Staphylococcal Alpha-hemolysin Research Articles

Influence of the lipid bilayer composition on interaction of polyanions with the alpha-toxin ion channel

The mechanism of interaction among polyanions and Staphylococcal alpha hemolysin (αHL) ion channel still was not elucidated completely. The initial interaction of polyanions with surface membrane of phospholipids is based in Ca2+ bridge formation. Such interaction increases polyanion concentration close to membrane surface that, in turn, increases the probability of polyanion enter into a channel and blocks it. Thus, this study proposed to investigate the role of the lipid membrane composition on interaction of polyanions, such as heparin, with αHL channel. It was found that the effectiveness of heparin to block αHL channels was significantly dependent on the lipid composition of the bilayers. The lipids on their ability to support heparin influence were ranked as follows: PC >> PI » PS > DPhPS ³ PE ³ DPhPC > OChol. These results indicate that the interaction of Ca2+ with lipid membranes depends on the exposure and density of phosphate groups in phospholipids at membrane surface. On the other hand, the effectiveness of heparin to block αHL channel was more strongly correlated with the length of the hydrocarbons chain of fatty acids of the phospholipids. Thus, we demonstrate that the polar head group of phospholipids in the membranes affects their interaction with divalent ions by changing their surface potential, and therefore influences the effectiveness of heparin blockage in the formed channels. The results might be of interest for pharmacology, biomedicine, and research aiming to design mesoscopic pore blockers.

The Role of Staphylococcus Aureus Types and Toxin-Producing Ability in Pediatric Atopic Dermatitis and its Association with Disease Severity

Background: The skin of patients with atopic dermatitis has a high susceptibility to staphylococcus aureus (S. aureus) colonization known to produce toxins with super antigen (SAgs) activity which are a family of potent immune-stimulatory exotoxins and may aggravate AD. The aim of this research was to evaluate the role of staphylococcus aureus types and toxin-producing ability in pediatric atopic dermatitis and its association with disease severity. Materials and Methods: In this cross-sectional study, fifty-two patients with AD were evaluated for clinical severity of disease using severity scoring of atopic dermatitis (SCORAD) index. Swabs were taken from their skin and S. aureus was isolated, then the mecA, SCCmec types and agr genes besides exotoxins with super antigen properties, Hla and TSST genes were evaluated by performing polymerase chain reaction (PCR). Results: Staphylococcus aureus was isolated in 38 (73.07%) out of 52 AD patients. The SCORAD index and AD severity were strongly correlated with S. aureus colonization (P=0.00). The staphylococcal alpha-hemolysin (Hla) was the predominant toxin gene found in AD patients, Hla was produced in 22 patients (57.9%). The toxic shock syndrome toxin-1 (TSST-1) gene was found in 12 (31.6%) isolates and, in 11 patients both TSST-1 and Hla toxin gene were detected. There was no significant relationship between the presence of TSST1 and Hla gene and the severity of the disease Hla (P=0.11 and P=0.08, respectively). Conclusion: AD severity based onthe SCORAD index was strongly correlated with S.aureus colonization, and the most frequent super antigen gene present in S. aureous isolates was that coding for (Hla).

Cages Driven Away from Equilibrium Binding by Electric Fields

Molecules and materials that are pushed away from equilibrium can produce unique dynamic and adaptive properties that cannot be attained when they are at rest. In this issue of Chem, Cockroft and co-workers study the transient binding and debinding of coordination cages to the nanopocket of α-hemolysin and show that the binding and debinding events are driven out of equilibrium by an external electric field. Strong applied fields can invert the cage-nanopore binding selectivities and can enhance enantio-inversion to enrich one chiral form of the cage over the other.

Protective effects of bacterial osmoprotectant ectoine on bovine erythrocytes subjected to staphylococcal alpha-haemolysin.

Ectoine (ECT) is a bacterial compatible solute with documented protective action however no data are available on its effects on various cells against bacterial toxins. Therefore, we determined the invitro influence of ECT on bovine erythrocytes subjected to staphylococcal α-haemolysin (HlyA). The cells exposed to HlyA alone showed a distinct haemolysis and reduced glutathione (GSH)/oxidised glutathione (GSSG) level, however the toxic effects were attenuated in the combinations of HlyA+ECT suggesting ECT-induced protection of erythrocytes from HlyA.

A biomimetic nanosponge that absorbs pore-forming toxins

Detoxification treatments such as toxin-targeted anti-virulence therapy1, 2 offer ways to cleanse the body of virulence factors that are caused by bacterial infections, venomous injuries, and biological weaponry. Because existing detoxification platforms such as antisera3, monoclonal antibodies4, small-molecule inhibitors5, 6, and molecularly imprinted polymers7 act by targeting the molecular structures of the toxins, customized treatments are required for different diseases. Here we show a biomimetic toxin nanosponge that functions as a toxin decoy in vivo. The nanosponge, which consists of a polymeric nanoparticle core surrounded by red blood cell membranes, absorbs membrane-damaging toxins and diverts them away from their cellular targets. In a mouse model, the nanosponges markedly reduce the toxicity of staphylococcal alpha-hemolysin (α-toxin) and thus improve the survival rate of toxin-challenged mice. This biologically inspired toxin nanosponge presents a detoxification treatment that can potentially treat a variety of injuries and diseases caused by pore-forming toxins.

203 Polymers through pores: single-molecule experiments with nucleic acids, polypeptides and polysaccharides

The translocation of polymers through pores has been examined for almost two decades with an emphasis on nucleic acids. There are also interesting circumstances in biology where polypeptides and polysaccharides pass through transmembrane pores, and our laboratory has been investigating examples of them. Single-molecule nucleic acid sequencing by nanopore technology is an emerging approach for ultrarapid genomics. Strand sequencing with engineered protein nanopores is a viable technology which has required advances in four areas: nucleic acid threading, nucleobase identification, controlled strand translocation, and nanopore arrays (Bayley, 2012). The latter remain a pressing need and our attempts to improve arrays will be described. In several physiological situations, folded proteins pass through transmembrane pores. We have developed a model system comprising mutant thioredoxins as the translocated proteins, and staphylococcal alpha-hemolysin, as the pore. Our findings support a mechanism in which there is local unfolding near the terminus of the polypeptide that enters the pore. The remainder of the protein then unfolds spontaneously and diffuses through the pore into the recipient compartment (Rodriguez-Larrea & Bayley, 2013). We have also examined the pore formed by the E. coli outer membrane protein Wza, which transports capsular polysaccharide from its site of synthesis to the outside of the cell. We made mutant open forms of the pore and screened blockers for them by electrical recording in planar bilayers. The most effective blocker binds in the alpha-helix barrel of Wza, a site accessible from the external medium, and therefore, a prospective target for antibiotics (Kong et al., 2013).

Generation and characterization of an inter-generic bivalent alpha domain fusion protein αCS from Clostridium perfringens and Staphylococcus aureus for concurrent diagnosis and therapeutic applications

To evaluate an inter-generic recombinant alpha domain fusion protein for simultaneous detection and neutralization of Clostridium perfringens and Staphylococcus aureus alpha toxins. Truncated portions of clostridial and staphylococcal alpha haemolysin genes were PCR amplified and linked to each other through a hydrophilic flexible Glycine linker sequence using overlap-extension PCR to form a chimeric gene αCS. The recombinant αCS fusion protein was expressed and characterized for its toxicity, cell binding capacity and haemolysis inhibition properties. The fusion protein was nontoxic and effectively retarded staphylococcal alpha haemolysis, probably by competitively interacting with putative staphylococcal alpha haemolysin receptors on erythrocytes. Murine hyperimmune polysera raised against r-αCS specifically detected 42-kDa and 33-kDa proteins when culture supernatants of Cl. perfringens (clostridial alpha toxin) and Staph. aureus (staphylococcal alpha toxin), respectively, were analysed in Western blot. The polyclonal antisera effectively diminished the haemolytic action of both the wild-type toxins in vitro. The r-αCS fusion protein was nontoxic competitive inhibitor of staphylococcal alpha haemolysin. The protein elicited specific immune response against Cl. perfringens and Staph. aureus alpha toxins. The antisera also neutralized the toxicities of both the native wild-type toxins in vitro. The bivalent recombinant αCS protein could be a novel intervention in the field of diagnostics and therapeutics against Cl. perfringens and Staph. aureus infections, particularly, in case of co-infections like gangrenous ischaemia, gangrenous mastitis, etc.

Reversing the Charges within the Lumen of Alpha-Hemolysin Protein Pore Affects its Robustness

Staphylococcal alpha-hemolysin (αHL), a heptameric transmembrane protein of known high-resolution crystal structure, has been targeted for engineering for many biotechnological applications due to its high stability in planar lipid bilayers. Using single-channel electrical recordings and standard protein engineering, we explored how reversed charges within the interior of the beta-barrel part of the pore alter the protein robustness. We replaced the lysines with aspartic acids. Reversing charges within the pore constriction, the structurally stiff region of the beta barrel, reduced the conductance of the pore, but produced current fluctuation-free channel behavior. In contrast, reversing charges at the structurally flexible glycine-rich turn of the beta barrel, increased the conductance of the pore and produced gating activity in the form of large-amplitude and frequent current fluctuations. Remarkably, reversing charges within both regions caused a large-amplitude permanent current blockade of the beta barrel. Our results suggest that these distant charge reversals are energetically coupled.

Impact of distant charge reversals within a robust beta-barrel protein pore.

Among all beta-barrel pores, staphylococcal alpha-hemolysin (alphaHL), a heptameric transmembrane protein of known high-resolution crystal structure, features a high stability in planar lipid bilayers under a wide range of harsh experimental conditions. Here, we employed single-channel electrical recordings and standard protein engineering to explore the impact of two distant charge reversals within the interior of the beta-barrel part of the pore. The charge reversals were replacements of lysines with aspartic acids. A charge reversal within the structurally stiff region of the beta barrel near the pore constriction reduced the open-state current of the pore, but produced a quiet pore, showing current fluctuation-free channel behavior. In contrast, a charge reversal on the trans entrance, within the structurally flexible glycine-rich turn of the beta barrel, increased the open-state current and produced gating activity of the pore in the form of large-amplitude and frequent current fluctuations. Remarkably, cumulative insertion of the two distant charge reversals resulted in a large-amplitude permanent blockade of the beta barrel, as judged by both single-channel and macroscopic current measurements. The results from this work suggest that these distant charge reversals are energetically coupled, producing different impacts on the ionic transport, the unitary conductance and the open-state probability of the pore.

Staphylococcus aureus α-Hemolysin Activates the NLRP3-Inflammasome in Human and Mouse Monocytic Cells

Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA) causes severe necrotizing infections of the skin, soft tissues, and lungs. Staphylococcal α-hemolysin is an essential virulence factor in mouse models of CA-MRSA necrotizing pneumonia. S. aureus α-hemolysin has long been known to induce inflammatory signaling and cell death in host organisms, however the mechanism underlying these signaling events were not well understood. Using highly purified recombinant α-hemolysin, we now demonstrate that α-hemolysin activates the Nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 protein (NLRP3)-inflammasome, a host inflammatory signaling complex involved in responses to pathogens and endogenous danger signals. Non-cytolytic mutant α-hemolysin molecules fail to elicit NLRP3-inflammasome signaling, demonstrating that the responses are not due to non-specific activation of this innate immune signaling system by bacterially derived proteins. In monocyte-derived cells from humans and mice, inflammasome assembly in response to α-hemolysin results in activation of the cysteine proteinase, caspase-1. We also show that inflammasome activation by α-hemolysin works in conjunction with signaling by other CA-MRSA-derived Pathogen Associated Molecular Patterns (PAMPs) to induce secretion of pro-inflammatory cytokines IL-1β and IL-18. Additionally, α-hemolysin induces cell death in these cells through an NLRP3-dependent program of cellular necrosis, resulting in the release of endogenous pro-inflammatory molecules, like the chromatin-associated protein, High-mobility group box 1 (HMGB1). These studies link the activity of a major S. aureus virulence factor to a specific host signaling pathway. The cellular events linked to inflammasome activity have clear relevance to the disease processes associated with CA-MRSA including tissue necrosis and inflammation.

Building And Controlling Networks Of Droplet Interface Bilayers

One goal of synthetic biology is the manufacture of micromachines from simple parts. Such machines would be motile, able to generate, store and use energy, capable of sensing and carrying out computation, and able to take up substrates and convert them to products. We have found that aqueous droplets can be connected by lipid bilayers to form networks in a hydrocarbon environment [1]. We propose to use these networks for the construction of “soft” micromachines (or “prototissues”, by analogy with efforts to build protocells). Proteins can be incorporated in to the bilayers of the networks [2]. Therefore, we expect that membrane proteins will play a role in the functioning of droplet-based micromachines, notably by allowing the droplets to communicate and exhibit emergent properties. Towards this end, we have engineered the staphylococcal alpha-hemolysin pore to endow it with a variety of capabilities. We have been able to alter the pore size, and its ion selectivity and rectification properties. We have also altered the pore so that it is regulated by chemicals, light and temperature. With these components, we have shown that droplet networks can behave like simple electrical circuits [3], be used to form tiny batteries [1] and respond to light [1]. With these subsystems in place, the manufacture of the proposed micromachines may soon be in the offing.1. Holden MA, Needham D, Bayley H: Functional bionetworks from nanoliter water droplets. J. Am. Chem. Soc. 2007, 129:8650-8655.2. Bayley H, Cronin B, Heron A, Holden MA, Hwang W, Syeda R, Thompson J, Wallace M: Droplet interface bilayers. Mol. BioSystems 2008:published ASAP, DOI: 10.1039/b808893d.3. Hwang WL, Holden MA, White S, Bayley H: Electrical analysis of protein pore insertion and blockade in droplet interface bilayer networks. J. Am. Chem. Soc. 2007, 129:11854-11864.

Simultaneous Measurement of Ionic Current and Fluorescence from Single Protein Pores

The ability to simultaneously monitor both the ionic current and fluorescence from membrane channels and pores has the potential to link structural changes with function in such proteins. We present a new method for simultaneously measuring single-channel electrical currents and fluorescence from membrane proteins by using water-in-oil droplet bilayers. We demonstrate the simultaneous fluorescence and electrical detection of stochastic blocking by cyclodextrin in multiple staphylococcal alpha-hemolysin pores. The combined fluorescence signal from individual pores exhibits the same sequence of blocking events as the total current recording, showing that the two signals from each pore are correlated.

Catalyzing the Translocation of Polypeptides through Attractive Interactions

Facilitated translocation of polypeptides through a protein pore is a ubiquitous and fundamental process in biology. Several translocation systems possess various well-defined binding sites within the pore lumen, but a clear mechanistic understanding of how the interaction of the polypeptides with the binding site alters the underlying kinetics is still missing. Here, we employed rational protein design and single-channel electrical recordings to obtain detailed kinetic signatures of polypeptide translocation through the staphylococcal alpha-hemolysin (alphaHL) transmembrane pore, a robust, tractable, and versatile beta-barrel protein. Acidic binding sites composed of rings of negatively charged aspartic acid residues, engineered at strategic positions within the beta barrel, produced dramatic changes in the functional properties of the alphaHL protein, facilitating the transport of cationic polypeptides from one side of the membrane to the other. When two electrostatic binding sites were introduced, at the entry and exit of the beta barrel, both the rate constants of association and dissociation increased substantially, diminishing the free energy barrier for translocation. By contrast, more hydrophobic polypeptides exhibited a considerable decrease in the rate constant of association to the pore lumen, having to overcome a greater energetic barrier because of the hydrophilic nature of the pore interior.

Single-Molecule Electrophoresis of β-Hairpin Peptides by Electrical Recordings and Langevin Dynamics Simulations

We used single-channel electrical recordings and Langevin molecular dynamics simulations to explore the electrophoretic translocation of various beta-hairpin peptides across the staphylococcal alpha-hemolysin (alphaHL) protein pore at single-molecule resolution. The beta-hairpin peptides, which varied in their folding properties, corresponded to the C terminal residues of the B1 domain of protein G. The translocation time was strongly dependent on the electric force and was correlated with the folding features of the beta-hairpin peptides. Highly unfolded peptides entered the pore in an extended conformation, resulting in fast single-file translocation events. In contrast, the translocation of the folded beta-hairpin peptides occurred more slowly. In this case, the beta-hairpin peptides traversed the alphaHL pore in a misfolded or fully folded conformation. This study demonstrates that the interaction between a polypeptide and a beta-barrel protein pore is dependent on the folding features of the polypeptide.

Role of the Amino Latch of Staphylococcal α-Hemolysin in Pore Formation: A CO-OPERATIVE INTERACTION BETWEEN THE N TERMINUS AND POSITION 217

Staphylococcal alpha-hemolysin (alphaHL) is a beta barrel pore-forming toxin that is secreted by the bacterium as a water-soluble monomeric protein. Upon binding to susceptible cells, alphaHL assembles via an inactive prepore to form a water-filled homoheptameric transmembrane pore. The N terminus of alphaHL, which in the crystal structure of the fully assembled pore forms a latch between adjacent subunits, has been thought to play a vital role in the prepore to pore conversion. For example, the deletion of two N-terminal residues produced a completely inactive protein that was arrested in assembly at the prepore stage. In the present study, we have re-examined assembly with a comprehensive set of truncation mutants. Surprisingly, we found that after truncation of up to 17 amino acids, the ability of alphaHL to form functional pores was diminished, but still substantial. We then discovered that the mutation Ser(217) --> Asn, which was present in our original set of truncations but not in the new ones, promotes complete inactivation upon truncation of the N terminus. Therefore, the N terminus of alphaHL cannot be critical for the prepore to pore transformation as previously thought. Residue 217 is involved in the assembly process and must interact indirectly with the distant N terminus during the last step in pore formation. In addition, we provide evidence that an intact N terminus prevents the premature oligomerization of alphaHL monomers in solution.

The leukocidin pore: Evidence for an octamer with four LukF subunits and four LukS subunits alternating around a central axis

The staphylococcal alpha-hemolysin (alphaHL) and leukocidin (Luk) polypeptides are members of a family of related beta-barrel pore-forming toxins. Upon binding to susceptible cells, alphaHL forms water-filled homoheptameric transmembrane pores. By contrast, Luk pores are formed by two classes of subunit, F and S, rendering a heptameric structure displeasing on symmetry grounds at least. Both the subunit stoichiometry and arrangement within the Luk pore have been contentious issues. Here we use chemical and genetic approaches to show that (1) the predominant, or perhaps the only, form of the Luk pore is an octamer; (2) the subunit stoichiometry is 1:1; and (3) the subunits are arranged in an alternating fashion about a central axis of symmetry, at least when a fused LukS-LukF construct is used. The experimental approaches we have used also open up new avenues for engineering the arrangement of the subunits of beta-barrel pore-forming toxins.

The Internal Cavity of the Staphylococcal α-Hemolysin Pore Accommodates ∼175 Exogenous Amino Acid Residues

The cavity within the cap domain of the transmembrane staphylococcal alpha-hemolysin (alphaHL) pore is roughly a sphere of diameter approximately 45 A (molecular surface volume approximately 39,500 A(3)). We tested the ability of the cavity to accommodate exogenous polypeptide chains. Concatemerized Gly/Ser-containing sequences ("loops", L; number of repeats = n; number of residues = 10n + 5, n = 0-21) were inserted at a position located within the cavity of the fully assembled heptameric alphaHL pore. Homomeric pores containing 25 or less residues in each loop (n <or= 2) could be assembled. The pores were protease-resistant, indicating that they had been formed correctly, and produced currents in planar lipid bilayers. The pores showed an up to 70% reduction in unitary conductance, depending on the length of the inserted loop. Protease-resistant heteromeric pores containing wild-type (W) and L subunits were also assembled: when n = 3, up to five L subunits were tolerated; when n = 4, three L subunits were tolerated; and when n = 5 or 6, two L subunits were tolerated. For n >or= 7, only one L subunit was incorporated. As the inserted loop was lengthened, transient closures were observed in planar bilayer experiments with single pores. However, L(1)W(6) pores with very long loops (n = 14 and 21) had unitary conductance values close to those of W(7), suggesting that the loop is extruded through the opening in the cap of the pore into the external medium. Further analysis of bilayer recordings and electrophoretic migration patterns indicates that the upper capacity of the cavity is approximately 175 amino acids. The findings suggest that small functional peptides or proteins might be assembled within the alphaHL pore.

Single-Molecule Observation of the Catalytic Subunit of cAMP-Dependent Protein Kinase Binding to an Inhibitor Peptide

An engineered version of the staphylococcal alpha-hemolysin protein pore, bearing a peptide inhibitor near the entrance to the beta barrel, interacts with the catalytic (C) subunit of cAMP-dependent protein kinase. By monitoring the ionic current through the pore, binding events are detected at the single-molecule level. The kinetic and thermodynamic constants governing the binding interaction and the synergistic effect of MgATP are comparable but not identical to the values in bulk solution. Further, the values are strongly dependent on the applied membrane potential. Additional exploration of these findings may lead to a better understanding of the properties of enzymes at the lipid/water interface. Despite the complications, we suggest that the engineered pore might be used as a sensor element to screen inhibitors that act at either the substrate or ATP binding sites of the C subunit.

Electroosmotic enhancement of the binding of a neutral molecule to a transmembrane pore.

The flux of solvent water coupled to the transit of ions through protein pores is considerable. The effect of this electroosmotic solvent flow on the binding of a neutral molecule [beta-cyclodextrin (betaCD)] to sites within the staphylococcal alpha-hemolysin pore was investigated. Mutant alpha-hemolysin pores were used to which betaCD can bind from either entrance and through which the direction of water flow can be controlled by choosing the charge selectivity of the pore and the polarity of the applied potential. The Kd values for betaCD for individual mutant pores varied by >100-fold with the applied potential over a range of -120 to +120 mV. In all cases, the signs of the changes in binding free energy and the influence of potential on the association and dissociation rate constants for betaCD were consistent with an electroosmotic effect.

Arresting and releasing Staphylococcal alpha-hemolysin at intermediate stages of pore formation by engineered disulfide bonds.

alpha-Hemolysin (alphaHL) is secreted by Staphylococcus aureus as a water-soluble monomer that assembles into a heptamer to form a transmembrane pore on a target membrane. The crystal structures of the LukF water-soluble monomer and the membrane-bound alpha-hemolysin heptamer show that large conformational changes occur during assembly. However, the mechanism of assembly and pore formation is still unclear, primarily because of the difficulty in obtaining structural information on assembly intermediates. Our goal is to use disulfide bonds to selectively arrest and release alphaHL from intermediate stages of the assembly process and to use these mutants to test mechanistic hypotheses. To accomplish this, we created four double cysteine mutants, D108C/K154C (alphaHL-A), M113C/K147C (alphaHL-B), H48C/ N121C (alphaHL-C), I5C/G130C (alphaHL-D), in which disulfide bonds may form between the pre-stem domain and the beta-sandwich domain to prevent pre-stem rearrangement and membrane insertion. Among the four mutants, alphaHL-A is remarkably stable, is produced at a level at least 10-fold greater than that of the wild-type protein, is monomeric in aqueous solution, and has hemolytic activity that can be regulated by the presence or absence of reducing agents. Cross-linking analysis showed that alphaHL-A assembles on a membrane into an oligomer, which is likely to be a heptamer, in the absence of a reducing agent, suggesting that oxidized alphaHL-A is halted at a heptameric prepore state. Therefore, conformational rearrangements at positions 108 and 154 are critical for the completion of alphaHL assembly but are not essential for membrane binding or for formation of an oligomeric prepore intermediate.