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Cholesterol and the activity of bacterial toxins
FEMS Microbiology Letters 238 (2004) 281–289 www.fems-microbiology.org
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Cholesterol and the activity of bacterial toxins Michael Palmer
*
Department of Chemistry, University of Waterloo, 200 University Avenue West, Waterloo, Ont., Canada N2L 3G1 Received 28 May 2004; received in revised form 15 July 2004; accepted 29 July 2004 First published online 20 August 2004 Edited by G. Ihler
Abstract Cholesterol may affect the activity of microbial toxins in a direct, specific way, or it may exert indirect effects because of its role in membrane fluidity, membrane line tension, and in the stabilization of rafts in the cytoplasmic membrane. The thiol-activated toxins of gram-positive bacteria, and the cytolysin of Vibrio cholerae are presented as examples of specific toxin–cholesterol interaction. Several mechanisms of indirect effects of cholesterol are discussed using examples such as Staphylococcus aureus a-hemolysin, aerolysin, and diphtheria toxin. Ó 2004 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies. Keywords: Cholesterol; Bacterial toxins; Toxin–cholesterol interaction; Line tension
1. Introduction Microbial toxins that act on animal cells will have some kind of interaction with membranes containing cholesterol. The membrane may be the primary target, as is the case with membrane-damaging toxins, or it may constitute a barrier on the way to the intracellular target, to be overcome by endocytosis and/or by some kind of protein translocation activity built into the toxin molecule itself. While both bacterial and animal cell membranes have a wide range of possible lipid compositions, a distinguishing feature of the latter is their content of cholesterol. The sterol, at the same time, modulates physical properties of the membranes such as membrane fluidity, lateral phase segregation, and the propensity to adopt non-bilayer structures such as the inverted hexagonal phase [1]. We might therefore expect two different effects of cholesterol on toxin activity: *
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A specific interaction with cholesterol, which will cause the toxin to be selectively active on animal cell membranes, and A modulation of toxin activity by the sterol that may be mimicked by structurally unrelated lipid molecules. We will consider these two possible cases in turn. In doing so, we will try to illustrate some of the principles and mechanisms govern cholesterol–toxin interaction, but not provide a complete enumeration of all toxins for which some effect of cholesterol has been observed.
2. Specific toxin–cholesterol interaction As criteria of a specific interaction of a toxin with cholesterol, we may consider: A strict requirement for cholesterol; A defined binding site – or several such sites – for the sterol on the toxin molecule. This implies a defined stoichiometry of toxin–cholesterol interaction
0378-1097/$22.00 Ó 2004 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies. doi:10.1016/j.femsle.2004.07.059
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A mutual structural selectivity: Other sterols should not or to a much reduced extent support toxin activity, and changes to the toxinÕs cholesterol binding site should inhibit interaction with the sterol. At present, there is no single toxin or toxin family for which all of the above criteria are fully met by available experimental evidence. The toxin family that comes closest are the so-called Ôthiol-activated toxinsÕ, a group of highly homologous pore-forming [2] toxins that is widespread among gram-positive bacteria [3–5]. Extensively studied members of this group are streptolysin O, listeriolysin, perfringolysin, alveolysin, and pneumolysin. These toxins therefore are a good starting point for the purpose of this review. As with most other pore-forming toxins, the action of the thiol-activated toxins involves three discernible successive steps: 1. Membrane binding of the toxin monomer [6], 2. assembly into an oligomeric pre-pore [7] on the target membrane, and 3. cooperative insertion of the oligomer into the lipid bilayer, leading to formation of the transmembrane pore. There is consensus about the requirement of cholesterol for activity; this requirement has given rise to the alternative name Ôcholesterol-binding cytolysinsÕ. However, there actually is some disagreement about whether cholesterol is involved in the binding, oligomerization, or membrane insertion steps of toxin action. 1 The strongest case for a requirement of cholesterol in binding of the toxin to membranes has been made using proteolytic and recombinant fragments of perfringolysin. These fragments are deficient for oligomerization and hence do not form pores, yet they retain the ability to bind to membranes with apparently unaltered affinity and even to interfere with oligomerization of the intact wild-type toxin. The fragments also bind to pure cholesterol but not to any other lipid or protein constituent of sheep erythrocyte membranes. Moreover, they bind to liposomes consisting of phosphatidylcholine, phosphatidylglycerol and cholesterol, but not to liposomes consisting of the two phospholipids only. Based on these data, their use as cytochemical probes of membrane cholesterol has been proposed [8,9]. Divergent results have been reported for the binding of intact toxin to cholesterol-free membranes. Listeriolysin was found to bind to liposomes consisting of phosphatidylcholine only, albeit less avidly than to 1 An alternative designation – Ôcholesterol-dependent cytolysinsÕ – is neutral with respect to this question but seems to suggest that a requirement for cholesterol is exclusive for this toxin family, which is not the case.
liposomes containing the sterol [10]. In a study employing perfringolysin, streptolysin O, and intermedilysin, reduced but still substantial binding was found with erythrocytes the membranes of which had been depleted of cholesterol by treatment with methyl-b-cyclodextrin [11]. The authors noted, however, that depletion of cholesterol was incomplete, so that the requirement for the sterol in binding cannot be reliably assessed from these experiments. Streptolysin O binds to cholesterol in suspension and then proceeds to form oligomers on the cholesterol particles; the oligomers can be readily detected by electron microscopy [12,13]. This observation indicates that no other ligand is specifically required to trigger oligomerization. Listeriolysin also binds to cholesterol in suspension; however, in contrast to streptolysin O it retains its monomeric state, as determined by gel permeation chromatography. In that experiment, 0.7 mol of cholesterol were recovered per mole of toxin [10]. While it is not clear from the available data how this ratio relates to the binding equilibrium, it does suggest a stoichiometric interaction of toxin and sterol, which had previously been inferred from a quantitative analysis of inhibition of the related toxin alveolysin by free cholesterol [14], and more recently was confirmed in a fluorescence study on pneumolysin [15]. Interestingly, listeriolysin that was first reacted with free cholesterol was found to bind with enhanced affinity to liposomes not containing the sterol. This increase in affinity might conceivably be due to a conformational change leading to an increased exposure of hydrophobic surfaces, or to surface-exposed hydrophobic features of the sterol molecule itself. Even after binding to red blood cells, i.e. to membranes containing cholesterol, the cholesterol-presaturated listeriolysin remained inactive. Hemolysis could only be induced by enzymatic depletion of the toxin-bound cholesterol with cholesterol oxidase. Therefore, while listeriolysin appears to bind to both free and membrane-embedded cholesterol, only the latter seems to induce proper oligomerization. In the results reported for both streptolysin and listeriolysin, there seems to be little room for ambiguity, suggesting that the two toxins indeed do behave differently, such that cholesterol alone suffices to induce oligomerization with streptolysin but not so with listeriolysin. It is worth noting that listeriolysin has a more acidic pH optimum than streptolysin O. The reported experiments were conducted at pH 7.4, which may have contributed to the suppression of oligomerization in this case. A role of cholesterol in the membrane insertion of the oligomer is apparent from the previously cited study on partially cholesterol-depleted cell membranes [11]. The toxin that bound to these membranes was arrested in a non-lytic, oligomeric stage, indicating that cholesterol has a role in supporting the co-operative membrane
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insertion of the oligomer. As noted by the authors, membrane insertion may be quite sensitive to ÔbulkÕ physical properties of the hydrophobic membrane core such as fluidity and lateral phase segregation. The available data therefore do not allow to discriminate between indirect and direct, ÔspecificÕ effects of cholesterol on membrane insertion (see Fig. 1). The structural specificity of the toxin–cholesterol interaction has been studied in some detail with a variety of cholesterol analogs. Four different approaches have been used in these experiments: Inhibition of hemolytic activity by sterols in suspension or solution [16,17]. Quantitative comparison between different sterols in this system is hampered by the fact that the sterols will have different free concentrations. On the other hand, the data detailed above for listeriolysin suggest that oligomerization is not required for inhibition to occur, so that this approach may give the best estimate of the effect of sterol structure on binding to the toxin, irrespective of the subsequent events of oligomerization and insertion. Permeabilization of liposomes containing the sterol in question in a background of phospholipids [18,19]. This approach reduces the heterogeneity in free concentration and physical state between the different sterols. However, this assay responds only if binding is also followed by oligomerization and membrane insertion. Also, in mixed bilayers, phase separation between sterol-enriched and-depleted domains may occur and be affected by sterol structure [20], which may then indirectly influence toxin activity. Surface pressure measurements on lipid monolayers floating atop solutions of the toxins [21]. The surface pressure will increase upon penetration of toxin molecules into the monolayer. In these experiments, the effects of binding and of oligomerization/insertion have not been distinguished. Electron microscopy of sterol particles incubated with the toxin. This will qualitatively detect binding to and oligomerization on the sterol particle surfaces [13].
H3C CH3
CH3
H H
CH3
CH3
In spite of their substantial inherent differences, all these approaches yield fairly consistent results with respect to the structural features of the cholesterol molecule required for toxin interaction. These findings can be summarized as follows: All variations of the 3-b-OH group of the sterol molecule – epimeric configuration (in epicholesterol), esterification (in cholesterol acetate), oxidation (in cholestenone), or replacement with a thiol group (thiocholesterol 2)-result in complete loss of interaction with the toxin. Introduction of hydroxyl substituents into the ring system strongly diminishes or abrogates activity. Deviant structures of the isooctyl side chain lead to distinct education but not abrogation of activity. All these findings suggest a fairly high degree of structural specificity of the toxin–cholesterol interaction. Against this background, it was quite surprising to observe that enantiomeric cholesterol [22] is only slightly less potent than cholesterol in supporting the activity of streptolysin O [19]. This finding suggests a considerably lower degree of structural specificity in the toxin– sterol interaction than was expected from the previous experiments. It is noteworthy that the two enantiomers of cholesterol closely resemble each other with respect to lipid–lipid interactions [23], so that any observed differences in toxin activity are likely due to differential interaction with the toxin molecules themselves. The location of the binding site for cholesterol on the toxin molecule is not precisely known. The smallest fragments of streptolysin O [24] and of perfringolysin O [9] that still retain the ability to bind to membranes (and, presumably, cholesterol) correspond to the C-terminal domain 4 [25], which has a molecular weight of approximately 15 kDa. Concomitantly with membrane binding, the fragment also becomes protected from proteolytic digestion, suggesting membrane insertion and/or significant conformational changes [9]. Sitedirected fluorescence labelling and quenching indicates that only a minor part of the domain actually becomes membrane-embedded [26]. Located within this domain is a highly conserved sequence motif that contains three tryptophan residues and one cysteine residue (Fig. 2). An increased intensity and spectral blue shift of tryptophan fluorescence upon binding [26,27], as well as the susceptibility of tryptophan fluorescence to quenching by brominated lipids [28] are consistent with the notion of membrane insertion of this motif. Mutations within this motif inhibit toxin activity, and chemical modification of the cysteine residue strongly reduces toxin 2
HO Fig. 1. Structure of cholesterol.
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Thiocholesterol monolayers actually did exhibit increased surface pressure in the presence of streptolysin O [21], whereas this compound did not exhibit any activity in the residual assays.
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Fig. 2. X-ray structure of perfringolysin [25]. The cholesterol-binding domain 4 is shown in space-filling mode. The tryptophan-rich motif involved in membrane binding is highlighted.
affinity for membranes [29]. However, cysteine modification does not prevent binding of streptolysin O to cholesterol [30]. One possible reason is that this motif is not immediately involved in binding to cholesterol. Another possible explanation is that the affinity of the toxin for free cholesterol is higher than that for cholesterol in mixed bilayers; this would be expected if the interaction of cholesterol with the toxin required partial or complete separation of the sterol from the residual membrane lipids (since the energy of sterol–lipid interaction would detract from that of sterol–protein interaction). With the peptide antibiotic gramicidin, a cluster of tryptophan residues has also been implicated in the interaction with membrane cholesterol. It has been observed that the indole side chain of tryptophan and the conjugated double bonds of amphotericin B and related polyene antibiotics are similar in being both rigid and polarizable, and these properties have been proposed
to be important for their interaction with sterols [31]. However, gramicidin is most active on bacterial membranes, which do not possess any sterols at all. The tryptophans found in integral membrane proteins usually reside fairly close to the membrane surface; this location would appear to restrict interaction with sterol molecules to the most superficially located parts of the latter. Tryptophans are found in membrane proteins not only of animals but also of prokaryotes, where they will not have a functional relationship with cholesterol. In sum, very little experimental evidence is available to support the idea of a preferential direct association of tryptophan residues with cholesterol. Apart from tryptophan clusters, another, different sequence motif that has been implicated in interaction with cholesterol. This motif was first described in a study on the peripheral benzodiazepine receptor, which is a membrane protein involved in the regulation of cholesterol transport within steroid-synthesizing cells [32]. The consensus sequence for this hypothetical cholesterol-binding motif is L/V–(X1–5)–Y–(X1–5)–R/K, i.e. it consists of a leucine or valine, a tyrosine, and an arginine or lysine, all separated by from 1 to 5 arbitrary intervening amino acid residues. This motif also occurs in NAP-22, a human neuronal membrane protein. Moreover, a sequence similarity was noted between NAP-22 and pneumolysin, and multiple instances of the consensus motif were found in pneumolysin; this was proposed to reflect the binding of multiple cholesterol molecules by the toxin [33]. While a participation of this sequence motif in cholesterol binding cannot be excluded, it certainly has insufficient predictive value for specific binding to cholesterol. This can be illustrated with the following, arbitrary example: The genome of Streptococcus agalactiae (GenBank Accession No. NC 004368) encodes 2094 known and hypothetical proteins, almost all of which will have no relationship to cholesterol whatsoever. Nevertheless, the putative cholesterol-binding motif occurs 5737 times among them. This corresponds to 2.7 occurrences per protein, or to 1 occurrence in every 112 amino acids. 3 Very similar numbers are found in the proteomes of Staphylococcus aureus and of Escherichia coli. Therefore, no conclusion can be drawn from the occurrence of this sequence motif in a protein as to its interaction with cholesterol. In fact, it has been shown recently that NAP-22 does not specifically require cholesterol, since it readily binds to sterol-free membranes that contain phosphatidylethanolamine [34]. Other pore-forming toxins that appear to have a strict requirement for cholesterol are the Vibrio cholerae 3 This was determined using a short Python program, which uses the regular expression Ô[LV][A–Z]{1,5}Y[A–Z]{1,5}[KR]Õ to find all instances of the protein motif in question. The program is available from the author upon request.
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cytolysin [35], and Streptococcus agalactiae CAMP factor [36]. These toxins have no significant sequence similarity to each other or to the thiol-activated toxins; no high-resolution structural information is available for either of them. Like streptolysin O, Vibrio cholerae cytolysin readily oligomerizes upon contact with cholesterol crystals, suggesting that the sterol has a role in oligomerization [37]. The toxin readily binds to membranes consisting of phosphatidylcholine only but does not form stable oligomers on these [38]. The structural selectivity of Vibrio cholerae cytolysin has been studied with several cholesterol analogues in model liposomes [18,19]. The most notable difference to streptolysin O consists in the fact that only very weak activity is observed with enantiomeric cholesterol. Ent-cholesterol, however, does induce oligomerization when added to the toxin in crystalline form, as do several other sterols that fail to support toxin activity in the liposome model. A hypothetical explanation of this discrepancy would again invoke the mutual interference of sterol interactions with the toxin and with other lipids in the bilayer, respectively, which would lower the affinity of the sterol for the toxin when embedded within mixed lipid membranes. With CAMP factor, there is no discernible difference at all between cholesterol and ent-cholesterol in their ability to support membrane permeabilization, suggesting a low degree of specificity in the toxin–cholesterol interaction (S. Lang, unpublished findings). Data for other sterols are not available.
3. Modulation of toxin activity by cholesterol Although with most bacterial toxins cholesterol is not strictly required for activity, a modulation, usually an enhancement of activity by cholesterol is a common observation. Such findings may relate to one or several effects the sterol has on the physical properties of lipid membranes [1]: Cholesterol modulates the fluidity of lipid bilayers, inducing a Ôliquid-orderedÕ state that is intermediate in fluidity between the gel state (which typically prevails at low temperature) and the liquid-crystalline state. Cholesterol, like phosphatidylethanolamine and diacylglycerol, is a Ôcone-shapedÕ lipid, 4 i.e. it occupies a larger cross-sectional area in the hydrophobic core of the membrane than within the head group layer (Fig. 3). Cone-shaped lipids promote the transition of lipid bilayers to the inverted hexagonal phase. 4 The terms Ôcone-shaped lipidsÕ and Ôinverted cone-shaped lipidsÕ are used inconsistently in the literature, so that cholesterol is sometimes referred to as an Ôinverted coneÕ. The choice of orientation is obviously arbitrary.
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(a)
(b)
(c)
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Fig. 3. Cone-shaped lipids and the inverted hexagonal phase. (a) Schematic of an inverted cone-shaped lipid (left), a cylindrical one (middle), and a cone-shaped lipid. (b)–(d) Bilayers rich in coneshaped lipids are prone to convert to the inverted hexagonal phase. This phase transition is favoured by high temperature.
Cholesterol influences the lateral segregation of lipids in mixed bilayers. In the cytoplasmic membrane, cholesterol promotes formation of microdomains also enriched in sphingolipids and GPI-anchored proteins. These domains, which have been dubbed Ôlipid raftsÕ [39], have a role in the function of several bacterial toxins. Cholesterol increases the line tension at membrane discontinuities [40]. This is possibly of relevance to the membrane defects induced by pore-forming toxins; however, this possibility has not been explored experimentally. The role of membrane fluidity has been documented for Staphylococcus aureus a-hemolysin [41]. This toxin inserts into pure phosphatidylcholine membranes above but not below their intrinsic gel-to-liquid phase transition temperatures. Inclusion of cholesterol at a molar fraction of 20%, which promotes formation of the liquid-ordered state even below the transition temperature, renders the membranes susceptible. Above a certain threshold temperature, lipid bilayers will undergo a transition to the inverted hexagonal phase (Fig. 3). Inclusion of Ôcone-shapedÕ lipids in mixed bilayers will lower this transition temperature, i.e. promote the formation of hexagonal phases [42]. Localized formation of structures similar to that of inverted
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hexagonal phases is believed to occur in membrane fusion. In keeping with this idea, cone-shaped lipids promote membrane fusion [43]. They also increase the susceptibility of liposome membranes to the pore-forming toxin aerolysin; this was observed with cholesterol, diacylglycerol, and phosphatidyethanolamine [44]. Like fusion, insertion of an extrinsic protein disrupts the continuity of the membrane, and in the study cited it was proposed that membrane insertion of aerolysin may again be facilitated by localized formation of hexagonal phase-like lipid structures. Alternatively, it is conceivable that even in the absence of hexagonal phase the thinned headgroup spacing induced by cone-shaped lipids may facilitate access of the toxin or enzyme molecules to the hydrophobic layer. Cone-shaped lipids also promote the activity of phospholipases A2 and C, which likewise have to penetrate the headgroup layer in order to reach their target sites within the membrane. With phospholipase C, no close correlation was observed between the increment of activity on the one hand and the decrement of the phase transition temperature or the extent of vesicle fusion on the other [45], which suggests that indeed actual transition to hexagonal phase may not be required. A pronounced effect of inverted cone-shaped lipids also been noted with the cholesterol-specific toxins streptolysin O and Vibrio cholerae cytolysin [18]. In this case, the cone-shaped lipids did not substitute but augment the membrane susceptibility conferred by cholesterol. This effect was remarkably similar between the two toxins, and substantially more pronounced than with staphylococcal a-hemolysin, which like aerolysin has no strict requirement for cholesterol. A possible explanation of their more pronounced effect observed with the cholesterol-specific toxins is that, by competing with cholesterol for headgroup coverage ÔborrowedÕ from adjacent polar lipids [46], cone-shaped lipids will raise the free energy of cholesterol in mixed bilayers; this should promote association of the sterol with specific binding sites on the toxins. The same mechanism has recently been proposed to account for the observation that ceramide displaces cholesterol from lipid rafts [47]; this suggests that it may be of considerable biological importance. Lipid rafts are liquid-ordered state membrane domains that are rich in cholesterol and saturated polar lipids such as sphingomyelin and glycosphingolipids [39]. They are ÔafloatÕ within surrounding fluid-state membrane domains that are enriched in unsaturated lipids. Lipid rafts can experimentally be detected by way of their insolubility in Triton X-100 [48]; this insolubility suggests a tight packing of the lipid molecules within the rafts. Of note, in a liposome model, various sterols were found to promote formation of rafts to different extents; most sterols stabilized rafts less strongly than cholesterol did [20]. Therefore, with toxins targeting rafts,
differences in activity might be observed experimentally with different sterols even in the absence of specific interaction with the sterol molecule. Preferential association of toxins with rafts may be due to their content of cholesterol, as is likely with perfringolysin, or to the enrichment of other receptor molecules in the rafts. Examples of the latter are aerolysin, which binds to the glycoside moiety of GPI-anchored proteins [49], and cholera toxin, which binds to the ganglioside GM1 [50]. Location of the receptors within the rafts may or may not be important in toxin function. Aerolysin is equally effective on model membranes with and without rafts, respectively, as long as either contains GPI anchors as receptors [51]. On the other hand, with Helicobacter pylori vacuolating toxin and with cholera toxin, raft intactness matters. Depletion of membrane cholesterol with b-cyclodextrins, which dissipates rafts, prevents uptake and intracellular trafficking of these toxins and accordingly disrupts their intracellular action [52,53]. It is presently not clear, however, whether the role of cholesterol in the intracellular fate of these toxins is limited to the stabilization of rafts. Diphtheria toxin is an AB toxin, i.e. its B subunit forms a transmembrane pore in the process of transporting the enzymatically active A subunit into the target cell cytoplasm. The toxin pore also permits the passage of other solutes in a non-specific way. Although the membrane topology of the B subunit is not known in complete detail, only between two and four helical segments appear to actually insert into the membrane [54]. Interestingly, there is good evidence for both monomeric [55] and oligomeric [56] channels formed by diphtheria toxin. Oligomeric pores appear to vary in stoichiometry, and the functional diameter to increase with the number of subunits; they permit permeation of dextran molecules of up to at least 10 kDa. Diphtheria toxin does not strictly require cholesterol for activity; however, intriguingly, the sterol increases the apparent pore size and the extent of oligomerization [56]. 5 If diphtheria toxin functions as a monomer, what drives its oligomerization? The oligomers do not seem to be stabilized by significant protein-protein interaction but only to exist on the membrane; this suggests that it is the membrane itself that stabilizes them (Fig. 4). Both its limited number of membrane-spanning segments and its ability to fuse with others into a larger membrane defect suggest the possibility that the monomer pore is not completely enclosed by protein but is lined in part by a free edge of the lipid membrane itself. Pores with such a molecular structure have been observed with both the terminal complement complex
5 In Fig. 6 of the study cited, the labels of panels B and C have been switched (E. London, personal communication).
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(a)
(b)
Fig. 4. Hypothetical model of diphtheria toxin pore structure. (a) Top view. The monomeric pore is lined in part by the protein and in part by a free edge of the lipid membrane. Several pores may coalesce, which will abolish the energetically unfavourable membrane edge and increase pore size. (b) Side view of the monomer pore. The lipids at the edge are likely to assume a micelle-like configuration. It is easy to see how cone-shaped lipids such as cholesterol would place additional strain on this assembly.
[57] and with streptolysin O [2,58]. Lipid molecules at a free membrane edge will be subject to an energetically unfavourable situation. This translates into a line tension, i.e. into a tendency of the edge to retract. It is plausible to assume that oligomerization should yield an edge of reduced or at least less than additive length. This relaxation of line tension could then create a driving force for oligomerization even in the absence of any favourable protein-protein interaction. Furthermore, the observation that cholesterol promotes oligomerization would be accounted for by the fact that it causes an increase in line tension. This explanation is admittedly entirely hypothetical, and the oligomerization of diphtheria toxin may not be essential to its function in vivo. However, considering the extreme scarcity of studies on the subject, this example may be acceptable to illustrate that cholesterol might affect the function of membrane-associated proteins by way of increasing the membrane line tension. The biological significance of this aspect of cholesterol function remains to be elucidated.
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[20] Xu, X. and London, E. (2000) The effect of sterol structure on membrane lipid domains reveals how cholesterol can induce lipid domain formation. Biochemistry 39, 843–849. [21] Alouf, J.E., Geoffroy, C., Pattus, F. and Verger, R. (1984) Surface properties of bacterial sulfhydryl-activated cytolytic toxins. Interaction with monomolecular films of phosphatidylcholine and various sterols. Eur. J. Biochem. 141, 205–210. [22] Kumar, A.S. and Covey, D.F. (1999) A new method for the preparation of ent-cholesterol from ent-testosterone. Tetrahedron Lett. 40, 823–826. [23] Westover, E.J., Covey, D.F., Brockman, H.L., Brown, R.E. and Pike, L.J. (2003) Cholesterol depletion results in site-specific increases in epidermal growth factor receptor phosphorylation due to membrane level effects. Studies with cholesterol enantiomers. J. Biol. Chem. 278, 51125–51133. [24] Weis, S. and Palmer, M. (2001) Streptolysin O: the C-terminal, tryptophan-rich domain carries functional sites for both membrane binding and self-interaction but not for stable oligomerization. Biochim. Biophys. Acta 1510, 292–299. [25] Rossjohn, J., Feil, S.C., McKinstry, W.J., Tweten, R.K. and Parker, M.W. (1997) Structure of a cholesterol-binding, thiolactivated cytolysin and a model of its membrane form. Cell 89, 685–692. [26] Ramachandran, R., Heuck, A.P., Tweten, R.K. and Johnson, A.E. (2002) Structural insights into the membrane-anchoring mechanism of a cholesterol-dependent cytolysin. Nat. Struct. Biol. 9, 823–827. [27] Nakamura, M., Sekino, N., Iwamoto, M. and Ohno-Iwashita, Y. (1995) Interaction of theta-toxin (perfringolysin O), a cholesterolbinding cytolysin, with liposomal membranes: change in the aromatic side chains upon binding and insertion. Biochemistry 34, 6513–6520. [28] Nakamura, M., Sekino-Suzuki, N., Mitsui, K. and Ohno-Iwashita, Y. (1998) Contribution of tryptophan residues to the structural changes in perfringolysin O during interaction with liposomal membranes. J. Biochem. (Tokyo) 123, 1145–1155. [29] Iwamoto, M., Ohno-Iwashita, Y. and Ando, S. (1987) Role of the essential thiol group in the thiol-activated cytolysin from Clostridium perfringens. Eur. J. Biochem. 167, 425–430. [30] Geoffroy, C. and Alouf, J.E. (1982) Interaction of alveolysin A sulfhydryl-activated bacterial cytolytic toxin with thiol group reagents and cholesterol. Toxicon 20, 239–241. [31] de Kruijff, B. (1990) Cholesterol as a target for toxins. Biosci. Rep. 10, 127–130. [32] Li, H. and Papadopoulos, V. (1998) Peripheral-type benzodiazepine receptor function in cholesterol transport. Identification of a putative cholesterol recognition/interaction amino acid sequence and consensus pattern. Endocrinology 139, 4991–4997. [33] Nollmann, M., Gilbert, R., Mitchell, T., Sferrazza, M. and Byron, O. (2004) The role of cholesterol in the activity of pneumolysin, a bacterial protein toxin. Biophys. J. 86, 3141–3151. [34] Terashita, A., Funatsu, N., Umeda, M., Shimada, Y., OhnoIwashita, Y., Epand, R.M. and Maekawa, S. (2002) Lipid binding activity of a neuron-specific protein NAP-22 studied in vivo and in vitro. J. Neurosci. Res. 70, 172–179. [35] Ikigai, H., Akatsuka, A., Tsujiyama, H., Nakae, T. and Shimamura, T. (1996) Mechanism of membrane damage by El Tor hemolysin of Vibrio cholerae O1. Infect. Immun. 64, 2968– 2973. [36] Fehrenbach, F.J., Schmidt, C.M., Sterzik, B. and Juergens, D. (1984) Interaction of amphiphilic bacterial polypeptides with artificial membranes In: Bacterial Protein Toxins (Alouf, J.E., Fehrenbach, F.J., Freer, J.H. and Jeljaszewicz, J., Eds.), pp. 317– 324. Academic Press, San Diego. [37] Harris, J.R., Bhakdi, S., Meissner, U., Scheffler, D., Bittman, R., Li, G., Zitzer, A. and Palmer, M. (2002) Interaction of the Vibrio cholerae cytolysin (VCC) with cholesterol, some cholesterol
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M. Palmer / FEMS Microbiology Letters 238 (2004) 281–289 probable relationship to oligomerization. J. Membr. Biol. 171, 209–221. [57] Bhakdi, S. and Tranum-Jensen, J. (1986) C5b-9 assembly: average binding of one C9 molecule to C5b-8 without poly-C9 formation generates a stable transmembrane pore. J. Immunol. 136, 2999–3005.
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MiniReview
Cholesterol and the activity of bacterial toxins Michael Palmer
*
Department of Chemistry, University of Waterloo, 200 University Avenue West, Waterloo, Ont., Canada N2L 3G1 Received 28 May 2004; received in revised form 15 July 2004; accepted 29 July 2004 First published online 20 August 2004 Edited by G. Ihler
Abstract Cholesterol may affect the activity of microbial toxins in a direct, specific way, or it may exert indirect effects because of its role in membrane fluidity, membrane line tension, and in the stabilization of rafts in the cytoplasmic membrane. The thiol-activated toxins of gram-positive bacteria, and the cytolysin of Vibrio cholerae are presented as examples of specific toxin–cholesterol interaction. Several mechanisms of indirect effects of cholesterol are discussed using examples such as Staphylococcus aureus a-hemolysin, aerolysin, and diphtheria toxin. Ó 2004 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies. Keywords: Cholesterol; Bacterial toxins; Toxin–cholesterol interaction; Line tension
1. Introduction Microbial toxins that act on animal cells will have some kind of interaction with membranes containing cholesterol. The membrane may be the primary target, as is the case with membrane-damaging toxins, or it may constitute a barrier on the way to the intracellular target, to be overcome by endocytosis and/or by some kind of protein translocation activity built into the toxin molecule itself. While both bacterial and animal cell membranes have a wide range of possible lipid compositions, a distinguishing feature of the latter is their content of cholesterol. The sterol, at the same time, modulates physical properties of the membranes such as membrane fluidity, lateral phase segregation, and the propensity to adopt non-bilayer structures such as the inverted hexagonal phase [1]. We might therefore expect two different effects of cholesterol on toxin activity: *
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A specific interaction with cholesterol, which will cause the toxin to be selectively active on animal cell membranes, and A modulation of toxin activity by the sterol that may be mimicked by structurally unrelated lipid molecules. We will consider these two possible cases in turn. In doing so, we will try to illustrate some of the principles and mechanisms govern cholesterol–toxin interaction, but not provide a complete enumeration of all toxins for which some effect of cholesterol has been observed.
2. Specific toxin–cholesterol interaction As criteria of a specific interaction of a toxin with cholesterol, we may consider: A strict requirement for cholesterol; A defined binding site – or several such sites – for the sterol on the toxin molecule. This implies a defined stoichiometry of toxin–cholesterol interaction
0378-1097/$22.00 Ó 2004 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies. doi:10.1016/j.femsle.2004.07.059
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A mutual structural selectivity: Other sterols should not or to a much reduced extent support toxin activity, and changes to the toxinÕs cholesterol binding site should inhibit interaction with the sterol. At present, there is no single toxin or toxin family for which all of the above criteria are fully met by available experimental evidence. The toxin family that comes closest are the so-called Ôthiol-activated toxinsÕ, a group of highly homologous pore-forming [2] toxins that is widespread among gram-positive bacteria [3–5]. Extensively studied members of this group are streptolysin O, listeriolysin, perfringolysin, alveolysin, and pneumolysin. These toxins therefore are a good starting point for the purpose of this review. As with most other pore-forming toxins, the action of the thiol-activated toxins involves three discernible successive steps: 1. Membrane binding of the toxin monomer [6], 2. assembly into an oligomeric pre-pore [7] on the target membrane, and 3. cooperative insertion of the oligomer into the lipid bilayer, leading to formation of the transmembrane pore. There is consensus about the requirement of cholesterol for activity; this requirement has given rise to the alternative name Ôcholesterol-binding cytolysinsÕ. However, there actually is some disagreement about whether cholesterol is involved in the binding, oligomerization, or membrane insertion steps of toxin action. 1 The strongest case for a requirement of cholesterol in binding of the toxin to membranes has been made using proteolytic and recombinant fragments of perfringolysin. These fragments are deficient for oligomerization and hence do not form pores, yet they retain the ability to bind to membranes with apparently unaltered affinity and even to interfere with oligomerization of the intact wild-type toxin. The fragments also bind to pure cholesterol but not to any other lipid or protein constituent of sheep erythrocyte membranes. Moreover, they bind to liposomes consisting of phosphatidylcholine, phosphatidylglycerol and cholesterol, but not to liposomes consisting of the two phospholipids only. Based on these data, their use as cytochemical probes of membrane cholesterol has been proposed [8,9]. Divergent results have been reported for the binding of intact toxin to cholesterol-free membranes. Listeriolysin was found to bind to liposomes consisting of phosphatidylcholine only, albeit less avidly than to 1 An alternative designation – Ôcholesterol-dependent cytolysinsÕ – is neutral with respect to this question but seems to suggest that a requirement for cholesterol is exclusive for this toxin family, which is not the case.
liposomes containing the sterol [10]. In a study employing perfringolysin, streptolysin O, and intermedilysin, reduced but still substantial binding was found with erythrocytes the membranes of which had been depleted of cholesterol by treatment with methyl-b-cyclodextrin [11]. The authors noted, however, that depletion of cholesterol was incomplete, so that the requirement for the sterol in binding cannot be reliably assessed from these experiments. Streptolysin O binds to cholesterol in suspension and then proceeds to form oligomers on the cholesterol particles; the oligomers can be readily detected by electron microscopy [12,13]. This observation indicates that no other ligand is specifically required to trigger oligomerization. Listeriolysin also binds to cholesterol in suspension; however, in contrast to streptolysin O it retains its monomeric state, as determined by gel permeation chromatography. In that experiment, 0.7 mol of cholesterol were recovered per mole of toxin [10]. While it is not clear from the available data how this ratio relates to the binding equilibrium, it does suggest a stoichiometric interaction of toxin and sterol, which had previously been inferred from a quantitative analysis of inhibition of the related toxin alveolysin by free cholesterol [14], and more recently was confirmed in a fluorescence study on pneumolysin [15]. Interestingly, listeriolysin that was first reacted with free cholesterol was found to bind with enhanced affinity to liposomes not containing the sterol. This increase in affinity might conceivably be due to a conformational change leading to an increased exposure of hydrophobic surfaces, or to surface-exposed hydrophobic features of the sterol molecule itself. Even after binding to red blood cells, i.e. to membranes containing cholesterol, the cholesterol-presaturated listeriolysin remained inactive. Hemolysis could only be induced by enzymatic depletion of the toxin-bound cholesterol with cholesterol oxidase. Therefore, while listeriolysin appears to bind to both free and membrane-embedded cholesterol, only the latter seems to induce proper oligomerization. In the results reported for both streptolysin and listeriolysin, there seems to be little room for ambiguity, suggesting that the two toxins indeed do behave differently, such that cholesterol alone suffices to induce oligomerization with streptolysin but not so with listeriolysin. It is worth noting that listeriolysin has a more acidic pH optimum than streptolysin O. The reported experiments were conducted at pH 7.4, which may have contributed to the suppression of oligomerization in this case. A role of cholesterol in the membrane insertion of the oligomer is apparent from the previously cited study on partially cholesterol-depleted cell membranes [11]. The toxin that bound to these membranes was arrested in a non-lytic, oligomeric stage, indicating that cholesterol has a role in supporting the co-operative membrane
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insertion of the oligomer. As noted by the authors, membrane insertion may be quite sensitive to ÔbulkÕ physical properties of the hydrophobic membrane core such as fluidity and lateral phase segregation. The available data therefore do not allow to discriminate between indirect and direct, ÔspecificÕ effects of cholesterol on membrane insertion (see Fig. 1). The structural specificity of the toxin–cholesterol interaction has been studied in some detail with a variety of cholesterol analogs. Four different approaches have been used in these experiments: Inhibition of hemolytic activity by sterols in suspension or solution [16,17]. Quantitative comparison between different sterols in this system is hampered by the fact that the sterols will have different free concentrations. On the other hand, the data detailed above for listeriolysin suggest that oligomerization is not required for inhibition to occur, so that this approach may give the best estimate of the effect of sterol structure on binding to the toxin, irrespective of the subsequent events of oligomerization and insertion. Permeabilization of liposomes containing the sterol in question in a background of phospholipids [18,19]. This approach reduces the heterogeneity in free concentration and physical state between the different sterols. However, this assay responds only if binding is also followed by oligomerization and membrane insertion. Also, in mixed bilayers, phase separation between sterol-enriched and-depleted domains may occur and be affected by sterol structure [20], which may then indirectly influence toxin activity. Surface pressure measurements on lipid monolayers floating atop solutions of the toxins [21]. The surface pressure will increase upon penetration of toxin molecules into the monolayer. In these experiments, the effects of binding and of oligomerization/insertion have not been distinguished. Electron microscopy of sterol particles incubated with the toxin. This will qualitatively detect binding to and oligomerization on the sterol particle surfaces [13].
H3C CH3
CH3
H H
CH3
CH3
In spite of their substantial inherent differences, all these approaches yield fairly consistent results with respect to the structural features of the cholesterol molecule required for toxin interaction. These findings can be summarized as follows: All variations of the 3-b-OH group of the sterol molecule – epimeric configuration (in epicholesterol), esterification (in cholesterol acetate), oxidation (in cholestenone), or replacement with a thiol group (thiocholesterol 2)-result in complete loss of interaction with the toxin. Introduction of hydroxyl substituents into the ring system strongly diminishes or abrogates activity. Deviant structures of the isooctyl side chain lead to distinct education but not abrogation of activity. All these findings suggest a fairly high degree of structural specificity of the toxin–cholesterol interaction. Against this background, it was quite surprising to observe that enantiomeric cholesterol [22] is only slightly less potent than cholesterol in supporting the activity of streptolysin O [19]. This finding suggests a considerably lower degree of structural specificity in the toxin– sterol interaction than was expected from the previous experiments. It is noteworthy that the two enantiomers of cholesterol closely resemble each other with respect to lipid–lipid interactions [23], so that any observed differences in toxin activity are likely due to differential interaction with the toxin molecules themselves. The location of the binding site for cholesterol on the toxin molecule is not precisely known. The smallest fragments of streptolysin O [24] and of perfringolysin O [9] that still retain the ability to bind to membranes (and, presumably, cholesterol) correspond to the C-terminal domain 4 [25], which has a molecular weight of approximately 15 kDa. Concomitantly with membrane binding, the fragment also becomes protected from proteolytic digestion, suggesting membrane insertion and/or significant conformational changes [9]. Sitedirected fluorescence labelling and quenching indicates that only a minor part of the domain actually becomes membrane-embedded [26]. Located within this domain is a highly conserved sequence motif that contains three tryptophan residues and one cysteine residue (Fig. 2). An increased intensity and spectral blue shift of tryptophan fluorescence upon binding [26,27], as well as the susceptibility of tryptophan fluorescence to quenching by brominated lipids [28] are consistent with the notion of membrane insertion of this motif. Mutations within this motif inhibit toxin activity, and chemical modification of the cysteine residue strongly reduces toxin 2
HO Fig. 1. Structure of cholesterol.
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Thiocholesterol monolayers actually did exhibit increased surface pressure in the presence of streptolysin O [21], whereas this compound did not exhibit any activity in the residual assays.
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Fig. 2. X-ray structure of perfringolysin [25]. The cholesterol-binding domain 4 is shown in space-filling mode. The tryptophan-rich motif involved in membrane binding is highlighted.
affinity for membranes [29]. However, cysteine modification does not prevent binding of streptolysin O to cholesterol [30]. One possible reason is that this motif is not immediately involved in binding to cholesterol. Another possible explanation is that the affinity of the toxin for free cholesterol is higher than that for cholesterol in mixed bilayers; this would be expected if the interaction of cholesterol with the toxin required partial or complete separation of the sterol from the residual membrane lipids (since the energy of sterol–lipid interaction would detract from that of sterol–protein interaction). With the peptide antibiotic gramicidin, a cluster of tryptophan residues has also been implicated in the interaction with membrane cholesterol. It has been observed that the indole side chain of tryptophan and the conjugated double bonds of amphotericin B and related polyene antibiotics are similar in being both rigid and polarizable, and these properties have been proposed
to be important for their interaction with sterols [31]. However, gramicidin is most active on bacterial membranes, which do not possess any sterols at all. The tryptophans found in integral membrane proteins usually reside fairly close to the membrane surface; this location would appear to restrict interaction with sterol molecules to the most superficially located parts of the latter. Tryptophans are found in membrane proteins not only of animals but also of prokaryotes, where they will not have a functional relationship with cholesterol. In sum, very little experimental evidence is available to support the idea of a preferential direct association of tryptophan residues with cholesterol. Apart from tryptophan clusters, another, different sequence motif that has been implicated in interaction with cholesterol. This motif was first described in a study on the peripheral benzodiazepine receptor, which is a membrane protein involved in the regulation of cholesterol transport within steroid-synthesizing cells [32]. The consensus sequence for this hypothetical cholesterol-binding motif is L/V–(X1–5)–Y–(X1–5)–R/K, i.e. it consists of a leucine or valine, a tyrosine, and an arginine or lysine, all separated by from 1 to 5 arbitrary intervening amino acid residues. This motif also occurs in NAP-22, a human neuronal membrane protein. Moreover, a sequence similarity was noted between NAP-22 and pneumolysin, and multiple instances of the consensus motif were found in pneumolysin; this was proposed to reflect the binding of multiple cholesterol molecules by the toxin [33]. While a participation of this sequence motif in cholesterol binding cannot be excluded, it certainly has insufficient predictive value for specific binding to cholesterol. This can be illustrated with the following, arbitrary example: The genome of Streptococcus agalactiae (GenBank Accession No. NC 004368) encodes 2094 known and hypothetical proteins, almost all of which will have no relationship to cholesterol whatsoever. Nevertheless, the putative cholesterol-binding motif occurs 5737 times among them. This corresponds to 2.7 occurrences per protein, or to 1 occurrence in every 112 amino acids. 3 Very similar numbers are found in the proteomes of Staphylococcus aureus and of Escherichia coli. Therefore, no conclusion can be drawn from the occurrence of this sequence motif in a protein as to its interaction with cholesterol. In fact, it has been shown recently that NAP-22 does not specifically require cholesterol, since it readily binds to sterol-free membranes that contain phosphatidylethanolamine [34]. Other pore-forming toxins that appear to have a strict requirement for cholesterol are the Vibrio cholerae 3 This was determined using a short Python program, which uses the regular expression Ô[LV][A–Z]{1,5}Y[A–Z]{1,5}[KR]Õ to find all instances of the protein motif in question. The program is available from the author upon request.
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cytolysin [35], and Streptococcus agalactiae CAMP factor [36]. These toxins have no significant sequence similarity to each other or to the thiol-activated toxins; no high-resolution structural information is available for either of them. Like streptolysin O, Vibrio cholerae cytolysin readily oligomerizes upon contact with cholesterol crystals, suggesting that the sterol has a role in oligomerization [37]. The toxin readily binds to membranes consisting of phosphatidylcholine only but does not form stable oligomers on these [38]. The structural selectivity of Vibrio cholerae cytolysin has been studied with several cholesterol analogues in model liposomes [18,19]. The most notable difference to streptolysin O consists in the fact that only very weak activity is observed with enantiomeric cholesterol. Ent-cholesterol, however, does induce oligomerization when added to the toxin in crystalline form, as do several other sterols that fail to support toxin activity in the liposome model. A hypothetical explanation of this discrepancy would again invoke the mutual interference of sterol interactions with the toxin and with other lipids in the bilayer, respectively, which would lower the affinity of the sterol for the toxin when embedded within mixed lipid membranes. With CAMP factor, there is no discernible difference at all between cholesterol and ent-cholesterol in their ability to support membrane permeabilization, suggesting a low degree of specificity in the toxin–cholesterol interaction (S. Lang, unpublished findings). Data for other sterols are not available.
3. Modulation of toxin activity by cholesterol Although with most bacterial toxins cholesterol is not strictly required for activity, a modulation, usually an enhancement of activity by cholesterol is a common observation. Such findings may relate to one or several effects the sterol has on the physical properties of lipid membranes [1]: Cholesterol modulates the fluidity of lipid bilayers, inducing a Ôliquid-orderedÕ state that is intermediate in fluidity between the gel state (which typically prevails at low temperature) and the liquid-crystalline state. Cholesterol, like phosphatidylethanolamine and diacylglycerol, is a Ôcone-shapedÕ lipid, 4 i.e. it occupies a larger cross-sectional area in the hydrophobic core of the membrane than within the head group layer (Fig. 3). Cone-shaped lipids promote the transition of lipid bilayers to the inverted hexagonal phase. 4 The terms Ôcone-shaped lipidsÕ and Ôinverted cone-shaped lipidsÕ are used inconsistently in the literature, so that cholesterol is sometimes referred to as an Ôinverted coneÕ. The choice of orientation is obviously arbitrary.
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(a)
(b)
(c)
(d)
Fig. 3. Cone-shaped lipids and the inverted hexagonal phase. (a) Schematic of an inverted cone-shaped lipid (left), a cylindrical one (middle), and a cone-shaped lipid. (b)–(d) Bilayers rich in coneshaped lipids are prone to convert to the inverted hexagonal phase. This phase transition is favoured by high temperature.
Cholesterol influences the lateral segregation of lipids in mixed bilayers. In the cytoplasmic membrane, cholesterol promotes formation of microdomains also enriched in sphingolipids and GPI-anchored proteins. These domains, which have been dubbed Ôlipid raftsÕ [39], have a role in the function of several bacterial toxins. Cholesterol increases the line tension at membrane discontinuities [40]. This is possibly of relevance to the membrane defects induced by pore-forming toxins; however, this possibility has not been explored experimentally. The role of membrane fluidity has been documented for Staphylococcus aureus a-hemolysin [41]. This toxin inserts into pure phosphatidylcholine membranes above but not below their intrinsic gel-to-liquid phase transition temperatures. Inclusion of cholesterol at a molar fraction of 20%, which promotes formation of the liquid-ordered state even below the transition temperature, renders the membranes susceptible. Above a certain threshold temperature, lipid bilayers will undergo a transition to the inverted hexagonal phase (Fig. 3). Inclusion of Ôcone-shapedÕ lipids in mixed bilayers will lower this transition temperature, i.e. promote the formation of hexagonal phases [42]. Localized formation of structures similar to that of inverted
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hexagonal phases is believed to occur in membrane fusion. In keeping with this idea, cone-shaped lipids promote membrane fusion [43]. They also increase the susceptibility of liposome membranes to the pore-forming toxin aerolysin; this was observed with cholesterol, diacylglycerol, and phosphatidyethanolamine [44]. Like fusion, insertion of an extrinsic protein disrupts the continuity of the membrane, and in the study cited it was proposed that membrane insertion of aerolysin may again be facilitated by localized formation of hexagonal phase-like lipid structures. Alternatively, it is conceivable that even in the absence of hexagonal phase the thinned headgroup spacing induced by cone-shaped lipids may facilitate access of the toxin or enzyme molecules to the hydrophobic layer. Cone-shaped lipids also promote the activity of phospholipases A2 and C, which likewise have to penetrate the headgroup layer in order to reach their target sites within the membrane. With phospholipase C, no close correlation was observed between the increment of activity on the one hand and the decrement of the phase transition temperature or the extent of vesicle fusion on the other [45], which suggests that indeed actual transition to hexagonal phase may not be required. A pronounced effect of inverted cone-shaped lipids also been noted with the cholesterol-specific toxins streptolysin O and Vibrio cholerae cytolysin [18]. In this case, the cone-shaped lipids did not substitute but augment the membrane susceptibility conferred by cholesterol. This effect was remarkably similar between the two toxins, and substantially more pronounced than with staphylococcal a-hemolysin, which like aerolysin has no strict requirement for cholesterol. A possible explanation of their more pronounced effect observed with the cholesterol-specific toxins is that, by competing with cholesterol for headgroup coverage ÔborrowedÕ from adjacent polar lipids [46], cone-shaped lipids will raise the free energy of cholesterol in mixed bilayers; this should promote association of the sterol with specific binding sites on the toxins. The same mechanism has recently been proposed to account for the observation that ceramide displaces cholesterol from lipid rafts [47]; this suggests that it may be of considerable biological importance. Lipid rafts are liquid-ordered state membrane domains that are rich in cholesterol and saturated polar lipids such as sphingomyelin and glycosphingolipids [39]. They are ÔafloatÕ within surrounding fluid-state membrane domains that are enriched in unsaturated lipids. Lipid rafts can experimentally be detected by way of their insolubility in Triton X-100 [48]; this insolubility suggests a tight packing of the lipid molecules within the rafts. Of note, in a liposome model, various sterols were found to promote formation of rafts to different extents; most sterols stabilized rafts less strongly than cholesterol did [20]. Therefore, with toxins targeting rafts,
differences in activity might be observed experimentally with different sterols even in the absence of specific interaction with the sterol molecule. Preferential association of toxins with rafts may be due to their content of cholesterol, as is likely with perfringolysin, or to the enrichment of other receptor molecules in the rafts. Examples of the latter are aerolysin, which binds to the glycoside moiety of GPI-anchored proteins [49], and cholera toxin, which binds to the ganglioside GM1 [50]. Location of the receptors within the rafts may or may not be important in toxin function. Aerolysin is equally effective on model membranes with and without rafts, respectively, as long as either contains GPI anchors as receptors [51]. On the other hand, with Helicobacter pylori vacuolating toxin and with cholera toxin, raft intactness matters. Depletion of membrane cholesterol with b-cyclodextrins, which dissipates rafts, prevents uptake and intracellular trafficking of these toxins and accordingly disrupts their intracellular action [52,53]. It is presently not clear, however, whether the role of cholesterol in the intracellular fate of these toxins is limited to the stabilization of rafts. Diphtheria toxin is an AB toxin, i.e. its B subunit forms a transmembrane pore in the process of transporting the enzymatically active A subunit into the target cell cytoplasm. The toxin pore also permits the passage of other solutes in a non-specific way. Although the membrane topology of the B subunit is not known in complete detail, only between two and four helical segments appear to actually insert into the membrane [54]. Interestingly, there is good evidence for both monomeric [55] and oligomeric [56] channels formed by diphtheria toxin. Oligomeric pores appear to vary in stoichiometry, and the functional diameter to increase with the number of subunits; they permit permeation of dextran molecules of up to at least 10 kDa. Diphtheria toxin does not strictly require cholesterol for activity; however, intriguingly, the sterol increases the apparent pore size and the extent of oligomerization [56]. 5 If diphtheria toxin functions as a monomer, what drives its oligomerization? The oligomers do not seem to be stabilized by significant protein-protein interaction but only to exist on the membrane; this suggests that it is the membrane itself that stabilizes them (Fig. 4). Both its limited number of membrane-spanning segments and its ability to fuse with others into a larger membrane defect suggest the possibility that the monomer pore is not completely enclosed by protein but is lined in part by a free edge of the lipid membrane itself. Pores with such a molecular structure have been observed with both the terminal complement complex
5 In Fig. 6 of the study cited, the labels of panels B and C have been switched (E. London, personal communication).
M. Palmer / FEMS Microbiology Letters 238 (2004) 281–289
(a)
(b)
Fig. 4. Hypothetical model of diphtheria toxin pore structure. (a) Top view. The monomeric pore is lined in part by the protein and in part by a free edge of the lipid membrane. Several pores may coalesce, which will abolish the energetically unfavourable membrane edge and increase pore size. (b) Side view of the monomer pore. The lipids at the edge are likely to assume a micelle-like configuration. It is easy to see how cone-shaped lipids such as cholesterol would place additional strain on this assembly.
[57] and with streptolysin O [2,58]. Lipid molecules at a free membrane edge will be subject to an energetically unfavourable situation. This translates into a line tension, i.e. into a tendency of the edge to retract. It is plausible to assume that oligomerization should yield an edge of reduced or at least less than additive length. This relaxation of line tension could then create a driving force for oligomerization even in the absence of any favourable protein-protein interaction. Furthermore, the observation that cholesterol promotes oligomerization would be accounted for by the fact that it causes an increase in line tension. This explanation is admittedly entirely hypothetical, and the oligomerization of diphtheria toxin may not be essential to its function in vivo. However, considering the extreme scarcity of studies on the subject, this example may be acceptable to illustrate that cholesterol might affect the function of membrane-associated proteins by way of increasing the membrane line tension. The biological significance of this aspect of cholesterol function remains to be elucidated.
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