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Interaction of the Vibrio cholerae cytolysin (VCC) with cholesterol, some cholesterol esters, and cholesterol derivatives: a TEM study
Journal of
Structural Biology Journal of Structural Biology 139 (2002) 122–135 www.academicpress.com
Interaction of the Vibrio cholerae cytolysin (VCC) with cholesterol, some cholesterol esters, and cholesterol derivatives: a TEM study J. Robin Harris,a,* Sucharit Bhakdi,b Ulrich Meissner,a Dirk Scheffler,a Robert Bittman,c Guoqing Li,c Alexander Zitzer,b and Michael Palmerd a Institute of Zoology, University of Mainz, 55099 Mainz, Germany Institute of Medical Microbiology and Hygiene, University of Mainz, D-55101 Mainz, Germany Department of Chemistry and Biochemistry, Queens College, City University of New York, Flushing, New York 11367-1597, USA d Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1 b
c
Received 12 June 2002, and in revised form 5 July 2002
Abstract The Vibrio cholerae cytolysin (VCC) 63-kDa monomer has been shown to interact in aqueous suspension with cholesterol microcystals to produce a ring/pore-like heptameric oligomer 8 nm in outer diameter. Transmission electron microscopy data were produced from cholesterol samples adsorbed to carbon support films, spread across the holes of holey carbon films, and negatively stained with ammonium molybdate. The VCC oligomers initially attach to the edge of the stacked cholesterol bilayers and with increasing time cover the two planar surfaces. VCC oligomers are also released into solution, with some tendency to cluster, possibly via the hydrophobic membrane-spanning domain. At the air/water interface, the VCC oligomers are likely to be selectively oriented with the hydrophobic domain facing the air. Despite some molecular disorder/plasticity within the oligomers, multivariate statistical analysis and rotational self-correlation using IMAGIC-5 strongly suggest the presence of sevenfold rotational symmetry. To correlate the electron microscopy data with on-going biochemical and permeability studies using liposomes of varying lipid composition, the direct interaction of VCC with several cholesterol derivatives and other steroids has been examined. 19Hydroxycholesterol and 7b-hydroxycholesterol both induce VCC oligomerization. b-Estradiol, which does not possess an aliphatic side chain, also efficiently induces VCC oligomer formation, as does cholesteryl acetate. Cholesteryl stearate and oleate and the C22 (2-trifluoroacetyl)naphthyloxy analogue of cholesterol fail to induce VCC oligomerization, but binding of the monomer to the surface of these steroids does occur. Stigmasterol has little tendency to induce oligomer formation, and oligomers are largely confined to the edge of the bilayers; ergosterol has even less oligomerization ability. Attempts to solubilize and stabilize the VCC oligomers from cholesterol suspensions have been pursued using the neutral surfactant octylglucoside. Although individual solubilized oligomers have been defined which exhibit a characteristic cytolysin channel conformation in the side-on orientation, a tendency remains for the oligomers to cluster via their hydrophobic domains. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Vibrio cholerae; Cytolysin; VCC; Oligomerization; Cholesterol; Negative staining; TEM
1. Introduction Enteropathogenic Vibrio cholerae produces the classic cholera toxin and also the zonula occludens and accessory cholera toxins and a cytolysin. The 63kDa V. cholerae cytolysin (VCC) monomer is readily *
Corresponding author. Fax: 49-6131-3924652. E-mail address: [email protected] (J. Robin Harris).
obtained from nontoxigenic strains of V. cholerae, such as the non-O1 serotypes and the E1 Tor biotype of O1 (Zitzer et al., 1995). It has the ability to form ring-like oligomeric pores that penetrate cholesterolcontaining cell membranes and thereby produce cellular lysis (Krasilnikov et al., 1992; Zitzer et al., 1997a,b; Ikagai et al., 1996, 1999). That VCC has a carbohydrate-binding domain that can regulate pore formation was shown by Saha and Banerjee (1997).
1047-8477/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 4 7 - 8 4 7 7 ( 0 2 ) 0 0 5 6 3 - 4
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VCC interacts with liposomes containing cholesterol (Ikagai et al., 1999), and the presence of ceramides has some influence upon the ability of the toxin monomer to access membrane cholesterol and oligomerize to form a cytolytic channel (Zitzer et al., 1999, 2000, 2001). To extend our transmission electron microscopy (TEM) study on the interaction and oligomerization of streptolysin O with pure cholesterol in the form of an aqueous microcrystal suspension and as carbon-immobilized planar cholesterol crystals (Harris, 1988; Harris et al., 1998a), we now report that VCC also binds to cholesterol alone, with the formation of oligomers. In addition, we have investigated the interaction of VCC with a range of cholesterol esters and cholesterol analogues, including a fluorescent analogue of cholesterol, to assess the ability of these compounds to promote oligomer formation and for correlation with our ongoing liposome permeability studies.
2. Materials and methods 2.1. Reagents VCC was isolated and purified from V. cholerae 01 E1 Tor 8731, as previously described (Zitzer et al., 1997b). Cholesterol, cholesterol esters, dihydroxycholesterol derivatives, other steroids, polyoxyethylchlesteryl sebacate/cholesterol-PEG 600 (soluble cholesterol), and n-octyl b-D -glucopyranoside/n-octyl glucoside (OG) were purchased from the Sigma Chemical. The chemical structures of some of the sterols used in this study are given in Fig. 1. Chemical synthesis of the fluorescent analogue of cholesterol is given in Scheme 1. 2.2. Steroid–VCC interaction VCC (0.1–0.5 mg/ml) was added to aqueous suspensions of steroids (1.0 mg/ml) prepared in advance by direct injection of an ethanolic solution of steroid (10 mg/ml) into distilled water, followed by removal of the alcohol by dialysis against Tris-buffered saline or distilled water (Harris, 1988). Toxin interaction was routinely performed at room temperature (22 °C), with some experiments at 4 °C, for periods of 10 min, 30 min, 1 h, and 24 h. For oligomer solubilization, cholesterol+VCC samples were treated with 10, 50, and 100 mM OG. Interaction of VCC with immobilized cholesterol was performed on microwells in a Teflon block, using small pieces of carbon film with bound planar cholesterol sheets floating cholesterol-side-down on 10 ll of VCC solution at room temperature (Harris, 1997; Harris et al., 1998a) for periods of 10 min, 1 h, and several hours.
Fig. 1. The chemical structure of some of the sterols used in this study.
2.3. Preparation of negatively stained specimens For the steroid aqueous suspensions, following interaction with VCC, 5-ll aliquots were taken for negative staining on continuous carbon support films using 2% ammonium molybdate (pH 7.0) and 5% ammonium molybdate containing 1% trehalose (pH 7.0) (Harris, 1997) and also on holey carbon support films (Harris, 1997; Harris et al., 1998b, 2001; Harris and Scheffler,
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Scheme 1. Synthesis of the C-22 (2-trifluoroacetyl)naphthyloxy cholesterol analogue. After the 3b-hydroxy group of 23,24-bisnor-5-cholenic acid-3bol (1) was protected as a THP ether, reduction with LiAlH4 gave alcohol 2 in 48% yield. Mitsunobu reaction of 2 with 6-trifluoroacetyl-2-naphthol (3) afforded intermediate 4, which was treated with catalytic p-toluenesulfonic acid (p-TsOH) in ethanol, affording the new fluorescent cholesterol probe 5 in 81% yield.
2002), using 5% ammonium molybdate (pH 7.0) containing 1.0 or 0.1% trehalose. 2.4. Electron microscopy Specimens were studied using a Zeiss EM 900, operated at 80 kV. Electron micrographs were recorded on Kodak Type 4489 EM film. Single-particle two-dimensional image processing and classification, together with rotational analysis of VCC oligomers, were performed using IMAGIC-5 software (Image Science GmbH, Berlin) (Van Heel et al., 1996).
3. Results and discussion 3.1. Cholesterol–VCC interaction The cytolytic action of VCC is temperature-dependent. It progresses rapidly (minutes) at both room temperature and 37 °C, but at 4 °C is much slower (Zitzer et al., 1995). Indeed, Zitzer et al. (1997b) showed that monomeric VCC was bound and spontaneously released from rabbit erythrocyte membranes that had been incubated at 4 °C, whereas at 37 °C stable VCC oligomers were formed and could be released by deoxycholate. With suspensions of cholesterol microcrystals, VCC interaction is somewhat slower than with liposomes and biological membranes, probably because of the lack of fluidity of the crystalline cholesterol bilayers (Craven, 1986; Huang et al., 1999). Following a 15-min interaction at room temperature (22 °C) with VCC monomer (0.1 mg/ml), oligomers were detected primarily on the edges of the cholesterol bilayers (Fig. 2a), and after 30 min the VCC oligomers were also detected on the planar surface of the cholesterol (Fig. 2b). Longer
incubation times led to the complete surface coverage of the cholesterol microcrystals by VCC oligomers (Figs. 2c and d) at 1 and 24 h, respectively. If, however, after mixing at room temperature the sample is rapidly refrigerated to 4 °C, even after a further 24-h incubation, the cholesterol bilayer edge-only VCC oligomerization predominates (data not shown). In all these cases some release of VCC oligomers occurs from the surface of the cholesterol into free solution. This suggests that the interaction between the oligomers and cholesterol is not strong or that the oligomers have the capacity to bind and remove molecular cholesterol from the crystalline bilayers, which may be unable to firmly anchor a fluid bilayer-penetrating VCC oligomer in this instance. Although cholesterol has a very low solubility in water (4:7 lM, maximum) (Haberland and Reynolds, 1973), it is possible that some soluble VCC oligomers might form by direct interaction with molecular cholesterol in solution. When samples with an increased concentration of VCC relative to the cholesterol are spread across holey carbon support films for negative staining, which provides superior imaging of the biological material (Harris and Scheffler, 2002), cholesterol microcrystals completely coated by VCC oligomers are visible, together with large numbers of free oligomers (Fig. 3a) and with a spread of smaller amorphous material which probably represents the excess VCC monomer that has not oligomerized (Fig. 3b). This suggests that if access to the cholesterol bilayers is prevented because of an existing complete layer of VCC oligomers, oligomerization ceases (any further interaction of VCC monomers and their subsequent oligomerization might be sterically hindered), and this argues against any significant formation of oligomers due to the presence of soluble cholesterol. This situation has been detected
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Fig. 2. Cholesterol microcrystals following: (a) interaction with VCC (0.1 mg/ml) for 15 min at room temperature (22 °C); (b) interaction with VCC for 30 min at room temperature; (c) and (d) interaction with VCC at room temperature for 1 and 24 h, respectively. Note that at the short incubation times, (a) and (b), VCC has oligomerized predominantly at the edge of the cholesterol microcrystals and that with increasing time the planar surface also becomes coated with oligomers. Specimens (a–c) were prepared by adsorption onto continuous carbon support films and (d) by the negative staining carbon film procedure, after spreading on mica and carbon coating. The samples were negatively stained with 2% ammonium molybdate (pH 7.0). The scale bars indicate 200 nm (a–c) and 100 nm (d).
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Fig. 3. Release of VCC oligomers: (a) A sample of cholesterol microcrystals following Incubation at room temperature with VCC (0.5 mg/ml) to promote formation and release of VCC oligomers. The solution was spread across a holey carbon support film and negatively stained with 5% ammonium molybdate containing 0.1% trehalose (pH 7.0); see Harris and Scheffler (2002). Most of the 8-nm-diameter free VCC oligomers are in essentially the same ‘‘face-on’’ orientation. In (b) the cholesterol-VCC sample shows the presence of disorganized amorphous protein (arrow), which is likely to represent toxin monomer. In both (a) and (b) a small cholesterol microcrystal is heavily coated with VCC oligomers (arrowheads). The scale bars indicate 200 nm.
only for samples stored at 4 °C, but at higher temperatures (22 °C), there is release of oligomers from the cholesterol surface, and the progressive oligomerization is more complete. 3.2. Image processing of the VCC oligomer Single-particle image processing with the spontaneously soluble VCC oligomers, using IMAGIC-5 software, presented some problems. The reason for this is apparent in Figs. 3 and 4a, where it can be seen that there is considerable variability between the individual images, which appear to have irregular projections extending outward from the main wall of each ring-like oligomer. These disordered projections probably represent the extended hydrophobic membrane-spanning domains of the individual VCC monomers, which would under physiological conditions prefer to be buried within a membrane lipid bilayer. In aqueous solution these oligomers are likely to be in a thermodynamically unstable state and tend to loosely cross-link by hydrophobic interaction, thereby forming a disorganized
2-dimensional array, within which all the particles are in essentially the same plane within the thin film of negative stain. The absence of a full range of tilted and side-on images from these VCC oligomers suggests that selective orientation is imposed at the air/water interface, since the hydrophobic-region of the oligomers would be directed toward the air (cf. W€ osten and de Vocht, 2000), as well as by intermolecular hydrophobic interaction. Nevertheless, the initial and final class averages obtained within IMAGIC-5 strongly indicate the presence of sevenfold rotational symmetry within the ring-like oligomers (Fig. 4b). Approximately 3000 similar VCC oligomer images were picked from digitized electron micrographs of ammonium molybdate-trehalose negatively stained VCC-cholesterol specimens on holey carbon micrographs (as in Figs. 3 and 4a) and used for the image processing. An intermediate bandpass-filtering (low pass ¼ 0.01, high pass ¼ 0.9) and alignment against a sum-image of 500 single particles led after treatment by multivariate statistical analysis (MSA) (Van Heel and Frank, 1981) to initial classaveraging, with 100 classes. The eigenimages (Fig. 5a)
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Fig. 4. Image analysis of the VCC oligomer: (a) VCC oligomers dispersed evenly across a hole, in the presence of ammonium molybdate and trehalose (as in Fig. 2), showing the presence of disorganized projections extending outward from the ring-like oligomers. Hydrophobic interaction between adjacent oligomers is likely to be responsible for the rows of oligomers. The scale bar indicates 100 nm. (b) A gallery of 20 of the final class averages of the VCC oligomer, obtained from individual images as shown in (a), within the IMAGIC-5 image processing suite. Despite some molecular disorder, a clear indication of sevenfold rotational symmetry is present (see also Fig. 5).
created during the MSA showed a sevenfold symmetry. Many of the single class averages following this first classification also suggest the presence of sevenfold symmetry (contrary to our earlier suggestion from indirect biochemical evidence for the presence of five subunits; Zitzer et al., 1999). One selected class average, showing this sevenfold symmetry (Fig. 5b), was taken as reference for further alignment. After another round of
MSA, now producing 300 class averages, 80% showed sevenfold symmetry (Fig. 4b). For additional rotational analysis, the ring of the initial reference class average (Fig. 5b) was opened by displaying cylindrical coordinates (Fig. 5c). Here the seven upper projections represent the side arms of the oligomer. Following rotational self-correlation (Van Heel et al., 1992), these projections are shown as seven transmission maxima (Fig. 5d),
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cholesterol attached to a carbon support film (Harris, 1997; Harris et al., 1998a). In this instance, the immobilized crystalline cholesterol sheets are incubated on-grid with the VCC solution, during which only the non-carbon-coated surface of the immobilized cholesterol crystals is available for interaction, which prevents oligomer superimposition in the images. Under these conditions, with a short time of incubation at room temperature, VCC oligomers again form first at the edge of the stacked cholesterol bilayers (Fig. 6a) and subsequently the whole of the planar cholesterol surface becomes coated with oligomers (Fig. 6b). At the bilayer edge of microcrystals in solution or with the immobilized planar cholesterol bilayers, the overall steroid molecule rather than just the 3b-hydroxyl group would be more readily accessible to the toxin monomers. In general, this interpretation agrees with our conclusions on the influence of nonsterol lipids on membrane permeabilization by VCC (Zitzer et al., 2001), where accessibility of the cytolysin monomer to the hydrophobic region of cholesterol is increased. 3.4. VCC interaction with some cholesteryl esters and other sterols
Fig. 5. Sevenfold rotational symmetry of the VCC oligomer: (a) The first five eigenimages created during the initial multivariate statistical analysis (MSA) within IMAGIC-5 show sevenfold rotational symmetry. An initial heptameric VCC oligomer class average used as a reference for the second MSA (b). When cut open to display cylindrical coordinates this image shows the seven vertical projections (c), which following rotational self-correlation are shown as seven transmission maxima (d). Expressed graphically, the profile from the rotational selfcorrelation shows seven peaks of intensity (e).
which when plotted graphically are presented as seven peaks of intensity (Fig. 5e). This analysis provides support for the presence of seven VCC subunits within the ring-like oligomer. 3.3. Interaction of VCC with immobilized cholesterol The tendency for the available cholesterol bilayer edge to interact with VCC more rapidly than the planar surface has been confirmed by using immobilized
To investigate the interaction of VCC with different steroids, studies have been performed using aqueous suspensions of 19-hydroxycholesterol (5-cholestene-3 b,19-diol), 7b-hydroxycholesterol (5-cholesten-3b, 7bdiol), b-estradiol (3,17b-dihydroxy-1,3,5[10]-estratriene), cholesteryl acetate (5-cholesten-3b-ol 3-acetate), cholesteryl stearate (5-cholesten-3b-ol 3-octadecanaoate), cholesteryl oleate (5-cholesten-3b-ol 3-oleate), stigmasterol (5,22-stigmastadiene-3b-ol), ergosterol (3bhydroxy-5,7,22-ergostatriene), and the C-22 (2-trifluoroacetyl)naphthyloxy analogue of cholesterol. The data shown in Figs. 7 and 8 were obtained following a 1-h incubation at room temperature. The interaction of VCC with 19-hydroxycholesterol is similar to that with cholesterol, but with the oligomer binding occurring predominantly at the edge of the microcrystals, even after 1 h (Fig. 7a). A large quantity of VCC monomer remains on the background, indicating that oligomerization is slowed. In aqueous suspension, 7b-hydroxycholesterol forms chains of globular particles, rather than microcrystals. Here again, VCC interaction produces lipid-bound and free oligomers (Fig. 7b). b-Estradiol, which does not possess a C17-isooctyl side chain, also has the ability to induce VCC oligomer formation (Fig. 7c), with most of the oligomers binding in a close-packed manner to the steroid surface. This indicates that the isooctyl chain is not by itself required for oligomerization. However, stigmasterol (which differs from cholesterol only in its isooctyl moiety) produces disorganized VCC oligomerization (Fig. 7d). Ergosterol shows even less ability to induce VCC
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Fig. 6. Carbon-immobilized planar cholesterol bilayers transferred from mica (Harris, 1988) following incubation with VCC at (a) 10 min and (b) 1 h. In (a) the predominant location of the VCC oligomers along the step-like edges of the cholesterol crystal is indicated (arrowheads). In (b) there is more complete yet random coverage of the planar cholesterol surface. The background, where cholesterol is absent, is devoid of VCC oligomers; only carbon-adsorbed VCC monomer is present here. Negatively stained with 2% ammonium molybdate (pH 7.0). The scale bars indicate 100 nm.
oligomer formation than stigmasterol, but again cytolysin binding occurs at the edge of the bilayers (data not shown). If the sterol isooctyl chain is replaced with the bulkier trifluoroacetylnaphthyloxy-containing group, oligomerization ceases completely; here the smoothsurfaced sterol globules become coated with VCC monomer rather than oligomers (Fig. 8a). The fact that this fluorescent analogue of cholesterol, which nevertheless has a 3b-hydroxy group available, fails to produce oligomers is not entirely unexpected, as its chemical properties may be markedly altered. With cholesteryl acetate, oligomerization of VCC occurs on the planar surface of the microcrystals as well as at the edge of the bilayers (Fig. 8b). Therefore, a substituent as small as the acetyl group at the 3b-position is compatible with oligomerization, which stands in marked contrast to the oligomerization properties of streptolysin O (see below). In the case of cholesteryl stearate, which forms small angular crystalline lipid particles in aqueous suspension, VCC interaction does not lead to oligomer formation. The cholesteryl stearate particles are coated with VCC monomer (Fig. 8c), with much residual VCC monomer and only an occasional oligomer visible on the background. Cholesteryl oleate
forms smooth-surfaced globular particles in aqueous suspension, which after incubation at room temperature with VCC show strong indications of clustering/ aggregation (Fig. 8d). No VCC oligomers can be detected, but there are indications that the surface of the cholesterol oleate globules become coated with VCC monomer. From the data presented in Figs. 7 and 8 it is clear that the presence of a second hydroxyl group in the cholesterol steroid ring and the absence of the C17-aliphatic chain do not interfere with the ability of the cholesterol to induce VCC oligomerization. However, introduction of a fatty acid side chain at the 3b-position does prevent VCC oligomer formation, whereas the short chain acetate ester does not. That stigmasterol can induce VCC oligomerization whereas ergosterol has less tendency to do so indicates that oligomer formation could be due to either the differences in the aliphatic side chain or the extra double bond in the ergosterol ring. The availability of the 3b-hydroxy group in both these sterols indicates that a more general sterol-specific interaction is also required. The binding of VCC monomer to cholesteryl stearate, cholesteryl oleate, and the trifluoroacetylnaphthyl analogue of cholesterol may be
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Fig. 7. The interaction of VCC with 19-hydroxycholesterol (a) and 7b-hydroxycholesterol (b). In both cases VCC oligomer formation has occurred, with binding to the sterol and release of individual oligomers. 19-Hydroxycholesterol forms microcrystals in aqueous suspension, whereas 7b-dihydroxycholesterol forms strands of globular particles. In (c) VCC interaction with b-estradiol is shown; irregularly shaped b-estradiol microcrystals are coated with VCC oligomers on their planar surface and around the edge. Addition of VCC to stigmasterol microcrystals does not readily produce oligomerization; disordered oligomers are present only at the edge of the microcrystals (d), and no oligomers are released onto the background. All samples were negatively stained on carbon support films with 2% ammonium molybdate (pH 7.0). The scale bars indicate 100 nm.
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Fig. 8. VCC addition to a C-17 trifluoroacetylnaphthyl analogue of cholesterol, which is present in aqueous suspension as smooth globular particles, does not produce VCC oligomers (a). However, binding of VCC monomer to the surface of the globules is apparent. When VCC is added to the hexagonal cholesteryl acetate microcrystals they become completely coated with close-packed VCC oligomers (b). VCC addition to cholesteryl stearate microcrystalline particles (c) produces no oligomers, but binding of the cytolysin to the surface of the angular microcrystal particles is clearly apparent, with VCC monomer dispersed on the background. With cholesteryl oleate, which forms smooth-surfaced globular particles in aqueous suspension, addition of VCC does not induce oligomers (d), but cytolysin binding is apparent. Some aggregation of the globular sterol particles occurs. All samples were negatively stained on carbon support films with 2% ammonium molybdate (pH 7.0). The scale bars indicate 100 nm.
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nonspecific (indeed VCC does bind to liposomes in the absence of cholesterol, but fails to oligomerize; Zitzer et al., 1997a). Alternatively, this monomer binding could be an indication of the inability of VCC to both bind and subsequently utilize these steroids in order to undergo the required shape and/or hydrophobicity change that potentiates monomer–monomer interaction and results in the formation of a stable oligomer. With the thiol-specific cholesterol-binding toxins of the perfringolysin/streptolysin O family it is generally considered that the availability of the 3b-hydroxy group of the cholesterol is an essential stereospecific requirement for oligomerization, rather than the hydrophobicity of the sterol rings or the hydrocarbon side chain (Pringent and Alouf, 1976; Watson and Kerr, 1974). From recent liposome permeabilization studies performed by Zitzer et al. (unpublished data) the situation with VCC appears to be more complex. That the C17 isooctyl chain is important is shown by the fact that ergosterol and stigmasterol are less efficient pore producers than cholesterol, as are both 7b- and 19-hydroxycholesterol. Thus, from this permeability data it seems that VCC may have specificity for both the 3b-hydroxyl group and a requirement for the C17 hydrocarbon chain. No liposome permeability data are currently available using b-estradiol. 3.5. Solubilization of VCC oligomers Solubilization of liposomal and erythrocyte membrane-bound VCC oligomers with the surfactant deoxycholate shown by Zitzer et al. (1997) yielded individual and small groups of oligomers, which clearly showed elongated channels, compatible with the side-on image of heptameric rings. Our data (Figs. 2 and 3) show that VCC oligomers released spontaneously from cholesterol have a tendency to adopt the ring-like face-on orientation (when adsorbed to carbon and at an air/water interface) with protein disorder apparent at one end of the channel. Presumably it is this region of the oligomer that attaches to the hydrophobic surface of cholesterol or penetrates a fluid lipid bilayer when the toxin interacts with a cellular membrane to produce cytolysis. Some macromolecular plasticity of the hydrophobic membrane-binding domain of VCC, together with bound sterol, is perhaps a requirement for the cytolysin to be able to penetrate the lipid bilayer as a heptameric pore, even though any consideration of a prepore state (Sellman et al., 1997; Valeva et al., 2001) may not be strictly appropriate in the more chemical context that has been presented here using pure sterols. Addition of the neutral surfactant OG was successfully used for the solubilization and crystallization of the staphylococal a-hemolysin (a-toxin) (Song et al., 1996). Addition of OG to VCC oligomer-coated cholesterol
microcrystals at subsolubilization and solubilization concentrations could increase the overall cholesterol mobility within a mixed OG–cholesterol micellar or vesicle system and improve the interaction among the seven disordered hydrophobic domains of the oligomer. Preliminary data using 10, 50, and 100 mM OG have shown that VCC oligomers remain attached to OG– cholesterol particles at the lower sub-cholesterol-solubilizing concentrations of OG. However, at 100 mM OG, many individual VCC oligomers are released, which possess the characteristic channel-like structure when viewed from the side (Fig. 9), but they still have a pronounced tendency to cluster as small groups via the putative membrane-spanning hydrophobic/cholesterolbinding domains. The presence of this relatively high concentration of surfactant during TEM specimen preparation presents considerable problems. Definition of mixed micellar OG–cholesterol particles alongside the cytolysin oligomers, which probably bind both molecular cholesterol and OG at their hydrophobic domain, needs to be carefully assessed by future studies. Recently obtained data indicate that at room temperature sodium deoxycholate alone has the ability to induce VCC oligomerization (data not shown), but as with OG solubilization of cholesterol-induced oligomers, there is a marked tendency for the oligomers to cluster. Considerations of the prepore state have been dealt with extensively in relation to the formation of the staphylococcal a-toxin pore (Walker et al., 1995; Vecsey-Semjen et al., 1999; Valeva et al., 2001) and the Clostridium septicum a-toxin (Sellman et al., 1997). Further development of a VCC oligomerization system using sodium deoxycholate, alone or in the presence of a low molar ratio of cholesterol, may generate soluble oligomer intermediates as well as the intact heptamer. The recent model of the VCC oligomer advanced by Yuldasheva et al. (2001) from single-channel conductance measurements and nonelectrolyte exclusion, based upon the earlier electrophysiological data of Menzl et al. (1996) and the TEM data of Zitzer et al. (1997b), provides a useful concept of the extracellular, bilayer-penetrating, and cytoplasmic domains of the VCC pore, upon which to base further more detailed structural studies. 3.6. General discussion The tendency for cholesterol, two hydroxycholesterols, and stigmasterol to initially promote VCC oligomerization at the edge of the sterol bilayers suggests that the availability of sterol molecules for interaction with the toxin may be somewhat greater than on the planar surface of the microcrystals. Nevertheless, VCC oligomerization on the planar surface of rigid cholesterol microcrystals progresses rapidly at room temperature. The fact that VCC oligomers are spontaneously released from liposomes (Zitzer et al., 2000), and also as shown
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Fig. 9. VCC oligomers released from cholesterol by solubilization with 100 mM OG. Oligomer clusters are present together with individual oligomers, which are oriented both end-on and side-on. The side-on images (arrowheads) show the characteristic channel/pore structure of the oligomer; this correlates well with the VCC oligomer images visible at the edge of cholesterol microcrystals (see Fig. 1). The presence of a large quantity of mixed cholesterol-OG micelles has produced a relatively coarse background alongside the oligomers. Negatively stained on a carbon support film with 2% ammonium molybdate (pH 7.0). The scale bar indicates 100 nm.
above for rigid cholesterol microcrystals, indicates some inability of the oligomers to become firmly anchored within these cholesterol-containing bilayers. Exactly how this observation relates to the release or nonrelease of VCC and other pore-forming toxin oligomers from biological membranes is not clear. Nevertheless, although disordered, many of the free VCC oligomers show sevenfold rotational symmetry.
Our unexpected finding that cholesteryl acetate readily promotes VCC oligomerization suggests that the sterol 3b-hydroxy group is not an essential requirement. In liposomes, cholesteryl acetate is much less effective than the free sterol, as is 5a-cholestane-3one (Ikigai et al., 1996). Furthermore, the 3b-hydroxy group available on the fluorescent analogue of cholesterol, which does not induce VCC oligomerization, is likely to
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be directed toward the surface of the globular particles in aqueous solutions. In conclusion, we have shown that water-soluble VCC monomer can be oligomerized by interaction with cholesterol, two dihydroxycholesterols, b-estradiol, cholesteryl acetate, and stigmasterol, whereas cholesteryl stearate, cholesteryl oleate, ergosterol, and a fluorescent analogue of cholesterol failed to produce oligomers. These results contribute to our understanding of the specificity of the VCC/sterol interaction, and they show that the structural requirements of VCC are different from those of the thiol-specific family of cholesterol-binding toxins. Although we have not yet succeeded in producing a stable suspension of single VCC oligomers by OG solubilization of cholesterol–VCC suspensions, this approach, possibly using other surfactants such as n-dodecyl b-D -maltoside, CHAPS, sodium deoxycholate, or polyoxyethyl cholesteryl sebacate/cholesterol-PEG 600 (soluble cholesterol), remains a potential method whereby stable well-ordered soluble VCC oligomers, probably with bound surfactant and/or cholesterol, might be produced for future single-particle EM analysis and 2-D and 3-D crystallization studies.
Acknowledgments This work was supported in part by grants from the Stiftung Innovation von Rheinland-Pfalz (GZ. 8312386261/281) and the Deutsche Forschungsgemeinschaft (SFB 490). TEM facilities were provided by Professor Albrecht Fischer, Institute of Zoology, University of Mainz.
References Craven, B.M., 1986. Crystal structure of cholesterol monohydrate. Nature 260, 727–729. Haberland, M.E., Reynolds, J.A., 1973. Self-association of cholesterol in aqueous solution. Proc. Natl. Acad.Sci. USA 70, 2313–2316. Harris, J.R., 1988. Electron microscopy of cholesterol. Micron Microsc. Acta 19, 19–32. Harris, J.R., 1997. Negative Staining and Cryoelectron Microscopy: The Thin Film Techniques RMS Handbook No. 35. BIOS Scientific Publications, Oxford. Harris, J.R., Adrian, M., Bhakdi, S., Palmer, M., 1998a. Cholesterol– streptolysin O interaction: An EM study of wild-type and mutant streptolysin O. J. Struct. Biol 121, 343–355. Harris, J.R., Gebauer, W., Adrian, M., Markl, J., 1998b. Keyhole limpet hemocyanin (KLH): Slow in vitro reassociation of KLH1 and KLH2. Immucothel Micron 29, 329–339. Harris, J.R., Scheffler, D., 2002. Routine preparation of air-dried negatively stained and unstained specimens on holey carbon support films: A review of applications. Micron 33, 461–480. Harris, J.R., Schr€ oder, E., Isupov, M.N., Scheffler, D., Kristensen, P., Littlechild, J.A., Vagin, A.A., Meissner, U., 2001. Comparison of the decameric structure of peroxiredoxin-II by transmission electron microscopy and X-ray crystallography. Biochim. Biophys. Acta 1547, 221–234.
Huang, J., Buboltz, J.T., Feigenson, G.W., 1999. Maximum solubility of cholesterol in phosphatidylcholine and phosphatidylethanolamine bilayers. Biochim. Biophys. Acta 1417, 89–100. Ikagai, H., Akatsuka, A., Tsujiyama, H., Nakae, T., Shimamura, T., 1996. Mechanism of membrane damage by E1 Tor hemolysin of Vibrio cholerae 01. Infect. Immun. 64, 2968–2973. Ikigai, H., Ono, T., Nakae, T., Otsuru, H., Shimamura, T., 1999. Two forms of Vibrio cholerae O1 E1 Tor hemolysin derived from identical precursor protein. Biochim. Biophys. Acta 141, 297– 305. Krasilnikov, O.V., Muratkhodjaev, J.N., Zitzer, A.O., 1992. The mode of action of Vibrio cholerae cytolysin. The influences of both erythrocytes and planar lipid bilayers. Biochim. Biophys. Acta 1111, 7–16. Menzl, K., Maier, E., Chakraborty, T., Benz, R., 1996. HlyA hemolysin of Vibrio cholerae O1 biotype El Tor. Identification of the haemolytic complex and evidence for the formation of anion-selective ion-permeable channels. Eur. J. Biochem. 240, 646– 654. Pringent, D., Alouf, J.E., 1976. Interaction of streptolyisin O with sterols. Biochim. Biophys. Acta 443, 288–300. Saha, N., Banerjee, K.K., 1997. Carbohydrate-mediated regulation of interaction of Vibrio cholerae hemolysin with erythrocyte and phospholipid vesicle. J. Biol. Chem. 272, 162–167. Sellman, B.R., Kagan, B.L., Tweten, R.K., 1997. Generation of a membrane-bound oligomerized pre-pore complex is necessary for pore formation by Clostridium septicum alpha toxin. Mol. Microbiol. 23, 551–558. Song, L., Hobaugh, M.R., Shustak, C., Cheley, S., Bayley, J., 1996. Structure of staphylococcal a-hemolysin, a heptameric transmembrane pore. Science 274, 1859–1865. Valeva, A., Schnabel, R., Walev, I., Boukhallouk, F., Bhakdi, S., Palmer, S., 2001. Membrane insertion of the heptameric staphylococcal a-toxin pore. J. Biol. Chem. 276, 14835–14841. Van Heel, M., Frank, J., 1981. Use of multivariate statistics in analysing the image of biological macromolecules. Ultramicroscopy 6, 187–194. Van Heel, M., Harauz, G., Orlova, E.V., Schmidt, R., Schatz, M., 1996. A new generation of IMAGIC image processing system. J. Struct. Biol. 116, 17–24. Van Heel, M., Schatz, M., Orlova, E., 1992. Correlation functions revisited. Ultramicroscopy 46, 307–316. Vecsey-Semjen, B., Knapp, S., M€ ollby, F., vandergoot, F.G., 1999. The staphylococcal a-toxin pore has a flexible conformation. Biochemistry 38, 4296–4302. Walker, B., Braha, O., Bayley, H., 1995. An intermediate in the assembly of a poreforming protein trapped with a geneticallyengineered switch. Chem. Biol. 2, 99–105. Watson, K.C., Kerr, E.J.C., 1974. Sterol requirements for inhibition of streptolysin O activity. Biochem. J. 140, 95–98. W€ osten, H.A.B., de Vocht, M.L., 2000. Hydrophobins, the fungal coat unravelled. Biochim. Biophys. Acta 1496, 79–86. Yuldasheva, L.N., Merzlyak, P.G., Zitzer, A.O., Rodrigues, C.G., Bhakdi, S., Krisilmmikov, O.V., 2001. Lumen geometry of ion channels formed by Vibrio cholerae EL Tor cytolysin elucidated by nonelectrolyte exclusion. Biochim. Biophys. Acta 1521, 53–63. Zitzer, A., Waklev, I., Palmer, M., Bhakdi, S., 1995. Characterization of Vibrio cholerae E1 Tor cytolysin as an oligomerizing poreforming toxin. Med. Microbiol. Immunol. 184, 37–44. Zitzer, A., Palmer, M., Weller, U., Wassenaar, T., Biermann, C., Tranum-Jensen, J., Bhakdi, S., 1997a. Mode of primary binding to target membranes and pore formation induced by Vibrio cholerae cytolysin hemolysin. Eur. J. Biochem. 247, 209–216. Zitzer, A., Wassenaar, T.M., Walev, I., Bhakdi, S., 1997b. Potent membrane-permeabilizing and cytocidal action of Vibrio cholerae cytolysin on human intestinal cells. Infect. Immun., 65,1293– 1298.
J. Robin Harris et al. / Journal of Structural Biology 139 (2002) 122–135 Zitzer, A., Zitzer, O., Bhakdi, S., Palmer, M., 1999. Oligomerization of Vibrio Cholerae cytolysin yields a pentameric pore and has dual specificity for cholesterol and sphingolipids in the target membrane. J. Biol. Chem. 274, 1375–1380. Zitzer, A., Harris, J.R., Kemminer, S.E., Zitzer, O., Bhakdi, S., Muething, J., Palmer, M., 2000. Vibrio cholerae cytolysin: Assembly and membrane insertion of the oligomeric pore are tightly
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linked and are not detectably restricted by membrane fluidity. Biochim. Biophys. Acta 1509, 264–274. Zitzer, A., Bittman, R., Verbicky, C.A., Erukulla, R.K., Bhakdi, S., Weis, S., Valeva, A., Palmer, M., 2001. Coupling of cholesterol and cone-shaped lipids in bilayers augments membrane permeabilization by the cholesterol-specific toxins streptolysin O and Vibrio cholera cytolysin. J. Biol. Chem. 276, 14628–14633.
Structural Biology Journal of Structural Biology 139 (2002) 122–135 www.academicpress.com
Interaction of the Vibrio cholerae cytolysin (VCC) with cholesterol, some cholesterol esters, and cholesterol derivatives: a TEM study J. Robin Harris,a,* Sucharit Bhakdi,b Ulrich Meissner,a Dirk Scheffler,a Robert Bittman,c Guoqing Li,c Alexander Zitzer,b and Michael Palmerd a Institute of Zoology, University of Mainz, 55099 Mainz, Germany Institute of Medical Microbiology and Hygiene, University of Mainz, D-55101 Mainz, Germany Department of Chemistry and Biochemistry, Queens College, City University of New York, Flushing, New York 11367-1597, USA d Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1 b
c
Received 12 June 2002, and in revised form 5 July 2002
Abstract The Vibrio cholerae cytolysin (VCC) 63-kDa monomer has been shown to interact in aqueous suspension with cholesterol microcystals to produce a ring/pore-like heptameric oligomer 8 nm in outer diameter. Transmission electron microscopy data were produced from cholesterol samples adsorbed to carbon support films, spread across the holes of holey carbon films, and negatively stained with ammonium molybdate. The VCC oligomers initially attach to the edge of the stacked cholesterol bilayers and with increasing time cover the two planar surfaces. VCC oligomers are also released into solution, with some tendency to cluster, possibly via the hydrophobic membrane-spanning domain. At the air/water interface, the VCC oligomers are likely to be selectively oriented with the hydrophobic domain facing the air. Despite some molecular disorder/plasticity within the oligomers, multivariate statistical analysis and rotational self-correlation using IMAGIC-5 strongly suggest the presence of sevenfold rotational symmetry. To correlate the electron microscopy data with on-going biochemical and permeability studies using liposomes of varying lipid composition, the direct interaction of VCC with several cholesterol derivatives and other steroids has been examined. 19Hydroxycholesterol and 7b-hydroxycholesterol both induce VCC oligomerization. b-Estradiol, which does not possess an aliphatic side chain, also efficiently induces VCC oligomer formation, as does cholesteryl acetate. Cholesteryl stearate and oleate and the C22 (2-trifluoroacetyl)naphthyloxy analogue of cholesterol fail to induce VCC oligomerization, but binding of the monomer to the surface of these steroids does occur. Stigmasterol has little tendency to induce oligomer formation, and oligomers are largely confined to the edge of the bilayers; ergosterol has even less oligomerization ability. Attempts to solubilize and stabilize the VCC oligomers from cholesterol suspensions have been pursued using the neutral surfactant octylglucoside. Although individual solubilized oligomers have been defined which exhibit a characteristic cytolysin channel conformation in the side-on orientation, a tendency remains for the oligomers to cluster via their hydrophobic domains. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Vibrio cholerae; Cytolysin; VCC; Oligomerization; Cholesterol; Negative staining; TEM
1. Introduction Enteropathogenic Vibrio cholerae produces the classic cholera toxin and also the zonula occludens and accessory cholera toxins and a cytolysin. The 63kDa V. cholerae cytolysin (VCC) monomer is readily *
Corresponding author. Fax: 49-6131-3924652. E-mail address: [email protected] (J. Robin Harris).
obtained from nontoxigenic strains of V. cholerae, such as the non-O1 serotypes and the E1 Tor biotype of O1 (Zitzer et al., 1995). It has the ability to form ring-like oligomeric pores that penetrate cholesterolcontaining cell membranes and thereby produce cellular lysis (Krasilnikov et al., 1992; Zitzer et al., 1997a,b; Ikagai et al., 1996, 1999). That VCC has a carbohydrate-binding domain that can regulate pore formation was shown by Saha and Banerjee (1997).
1047-8477/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 4 7 - 8 4 7 7 ( 0 2 ) 0 0 5 6 3 - 4
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VCC interacts with liposomes containing cholesterol (Ikagai et al., 1999), and the presence of ceramides has some influence upon the ability of the toxin monomer to access membrane cholesterol and oligomerize to form a cytolytic channel (Zitzer et al., 1999, 2000, 2001). To extend our transmission electron microscopy (TEM) study on the interaction and oligomerization of streptolysin O with pure cholesterol in the form of an aqueous microcrystal suspension and as carbon-immobilized planar cholesterol crystals (Harris, 1988; Harris et al., 1998a), we now report that VCC also binds to cholesterol alone, with the formation of oligomers. In addition, we have investigated the interaction of VCC with a range of cholesterol esters and cholesterol analogues, including a fluorescent analogue of cholesterol, to assess the ability of these compounds to promote oligomer formation and for correlation with our ongoing liposome permeability studies.
2. Materials and methods 2.1. Reagents VCC was isolated and purified from V. cholerae 01 E1 Tor 8731, as previously described (Zitzer et al., 1997b). Cholesterol, cholesterol esters, dihydroxycholesterol derivatives, other steroids, polyoxyethylchlesteryl sebacate/cholesterol-PEG 600 (soluble cholesterol), and n-octyl b-D -glucopyranoside/n-octyl glucoside (OG) were purchased from the Sigma Chemical. The chemical structures of some of the sterols used in this study are given in Fig. 1. Chemical synthesis of the fluorescent analogue of cholesterol is given in Scheme 1. 2.2. Steroid–VCC interaction VCC (0.1–0.5 mg/ml) was added to aqueous suspensions of steroids (1.0 mg/ml) prepared in advance by direct injection of an ethanolic solution of steroid (10 mg/ml) into distilled water, followed by removal of the alcohol by dialysis against Tris-buffered saline or distilled water (Harris, 1988). Toxin interaction was routinely performed at room temperature (22 °C), with some experiments at 4 °C, for periods of 10 min, 30 min, 1 h, and 24 h. For oligomer solubilization, cholesterol+VCC samples were treated with 10, 50, and 100 mM OG. Interaction of VCC with immobilized cholesterol was performed on microwells in a Teflon block, using small pieces of carbon film with bound planar cholesterol sheets floating cholesterol-side-down on 10 ll of VCC solution at room temperature (Harris, 1997; Harris et al., 1998a) for periods of 10 min, 1 h, and several hours.
Fig. 1. The chemical structure of some of the sterols used in this study.
2.3. Preparation of negatively stained specimens For the steroid aqueous suspensions, following interaction with VCC, 5-ll aliquots were taken for negative staining on continuous carbon support films using 2% ammonium molybdate (pH 7.0) and 5% ammonium molybdate containing 1% trehalose (pH 7.0) (Harris, 1997) and also on holey carbon support films (Harris, 1997; Harris et al., 1998b, 2001; Harris and Scheffler,
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Scheme 1. Synthesis of the C-22 (2-trifluoroacetyl)naphthyloxy cholesterol analogue. After the 3b-hydroxy group of 23,24-bisnor-5-cholenic acid-3bol (1) was protected as a THP ether, reduction with LiAlH4 gave alcohol 2 in 48% yield. Mitsunobu reaction of 2 with 6-trifluoroacetyl-2-naphthol (3) afforded intermediate 4, which was treated with catalytic p-toluenesulfonic acid (p-TsOH) in ethanol, affording the new fluorescent cholesterol probe 5 in 81% yield.
2002), using 5% ammonium molybdate (pH 7.0) containing 1.0 or 0.1% trehalose. 2.4. Electron microscopy Specimens were studied using a Zeiss EM 900, operated at 80 kV. Electron micrographs were recorded on Kodak Type 4489 EM film. Single-particle two-dimensional image processing and classification, together with rotational analysis of VCC oligomers, were performed using IMAGIC-5 software (Image Science GmbH, Berlin) (Van Heel et al., 1996).
3. Results and discussion 3.1. Cholesterol–VCC interaction The cytolytic action of VCC is temperature-dependent. It progresses rapidly (minutes) at both room temperature and 37 °C, but at 4 °C is much slower (Zitzer et al., 1995). Indeed, Zitzer et al. (1997b) showed that monomeric VCC was bound and spontaneously released from rabbit erythrocyte membranes that had been incubated at 4 °C, whereas at 37 °C stable VCC oligomers were formed and could be released by deoxycholate. With suspensions of cholesterol microcrystals, VCC interaction is somewhat slower than with liposomes and biological membranes, probably because of the lack of fluidity of the crystalline cholesterol bilayers (Craven, 1986; Huang et al., 1999). Following a 15-min interaction at room temperature (22 °C) with VCC monomer (0.1 mg/ml), oligomers were detected primarily on the edges of the cholesterol bilayers (Fig. 2a), and after 30 min the VCC oligomers were also detected on the planar surface of the cholesterol (Fig. 2b). Longer
incubation times led to the complete surface coverage of the cholesterol microcrystals by VCC oligomers (Figs. 2c and d) at 1 and 24 h, respectively. If, however, after mixing at room temperature the sample is rapidly refrigerated to 4 °C, even after a further 24-h incubation, the cholesterol bilayer edge-only VCC oligomerization predominates (data not shown). In all these cases some release of VCC oligomers occurs from the surface of the cholesterol into free solution. This suggests that the interaction between the oligomers and cholesterol is not strong or that the oligomers have the capacity to bind and remove molecular cholesterol from the crystalline bilayers, which may be unable to firmly anchor a fluid bilayer-penetrating VCC oligomer in this instance. Although cholesterol has a very low solubility in water (4:7 lM, maximum) (Haberland and Reynolds, 1973), it is possible that some soluble VCC oligomers might form by direct interaction with molecular cholesterol in solution. When samples with an increased concentration of VCC relative to the cholesterol are spread across holey carbon support films for negative staining, which provides superior imaging of the biological material (Harris and Scheffler, 2002), cholesterol microcrystals completely coated by VCC oligomers are visible, together with large numbers of free oligomers (Fig. 3a) and with a spread of smaller amorphous material which probably represents the excess VCC monomer that has not oligomerized (Fig. 3b). This suggests that if access to the cholesterol bilayers is prevented because of an existing complete layer of VCC oligomers, oligomerization ceases (any further interaction of VCC monomers and their subsequent oligomerization might be sterically hindered), and this argues against any significant formation of oligomers due to the presence of soluble cholesterol. This situation has been detected
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Fig. 2. Cholesterol microcrystals following: (a) interaction with VCC (0.1 mg/ml) for 15 min at room temperature (22 °C); (b) interaction with VCC for 30 min at room temperature; (c) and (d) interaction with VCC at room temperature for 1 and 24 h, respectively. Note that at the short incubation times, (a) and (b), VCC has oligomerized predominantly at the edge of the cholesterol microcrystals and that with increasing time the planar surface also becomes coated with oligomers. Specimens (a–c) were prepared by adsorption onto continuous carbon support films and (d) by the negative staining carbon film procedure, after spreading on mica and carbon coating. The samples were negatively stained with 2% ammonium molybdate (pH 7.0). The scale bars indicate 200 nm (a–c) and 100 nm (d).
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Fig. 3. Release of VCC oligomers: (a) A sample of cholesterol microcrystals following Incubation at room temperature with VCC (0.5 mg/ml) to promote formation and release of VCC oligomers. The solution was spread across a holey carbon support film and negatively stained with 5% ammonium molybdate containing 0.1% trehalose (pH 7.0); see Harris and Scheffler (2002). Most of the 8-nm-diameter free VCC oligomers are in essentially the same ‘‘face-on’’ orientation. In (b) the cholesterol-VCC sample shows the presence of disorganized amorphous protein (arrow), which is likely to represent toxin monomer. In both (a) and (b) a small cholesterol microcrystal is heavily coated with VCC oligomers (arrowheads). The scale bars indicate 200 nm.
only for samples stored at 4 °C, but at higher temperatures (22 °C), there is release of oligomers from the cholesterol surface, and the progressive oligomerization is more complete. 3.2. Image processing of the VCC oligomer Single-particle image processing with the spontaneously soluble VCC oligomers, using IMAGIC-5 software, presented some problems. The reason for this is apparent in Figs. 3 and 4a, where it can be seen that there is considerable variability between the individual images, which appear to have irregular projections extending outward from the main wall of each ring-like oligomer. These disordered projections probably represent the extended hydrophobic membrane-spanning domains of the individual VCC monomers, which would under physiological conditions prefer to be buried within a membrane lipid bilayer. In aqueous solution these oligomers are likely to be in a thermodynamically unstable state and tend to loosely cross-link by hydrophobic interaction, thereby forming a disorganized
2-dimensional array, within which all the particles are in essentially the same plane within the thin film of negative stain. The absence of a full range of tilted and side-on images from these VCC oligomers suggests that selective orientation is imposed at the air/water interface, since the hydrophobic-region of the oligomers would be directed toward the air (cf. W€ osten and de Vocht, 2000), as well as by intermolecular hydrophobic interaction. Nevertheless, the initial and final class averages obtained within IMAGIC-5 strongly indicate the presence of sevenfold rotational symmetry within the ring-like oligomers (Fig. 4b). Approximately 3000 similar VCC oligomer images were picked from digitized electron micrographs of ammonium molybdate-trehalose negatively stained VCC-cholesterol specimens on holey carbon micrographs (as in Figs. 3 and 4a) and used for the image processing. An intermediate bandpass-filtering (low pass ¼ 0.01, high pass ¼ 0.9) and alignment against a sum-image of 500 single particles led after treatment by multivariate statistical analysis (MSA) (Van Heel and Frank, 1981) to initial classaveraging, with 100 classes. The eigenimages (Fig. 5a)
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Fig. 4. Image analysis of the VCC oligomer: (a) VCC oligomers dispersed evenly across a hole, in the presence of ammonium molybdate and trehalose (as in Fig. 2), showing the presence of disorganized projections extending outward from the ring-like oligomers. Hydrophobic interaction between adjacent oligomers is likely to be responsible for the rows of oligomers. The scale bar indicates 100 nm. (b) A gallery of 20 of the final class averages of the VCC oligomer, obtained from individual images as shown in (a), within the IMAGIC-5 image processing suite. Despite some molecular disorder, a clear indication of sevenfold rotational symmetry is present (see also Fig. 5).
created during the MSA showed a sevenfold symmetry. Many of the single class averages following this first classification also suggest the presence of sevenfold symmetry (contrary to our earlier suggestion from indirect biochemical evidence for the presence of five subunits; Zitzer et al., 1999). One selected class average, showing this sevenfold symmetry (Fig. 5b), was taken as reference for further alignment. After another round of
MSA, now producing 300 class averages, 80% showed sevenfold symmetry (Fig. 4b). For additional rotational analysis, the ring of the initial reference class average (Fig. 5b) was opened by displaying cylindrical coordinates (Fig. 5c). Here the seven upper projections represent the side arms of the oligomer. Following rotational self-correlation (Van Heel et al., 1992), these projections are shown as seven transmission maxima (Fig. 5d),
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cholesterol attached to a carbon support film (Harris, 1997; Harris et al., 1998a). In this instance, the immobilized crystalline cholesterol sheets are incubated on-grid with the VCC solution, during which only the non-carbon-coated surface of the immobilized cholesterol crystals is available for interaction, which prevents oligomer superimposition in the images. Under these conditions, with a short time of incubation at room temperature, VCC oligomers again form first at the edge of the stacked cholesterol bilayers (Fig. 6a) and subsequently the whole of the planar cholesterol surface becomes coated with oligomers (Fig. 6b). At the bilayer edge of microcrystals in solution or with the immobilized planar cholesterol bilayers, the overall steroid molecule rather than just the 3b-hydroxyl group would be more readily accessible to the toxin monomers. In general, this interpretation agrees with our conclusions on the influence of nonsterol lipids on membrane permeabilization by VCC (Zitzer et al., 2001), where accessibility of the cytolysin monomer to the hydrophobic region of cholesterol is increased. 3.4. VCC interaction with some cholesteryl esters and other sterols
Fig. 5. Sevenfold rotational symmetry of the VCC oligomer: (a) The first five eigenimages created during the initial multivariate statistical analysis (MSA) within IMAGIC-5 show sevenfold rotational symmetry. An initial heptameric VCC oligomer class average used as a reference for the second MSA (b). When cut open to display cylindrical coordinates this image shows the seven vertical projections (c), which following rotational self-correlation are shown as seven transmission maxima (d). Expressed graphically, the profile from the rotational selfcorrelation shows seven peaks of intensity (e).
which when plotted graphically are presented as seven peaks of intensity (Fig. 5e). This analysis provides support for the presence of seven VCC subunits within the ring-like oligomer. 3.3. Interaction of VCC with immobilized cholesterol The tendency for the available cholesterol bilayer edge to interact with VCC more rapidly than the planar surface has been confirmed by using immobilized
To investigate the interaction of VCC with different steroids, studies have been performed using aqueous suspensions of 19-hydroxycholesterol (5-cholestene-3 b,19-diol), 7b-hydroxycholesterol (5-cholesten-3b, 7bdiol), b-estradiol (3,17b-dihydroxy-1,3,5[10]-estratriene), cholesteryl acetate (5-cholesten-3b-ol 3-acetate), cholesteryl stearate (5-cholesten-3b-ol 3-octadecanaoate), cholesteryl oleate (5-cholesten-3b-ol 3-oleate), stigmasterol (5,22-stigmastadiene-3b-ol), ergosterol (3bhydroxy-5,7,22-ergostatriene), and the C-22 (2-trifluoroacetyl)naphthyloxy analogue of cholesterol. The data shown in Figs. 7 and 8 were obtained following a 1-h incubation at room temperature. The interaction of VCC with 19-hydroxycholesterol is similar to that with cholesterol, but with the oligomer binding occurring predominantly at the edge of the microcrystals, even after 1 h (Fig. 7a). A large quantity of VCC monomer remains on the background, indicating that oligomerization is slowed. In aqueous suspension, 7b-hydroxycholesterol forms chains of globular particles, rather than microcrystals. Here again, VCC interaction produces lipid-bound and free oligomers (Fig. 7b). b-Estradiol, which does not possess a C17-isooctyl side chain, also has the ability to induce VCC oligomer formation (Fig. 7c), with most of the oligomers binding in a close-packed manner to the steroid surface. This indicates that the isooctyl chain is not by itself required for oligomerization. However, stigmasterol (which differs from cholesterol only in its isooctyl moiety) produces disorganized VCC oligomerization (Fig. 7d). Ergosterol shows even less ability to induce VCC
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Fig. 6. Carbon-immobilized planar cholesterol bilayers transferred from mica (Harris, 1988) following incubation with VCC at (a) 10 min and (b) 1 h. In (a) the predominant location of the VCC oligomers along the step-like edges of the cholesterol crystal is indicated (arrowheads). In (b) there is more complete yet random coverage of the planar cholesterol surface. The background, where cholesterol is absent, is devoid of VCC oligomers; only carbon-adsorbed VCC monomer is present here. Negatively stained with 2% ammonium molybdate (pH 7.0). The scale bars indicate 100 nm.
oligomer formation than stigmasterol, but again cytolysin binding occurs at the edge of the bilayers (data not shown). If the sterol isooctyl chain is replaced with the bulkier trifluoroacetylnaphthyloxy-containing group, oligomerization ceases completely; here the smoothsurfaced sterol globules become coated with VCC monomer rather than oligomers (Fig. 8a). The fact that this fluorescent analogue of cholesterol, which nevertheless has a 3b-hydroxy group available, fails to produce oligomers is not entirely unexpected, as its chemical properties may be markedly altered. With cholesteryl acetate, oligomerization of VCC occurs on the planar surface of the microcrystals as well as at the edge of the bilayers (Fig. 8b). Therefore, a substituent as small as the acetyl group at the 3b-position is compatible with oligomerization, which stands in marked contrast to the oligomerization properties of streptolysin O (see below). In the case of cholesteryl stearate, which forms small angular crystalline lipid particles in aqueous suspension, VCC interaction does not lead to oligomer formation. The cholesteryl stearate particles are coated with VCC monomer (Fig. 8c), with much residual VCC monomer and only an occasional oligomer visible on the background. Cholesteryl oleate
forms smooth-surfaced globular particles in aqueous suspension, which after incubation at room temperature with VCC show strong indications of clustering/ aggregation (Fig. 8d). No VCC oligomers can be detected, but there are indications that the surface of the cholesterol oleate globules become coated with VCC monomer. From the data presented in Figs. 7 and 8 it is clear that the presence of a second hydroxyl group in the cholesterol steroid ring and the absence of the C17-aliphatic chain do not interfere with the ability of the cholesterol to induce VCC oligomerization. However, introduction of a fatty acid side chain at the 3b-position does prevent VCC oligomer formation, whereas the short chain acetate ester does not. That stigmasterol can induce VCC oligomerization whereas ergosterol has less tendency to do so indicates that oligomer formation could be due to either the differences in the aliphatic side chain or the extra double bond in the ergosterol ring. The availability of the 3b-hydroxy group in both these sterols indicates that a more general sterol-specific interaction is also required. The binding of VCC monomer to cholesteryl stearate, cholesteryl oleate, and the trifluoroacetylnaphthyl analogue of cholesterol may be
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Fig. 7. The interaction of VCC with 19-hydroxycholesterol (a) and 7b-hydroxycholesterol (b). In both cases VCC oligomer formation has occurred, with binding to the sterol and release of individual oligomers. 19-Hydroxycholesterol forms microcrystals in aqueous suspension, whereas 7b-dihydroxycholesterol forms strands of globular particles. In (c) VCC interaction with b-estradiol is shown; irregularly shaped b-estradiol microcrystals are coated with VCC oligomers on their planar surface and around the edge. Addition of VCC to stigmasterol microcrystals does not readily produce oligomerization; disordered oligomers are present only at the edge of the microcrystals (d), and no oligomers are released onto the background. All samples were negatively stained on carbon support films with 2% ammonium molybdate (pH 7.0). The scale bars indicate 100 nm.
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Fig. 8. VCC addition to a C-17 trifluoroacetylnaphthyl analogue of cholesterol, which is present in aqueous suspension as smooth globular particles, does not produce VCC oligomers (a). However, binding of VCC monomer to the surface of the globules is apparent. When VCC is added to the hexagonal cholesteryl acetate microcrystals they become completely coated with close-packed VCC oligomers (b). VCC addition to cholesteryl stearate microcrystalline particles (c) produces no oligomers, but binding of the cytolysin to the surface of the angular microcrystal particles is clearly apparent, with VCC monomer dispersed on the background. With cholesteryl oleate, which forms smooth-surfaced globular particles in aqueous suspension, addition of VCC does not induce oligomers (d), but cytolysin binding is apparent. Some aggregation of the globular sterol particles occurs. All samples were negatively stained on carbon support films with 2% ammonium molybdate (pH 7.0). The scale bars indicate 100 nm.
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nonspecific (indeed VCC does bind to liposomes in the absence of cholesterol, but fails to oligomerize; Zitzer et al., 1997a). Alternatively, this monomer binding could be an indication of the inability of VCC to both bind and subsequently utilize these steroids in order to undergo the required shape and/or hydrophobicity change that potentiates monomer–monomer interaction and results in the formation of a stable oligomer. With the thiol-specific cholesterol-binding toxins of the perfringolysin/streptolysin O family it is generally considered that the availability of the 3b-hydroxy group of the cholesterol is an essential stereospecific requirement for oligomerization, rather than the hydrophobicity of the sterol rings or the hydrocarbon side chain (Pringent and Alouf, 1976; Watson and Kerr, 1974). From recent liposome permeabilization studies performed by Zitzer et al. (unpublished data) the situation with VCC appears to be more complex. That the C17 isooctyl chain is important is shown by the fact that ergosterol and stigmasterol are less efficient pore producers than cholesterol, as are both 7b- and 19-hydroxycholesterol. Thus, from this permeability data it seems that VCC may have specificity for both the 3b-hydroxyl group and a requirement for the C17 hydrocarbon chain. No liposome permeability data are currently available using b-estradiol. 3.5. Solubilization of VCC oligomers Solubilization of liposomal and erythrocyte membrane-bound VCC oligomers with the surfactant deoxycholate shown by Zitzer et al. (1997) yielded individual and small groups of oligomers, which clearly showed elongated channels, compatible with the side-on image of heptameric rings. Our data (Figs. 2 and 3) show that VCC oligomers released spontaneously from cholesterol have a tendency to adopt the ring-like face-on orientation (when adsorbed to carbon and at an air/water interface) with protein disorder apparent at one end of the channel. Presumably it is this region of the oligomer that attaches to the hydrophobic surface of cholesterol or penetrates a fluid lipid bilayer when the toxin interacts with a cellular membrane to produce cytolysis. Some macromolecular plasticity of the hydrophobic membrane-binding domain of VCC, together with bound sterol, is perhaps a requirement for the cytolysin to be able to penetrate the lipid bilayer as a heptameric pore, even though any consideration of a prepore state (Sellman et al., 1997; Valeva et al., 2001) may not be strictly appropriate in the more chemical context that has been presented here using pure sterols. Addition of the neutral surfactant OG was successfully used for the solubilization and crystallization of the staphylococal a-hemolysin (a-toxin) (Song et al., 1996). Addition of OG to VCC oligomer-coated cholesterol
microcrystals at subsolubilization and solubilization concentrations could increase the overall cholesterol mobility within a mixed OG–cholesterol micellar or vesicle system and improve the interaction among the seven disordered hydrophobic domains of the oligomer. Preliminary data using 10, 50, and 100 mM OG have shown that VCC oligomers remain attached to OG– cholesterol particles at the lower sub-cholesterol-solubilizing concentrations of OG. However, at 100 mM OG, many individual VCC oligomers are released, which possess the characteristic channel-like structure when viewed from the side (Fig. 9), but they still have a pronounced tendency to cluster as small groups via the putative membrane-spanning hydrophobic/cholesterolbinding domains. The presence of this relatively high concentration of surfactant during TEM specimen preparation presents considerable problems. Definition of mixed micellar OG–cholesterol particles alongside the cytolysin oligomers, which probably bind both molecular cholesterol and OG at their hydrophobic domain, needs to be carefully assessed by future studies. Recently obtained data indicate that at room temperature sodium deoxycholate alone has the ability to induce VCC oligomerization (data not shown), but as with OG solubilization of cholesterol-induced oligomers, there is a marked tendency for the oligomers to cluster. Considerations of the prepore state have been dealt with extensively in relation to the formation of the staphylococcal a-toxin pore (Walker et al., 1995; Vecsey-Semjen et al., 1999; Valeva et al., 2001) and the Clostridium septicum a-toxin (Sellman et al., 1997). Further development of a VCC oligomerization system using sodium deoxycholate, alone or in the presence of a low molar ratio of cholesterol, may generate soluble oligomer intermediates as well as the intact heptamer. The recent model of the VCC oligomer advanced by Yuldasheva et al. (2001) from single-channel conductance measurements and nonelectrolyte exclusion, based upon the earlier electrophysiological data of Menzl et al. (1996) and the TEM data of Zitzer et al. (1997b), provides a useful concept of the extracellular, bilayer-penetrating, and cytoplasmic domains of the VCC pore, upon which to base further more detailed structural studies. 3.6. General discussion The tendency for cholesterol, two hydroxycholesterols, and stigmasterol to initially promote VCC oligomerization at the edge of the sterol bilayers suggests that the availability of sterol molecules for interaction with the toxin may be somewhat greater than on the planar surface of the microcrystals. Nevertheless, VCC oligomerization on the planar surface of rigid cholesterol microcrystals progresses rapidly at room temperature. The fact that VCC oligomers are spontaneously released from liposomes (Zitzer et al., 2000), and also as shown
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Fig. 9. VCC oligomers released from cholesterol by solubilization with 100 mM OG. Oligomer clusters are present together with individual oligomers, which are oriented both end-on and side-on. The side-on images (arrowheads) show the characteristic channel/pore structure of the oligomer; this correlates well with the VCC oligomer images visible at the edge of cholesterol microcrystals (see Fig. 1). The presence of a large quantity of mixed cholesterol-OG micelles has produced a relatively coarse background alongside the oligomers. Negatively stained on a carbon support film with 2% ammonium molybdate (pH 7.0). The scale bar indicates 100 nm.
above for rigid cholesterol microcrystals, indicates some inability of the oligomers to become firmly anchored within these cholesterol-containing bilayers. Exactly how this observation relates to the release or nonrelease of VCC and other pore-forming toxin oligomers from biological membranes is not clear. Nevertheless, although disordered, many of the free VCC oligomers show sevenfold rotational symmetry.
Our unexpected finding that cholesteryl acetate readily promotes VCC oligomerization suggests that the sterol 3b-hydroxy group is not an essential requirement. In liposomes, cholesteryl acetate is much less effective than the free sterol, as is 5a-cholestane-3one (Ikigai et al., 1996). Furthermore, the 3b-hydroxy group available on the fluorescent analogue of cholesterol, which does not induce VCC oligomerization, is likely to
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be directed toward the surface of the globular particles in aqueous solutions. In conclusion, we have shown that water-soluble VCC monomer can be oligomerized by interaction with cholesterol, two dihydroxycholesterols, b-estradiol, cholesteryl acetate, and stigmasterol, whereas cholesteryl stearate, cholesteryl oleate, ergosterol, and a fluorescent analogue of cholesterol failed to produce oligomers. These results contribute to our understanding of the specificity of the VCC/sterol interaction, and they show that the structural requirements of VCC are different from those of the thiol-specific family of cholesterol-binding toxins. Although we have not yet succeeded in producing a stable suspension of single VCC oligomers by OG solubilization of cholesterol–VCC suspensions, this approach, possibly using other surfactants such as n-dodecyl b-D -maltoside, CHAPS, sodium deoxycholate, or polyoxyethyl cholesteryl sebacate/cholesterol-PEG 600 (soluble cholesterol), remains a potential method whereby stable well-ordered soluble VCC oligomers, probably with bound surfactant and/or cholesterol, might be produced for future single-particle EM analysis and 2-D and 3-D crystallization studies.
Acknowledgments This work was supported in part by grants from the Stiftung Innovation von Rheinland-Pfalz (GZ. 8312386261/281) and the Deutsche Forschungsgemeinschaft (SFB 490). TEM facilities were provided by Professor Albrecht Fischer, Institute of Zoology, University of Mainz.
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