Beta-barrel Pore-forming Toxins Research Articles

Implication of Cholesterol in Regulating the Membrane-Interaction Mechanism of Vibrio cholerae Cytolysin, a Beta-Barrel Pore-Forming Toxin

Implication of Cholesterol in Regulating the Membrane-Interaction Mechanism of Vibrio cholerae Cytolysin, a Beta-Barrel Pore-Forming Toxin

Revisiting the oligomerization mechanism of Vibrio cholerae cytolysin, a beta-barrel pore-forming toxin

Vibrio cholerae cytolysin (VCC) is a membrane-damaging beta-barrel pore-forming toxin (beta-PFT). VCC causes permeabilization of the target membranes by forming transmembrane oligomeric beta-barrel pores. Oligomerization is a key step in the mode of action of any beta-PFT, including that of VCC. Earlier studies have identified some of the key residues in VCC that are directly involved in the generation of the inter-protomer contacts, thus playing critical roles in the oligomerization of the membrane-bound toxin. Analysis of the VCC oligomeric pore structure reveals a potential hydrogen-bond network that appears to connect the sidechain of an asparagine residue (Asn582; located within an inter-domain linker sequence) from one protomer to the backbone CO- and NH-groups of the neighbouring protomer, indirectly through water molecules at most of the inter-protomer interfaces. In the present study, we show that the mutation of Asn582Ala affects the oligomerization and the pore-forming activity of VCC in the membrane lipid bilayer of the synthetic lipid vesicles, while the replacement of Asn582Gln results into the restoration of the oligomeric pore-forming ability of the toxin. Using a number of truncated variants of VCC, having deletion in the C-terminal region of the toxin starting from the Asn582 residue or beyond, we also show that the presence of Asn582 is critically required for the oligomerization of the truncated form of the protein.

Staphylococcus aureus Pore-Forming Toxins.

Staphylococcus aureus (S. aureus) is a formidable foe equipped with an armamentarium of virulence factors to thwart host defenses and establish a successful infection. Among these virulence factors, S. aureus produces several potent secreted proteins that act as cytotoxins, predominant among them the beta-barrel pore-forming toxins. These toxins play several roles in pathogenesis, including disruption of cellular adherens junctions at epithelial barriers, alteration of intracellular signaling events, modulation of host immune responses, and killing of eukaryotic immune and non-immune cells. This chapter provides an updated overview on the S. aureus beta-barrel pore-forming cytotoxins, the identification of toxin receptors on host cells, and their roles in pathogenesis.

Physicochemical constraints of elevated pH affect efficient membrane interaction and arrest an abortive membrane-bound oligomeric intermediate of the beta-barrel pore-forming toxin Vibrio cholerae cytolysin

Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging cytotoxic protein. VCC causes permeabilization of the target cell membranes by forming transmembrane oligomeric beta-barrel pores. Membrane pore formation by VCC involves following key steps: (i) membrane binding, (ii) formation of a pre-pore oligomeric intermediate, (iii) membrane insertion of the pore-forming motifs, and (iv) formation of the functional transmembrane pore. Membrane binding, oligomerization, and subsequent pore-formation process of VCC appear to be facilitated by multiple regulatory mechanisms that are only partly understood. Here, we have explored the role(s) of the physicochemical constraints, specifically imposed by the elevated pH conditions, on the membrane pore-formation mechanism of VCC. Elevated pH abrogates efficient interaction of VCC with the target membranes, and blocks its pore-forming activity. Under the elevated pH conditions, membrane-bound fractions of VCC remain trapped in the form of abortive oligomeric species that fail to generate the functional transmembrane pores. Such an abortive oligomeric assembly appears to represent a distinct, more advanced intermediate state than the pre-pore state. The present study offers critical insights regarding the implications of the physicochemical constraints for regulating the efficient membrane interaction and pore formation by VCC.

Programmed Cellular Necrosis Mediated by the Pore-Forming α-Toxin from Clostridium septicum

Programmed necrosis is a mechanism of cell death that has been described for neuronal excitotoxicity and ischemia/reperfusion injury, but has not been extensively studied in the context of exposure to bacterial exotoxins. The α-toxin of Clostridium septicum is a β-barrel pore-forming toxin and a potent cytotoxin; however, the mechanism by which it induces cell death has not been elucidated in detail. We report that α-toxin formed Ca2+-permeable pores in murine myoblast cells, leading to an increase in intracellular Ca2+ levels. This Ca2+ influx did not induce apoptosis, as has been described for other small pore-forming toxins, but a cascade of events consistent with programmed necrosis. Ca2+ influx was associated with calpain activation and release of cathepsins from lysosomes. We also observed deregulation of mitochondrial activity, leading to increased ROS levels, and dramatically reduced levels of ATP. Finally, the immunostimulatory histone binding protein HMGB1 was found to be released from the nuclei of α-toxin-treated cells. Collectively, these data show that α-toxin initiates a multifaceted necrotic cell death response that is consistent with its essential role in C. septicum-mediated myonecrosis and sepsis. We postulate that cellular intoxication with pore-forming toxins may be a major mechanism by which programmed necrosis is induced.

A covalent S‐F heterodimer of leucotoxin reveals molecular plasticity of β‐barrel pore‐forming toxins

Staphylococcal leucotoxins, leucocidins, and gamma-hemolysins are bicomponent beta-barrel pore-forming toxins (beta-PFTs). Their production is associated with several clinical diseases. They have cytotoxic activity due to the synergistic action of a class S component and a class F component, which are secreted as water-soluble monomers and form hetero-oligomeric transmembrane pores, causing the lysis of susceptible cells. Structural information is currently available for the monomeric S and F proteins and the homoheptamer formed by the related alpha-hemolysin. These structures illustrate the start and end points in the mechanistic framework of beta-PFT assembly. Only limited structural data exist for the intermediate stages, including hetero-oligomeric complexes of leucotoxins. We investigated the protein-protein interactions responsible for maintaining the final bipartite molecular architecture and describe here the high-resolution crystal structure and low-resolution solution structure of a site-specific cross-linked heterodimer of gamma-hemolysin (HlgA T28C-HlgB N156C), which were solved by X-ray crystallography and small angle X-ray scattering, respectively. These structures reveal a molecular plasticity of beta-PFTs, which may facilitate the transition from membrane-bound monomers to heterodimers.

Enhanced Stability and Fluidity in Droplet on Hydrogel Bilayers for Measuring Membrane Protein Diffusion

We form artificial lipid bilayers suitable for single-molecule fluorescence microscopy by contacting an aqueous droplet with a hydrogel support immersed in a solution of lipid in oil. Our results show that droplet on hydrogel bilayers (DHBs) have high lipid mobilities, similar to those observed in unsupported lipid bilayers. DHBs are also stable over a period of several weeks. We examine membrane protein diffusion in these bilayers and report a decreased lateral mobility of the heptameric beta-barrel pore-forming toxin alpha-hemolysin versus that of its monomeric precursor. These results corroborate previous models of the alpha-hemolysin insertion mechanism where the monomer binds to the lipid bilayer without insertion.

Identification of a prepore large-complex stage in the mechanism of action of Clostridium perfringens enterotoxin.

Clostridium perfringens enterotoxin (CPE) is the etiological agent of the third most common food-borne illness in the United States. The enteropathogenic effects of CPE result from formation of large CPE-containing complexes in eukaryotic cell membranes. Formation of these approximately 155- and approximately 200-kDa complexes coincides with plasma membrane permeability changes in eukaryotic cells, causing a Ca2+ influx that drives cell death pathways. CPE contains a stretch of amino acids (residues 81 to 106) that alternates markedly in side chain polarity (a pattern shared by the transmembrane domains of the beta-barrel pore-forming toxin family). The goal of this study, therefore, was to investigate whether this CPE region is involved in pore formation. Complete deletion of the CPE region from 81 to 106 produced a CPE variant that was noncytotoxic for Caco-2 cells and was unable to form CPE pores. However, this variant maintained the ability to form the approximately 155-kDa large complex. This large complex appears to be a prepore present on the plasma membrane surface since it showed greater susceptibility to proteases, increased complex instability, and a higher degree of dissociation from membranes compared to the large complex formed by recombinant CPE. When a D48A mutation was engineered into this prepore-forming CPE variant, the resultant variant was unable to form any prepore approximately 155-kDa large complex. Collectively these findings reveal a new step in CPE action, whereby receptor binding is followed by formation of a prepore large complex, which then inserts into membranes to form a pore.

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.

Cooperative Assembly of -Barrel Pore-Forming Toxins

Bacterial beta-barrel pore-forming toxins are secreted as water-soluble monomeric proteins and assemble into beta-barrel-shaped pores/channels through membranes of target cells, causing cell death and lysis. The pore assemblies that undergo various intermediate stages are symbolized by the association of multi-subunit structures in cells. Crystal structures of water-soluble monomers and membrane-embedded oligomeric pores, and recent studies involving biochemical detection and direct visualization of the sequential assembly of the toxin monomers have solved the mystery of how the pores are formed. Here, we review the mechanism of the cooperative assembly of several toxins of interest to explain the nature of the activities of the toxins.

Subunit composition of a bicomponent toxin: staphylococcal leukocidin forms an octameric transmembrane pore.

Staphylococcal leukocidin pores are formed by the obligatory interaction of two distinct polypeptides, one of class F and one of class S, making them unique in the family of beta-barrel pore-forming toxins (beta-PFTs). By contrast, other beta-PFTs form homo-oligomeric pores; for example, the staphylococcal alpha-hemolysin (alpha HL) pore is a homoheptamer. Here, we deduce the subunit composition of a leukocidin pore by two independent methods: gel shift electrophoresis and site-specific chemical modification during single-channel recording. Four LukF and four LukS subunits coassemble to form an octamer. This result in part explains properties of the leukocidin pore, such as its high conductance compared to the alpha HL pore. It is also pertinent to the mechanism of assembly of beta-PFT pores and suggests new possibilities for engineering these proteins.

CytK toxin of Bacillus cereus forms pores in planar lipid bilayers and is cytotoxic to intestinal epithelia.

CytK is a cytotoxin isolated from a strain of Bacillus cereus cultured from cases of necrotic enteritis and the amino acid sequence of the protein suggests that it may belong to the family of beta-barrel pore-forming toxins. We show here in planar lipid bilayers the toxin is able to form pores which are weakly anion selective and exhibit an open channel probability close to one. The predicted minimum pore diameter is approximately 7 A. We also show that cytK is a potent cytotoxin against human intestinal Caco-2 epithelia. CytK, like other beta-barrel pore-forming toxins, spontaneously forms oligomers which are resistant to sodium dodecyl sulphate (SDS), but not to boiling. CytK represents a pore-forming toxin linked with human cases of necrotic enteritis.