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.
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+-cal-modulin 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.
Vibrio cholerae cytolysin (VCC) forms SDS-stable heptameric β-barrel transmembrane pores in mammalian cell membranes. In contrast to structurally related pore formers of gram-positive organisms, no ...oligomeric prepore stage of assembly has been detected to date. In the present study, disulfide bonds were engineered to tie the pore-forming amino acid sequence to adjacent domains. In their nonreduced form, mutants were able to bind to rabbit erythrocytes and to native erythrocyte membranes suspended in PBS solution and form SDS-labile oligomers. These remained nonfunctional and represented the long-sought VCC prepores. Disulfide bond reduction in these oligomers released the pore-forming sequence from its locked position, and subsequent membrane insertion led to formation of SDS-stable pores and hemolysis. Addition of increasing amounts of an inactive mutant to wild-type toxin resulted in the formation of mixed oligomers with progressively reduced SDS stability on membranes. Membrane insertion of active monomers in these hybrid oligomers was still observed, but the functional pore diameter was reduced. These findings indicate that formation of an oligomeric prepore precedes membrane insertion of the pore-forming amino acid sequence and demonstrate that pore formation by VCC follows the same archetypical pathway as β-barrel cytolysins of gram-positive organisms such as staphylococcal α-toxin.--Löhner, S., Walev, I., Boukhallouk, F., Palmer, M., Bhakdi, S., Valeva, A. Pore formation by Vibrio cholerae cytolysin follows the same archetypical mode as β-barrel toxins from gram-positive organisms.
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.
: Mast cells are known to be important effector cells in innate immune responses to bacterial infections. However, up to now, neither the mechanisms nor the relevance of mast cell degranulation in ...innate skin immune responses to bacteria have been adequately addressed. In this article, we show that the bacterial toxins streptolysin O (SLO) and α‐toxin potently induce degranulation of mast cells in vitro and in vivo. Furthermore, intradermal injection of the toxins results in pronounced skin inflammation, which either resolves quickly within a few h (SLO‐induced inflammation) or presents a chronic process with ongoing inflammation for weeks (α‐toxin). Interestingly, mast cells mediated the inflammatory effects of SLO, but in contrast limited inflammatory skin responses to α‐toxin. These findings further support the hypothesis that mast cells are critically involved in initiating and modulating optimal host responses to bacteria by either inflammatory or anti‐inflammatory effects, depending on the course of the host reaction induced by the pathogen.
Many strains of Vibrio cholerae produce a cytolysin (VCC) that forms oligomeric transmembrane pores in animal cells. The molecule is secreted as a procytolysin (pro-VCC) of 79 kDa that must be ...cleaved at the N terminus to generate the active 65-kDa toxin. Processing can occur in solution, and previous studies have described the action of mature VCC thus generated. However, little is known about the properties of pro-VCC itself. In this study, it is shown that pro-VCC exist as a monomer in solution and binds as a monomer to eukaryotic cells. Bound pro-VCC can then be activated either by exogenous, extracellular, or by endogenous, cell-bound proteases. In both cases, cleavage generates the 65-kDa VCC that oligomerizes to form transmembrane pores. A wide variety of exogenous proteinases can mediate activation. In contrast, the activating cellular protease is selectively inhibited by the hydroxamate inhibitor TAPI, and thus probable candidates are members of the ADAM-metalloproteinase family. Furin, MMP-2, MMP-9, and serine proteinases were excluded. Cells over-expressing ADAM-17, also known as tumor necrosis factor α converting enzyme, displayed increased activation of VCC, and knockout cells lacking ADAM-17 had a markedly decreased capacity to cleave the protoxin. The possibility is raised that pro-VCC is targeted to membrane sites that selectively contain or are accessible to cellular ADAM-metalloproteinases. Although many microbial toxins are activated by furin, this is the first evidence for processing by a cellular metalloproteinase. We identified ADAM-17 as a potent activator of pro-VCC.
Vibrio cholerae cytolysin (VCC) forms oligomeric pores in lipid bilayers containing cholesterol. Membrane permeabilization is inefficient if the sterol is embedded within bilayers prepared from ...phosphatidylcholine only but is greatly enhanced if the target membrane also contains ceramide. Although the enhancement of VCC action is stereospecific with respect to cholesterol, we show here that no such specificity applies to the two stereocenters in ceramide; all four stereoisomers of ceramide enhanced VCC activity in cholesterol-containing bilayers. A wide variety of ceramide analogs were as effective asd-erythro-ceramide, as was diacylglycerol, suggesting that the effect of ceramide exemplifies a general trend of lipids with a small headgroup to augment the activity of VCC. Incorporation of these cone-shaped lipids into cholesterol-containing bilayers also gave similar effects with streptolysin O, another cholesterol-specific but structurally unrelated cytolysin. In contrast, the activity of staphylococcal α-hemolysin, which does not share with the other toxins the requirement for cholesterol, was far less affected by the presence of lipids with a conical shape. The collective data indicate that sphingolipids and glycerolipids do not interact with the cytolysins specifically. Instead, lipids that have a conical molecular shape appear to effect a change in the energetic state of membrane cholesterol that in turn augments the interaction of the sterol with the cholesterol-specific cytolysins.
Staphylococcal α -toxin is a 293-residue, single-chain polypeptide that spontaneously assembles into a heptameric pore in target cell membranes. To identify the pore-forming domain, substitution ...mutants have been produced in which single cysteine residues were introduced throughout the toxin molecule. By attaching the environmentally sensitive dye acrylodan to the sulfhydryl groups, the environment of individual amino acid side chains could be probed. In liposomes, a single 23-amino acid sequence (residues 118-140) was found to move from a polar to a nonpolar environment, indicating that this sequence forms the walls of the pore. However, periodicity in side chain environmental polarity could not be detected in the liposomal system. In the present study, the fluorimetric analyses were extended to physiological target cells. With susceptible cells such as rabbit erythrocytes and human lymphocytes, the 23 central amino acids 118-140 were again found to insert into the membrane; in contrast to the previous study with liposomes, the expected periodicity was now detected. Thus, every other residue in the sequence 126-140 entered a nonpolar environment in a striking display of an amphipathic transmembrane β -barrel. In contrast, human granulocytes were found to bind α -toxin to a similar extent as lymphocytes, but the heptamers forming on these cells failed to insert their pore-forming domain into the membrane. As a consequence, nonfunctional heptamers assembled and the cells remained viable. The data resolve the molecular organization of a pore-forming toxin domain in living cells and reveal that resistant cells can prevent insertion of the functional domain into the bilayer.
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.
Streptolysin O (SLO), a polypeptide of 571 amino acids, belongs to a family of highly homologous toxins that bind to cell
membranes containing cholesterol and then polymerize to form large ...transmembrane pores. A conserved region close to the C
terminus contains the single cysteine residue of SLO and has been implicated in membrane binding, which has been the only
clear assignment of function to a part of the sequence. We have used a cysteine-less active mutant of SLO to introduce single
cysteine residues at 19 positions distributed throughout the sequence. The cysteines were derivatized with the polarity-sensitive
fluorophore acrylodan, and the fluorescence emission of the label was examined at the different stages of SLO pore assembly.
With several mutants, oligomerization on membranes was accompanied by emission blue-shifts, indicating movement of the label
into a more hydrophobic environment. These effects were essentially confined to the range of amino acids 213-305. With oligomeric
mutants L274C, S286C, and S305C, additional environmental alterations were induced when different nondenaturing detergents
were used to dislodge the membrane lipids from the oligomers. The corresponding amino acid residues thus insert into the lipid
bilayer during pore formation. Conversely, the spectra of oligomeric mutants A213C and T245C were not affected by detergents.
Devoid of contact with the lipid bilayer, these amino acid residues probably participate in the interaction of SLO molecules
within the oligomer.
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