High susceptibility of rabbit erythrocytes toward the poreforming action of staphylococcal ␣-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 ␣-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 Hla (α-toxin) from Staphylococcus aureus is an important pathogenic factor of the bacterium S. aureus and also a model system for the process of membrane-induced protein oligomerisation and pore formation. It has been shown that binding to lipid membranes at neutral or basic pH requires the presence of a phosphocholine-headgroup. Thus, sphingomyelin and phosphatidylcholine may serve as interaction partners in cellular membranes. Based on earlier studies it has been suggested that rafts of sphingomyelin are particularly efficient in toxin binding. In this study we compared the oligomerisation of Hla on liposomes of various lipid compositions in order to identify the preferred interaction partners and conditions. Hla seems to have an intrinsic preference for sphingomyelin compared to phosphatidylcholine due to a higher probability of oligomerisation of membrane bound monomer. We also can show that increasing the surface density of Hla-binding sites enhances the oligomerisation efficiency. Thus, preferential binding to lipid rafts can be expected in the cellular context. On the other hand, sphingomyelin in the liquid disordered phase is a more favourable binding partner for Hla than sphingomyelin in the liquid ordered phase, which makes the membrane outside of lipid rafts the more preferred region of interaction. Thus, the partitioning of Hla is expected to strongly depend on the exact composition of raft and non-raft domains in the membrane.
Objective: In an experimental setting, human basophil degranulation was triggered by anti-IgE to measure the effects from homeopathic solutions in an in-vitro cell system. A 3-color flow cytometric method with enhanced accuracy was established. As an example we looked at the influence of histamine on anti-IgE activation of basophils. Methods: Basophils were identified in the flow cytometer by their physical properties in the forward and side scatter light depiction and by gating on CD2–, CD14–, CD16–, CD19–, HLA-DR– negative and CD123-positive cells. CD63 expression on the cell surface of the anti-IgE-activated basophils served as an activation marker. Results: With this method we were able to study basophil function of the 0.6–3.9% basophils out of the mononuclear blood cell fraction and to document their activation status upon anti-IgE activation. Optimal activation occurs at 0.6 µg/ml final anti-IgE concentration; not less than 10,000 basophils have to be counted per batch to reduce the variation of the measurement. The fixation method was able to stabilize activation for two days. After investigation and reduction of the source of measurement variability, an unequivocally inhibited basophil activation was documented in a partly optimized system with homeopathic dilutions of histamine (10–22M, 10–23M, 10–24M, and 10–25M histamine). Dilutions greater than 10–20M histamine (Avogadro’s number 6.02 × 1023) account for less than 1.36 molecules of histamine in the test sample, indicating a true homeopathic effect. Conclusions: This test system is adequate for studying the effects of highly diluted mediators on basophil activation by anti-IgE. The systematic application of this experimental arrangement is recommended to study the effects of homeopathic dilutions on basophils.
Alpha-hemolysin from Staphylococcus aureus is secreted as a water-soluble monomer and assembles on membranes to oligomerize into a homo-heptameric, water-filled pore. These pores lead to lysis and cell death. Although the structure of the heptameric pore is solved by means of X-ray crystallography, structures of intermediate states-from the soluble monomer to all potential "pre-pore" structures-are yet unknown. Here, we propose a model of the monomeric alpha-hemolysin in solution based on molecular modeling, verified by small angle X-ray scattering data. This structure reveals details of the monomeric conformation of the alpha-hemolysin, for example inherent flexibility, along with definite differences in comparison to the structures used as templates.
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