Angela Valeva

Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O

The pore-forming toxin streptolysin O (SLO) can be used to reversibly permeabilize adherent and nonadherent cells, allowing delivery of molecules with up to 100 kDa mass to the cytosol. Using FITC-labeled albumin, 105–106 molecules were estimated to be entrapped per cell. Repair of toxin lesions depended on Ca2+-calmodulin and on intact microtubules, but was not sensitive to actin disruption or to inhibition of protein synthesis. Resealed cells were viable for days and retained the capacity to endocytose and to proliferate. The active domains of large clostridial toxins were introduced into three different cell lines. The domains were derived from Clostridium difficile B-toxin and Clostridium sordelli lethal toxin, which glycosylate small G-proteins, and from Clostridium botulinum C2 toxin, which ADP-ribosylates actin. After delivery with SLO, all three toxins disrupted the actin cytoskeleton to cause rounding up of the cells. Glucosylation assays demonstrated that G-proteins Rho and Ras were retained in the permeabilized cells and were modified by the respective toxins. Inactivation of these G-proteins resulted in reduced stimulus-dependent granule secretion, whereas ADP-ribosylation of actin by the C. botulinum C2-toxin resulted in enhanced secretion in cells. The presented method for introducing proteins into living cells should find multifaceted application in cell biology.

Staphylococcal alpha-toxin, streptolysin-O, and Escherichia coli hemolysin: prototypes of pore-forming bacterial cytolysins.

Abstract Staphylococcal alpha-toxin, streptolysin-O, and Escherichia coli hemolysin are well-studied prototypes of pore-forming bacterial cytotoxins. Each is produced as a water-soluble single-chain polypeptide that inserts into target membranes to form aqueous transmembrane pores. This review will compare properties of the three toxin prototypes, highlighting the similarities and also the differences in their structure, mode of binding, mechanism of pore formation, and the responses they elicit in target cells. Pore-forming toxins represent the most potent and versatile weapons with which invading microbes damage the host macroorganism.

Potassium-inhibited processing of IL-1 beta in human monocytes.

Agents that deplete cells of K+ without grossly disrupting the plasma membrane were found to stimulate the cleavage of pro‐interleukin (IL)‐1 beta to mature IL‐1 beta. Agents examined in this study included staphylococcal alpha‐toxin and gramicidin, both of which selectively permeabilize plasma membranes for monovalent ions, the ionophores nigericin and valinomycin, and the Na+/K+ ATPase inhibitor ouabain. K+ depletion by brief hypotonic shock also triggered processing of pro‐IL‐1 beta. The central role of K+ depletion for inducing IL‐1 beta maturation was demonstrated in cells permeabilized with alpha‐toxin: processing of pro‐IL‐1 beta was totally blocked when cells were suspended in medium that contained high K+, but could be induced by replacing extracellular K+ with Na+, choline+ or sucrose. To test whether K+ flux might also be important in physiological situations, monocytes were stimulated with lipopolysaccharide (LPS) for 1‐2 h to trigger pro‐IL‐1 beta synthesis, and transferred to K(+)‐rich medium. This maneuver totally suppressed IL‐1 beta maturation. Even after 16 h, however, removal of K+ from the medium resulted in rapid processing and export of IL‐1 beta. Ongoing export of mature IL‐1 beta from cells stimulated with LPS for 2‐6 h could also be arrested by transfer to K(+)‐rich medium. Moreover, a combination of two K+ channel blockers inhibited processing of IL‐1 beta in LPS‐stimulated monocytes. We hypothesize that K+ movement and local K+ concentrations directly or indirectly influence the action of interleukin‐1 beta‐converting enzyme (ICE) and, possibly, of related intracellular proteases.

Molecular architecture of a toxin pore: a 15-residue sequence lines the transmembrane channel of staphylococcal alpha-toxin.

Staphylococcus aureus alpha‐toxin is a hydrophilic polypeptide of 293 amino acids that produces heptameric transmembrane pores. During assembly, the formation of a pre‐pore precedes membrane permeabilization; the latter is linked to a conformational change in the oligomer. Here, 41 single‐cysteine replacement toxin mutants were thiol‐specifically labelled with the polarity‐sensitive fluorescent probe acrylodan. After oligomerization on membranes, only the mutants with acrylodan attached to residues in the sequence 118–140 exhibited a marked blue shift in the fluorescence emission maximum, indicative of movement of the fluorophore to a hydrophobic environment. Within this region, two functionally distinct parts could be identified. For mutants at positions 126–140, the shifts were partially reversed after membrane solubilization by detergents, indicating a direct interaction of the label with the membrane lipids. Membrane insertion of this sequence occurred together with the final pre‐pore to pore transition of the heptamer. Thus residues 126–140 constitute a transmembrane sequence in the pore. With labelled residues 118–124, pre‐pore assembly was the critical event to induce the spectral shifts, which persisted after the removal of membrane lipids and hence probably reflects protomer‐protomer contacts within the heptamer. Finally, a derivative of the mutant N121C yielded occluded pores which could be opened by reductive reversal of the modification. Therefore this residue probably lines the lumen of the pore.

Evidence That Clustered Phosphocholine Head Groups Serve as Sites for Binding and Assembly of an Oligomeric Protein Pore

High susceptibility of rabbit erythrocytes toward the poreforming action of staphylococcal α-toxin correlates with the presence of saturable, high affinity binding sites. All efforts to identify a protein or glycolipid receptor have failed, and the fact that liposomes composed solely of phosphatidylcholine are efficiently permeabilized adds to the enigma. A novel concept is advanced here to explain the puzzle. We propose that low affinity binding moieties can assume the role of high affinity binding sites due to their spatial arrangement in the membrane. Evidence is presented that phosphocholine head groups of sphingomyelin, clustered in sphingomyelin-cholesterol microdomains, serve this function for α-toxin. Clustering is required so that oligomerization, which is prerequisite for stable attachment of the toxin to the membrane, can efficiently occur. Outside these clusters, binding to phosphocholine is too transient for toxin monomers to find each other. The principle of membrane targeting in the absence of any genuine, high affinity receptor may also underlie the assembly of other lipid-inserted oligomers including cytotoxic peptides, protein toxins, and immune effector molecules.

Potassium Regulates IL-1β Processing Via Calcium-Independent Phospholipase A2

We report that potassium leakage from cells leads to activation of the Ca2+-independent phospholipase A2 (iPLA2), and the latter plays a pivotal role in regulating the cleavage of pro-IL-1β by the IL-converting enzyme caspase-1 in human monocytes. K+ efflux led to increases of cellular levels of glycerophosphocholine, an unambiguous indicator of phospholipase A2 activation. Both maturation of IL-1β and formation of glycerophosphocholine were blocked by bromoenol lactone, the specific iPLA2 inhibitor. Bromoenol lactone-dependent inhibition of IL-1β processing was not due to perturbation of the export machinery for pro-IL-1β and IL-1β or to caspase-1 suppression. Conspicuously, activation of Ca2+-dependent phospholipase A2 did not support but rather suppressed IL-1β processing. Thus, our findings reveal a specific role for iPLA2 activation in the sequence of events underlying IL-1β maturation.

Why Escherichia coli α-hemolysin induces calcium oscillations in mammalian cells—the pore is on its own

Escherichia coli α‐hemolysin (HlyA), archetype of a bacterial pore‐forming toxin, has been reported to deregulate physiological Ca2+ channels, thus inducing periodic low‐frequency Ca2+ oscillations that trigger transcriptional processes in mammalian cells. The present study was undertaken to delineate the mechanisms underlying the Ca2+ oscillations. Patch‐clamp experiments were combined with single cell measurements of intracellular Ca2+ and with flow‐cytometric analyses. Application of HlyA at subcytocidal concentrations provoked Ca2+ oscillations in human renal and endothelial cells. However, contrary to the previous report, the phenomenon could not be inhibited by the Ca2+ channel blocker nifedipine and Ca2+ oscillations showed no constant periodicity at all. Ca2+ oscillations were dependent on the pore‐forming activity of HlyA: application of a nonhemolytic but bindable toxin had no effect. Washout experiments revealed that Ca2+ oscillations could not be maintained in the absence of toxin in the medium. Analogously, propidium iodide flux into cells occurred in the presence of HlyA, but cells rapidly became impermeable toward the dye after toxin washout, indicating resealing or removal of the membrane lesions. Finally, patch‐clamp experiments revealed temporal congruence between pore formation and Ca2+ influx. We conclude that the nonperiodic Ca2+ oscillations induced by HlyA are not due to deregulation of physiological Ca2+ channels but derive from pulsed influxes of Ca2+ as a consequence of formation and rapid closure of HlyA pores in mammalian cell membranes.—Koschinski, A., Repp, H., Ünver, B., Dreyer, F., Brockmeier, D., Valeva, A., Bhakdi, S., and Walev, I. Why Escherichia coli α‐hemolysin induces calcium oscillations in mammalian cells—the pore is on its own. FASEB J. 20, E80‐E87 (2006)

Binding of Escherichia coli Hemolysin and Activation of the Target Cells Is Not Receptor-dependent

Production of a single cysteine substitution mutant, S177C, allowed Escherichia coli hemolysin (HlyA) to be radioactively labeled with tritiated N-ethylmaleimide without affecting biological activity. It thus became possible to study the binding characteristics of HlyA as well as of toxin mutants in which one or both acylation sites were deleted. All toxins bound to erythrocytes and granulocytes in a nonsaturable manner. Only wild-type toxin and the lytic monoacylated mutant stimulated production of superoxide anions in granulocytes. An oxidative burst coincided with elevation of intracellular Ca2+, which was likely because of passive influx of Ca2+ through the toxin pores. Competition experiments showed that binding to the cells was receptor-independent, and preloading of cells with a nonlytic HlyA mutant did not abrogate the respiratory burst provoked by a subsequent application of wild-type HlyA. In contrast to a previous report, expression or activation of the β2 integrin lymphocyte function-associated antigen-1 did not affect binding of HlyA. We conclude that HlyA binds nonspecifically to target cells and a receptor is involved neither in causing hemolysis nor in triggering cellular reactions.

Electrophysiological evidence for heptameric stoichiometry of ion channels formed by Staphylococcus aureus alpha‐toxin in planar lipid bilayers

Staphylococcal alpha‐toxin forms homo‐oligomeric channels in lipid bilayers and cell membranes. Here, we report that electrophysiological monitoring of single‐channel function using a derivatized cysteine substitution mutant allows accurate determination of the subunit stoichiometry of the oligomer in situ. The electrophysiological phenotype of channels formed in planar lipid bilayers with the cysteine replacement mutant I7C is equal to that of the wild type. When pores were formed with I7C, alterations of several channel properties were observed upon modification with SH reagents. Decreases in conductance then occurred that were seen only as negative voltage was applied. At the level of single channels, these were manifest as stepwise changes in conductance, each step most probably reflecting modification of a single SH group within the oligomer. Because seven steps were observed, the functional channel formed by alpha‐toxin in planar lipid membranes is a heptamer.

STAPHYLOCOCCAL ALPHA -TOXIN: THE ROLE OF THE N-TERMINUS IN FORMATION OF THE HEPTAMERIC PORE: A FLUORESCENCE STUDY

Staphylococcus aureus alpha-toxin forms heptameric pores on eukaryotic cell membranes. Assembly of the heptamer precedes formation of the transmembrane pore. The latter event depends on a conformational change that drives a centrally located stretch of 15 amino acid residues into the lipid bilayer. A second region of the molecule that has been implicated in the pre-pore to pore transition is the far N-terminus. Here, we used fluorescently labeled single cysteine replacement mutants to analyze the functional role of the far N-terminus of alpha-toxin. Pyrene attached to mutants S3C, I5C and 17C forms excimers within the toxin pore complex. This indicates that the distance of adjacent N-termini is less than 10-12 Angstrom. By labeling with the polarity-sensitive fluorophore acrylodan, pore formation is shown to cause distinct environmental changes in the N-terminus. Removal of membrane lipids from the labeled heptamers has no effect upon the acrylodan spectrum, indicating lack of direct contact of the N-terminus with the target membrane. The environmental alterations to the N-terminus are thus due to altered protein structure only. Both acrylodan emission shifts and pyrene excimers were shown to be absent in toxin heptamers that were arrested at the pre-pore stage. Therefore, while not being directly involved in membrane penetration, the N-termini of the alpha-toxin heptamer subunits move into immediate mutual proximity concomitantly with transmembrane pore formation.