The family of thiol-activated, cholesterol-binding cytolysins
Section snippets
Protein sequence diversity and homology
All toxins in this family (Table 1) consist of a single polypeptide chain, the length of which ranges from 471 with pneumolysin (Walker et al., 1987) to 571 with streptolysin O (Kehoe et al., 1987) amino acid residues (aa). The conserved core shared by all molecules and essential for cytolytic activity corresponds to the sequence of the smallest family member, pneumolysin. This toxin deviates from the residual ones by lacking a secretory signal peptide (Walker et al., 1987) and, accordingly, is
Mode of membrane damage
As indicated above, the thiol-activated toxins form discrete, oligomeric pores in membranes containing cholesterol (Bhakdi et al., 1985). These pores are exceptionally large, assuming an internal diameter of up to 30 nm and comprising around 50 monomer subunits (Bhakdi et al., 1985, Olofsson et al., 1993, Morgan et al., 1995). The ring-shaped, non-covalently bonded oligomers had been visualized by electron microscopy (Duncan and Schlegel, 1975) before they were functionally characterized as
Mode of interaction with cholesterol
The toxins do not bind to membranes that do not contain cholesterol or a closely related sterol (Ohno Iwashita et al., 1992). Interaction with cholesterol also occurs in the absence of any other lipids, i.e. with the pure sterol in solution or suspension (Duncan and Schlegel, 1975), and leads to inhibition of lytic activity (Prigent and Alouf, 1976). Structural features of the cholesterol molecule required for interaction with the toxins include the 3β-hydroxy-group, the stereochemistry of the
Role of membrane components other than cholesterol
The individual toxins in this family display varying—and sometimes opposite—preferences for target erythrocytes from different mammalian species (Smyth and Duncan, 1978). The most pronounced selectivity apparently is displayed by intermediolysin, a novel member of the class that lacks the conserved unique cysteine residue; this toxin reportedly is largely specific for human cells (Nagamune et al., 1996). While these findings clearly indicate that membrane components other than cholesterol must
3D-structure
The crystal structure of the toxin monomer has been solved for perfringolysin O (Rossjohn et al., 1997). This structure covers the amino acid residues 37—500 of that molecule, which comprise the region of structural homology. The molecule has an elongated structure comprised of four domains. Domains 1, 2 and 4 are arranged along the longitudinal axis. Domain 3, which is covalently connected to domain 1, is laterally packed against domain 2. The polypeptide chain runs back and forth several
Structure to function relationships
A host of biochemical techniques such as limited proteolysis (Ohno Iwashita et al., 1986, Ohno Iwashita et al., 1988, Tweten et al., 1991), chemical modification (Iwamoto et al., 1987), epitope mapping of inhibitory monoclonal antibodies (Darji et al., 1996), site directed (Boulnois et al., 1991, Palmer et al., 1998a) or truncation mutagenesis (Shimada et al., 1999) and fluorescence spectroscopy (Nakamura et al., 1995, Palmer et al., 1996, Shepard et al., 1998, Shatursky et al., 1999) have been
Kinetic and mechanistic aspects of pore formation
As noted above, the first step toward pore formation consists in binding of the monomeric toxin to membranes containing cholesterol. Binding is very fast at both low and high temperature, and it is fully reversible, provided that subsequent oligomerization is prevented by either low temperature or low toxin concentration. It is of first order with respect to the monomeric toxin in solution (Palmer et al., 1995), and it thus probably does not involve mutual interaction of the toxin molecules.
Biological role of the thiol-activated toxins
The thiol-activated toxins occur in a wide variety of bacteria that have adopted similarly varied lifestyles. Several of them such as Listeriae, streptococci and pneumococci cause invasive disease. In these cases, the toxins represent determinants of pathogenicity, as shown with isogenic bacterial strains and animal models of infection (Limbago et al., 2000, Berry et al., 1989, Berry et al., 1995, Berry et al., 1999, Mengaud et al., 1989, Cossart et al., 1989). Others, such as Bacillus alvei
Vaccination studies
Immunization is particularly important with pneumococci, since these are very prevalent agents of bacterial pneumonia, meningitis, and septicemia. Pneumolysin has been employed as a carrier protein for conjugates with pneumococcal capsular polysaccharides (Michon et al., 1998), and in this application reported to be superior to the traditional carrier protein tetanus toxoid. Pneumolysin has also been found effective experimentally when used in combination with other proteinaceous pneumococcal
Cell biological applications
These toxins are very useful as tools in cell biological experiments, as witnessed by a large number of published applications. The extraordinarily large pore diameter renders the cytoplasmic membrane permeable to macromolecules that may be retrieved from or shuttled into the cells of interest (Lafont et al., 1995). As a rule, this will result in cell death, although experimental protocols have been elaborated that permit recovery of the permeabilized cell after uptake of the molecule of
Acknowledgements
I thank A.E. Johnson for useful suggestions on the manuscript.
References (87)
Cholesterol-binding cytolytic protein toxins
Int. J. Med. Microbiol.
(2000)- et al.
Permeabilization of the erythrocyte membrane with streptolysin O allows access to the vacuolar membrane of Plasmodium falciparum and a molecular analysis of membrane topology
Mol. Biochem. Parasitol.
(1997) - et al.
Structural analysis of the protein/lipid complexes associated with pore formation by the bacterial toxin pneumolysin
J. Biol. Chem.
(2001) - et al.
The potential to use PspA and other pneumococcal proteins to elicit protection against pneumococcal infection
Vaccine
(2000) - et al.
The use of host cell machinery in the pathogenesis of Listeria monocytogenes
Curr. Opin. Immunol.
(2001) - et al.
Secretory proteins move through the endoplasmic reticulum membrane via an aqueous, gated pore
Cell
(1994) - et al.
The effect of Streptococcus pneumoniae pneumolysin on human respiratory epithelium in vitro
Microb. Pathog.
(1990) - et al.
Selective purification by thiol-disulfide interchange chromatography of alveolysin, a sulfhydryl-activated toxin of Bacillus alvei. Toxin properties and interaction with cholesterol and liposomes
J. Biol. Chem.
(1983) - et al.
Two structural transitions in membrane pore formation by pneumolysin, the pore-forming toxin of Streptococcus pneumoniae
Cell
(1999) - et al.
The aqueous pore through the translocon has a diameter of 40–60 A during cotranslational protein translocation at the ER membrane
Cell
(1997)
Cholesterol-Streptolysin O-interaction: an EM study of wild-type and mutant Streptolysin O
J. Struct. Biol.
Mechanism of membrane insertion of a multimeric beta-barrel protein. Perfringolysin O creates a pore using ordered and coupled conformational changes
Mol. Cell
Arresting pore formation of a cholesterol-dependent cytolysin by disulfide trapping synchronizes the insertion of the transmembrane β-sheet from a prepore intermediate
J. Biol. Chem.
Structure and molecular mechanism of a functional form of pneumolysin: a cholesterol-dependent cytolysin from Streptococcus pneumoniae
J. Struct. Biol.
Multivalent pneumococcal capsular polysaccharide conjugate vaccines employing genetically detoxified pneumolysin as a carrier protein
Vaccine
Subunit organisation and symmetry of pore-forming, oligomeric pneumolysin
FEBS Lett.
Effect of lipidic factors on membrane cholesterol topology-mode of binding of theta-toxin to cholesterol in liposomes
Biochim. Biophys. Acta
The projection structure of perfringolysin O (Clostridium perfringens theta-toxin)
FEBS Lett.
Membrane-inserting domain of streptolysin O identified by cysteine scanning mutagenesis
J. Biol. Chem.
Interaction of steptolysin O with sterols
Biochim. Biophys. Acta
PEST sequences and regulation by proteolysis
Trends Biochem. Sci.
Structure of a cholesterol-binding. Thiol-activated cytolysin and a model of its membrane form
Cell
The mechanism of membrane insertion for a cholesterol-dependent cytolysin: a novel paradigm for pore-forming toxins
Cell
C-terminal amino acid residues are required for the folding and cholesterol binding property of perfringolysin O, a pore-forming cytolysin
J. Biol. Chem.
Ultrastructure of the membrane attack complex of complement. Heterogeneity of the complex caused by different degree of C9 polymerization
J. Biol. Chem.
Isolation of a tryptic fragment from Clostridium perfringens theta-toxin that contains sites for membrane binding and self- aggregation
J. Biol. Chem.
Assembly of the oligomeric membrane pore formed by Staphylococcal alpha-hemolysin examined by truncation mutagenesis
J. Biol. Chem.
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
Coupling of cholesterol and cone-shaped lipids in bilayers augments membrane permeabilization by the cholesterol-specific toxins Streptolysin O and Vibrio cholerae cytolysin
J. Biol. Chem.
Streptolysin O: inhibition of the conformational change during membrane binding of the monomer prevents oligomerization and pore formation
Biochemistry
Protein sorting in Plasmodium falciparum-infected red blood cells permeabilized with the pore-forming protein streptolysin O
Biochem. J.
pH-dependent perforation of macrophage phagosomes by listeriolysin O from Listeria monocytogenes
J. Exp. Med.
The Toxoplasma gondii rhoptry protein ROP 2 is inserted into the parasitophorous vacuole membrane, surrounding the intracellular parasite, and is exposed to the host cell cytoplasm
J. Cell Biol.
Reduced virulence of a defined pneumolysin-negative mutant of Streptococcus pneumoniae
Infect. Immun.
Effect of defined point mutations in the pneumolysin gene on the virulence of Streptococcus pneumoniae
Infect. Immun.
Comparative virulence of Streptococcus pneumoniae strains with insertion-duplication, point, and deletion mutations in the pneumolysin gene
Infect. Immun.
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.
Mechanism of membrane damage by streptolysin-O
Infect. Immun.
The Arcanobacterium (Actinomyces) pyogenes hemolysin, pyolysin, is a novel member of the thiol-activated cytolysin family
J. Bacteriol.
Structure and function of pneumolysin, the multifunctional, thiol-activated toxin of Streptococcus pneumoniae
Mol. Microbiol.
Pneumolysin, a protein toxin of Streptococcus pneumoniae, induces nitric oxide production from macrophages
Infect. Immun.
Genetic immunization of mice against Listeria monocytogenes using plasmid DNA encoding listeriolysin O
J. Immunol.
Listeriolysin O is essential for virulence of Listeria monocytogenes: direct evidence obtained by gene complementation
Infect. Immun.
Cited by (157)
The Bacillus cereus group
2023, Molecular Medical Microbiology, Third EditionBiomimetic cell model for fluorometric and smartphone colorimetric dual-signal readout detection of bacterial toxin
2020, Sensors and Actuators, B: ChemicalAddressable Cholesterol Analogs for Live Imaging of Cellular Membranes
2018, Cell Chemical BiologyCitation Excerpt :As cholesterol does not show an intrinsic fluorescence, modifications of the cholesterol body introducing a fluorescent moiety or cholesterol binding molecules, which bear a fluorescent moiety, were developed to characterize its steady-state distribution (Gimpl and Gehrig-Burger, 2007; Maxfield and Wüstner, 2012). These include cholesterol binding agents such as filipin and perfringolysin O (Lopes et al., 2004; Palmer, 2001), chemically related steroids such as dehydroergosterol, which are intrinsically fluorescent (Rogers et al., 1979; Bar et al., 1989; Wüstner, 2007), and fluorescently labeled cholesterol derivatives such as those containing an azi group as a fluorescent moiety at the double bond (Thiele et al., 2000) or BODIPY coupled to the end of the alkyl chain (Li et al., 2006; Höltta-Vuori et al., 2008). However, the visualization of cholesterol organization and dynamics in live cells using these markers has been hampered by the poor delivery of the labeled derivatives into cells, the prerequisite for cell fixation, the relatively weak staining and resolution, and the perturbation of the native membrane organization, e.g., because of less efficient packing (Sezgin et al., 2017).
Degradation of nuclear Ubc9 induced by listeriolysin O is dependent on K<sup>+</sup> efflux
2017, Biochemical and Biophysical Research CommunicationsCitation Excerpt :Cholesterol-dependent cytolysins (CDCs) constitute a large family of pore-forming toxin produced by many gram-positive pathogenic bacteria, such as Bacillus, Clostridium and Streptococcus [1].