Abstract
The cholesterol-dependent cytolysins (CDCs) are part of a large family of pore-forming proteins that include the human proteins perforin and the complement membrane attack complex. The activity of all family members is focused on membranes, but the proteins are themselves involved in a diverse range of phenomena. An overview of some of these phenomena is provided here, along with an historical perspective of CDCs themselves and how our understanding of their mechanism of action has developed over the years. The way in which pore formation depends on specific characteristics of the membrane under attack as well as of the protein doing the attacking is emphasised.
The cholesterol-dependent cytolysins (CDCs) have been the focus of a renewed keen research interest for over ten years now.1–4 Their importance has been even further enhanced by the homology now identified between them and the membrane attack complex/perforin (MACPF) family of proteins, which includes several components of the complement cascade as well as perforin itself.5–9 In this chapter I aim to provide an overview of our understanding of the interaction between CDCs and other members of what is now called the MACPF/CDC superfamily, with their target membranes.
CDCs (also in the past known as thiol-activated toxins or cholesterol-binding toxins) were originally identified from four Gram-positive bacterial genera (Clostridium, Listeria, Bacillus and Streptococcus). Well-known examples include listeriolysin, perfringolysin, streptolysin and pneumoysin. Listeriolysin from L. monocytogenes is responsible for the escape of bacteria from the phagosome to colonise the cytoplasm 10 and has been applied as a protein adjuvant in the development of vaccines against cancer and tuberculosis, for example.11–13 Perfringolysin from C. perfringens (Fig. 1A) has become perhaps the most studied CDC4 and has an important role in pathology associated with infection (gangrene).14–16 Streptolysin from S. pyogenes is another intensely studied CDC and has been applied widely in experimental permeabilisation of biological membranes.17,18 Pneumolysin is a major virulence determinant for S. pneumoniae, allowing bacterial invasion of tissues and mediating inflammation and the activation of the complement cascade.19,20 However, CDCs have now, for example, been identified in the bacteria Arcanobacterium pyogenes and Gardnerella vaginalis 8 and there also appear to be homologues outside prokaryotes such as the sea anemone Metridium senile pore-forming toxin metridiolysin. 21 The homology with the MACPF family was unknown until the first structures of the canonical fold of that family were solved, revealing the now characteristic MACPF/CDC fold of a twisted ß-sheet around which helices are clustered (Fig. 1A and D). Without any significant other sequence homology, the fold of this superfamily of pore-forming and membrane-binding proteins has been conserved by compensatory mutation around a handful of key conserved glycines.6,8,9 The glycines presumably act as critical hinges during the dramatic refolding that CDCs are known to undergo and which is presumably the selective advantage of this specific structure that has caused it to be conserved over such a vast evolutionary timescale. While not all MACPF domains are involved in pore formation—for example, C6 and C8ß—they are all apparently involved in action on membranes.8,22 The dramatic refolding undergone by CDCs is tightly coupled to their oligomerisation and results in the conversion of the helices hemming the core ß-sheet of the MACPF/CDC domain into a pair of ß-hairpins which in tandem and alongside those from other subunits within the oligomer insert into the membrane to create a pore2,4,23–27 (Fig. 1A-C). It is obviously the basic assumption that where nonCDC members of the superfamily—such as complement proteins and perforin— act on membranes they do so by a mechanism involving similar refolding.5,8 Even where a member of the MACPF/CDC superfamily is not known to form a pore, or has been shown not to—at least alone—the same conformational change could have other adaptive functions during activity on or at membranes. However, the bicomponent nature of some pore-forming toxins28 alerts us that showing an absence of activity for one pure protein does not mean that they do not contribute to pore formation quite directly, since that may require the presence of another MACPF/CDC family member or members from the same specific system. Complement acts by a combination of the C5b-8 complex of proteins preassembled on a target membrane recruiting C9 to form a lesion, which may be a complete ring of C9 associated with the C5b-8 or an arc—electron microscopy images show both possibilities.29,30 Perforin acts in concert with granzymes, to trigger apoptosis when delivered by cytotoxic cells at their targets (damaged, transformed and infected host cells). Incomplete rings are visible for perforin also31–33 and there are many unresolved issues concerning its mechanism and the dependence of granzymes on it for their delivery.34–38
A) Atomic structure of perfringolysin78 depicted as a grey ribbon. The two helical regions which refold into the membrane as a ß-hairpin are coloured red, the loops at the base of domain 4 implicated in cholesterol binding blue with an expanded coil radius and the tryptophan-rich loop upon which cholesterol impacts in pore formation in cyan. B) Atomic model of the prepore state of CDCs when a full ring oligomer is formed, modeling the perfringolysin crystal structure into a cryo-EM map for the prepore.27 Regions of the structure are coloured as in panel (A). C) Atomic model of the pore state of CDCs as a full ring, as in (B) for the prepore. D) Atomic models of complement protein C8α (left), a complex of C8α and C8γ (center) and the Plu-MACPF domain, a prokaryotic MACPF protein (right). The regions of each structure correlating to the membrane-inserting regions of CDCs are coloured red in each case and in the central panel C8γ is coloured a lighter shade of grey.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
Gilbert RJ. Pore-forming toxins. Cell Mol Life Sci 2002; 59:832–844.
Gilbert RJ. Inactivation and activity of cholesterol-dependent cytolysins: what structural studies tell us. Structure 2005; 13:1097–1106.
Tilley SJ, Saibil HR. The mechanism of pore formation by bacterial toxins. Curr Opin Struct Biol 2006; 16:230–236.
Tweten RK. Cholesterol-dependent cytolysins, a family of versatile pore-forming toxins. Infect Immun 2005; 73:6199–6209.
Anderluh G, Lakey JH. Disparate proteins use similar architectures to damage membranes. Trends Biochem Sci 2008; 33:482–490.
Hadders MA, Beringer DX, Gros P. Structure of C8 alpha-MACPF reveals mechanism of membrane attack in complement immune defense. Science 2007; 317:1552–1554.
Rosado CJ, Buckle AM, Law RH et al. A common fold mediates vertebrate defense and bacterial attack. Science 2007; 317:1548–1551.
Rosado CJ, Kondos S, Bull TE et al. The MACPF/CDC family of pore-forming toxins. Cell Microbiol 2008; 10:1765–1774.
Slade DJ, Lovelace LL, Chruszcz M et al. Crystal structure of the MACPF domain of human complement protein C8 alpha in complex with the C8 gamma subunit. J Mol Biol 2008; 379:331–342.
Birmingham CL, Canadien V, Kaniuk NA et al. Listeriolysin O allows Listeria monocytogenes replication in macrophage vacuoles. Nature 2008; 451:350–354.
Grode L, Seiler P, Baumann S et al. Increased vaccine efficacy against tuberculosis of recombinant mycobacterium bovis bacille calmette-guerin mutants that secrete listeriolysin. J Clin Invest 2005; 115:2472–2479.
Neeson P, Pan ZK, Paterson Y. Listeriolysin O is an improved protein carrier for lymphoma immunoglobulin idiotype and provides systemic protection against 38C13 lymphoma. Cancer Immunol Immunother 2008; 57:493–505.
Peng X, Treml J, Paterson Y. Adjuvant properties of listeriolysin O protein in a DNA vaccination strategy. Cancer Immunol Immunother 2007; 56:797–806.
Awad MM, Ellemor DM, Boyd RL et al. Synergistic effects of alpha-toxin and perfringolysin O in Clostridium perfringens-mediated gas gangrene. Infect Immun 2001; 69:7904–7910.
Christianson KK, Tweten RK, Iandolo JJ. Transport and processing of staphylococcal enterotoxin A. Appl Environ Microbiol 1985; 50:696–697.
Rood JI. Virulence genes of clostridium perfringens. Annu Rev Microbiol 1998; 52:333–360.
Bhakdi S, Weller U, Walev I et al. A guide to the use of pore-forming toxins for controlled permeabilization of cell membranes. Med Microbiol Immunol 1993; 182:167–175.
Weimbs T, Low SH, Li X et al. SNAREs and epithelial cells. Methods 2003; 30:191–197.
Kadioglu A, Weiser JN, Paton JC et al. The role of streptococcus pneumoniae virulence factors in host respiratory colonization and disease. Nat Rev Microbiol 2008; 6:288–301.
Marriott HM, Mitchell TJ, Dockrell DH. Pneumolysin: a double-edged sword during the host-pathogen interaction. Curr Mol Med 2008; 8:497–509.
Bernheimer AW, Rudy B. Interactions between membranes and cytolytic peptides. Biochim Biophys Acta 1986; 864:123–141.
Ponting CP. Chlamydial homologues of the MACPF (MAC/perforin) domain. Curr Biol 1999; 9:R911–913.
Heuck AP, Hotze EM, Tweten RK et al. Mechanism of membrane insertion of a multimeric beta-barrel protein: perfringolysin O creates a pore using ordered and coupled conformational changes. Mol Cell 2000; 6:1233–1242.
Hotze EM, Heuck AP, Czajkowsky DM et al. Monomer-monomer interactions drive the prepore to pore conversion of a beta-barrel-forming cholesterol-dependent cytolysin. J Biol Chem 2002; 277:11597–11605.
Hotze EM, Wilson-Kubalek EM, Rossjohn J et al. Arresting pore formation of a cholesterol-dependent cytolysin by disulfide trapping synchronizes the insertion of the transmembrane beta-sheet from a prepore intermediate. J Biol Chem 2001; 276:8261–8268.
Shatursky O, Heuck AP, Shepard LA et al. The mechanism of membrane insertion for a cholesterol-dependent cytolysin: a novel paradigm for pore-forming toxins. Cell 1999; 99:293–299.
Tilley SJ, Orlova EV, Gilbert RJ et al. Structural basis of pore formation by the bacterial toxin pneumolysin. Cell 2005; 121:247–256.
Menestrina G, Dalla Serra M, Comai M et al. Ion channels and bacterial infection: the case of beta-barrel pore-forming protein toxins of Staphylococcus aureus. FEBS Lett 2003; 552:54–60.
Bhakdi S, Tranum-Jensen J. On the cause and nature of C9-related heterogeneity of terminal complement complexes generated on target erythrocytes through the action of whole serum. J Immunol 1984; 133:1453–1463.
Bhakdi S, Tranum-Jensen J. C5b-9 assembly: average binding of one C9 molecule to C5b-8 without poly-C9 formation generates a stable transmembrane pore. J Immunol 1986; 136:2999–3005.
Podack ER, Dennert G. Assembly of two types of tubules with putative cytolytic function by cloned natural killer cells. Nature 1983; 302:442–445.
Young JD, Hengartner H, Podack ER et al. Purification and characterization of a cytolytic pore-forming protein from granules of cloned lymphocytes with natural killer activity. Cell 1986; 44:849–859.
Young LH, Joag SV, Zheng LM et al. Perforin-mediated myocardial damage in acute myocarditis. Lancet 1990; 336:1019–1021.
Froelich CJ, Pardo J, Simon MM. Granule-associated serine proteases: granzymes might not just be killer proteases. Trends Immunol 2009; 30:117–123.
Metkar SS, Wang B, Aguilar-Santelises M et al. Cytotoxic cell granule-mediated apoptosis: perforin delivers granzyme B-serglycin complexes into target cells without plasma membrane pore formation. Immunity 2002; 16:417–428.
Trapani JA, Voskoboinik I. The complex issue of regulating perforin expression. Trends Immunol 2007; 28:243–245.
Voskoboinik I, Smyth MJ, Trapani JA. Perforin-mediated target-cell death and immune homeostasis. Nat Rev Immunol 2006; 6:940–952.
Keefe D, Shi L, Feske S et al. Perforin triggers a plasma membrane-repair response that facilitates CTL induction of apoptosis. Immunity 2005; 23:249–262.
Bhakdi S, Tranum-Jensen J, Sziegoleit A. Mechanism of membrane damage by streptolysin-O. Infect Immun 1985; 47:52–60.
Harris RW, Sims PJ, Tweten RK. Kinetic aspects of the aggregation of Clostridium perfringens theta-toxin on erythrocyte membranes. A fluorescence energy transfer study. J Biol Chem 1991; 266:6936–6941.
Palmer M, Valeva A, Kehoe M et al. Kinetics of streptolysin O self-assembly. Eur J Biochem 1995; 231:388–395.
Palmer M, Harris R, Freytag C et al. Assembly mechanism of the oligomeric streptolysin O pore: the early membrane lesion is lined by a free edge of the lipid membrane and is extended gradually during oligomerization. EMBO J 1998; 17:1598–1605.
Czajkowsky DM, Hotze EM, Shao Z et al. Vertical collapse of a cytolysin prepore moves its transmembrane beta-hairpins to the membrane. EMBO J 2004; 23:3206–3215.
Cohen B, Schwachman H, Perkins ME. Inactivation of pneumococcal hemolysin by certain sterols. Proc Soc Exp Biol Med 1937; 35:586–591.
Saunders FK, Mitchell TJ, Walker JA et al. Pneumolysin, the thiol-activated toxin of Streptococcus pneumoniae, does not require a thiol group for in vitro activity. Infect Immun 1989; 57:2547–2552.
Gilbert RJ, Heenan RK, Timmins PA et al. Studies on the structure and mechanism of a bacterial protein toxin by analytical ultracentrifugation and small-angle neutron scattering. J Mol Biol 1999; 293:1145–1160.
Gilbert RJ, Jimenez JL, Chen S et al. Two structural transitions in membrane pore formation by pneumolysin, the pore-forming toxin of Streptococcus pneumoniae. Cell 1999; 97:647–655.
Nagamune H, Whiley RA, Goto T et al. Distribution of the intermedilysin gene among the anginosus group streptococci and correlation between intermedilysin production and deep-seated infection with Streptococcus intermedius. J Clin Microbiol 2000; 38:220–226.
Giddings KS, Zhao J, Sims PJ et al. Human CD59 is a receptor for the cholesterol-dependent cytolysin intermedilysin. Nat Struct Mol Biol 2004; 11:1173–1178.
Soltani CE, Hotze EM, Johnson AE et al. Structural elements of the cholesterol-dependent cytolysins that are responsible for their cholesterol-sensitive membrane interactions. Proc Natl Acad Sci USA 2007; 104:20226–20231.
Giddings KS, Johnson AE, Tweten RK. Redefining cholesterol’s role in the mechanism of the cholesterol-dependent cytolysins. Proc Natl Acad Sci USA 2003; 100:11315–11320.
Geoffroy C, Alouf JE. Interaction of alveolysin A sulfhydryl-activated bacterial cytolytic toxin with thiol group reagents and cholesterol. Toxicon 1982; 20:239–241.
Ohno-Iwashita Y, Iwamoto M, Mitsui K et al. Protease-nicked theta-toxin of Clostridium perfringens, a new membrane probe with no cytolytic effect, reveals two classes of cholesterol as toxin-binding sites on sheep erythrocytes. Eur J Biochem 1988; 176:95–101.
Harris JR, Adrian M, Bhakdi S et al. Cholesterol-streptolysin O interaction: an EM study of wild-type and mutant streptolysin O. J Struct Biol 1998; 121:343–355.
Sonnen AF, Rowe AJ, Andrew PW et al. Oligomerisation of pneumolysin on cholesterol crystals: similarities to the behaviour of polyene antibiotics. Toxicon 2008; 51:1554–1559.
Howard JG, Wallace KR, Wright GP. The inhibitory effects of cholesterol and related sterols on haemolysis by streptolysin O. Br J Exp Pathol 1953; 34:174–180.
Nollmann M, Gilbert R, Mitchell T et al. The role of cholesterol in the activity of pneumolysin, a bacterial protein toxin. Biophys J 2004; 86:3141–3151.
Ohno-Iwashita Y, Iwamoto M, Ando S et al. Effect of lipidic factors on membrane cholesterol topology— mode of binding of theta-toxin to cholesterol in liposomes. Biochim Biophys Acta 1992; 1109:81–90.
Ohno-Iwashita Y, Iwamoto M, Mitsui K et al. A cytolysin, theta-toxin, preferentially binds to membrane cholesterol surrounded by phospholipids with 18-carbon hydrocarbon chains in cholesterol-rich region. J Biochem 1991; 110:369–375.
Delattre J, Badin J, Canal J et al. Influence of lecithins on the inhibitory effect of cholesterol towards streptolysin O. C R Acad Sci Hebd Seances Acad Sci D 1973; 277:441–443.
Flanagan JJ, Tweten RK, Johnson AE et al. Cholesterol exposure at the membrane surface is necessary and sufficient to trigger perfringolysin O binding. Biochemistry 2009; 48:3977–3987.
El-Rachkidy RG, Davies NW, Andrew PW. Pneumolysin generates multiple conductance pores in the membrane of nucleated cells. Biochem Biophys Res Commun 2008; 368:786–792.
Hill J, Andrew PW, Mitchell TJ. Amino acids in pneumolysin important for hemolytic activity identified by random mutagenesis. Infect Immun 1994; 62:757–758.
Korchev YE, Bashford CL, Pederzolli C et al. A conserved tryptophan in pneumolysin is a determinant of the characteristics of channels formed by pneumolysin in cells and planar lipid bilayers. Biochem J 1998; 329:571–577.
Heuck AP, Tweten RK, Johnson AE. Assembly and topography of the prepore complex in cholesterol-dependent cytolysins. J Biol Chem 2003; 278:31218–31225.
Menestrina G, Bashford CL, Pasternak CA. Pore-forming toxins: experiments with S. aureus alpha-toxin C. perfringens theta-toxin and E. coli haemolysin in lipid bilayers, liposomes and intact cells. Toxicon 1990; 28:477–491.
Almers W. Fusion needs more than SNAREs. Nature 2001; 409:567–568.
Kielian M, Rey FA. Virus membrane-fusion proteins: more than one way to make a hairpin. Nat Rev Microbiol 2006; 4:67–76.
Peters C, Bayer MJ, Buhler S et al. Trans-complex formation by proteolipid channels in the terminal phase of membrane fusion. Nature 2001; 409:581–288.
Anderluh G, Dalla Serra M, Viero G et al. Pore formation by equinatoxin II, a eukaryotic protein toxin, occurs by induction of nonlamellar lipid structures. J Biol Chem 2003; 278:45216–45223.
Weaver JC. Molecular basis for cell membrane electroporation. Ann N Y Acad Sci 1994; 720:141–152.
Qian S, Wang W, Yang L et al. Structure of transmembrane pore induced by Bax-derived peptide: evidence for lipidic pores. Proc Natl Acad Sci USA 2008; 105:17379–17383.
Morgan PJ, Hyman SC, Byron O et al. Modeling the bacterial protein toxin, pneumolysin, in its monomeric and oligomeric form. J Biol Chem 1994; 269:25315–25320.
Olofsson A, Hebert H, Thelestam M. The projection structure of perfringolysin O (Clostridium perfringens theta-toxin). FEBS Lett 1993; 319:125–127.
Morgan PJ, Hyman SC, Rowe AJ et al. Subunit organisation and symmetry of pore-forming, oligomeric pneumolysin. FEBS Lett 1995; 371:77–80.
Shepard LA, Heuck AP, Hamman BD et al. Identification of a membrane-spanning domain of the thiol-activated pore-forming toxin Clostridium perfringens perfringolysin O: an alpha-helical to beta-sheet transition identified by fluorescence spectroscopy. Biochemistry 1998; 37:14563–14574.
Shepard LA, Shatursky O, Johnson AE et al. The mechanism of pore assembly for a cholesterol-dependent cytolysin: formation of a large prepore complex precedes the insertion of the transmembrane beta-hairpins. Biochemistry 2000; 39:10284–10293.
Rossjohn J, Feil SC, McKinstry WJ et al. Structure of a cholesterol-binding, thiol-activated cytolysin and a model of its membrane form. Cell 1997; 89:685–692.
Solovyova AS, Nollmann M, Mitchell TJ et al. The solution structure and oligomerization behavior of two bacterial toxins: pneumolysin and perfringolysin O. Biophys J 2004; 87:540–552.
Rossjohn J, Polekhina G, Feil SC et al. Structures of perfringolysin O suggest a pathway for activation of cholesterol-dependent cytolysins. J Mol Biol 2007; 367:1227–1236.
Bourdeau RW, Malito E, Chenal A et al. Cellular functions and X-ray structure of anthrolysin O, a cholesterol-dependent cytolysin secreted by Bacillus anthracis. J Biol Chem 2009; 284:14645–14656.
Bonev B, Gilbert R, Watts A. Structural investigations of pneumolysin/lipid complexes. Mol Membr Biol 2000; 17:229–235.
Bonev BB, Gilbert RJ, Andrew PW et al. Structural analysis of the protein/lipid complexes associated with pore formation by the bacterial toxin pneumolysin. J Biol Chem 2001; 276:5714–5719.
Idone V, Tam C, Goss JW et al. Repair of injured plasma membrane by rapid Ca2+-dependent endocytosis. J Cell Biol 2008; 180:905–914.
Mitchell TJ. Virulence factors and the pathogenesis of disease caused by Streptococcus pneumoniae. Res Microbiol 2000; 151:413–419.
Kafsack BF, Pena JD, Coppens I et al. Rapid membrane disruption by a perforin-like protein facilitates parasite exit from host cells. Science 2009; 323:530–533.
Amino R, Giovannini D, Thiberge S et al. Host cell traversal is important for progression of the malaria parasite through the dermis to the liver. Cell Host Microbe 2008; 3:88–96.
Ecker A, Pinto SB, Baker KW et al. Plasmodium berghei: plasmodium perforin-like protein 5 is required for mosquito midgut invasion in Anopheles stephensi. Exp Parasitol 2007; 116:504–508.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Landes Bioscience and Springer Science+Business Media
About this chapter
Cite this chapter
Gilbert, R.J.C. (2010). Cholesterol-Dependent Cytolysins. In: Anderluh, G., Lakey, J. (eds) Proteins Membrane Binding and Pore Formation. Advances in Experimental Medicine and Biology, vol 677. Springer, New York, NY. https://doi.org/10.1007/978-1-4419-6327-7_5
Download citation
DOI: https://doi.org/10.1007/978-1-4419-6327-7_5
Publisher Name: Springer, New York, NY
Print ISBN: 978-1-4419-6326-0
Online ISBN: 978-1-4419-6327-7
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)