interaction irrevocably commits it to the formation of the membrane-inserted giant ß-barrel pore. This interaction supplies the free energy to overcome the energy barrier (determined here to be ∼ 19 kcal/mol) to the prepore-to-pore transition by the coordinated disruption of a critical interface within each monomer. These studies provide the first insight to our knowledge into the molecular mechanism that controls the prepore-to-pore transition for a ßPFT.

Stonefish toxin defines an ancient branch of the perforin-like superfamily.

The lethal factor in stonefish venom is stonustoxin (SNTX), a heterodimeric cytolytic protein that induces cardiovascular collapse in humans and native predators. Here, using X-ray crystallography, we make the unexpected finding that SNTX is a pore-forming member of an ancient branch of the Membrane Attack Complex-Perforin/Cholesterol-Dependent Cytolysin (MACPF/CDC) superfamily. SNTX comprises two homologous subunits (α and ß), each of which comprises an N-terminal pore-forming MACPF/CDC domain, a central focal adhesion-targeting domain, a thioredoxin domain, and a C-terminal tripartite motif family-like PRY SPla and the RYanodine Receptor immune recognition domain. Crucially, the structure reveals that the two MACPF domains are in complex with one another and arranged into a stable early prepore-like assembly. These data provide long sought after near-atomic resolution insights into how MACPF/CDC proteins assemble into prepores on the surface of membranes. Furthermore, our analyses reveal that SNTX-like MACPF/CDCs are distributed throughout eukaryotic life and play a broader, possibly immune-related function outside venom.

The Cholesterol-dependent Cytolysin Membrane-binding Interface Discriminates Lipid Environments of Cholesterol to Support ß-Barrel Pore Insertion.

The majority of cholesterol-dependent cytolysins (CDCs) utilize cholesterol as a membrane receptor, whereas a small number are restricted to the GPI-anchored protein CD59 for initial membrane recognition. Two cholesterol-binding CDCs, perfringolysin O (PFO) and streptolysin O (SLO), were found to exhibit strikingly different binding properties to cholesterol-rich natural and synthetic membranes. The structural basis for this difference was mapped to one of the loops (L3) in the membrane binding interface that help anchor the toxin monomers to the membrane after receptor (cholesterol) binding by the membrane insertion of its amino acid side chains. A single point mutation in this loop conferred the binding properties of SLO to PFO and vice versa. Our studies strongly suggest that changing the side chain structure of this loop alters its equilibrium between membrane-inserted and uninserted states, thereby affecting the overall binding affinity and total bound toxin. Previous studies have shown that the lipid environment of cholesterol has a dramatic effect on binding and activity. Combining this data with the results of our current studies on L3 suggests that the structure of this loop has evolved in the different CDCs to preferentially direct binding to cholesterol in different lipid environments. Finally, the efficiency of ß-barrel pore formation was inversely correlated with the increased binding and affinity of the PFO L3 mutant, suggesting that selection of a compatible lipid environment impacts the efficiency of membrane insertion of the ß-barrel pore.

Mouse, but not human, ApoB-100 lipoprotein cholesterol is a potent innate inhibitor of Streptococcus pneumoniae pneumolysin.

Streptococcus pneumoniae produces the pore-forming toxin pneumolysin (PLY), which is a member of the cholesterol-dependent cytolysin (CDC) family of toxins. The CDCs recognize and bind the 3ß-hydroxyl group of cholesterol at the cell surface, which initiates membrane pore formation. The cholesterol transport lipoproteins, which carry cholesterol in their outer monolayer, are potential off-pathway binding targets for the CDCs and are present at significant levels in the serum and the interstitial spaces of cells. Herein we show that cholesterol carried specifically by the ApoB-100-containing lipoprotein particles (CH-ApoB-100) in the mouse, but not that carried by human or guinea pig particles, is a potent inhibitor of the PLY pore-forming mechanism. Cholesterol present in the outer monolayer of mouse ApoB-100 particles is recognized and bound by PLY, which stimulates premature assembly of the PLY oligomeric complex thereby inactivating PLY. These studies further suggest that the vast difference in the inhibitory capacity of mouse CH-ApoB-100 and that of the human and the guinea pig is due to differences in the presentation of cholesterol in the outer monolayer of their ApoB-100 particles. Therefore mouse CH-ApoB-100 represents a significant innate CDC inhibitor that is absent in humans, which may underestimate the contribution of CDCs to human disease when utilizing mouse models of disease.

Disulfide-bond scanning reveals assembly state and ß-strand tilt angle of the PFO ß-barrel.

Perfringolysin O (PFO), a bacterial cholesterol-dependent cytolysin, binds a mammalian cell membrane, oligomerizes into a circular prepore complex (PPC) and forms a 250-Å transmembrane ß-barrel pore in the cell membrane. Each PFO monomer has two sets of three short α-helices that unfold and ultimately refold into two transmembrane ß-hairpin (TMH) components of the membrane-embedded ß-barrel. Interstrand disulfide-bond scanning revealed that ß-strands in a fully assembled PFO ß-barrel were strictly aligned and tilted at 20° to the membrane perpendicular. In contrast, in a low temperature-trapped PPC intermediate, the TMHs were unfolded and had sufficient freedom of motion to interact transiently with each other, yet the TMHs were not aligned or stably hydrogen bonded. The PFO PPC-to-pore transition therefore converts TMHs in a dynamic folding intermediate far above the membrane into TMHs that are hydrogen bonded to those of adjacent subunits in the bilayer-embedded ß-barrel.

Broadly protective protein-based pneumococcal vaccine composed of pneumolysin toxoid-CbpA peptide recombinant fusion protein.

BACKGROUND: Pneumococcus, meningococcus, and Haemophilus influenzae cause a similar spectrum of infections in the ear, lung, blood, and brain. They share cross-reactive antigens that bind to the laminin receptor of the blood-brain barrier as a molecular basis for neurotropism, and this step in pathogenesis was addressed in vaccine design. METHODS: Biologically active peptides derived from choline-binding protein A (CbpA) of pneumococcus were identified and then genetically fused to L460D pneumolysoid. The fusion construct was tested for vaccine efficacy in mouse models of nasopharyngeal carriage, otitis media, pneumonia, sepsis, and meningitis. RESULTS: The CbpA peptide-L460D pneumolysoid fusion protein was more broadly immunogenic than pneumolysoid alone, and antibodies were active in vitro against Streptococcus pneumoniae, Neisseria meningitidis, and H. influenzae. Passive and active immunization protected mice from pneumococcal carriage, otitis media, pneumonia, bacteremia, meningitis, and meningococcal sepsis. CONCLUSIONS: The CbpA peptide-L460D pneumolysoid fusion protein was broadly protective against pneumococcal infection, with the potential for additional protection against other meningeal pathogens.

The cholesterol-dependent cytolysin signature motif: a critical element in the allosteric pathway that couples membrane binding to pore assembly.

The cholesterol-dependent cytolysins (CDCs) constitute a family of pore-forming toxins that contribute to the pathogenesis of a large number of Gram-positive bacterial pathogens.The most highly conserved region in the primary structure of the CDCs is the signature undecapeptide sequence (ECTGLAWEWWR). The CDC pore forming mechanism is highly sensitive to changes in its structure, yet its contribution to the molecular mechanism of the CDCs has remained enigmatic. Using a combination of fluorescence spectroscopic methods we provide evidence that shows the undecapeptide motif of the archetype CDC, perfringolysin O (PFO), is a key structural element in the allosteric coupling of the cholesterol-mediated membrane binding in domain 4 (D4) to distal structural changes in domain 3 (D3) that are required for the formation of the oligomeric pore complex. Loss of the undecapeptide function prevents all measurable D3 structural transitions, the intermolecular interaction of membrane bound monomers and the assembly of the oligomeric pore complex. We further show that this pathway does not exist in intermedilysin (ILY), a CDC that exhibits a divergent undecapeptide and that has evolved to use human CD59 rather than cholesterol as its receptor. These studies show for the first time that the undecapeptide of the cholesterol-binding CDCs forms a critical element of the allosteric pathway that controls the assembly of the pore complex.

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.

Monomer-monomer interactions propagate structural transitions necessary for pore formation by the cholesterol-dependent cytolysins.

The assembly of the cholesterol-dependent cytolysin (CDC) oligomeric pore complex requires a complex choreography of secondary and tertiary structural changes in domain 3 (D3) of the CDC monomer structure. A point mutation was identified in the archetype CDC, perfringolysin O, that blocks detectable D3 structural changes and traps the membrane-bound monomers in an early and reversible stage of oligomer assembly. Using this and other mutants we show that specific D3 structural changes are propagated from one membrane-bound monomer to another. Propagation of these structural changes results in the exposure of a ß-strand in D3 that allows it to pair and form edge-on interactions with a second ß-strand of a free membrane-bound monomer. Pairing of these strands establishes the final geometry of the pore complex and is necessary to drive the formation of the ß-barrel pore. These studies provide new insights into how structural information is propagated between membrane-bound monomers of a self-assembling system and the interactions that establish the geometry of the final pore complex.

Membrane assembly of the cholesterol-dependent cytolysin pore complex.

The cholesterol-dependent cytolysins (CDCs) are a large family of pore-forming toxins that are produced, secreted and contribute to the pathogenesis of many species of Gram-positive bacteria. The assembly of the CDC pore-forming complex has been under intense study for the past 20 years. These studies have revealed a molecular mechanism of pore formation that exhibits many novel features. The CDCs form large ß-barrel pore complexes that are assembled from 35 to 40 soluble CDC monomers. Pore formation is dependent on the presence of membrane cholesterol, which functions as the receptor for most CDCs. Cholesterol binding initiates significant secondary and tertiary structural changes in the monomers, which lead to the assembly of a large membrane embedded ß-barrel pore complex. This review will focus on the molecular mechanism of assembly of the CDC membrane pore complex and how these studies have led to insights into the mechanism of pore formation for other pore-forming proteins. This article is part of a Special Issue entitled: Protein Folding in Membranes.

Identification and characterization of the first cholesterol-dependent cytolysins from Gram-negative bacteria.

The cholesterol-dependent cytolysins (CDCs) are pore-forming toxins that have been exclusively associated with a wide variety of bacterial pathogens and opportunistic pathogens from the Firmicutes and Actinobacteria, which exhibit a Gram-positive type of cell structure. We have characterized the first CDCs from Gram-negative bacterial species, which include Desulfobulbus propionicus type species Widdel 1981 (DSM 2032) (desulfolysin [DLY]) and Enterobacter lignolyticus (formerly Enterobacter cloacae) SCF1 (enterolysin [ELY]). The DLY and ELY primary structures show that they maintain the signature motifs of the CDCs but lack an obvious secretion signal. Recombinant, purified DLY (rDLY) and ELY (rELY) exhibited cholesterol-dependent binding and cytolytic activity and formed the typical large CDC membrane oligomeric pore complex. Unlike the CDCs from Gram-positive species, which are human- and animal-opportunistic pathogens, neither D. propionicus nor E. lignolyticus is known to be a pathogen or commensal of humans or animals: the habitats of both organisms appear to be restricted to anaerobic soils and/or sediments. These studies reveal for the first time that the genes for functional CDCs are present in bacterial species that exhibit a Gram-negative cell structure. These are also the first bacterial species containing a CDC gene that are not known to inhabit or cause disease in humans or animals, which suggests a role of these CDCs in the defense against eukaryote bacterial predators.

The pore-forming toxin listeriolysin O mediates a novel entry pathway of L. monocytogenes into human hepatocytes.

Intracellular pathogens have evolved diverse strategies to invade and survive within host cells. Among the most studied facultative intracellular pathogens, Listeria monocytogenes is known to express two invasins-InlA and InlB-that induce bacterial internalization into nonphagocytic cells. The pore-forming toxin listeriolysin O (LLO) facilitates bacterial escape from the internalization vesicle into the cytoplasm, where bacteria divide and undergo cell-to-cell spreading via actin-based motility. In the present study we demonstrate that in addition to InlA and InlB, LLO is required for efficient internalization of L. monocytogenes into human hepatocytes (HepG2). Surprisingly, LLO is an invasion factor sufficient to induce the internalization of noninvasive Listeria innocua or polystyrene beads into host cells in a dose-dependent fashion and at the concentrations produced by L. monocytogenes. To elucidate the mechanisms underlying LLO-induced bacterial entry, we constructed novel LLO derivatives locked at different stages of the toxin assembly on host membranes. We found that LLO-induced bacterial or bead entry only occurs upon LLO pore formation. Scanning electron and fluorescence microscopy studies show that LLO-coated beads stimulate the formation of membrane extensions that ingest the beads into an early endosomal compartment. This LLO-induced internalization pathway is dynamin-and F-actin-dependent, and clathrin-independent. Interestingly, further linking pore formation to bacteria/bead uptake, LLO induces F-actin polymerization in a tyrosine kinase-and pore-dependent fashion. In conclusion, we demonstrate for the first time that a bacterial pathogen perforates the host cell plasma membrane as a strategy to activate the endocytic machinery and gain entry into the host cell.

Only two amino acids are essential for cytolytic toxin recognition of cholesterol at the membrane surface.

The recognition and binding of cholesterol is an important feature of many eukaryotic, viral, and prokaryotic proteins, but the molecular details of such interactions are understood only for a few proteins. The pore-forming cholesterol-dependent cytolysins (CDCs) contribute to the pathogenic mechanisms of a large number of Gram-positive bacteria. Cholesterol dependence of the CDC mechanism is a hallmark of these toxins, yet the identity of the CDC cholesterol recognition motif has remained elusive. A detailed analysis of membrane interactive structures at the tip of perfringolysin O (PFO) domain 4 reveals that a threonine-leucine pair mediates CDC recognition of and binding to membrane cholesterol. This motif is conserved in all known CDCs and conservative changes in its sequence or order are not well tolerated. Thus, the Thr-Leu pair constitutes a common structural basis for mediating CDC-cholesterol recognition and binding, and defines a unique paradigm for membrane cholesterol recognition by surface-binding proteins.

Mapping the intermedilysin-human CD59 receptor interface reveals a deep correspondence with the binding site on CD59 for complement binding proteins C8alpha and C9.

CD59 is a glycosylphosphatidylinositol-anchored protein that inhibits the assembly of the terminal complement membrane attack complex (MAC) pore, whereas Streptococcus intermedius intermedilysin (ILY), a pore forming cholesterol-dependent cytolysin (CDC), specifically binds to human CD59 (hCD59) to initiate the formation of its pore. The identification of the residues of ILY and hCD59 that form their binding interface revealed a remarkably deep correspondence between the hCD59 binding site for ILY and that for the MAC proteins C8α and C9. ILY disengages from hCD59 during the prepore to pore transition, suggesting that loss of this interaction is necessary to accommodate specific structural changes associated with this transition. Consistent with this scenario, mutants of hCD59 or ILY that increased the affinity of this interaction decreased the cytolytic activity by slowing the transition of the prepore to pore but not the assembly of the prepore oligomer. A signature motif was also identified in the hCD59 binding CDCs that revealed a new hCD59-binding member of the CDC family. Although the binding site on hCD59 for ILY, C8α, and C9 exhibits significant homology, no similarity exists in their binding sites for hCD59. Hence, ILY and the MAC proteins interact with common amino acids of hCD59 but lack detectable conservation in their binding sites for hCD59.