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Botulinolysin (BLY) is an oxygen-labile hemolysin produced by Clostridium botulinum (1-4, 13) that is a member of the family of thiol-activated cytotoxins (1-4, 32, 34). This family consists, at present, of about 20 different toxins that are produced by four genera of gram-positive bacteria, namely, Streptococcus, Bacillus, Clostridium, and Listeria (1-4, 8, 10, 27, 34). Formation of ring-shaped and arc-shaped structures has been observed under the electron microscope on erythrocyte membranes that have been treated with some of these toxins (5, 11, 12, 20, 24, 29). Members of our group reported a molecular model for the formation of rings on erythrocyte membranes by streptolysin O (SLO) from Streptococcus pyogenes (29). The various toxins bind cholesterol and their cytolytic activity is inhibited by treatment with cholesterol (12, 15, 16, 20, 31). Recently, the three-dimensional structure of perfringolysin O (PFO; -toxin), another thiol-activated cytolysin, was reported, and the fine structures of the cholesterol-binding site and of the region required for formation of oligomers were described (28). Thiol-activated toxins have potent lytic activity against mammalian cells and other eukaryotic cells (3, 35). Purified BLY also has a lethal effect on mice and a cytotoxic effect on Vero cells (13, 33). Thus, membrane damage by thiol-activated toxins might play an important role in the pathogenesis (9) of various diseases if the causative microorganisms produce thiol-activated cytolysins. We are interested in the similarities and differences between toxins from different genera, and we are attempting to clarify the characteristics of various toxins. In this study, we examined the fine-structural changes in erythrocyte membranes induced by BLY, focusing on the formation of ring-shaped structures, as part of an effort to elucidate the mechanism of membrane damage by BLY of erythrocyte membranes.

BLY was purified as described by Haque et al., with slight modification (13). The hemolytic activity of BLY was expressed as the 50% hemolytic dose per microgram of protein. The specific activity of our preparation of BLY was approximately 1,500 hemolytic units (HU)/µg of protein with rabbit erythrocytes as the assay material. Erythrocyte and ghost membranes were prepared from rabbit erythrocytes by the previously described method, with slight modification (29, 30). Preparations of membranes that had been treated with BLY as described below were washed with physiological saline (Ohtsuka Pharmaceutical Co., Tokyo, Japan), fixed with 2.5% glutaraldehyde for 1 min, washed with distilled water (DW; Ohtsuka Pharmaceutical Co.), and negatively stained with 2% phosphotungstic acid. Specimens were observed under a transmission electron microscope (Hitachi H-500, JEOL 2000 EX, JEM 1010, or Carl Zeiss CEM-902A).

Sizes and structures of BLY rings on erythrocyte membranes. Twenty microliters of a 10% suspension of erythrocytes that had been diluted with physiological saline was mixed with 10 µl of BLY (170,000 HU/ml) in a test tube. The mixture was incubated at 37°C for 5 min. Then 10 µl of the mixture of lysed erythrocytes was dropped onto 100 µl of DW on Sealon film (Fuji Film Co., Tokyo, Japan) and mixed gently with a micropipette. After 30 to 40 min, the membrane fragments that floated at the air-water interface were mounted on supporting Butval-98 films (Nissin EM, Tokyo, Japan) on a grid for electron microscopy. Negative staining of erythrocyte membranes treated with BLY revealed numerous ring-shaped structures (BLY rings [Fig. 1A]). Semicircular structures were also observed. The inner diameters of BLY rings were 32 ± 2.4 nm (n = 50), as determined from radii of semicircular rings, and the rings themselves were 6.7 ± 0.6 nm (n = 50) wide, as observed in the case of SLO rings. The dimensions of the rings, together with those of rings formed by other toxins, are summarized in Table 1. Higher magnification revealed a double-layered structure (29, 30) (Fig. 1B). Crown-shaped structures (29) were also observed on rings at folded edges of erythrocyte membranes (data not shown).

View larger version (132K): [in this window] [in a new window]   FIG. 1.   Electron micrographs of rings formed on an erythrocyte membrane that had been treated with BLY at 37°C for 5 min. Note rings with pores in panel A and double-layered structures indicated by the arrow in panel B.

Effects of temperature on binding and formation of pores by BLY on erythrocyte membranes. Ghost membranes were thawed at room temperature after storage at 80°C. Floating ghost membranes on the surface of DW were prepared as described previously (23, 29) and mounted on supporting Butval-98 films on a grid. Purified BLY was diluted 25-fold with 5 mM Tris-HCl (pH 7.2) that contained 0.85% NaCl and 5 mM cysteine (Tris NaCl-cysteine buffer). The ghost membranes on supporting films were treated with 10 µl of diluted BLY (6,800 HU/ml). After the treatment with BLY at room temperature for 3 to 5 min, we observed numerous BLY rings (results resembled those in Fig. 1A). By contrast, we observed no ring-shaped structures on ghost membranes treated with BLY at 0°C (data not shown). Binding of BLY to ghost membranes was confirmed by immunogold labeling with a pooled preparation of four mouse monoclonal antibodies against BLY that had almost the same affinities. For visualization, we used 5-nm-diameter colloidal-gold-conjugated goat antibodies against mouse immunoglobulin G (Zymed Laboratories Inc., San Francisco, Calif.) (data not shown). Controls were treated with only gold-conjugated second antibody, and no gold particles were found on the membranes. Rings with pores formed on membranes that had been incubated with BLY at 0°C for 3 to 5 min, washed to remove excess BLY, and then incubated at 37°C for 1 min (results resembled those in Fig. 1A).

Effects of glutaraldehyde on pore formation. When ghost membranes were treated with BLY at 0°C for 3 to 5 min, fixed with 1% glutaraldehyde at 0°C for 1 min, and then incubated at 37°C for 1 to 3 min, we observed very few BLY rings on the membranes (data not shown). However, BLY formed numerous rings with pores on membranes that had been fixed with glutaraldehyde for 1 min before exposure to BLY at room temperature for 5 min (results resembled those in Fig. 1A).

Effects of Zn²⁺ ions on pore formation. When the preparation of BLY was diluted with Tris NaCl-cysteine buffer that contained 10 mM Zn²⁺ ions and applied to membranes at room temperature for 5 min, no BLY rings were observed. However, gold particles were found on these membranes after immunogold labeling (data not shown).