Staphylococcal Pore-forming Toxins Research Articles

Development of Combination Vaccine Conferring Optimal Protection against Six Pore-Forming Toxins of Staphylococcus aureus.

In the Gram-positive pathogen Staphylococcus aureus, pore-forming toxins (PFTs), such as leukocidins and hemolysins, play prominent roles in staphylococcal pathogenesis by killing host immune cells and red blood cells (RBCs). However, it remains unknown which combination of toxin antigens would induce the broadest protective immune response against those toxins. In this study, by targeting six major staphylococcal PFTs (i.e., gamma-hemolysin AB [HlgAB], gamma-hemolysin CB [HlgCB], leukocidin AB [LukAB], leukocidin ED [LukED], Panton-Valentine leukocidin [LukSF-PV], and alpha-hemolysin [Hla]), we generated 10 recombinant toxins or toxin subunits, 3 toxoids, and their rabbit antibodies. Using the cytolytic assay for RBCs and polymorphonuclear cells (PMNs), we determined the best combination of toxin antibodies conferring the broadest protection against those staphylococcal PFTs. Although anti-HlgA IgG (HlgA-IgG) showed low cross-reactivity to other toxin components, it was essential to protect rabbit and human RBCs and human PMNs. For the protection of rabbit RBCs, HlaH35L toxoid-IgG was also required, whereas for human PMNs, LukS-IgG and LukAE323AB-IgG were essential too. When the toxin/toxoid antigens HlgA, LukS-PV, HlaH35L, and LukAE323AB were used to immunize rabbits, they increased rabbit survival; however, they did not block staphylococcal abscess formation in kidneys. Based on these results, we proposed that the combination of HlgA, LukS, HlaH35L, and LukAE323AB is the optimal vaccine component to protect human RBCs and PMNs from staphylococcal PFTs. We also concluded that a successful S. aureus vaccine requires not only those toxin antigens but also other antigens that can induce immune responses blocking staphylococcal colonization.

Targeting NLRP3 and Staphylococcal pore-forming toxin receptors in human-induced pluripotent stem cell-derived macrophages.

Staphylococcus aureus causes necrotizing pneumonia by secreting toxins such as leukocidins that target front-line immune cells. The mechanism by which leukocidins kill innate immune cells and trigger inflammation during S. aureus lung infection, however, remains unresolved. Here, we explored human-induced pluripotent stem cell-derived macrophages (hiPSC-dMs) to study the interaction of the leukocidins Panton-Valentine leukocidin (PVL) and LukAB with lung macrophages, which are the initial leukocidin targets during S. aureus lung invasion. hiPSC-dMs were susceptible to the leukocidins PVL and LukAB and both leukocidins triggered NLPR3 inflammasome activation resulting in IL-1β secretion. hiPSC-dM cell death after LukAB exposure, however, was only temporarily dependent of NLRP3, although NLRP3 triggered marked cell death after PVL treatment. CRISPR/Cas9-mediated deletion of the PVL receptor, C5aR1, protected hiPSC-dMs from PVL cytotoxicity, despite the expression of other leukocidin receptors, such as CD45. PVL-deficient S. aureus had reduced ability to induce lung IL-1β levels in human C5aR1 knock-in mice. Unexpectedly, inhibiting NLRP3 activity resulted in increased wild-type S. aureus lung burdens. Our findings suggest that NLRP3 induces macrophage death and IL-1β secretion after PVL exposure and controls S. aureus lung burdens.

Human Monocyte-Derived Osteoclasts Are Targeted by Staphylococcal Pore-Forming Toxins and Superantigens.

Staphylococcus aureus is the leading cause of bone and joint infections (BJIs). Staphylococcal pathogenesis involves numerous virulence factors including secreted toxins such as pore-forming toxins (PFTs) and superantigens. The role of these toxins on BJI outcome is largely unknown. In particular, few studies have examined how osteoclasts, the bone-resorbing cells, respond to exposure to staphylococcal PFTs and superantigens. We investigated the direct impact of recombinant staphylococcal toxins on human primary mature monocyte-derived osteoclasts, in terms of cytotoxicity and cell activation with cell death and bone resorption assays, using macrophages of the corresponding donors as a reference. Monocyte-derived osteoclasts displayed similar toxin susceptibility profiles compared to macrophages. Specifically, we demonstrated that the Panton-Valentine leukocidin, known as one of the most powerful PFT which lyses myeloid cells after binding to the C5a receptor, was able to induce the death of osteoclasts. The archetypal superantigen TSST-1 was not cytotoxic but enhanced the bone resorption activity of osteoclasts, suggesting a novel mechanism by which superantigen-producing S. aureus can accelerate the destruction of bone tissue during BJI. Altogether, our data indicate that the diverse clinical presentations of BJIs could be related, at least partly, to the toxin profiles of S. aureus isolates involved in these severe infections.

Structural basis for pore-forming mechanism of staphylococcal α-hemolysin

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

Molecular basis of transmembrane beta-barrel formation of staphylococcal pore-forming toxins.

Pathogenic bacteria secrete pore-forming toxins (PFTs) to attack target cells. PFTs are expressed as water-soluble monomeric proteins, which oligomerize into nonlytic prepore intermediates on the target cell membrane before forming membrane-spanning pores. Despite a wealth of biochemical data, the lack of high-resolution prepore structural information has hampered understanding of the β-barrel formation process. Here, we report crystal structures of staphylococcal γ-haemolysin and leucocidin prepores. The structures reveal a disordered bottom half of the β-barrel corresponding to the transmembrane region, and a rigid upper extramembrane half. Spectroscopic analysis of fluorescently labelled mutants confirmed that the prepore is distinct from the pore within the transmembrane region. Mutational analysis also indicates a pivotal role for the glycine residue located at the lipid-solvent interface as a 'joint' between the two halves of the β-barrel. These observations suggest a two-step transmembrane β-barrel pore formation mechanism in which the upper extramembrane and bottom transmembrane regions are formed independently.

Structural Insights into Clostridium perfringens Delta Toxin Pore Formation

Clostridium perfringens Delta toxin is one of the three hemolysin-like proteins produced by C. perfringens type C and possibly type B strains. One of the others, NetB, has been shown to be the major cause of Avian Nectrotic Enteritis, which following the reduction in use of antibiotics as growth promoters, has become an emerging disease of industrial poultry. Delta toxin itself is cytotoxic to the wide range of human and animal macrophages and platelets that present GM2 ganglioside on their membranes. It has sequence similarity with Staphylococcus aureus β-pore forming toxins and is expected to heptamerize and form pores in the lipid bilayer of host cell membranes. Nevertheless, its exact mode of action remains undetermined. Here we report the 2.4 Å crystal structure of monomeric Delta toxin. The superposition of this structure with the structure of the phospholipid-bound F component of S. aureus leucocidin (LukF) revealed that the glycerol molecules bound to Delta toxin and the phospholipids in LukF are accommodated in the same hydrophobic clefts, corresponding to where the toxin is expected to latch onto the membrane, though the binding sites show significant differences. From structure-based sequence alignment with the known structure of staphylococcal α-hemolysin, a model of the Delta toxin pore form has been built. Using electron microscopy, we have validated our model and characterized the Delta toxin pore on liposomes. These results highlight both similarities and differences in the mechanism of Delta toxin (and by extension NetB) cytotoxicity from that of the staphylococcal pore-forming toxins.

Application of photoactive yellow protein as a photoresponsive module for controlling hemolytic activity of staphylococcal α-hemolysin

A chimeric protein (N-PYP-Hla), consisting of staphylococcal pore-forming toxin α-hemolysin (Hla) and photoactive yellow protein (PYP), exhibited photoresponsive hemolytic activities, where visible light irradiation gave rise to retardation of hemolysis at 25 °C.

Rapid Assembly of a Multimeric Membrane Protein Pore Observed by Single Molecule Fluorescence

Pore-forming proteins play a vital role in the eukaryotic immune response, where immune surveillance can lead to the targeted-attack of infected cells and bacteria via pore generation. Conversely, many bacteria use similar protein pores to kill target cells and acquire nutrients from their host. However, the mechanisms by which many of these multimeric membrane proteins assemble to form pores are not well understood. We have observed the assembly of the staphylococcal pore-forming toxin α-hemolysin using single-molecule fluorescence imaging. Surprisingly, assembly from the monomer to the complete heptamer is extremely rapid, occurring in less than 5 ms. No lower-order oligomeric intermediates are detected. Monte Carlo simulation of our experiment shows that assembly is diffusion-limited, and pore formation is dependent on the stability of intermediate species. There are close similarities between bacterial pore-forming toxins, such as staphylococcal α-hemolysin, the anthrax protective antigen and the cholesterol-dependent cytolysins (CDCs), and their eukaryotic analogues, such as the complement pore - membrane attack complex and perforin domain (MACPF). The assembly mechanism we have observed for α-hemolysin provides a simple model that aids our understanding of these important pore-formers. To image rapidly diffusing α-hemolysin monomers and observe their assembly into higher-order oligomers requires bilayer longevity, high signal-to-noise, high time resolution and control of lipid composition. We have recently developed a new synthetic mimic of the cell membrane that fulfils these requirements; we form a Droplet Interface Bilayer by contact of two lipid monolayers between a nanolitre aqueous droplet and a hydrogel support immersed in a solution of phospholipid in hexadecane. To monitor the entire assembly process we introduced Cy3b-labeled α-hemolysin monomers into the droplet using a piezo-driven nanoinjector. The labeled α-hemolysin species were imaged on the bilayer using total internal reflection fluorescence (TIRF) microscopy.

Rapid Assembly of a Multimeric Membrane Protein Pore

We have observed the assembly of the staphylococcal pore-forming toxin α-hemolysin using single-molecule fluorescence imaging. Surprisingly, assembly from the monomer to the complete heptamer is extremely rapid, occurring in <5 ms. No lower order oligomeric intermediates are detected. Monte Carlo simulation of our experiment shows that assembly is diffusion limited, and pore formation is dependent on the stability of intermediate species. There are close similarities between bacterial pore-forming toxins, such as staphylococcal α-hemolysin, the anthrax protective antigen, and the cholesterol-dependent cytolysins, and their eukaryotic analogs, such as the complement pore membrane attack complex and perforin domain. The assembly mechanism we have observed for α-hemolysin provides a simple model that aids our understanding of these important pore formers.

Standardizing the atomic description, axis and centre of biological ion channels

A general representation of the atomic co-ordinates of a biological ion channel is obtained from a definition of channel axis and centre. Through rotation and translation of the channel, its centre becomes the origin of the standard co-ordinate system, and the channel axis becomes the system's z-axis. A method for determining the channel axis and centre based on the concepts of mass centre and mass moment of inertia is presented. The method for determining the channel axis can be directly applied to channels that adhere to two specific conditions regarding their geometry and mass distribution. Specific examples are given for Gramicidin A (GA), and the mammalian potassium channel Kv1.2. For channels that do not adhere to these conditions, minor modifications of these procedures can be applied in determining the channel axis. Specific examples are given for the outer membrane bacterial porin OmpF, and for the staphylococcal pore-forming toxin α-hemolysin (αHL). The definitions and procedures presented are made in an effort to establish a standard basis for performing, sharing, and comparing computations in a consistent manner.

Crystal Structure of the Vibrio cholerae Cytolysin (VCC) Pro-toxin and its Assembly into a Heptameric Transmembrane Pore

Pathogenic Vibrio cholerae secrete V. cholerae cytolysin (VCC), an 80 kDa pro-toxin that assembles into an oligomeric pore on target cell membranes following proteolytic cleavage and interaction with cell surface receptors. To gain insight into the activation and targeting activities of VCC, we solved the crystal structure of the pro-toxin at 2.3A by X-ray diffraction. The core cytolytic domain of VCC shares a fold similar to the staphylococcal pore-forming toxins, but in VCC an amino-terminal pro-domain and two carboxy-terminal lectin domains decorate the cytolytic domain. The pro-domain masks a protomer surface that likely participates in inter-protomer interactions in the cytolytic oligomer, thereby explaining why proteolytic cleavage and movement of the pro-domain is necessary for toxin activation. A single beta-octyl glucoside molecule outlines a possible receptor binding site on one lectin domain, and removal of this domain leads to a tenfold decrease in lytic activity toward rabbit erythrocytes. VCC activated by proteolytic cleavage assembles into an oligomeric species upon addition of soybean asolectin/cholesterol liposomes and this oligomer was purified in detergent micelles. Analytical ultracentrifugation and crystallographic analysis indicate that the resulting VCC oligomer is a heptamer. Taken together, these studies define the architecture of a pore forming toxin and associated lectin domains, confirm the stoichiometry of the assembled oligomer as heptameric, and suggest a common mechanism of assembly for staphylococcal and Vibrio cytolytic toxins.

Use of Fourier-transformed infrared spectroscopy for secondary structure determination of staphylococcal pore-forming toxins.

Pathogenic strains of Staphylococcus aureus produce a large number of exotoxins (1). Among these a family of single-chain leukotoxins of 32–34 kDa has been identified. They include α-toxin (or α-hemolysin), which is probably one of the best studied toxins (2), and a large group of bicomponent toxins formed by the leukocidins and the Γ-hemolysins (3). All these toxins share sequence homology (4) as well as functional similarity (5) which altogether suggest they probably have a significantly similar structural organization (6).