Elsevier

Toxicon

Volume 39, Issue 11, November 2001, Pages 1681-1689
Toxicon

The family of thiol-activated, cholesterol-binding cytolysins

https://doi.org/10.1016/S0041-0101(01)00155-6Get rights and content

Abstract

Several species of both pathogenic and non-pathogenic grampositive bacteria within the genera Streptococcus, Clostridium and Bacillus secrete cytolytic proteins that belong to a single, highly homologous family. The most widely known members of this family are streptolysin O, listeriolysin, perfringolysin, and pneumolysin. These toxins specifically require membrane cholesterol but, apparently, do not depend on any other specific cell surface receptor, so that they are able to lyse the cytoplasmic membranes of virtually any animal cell. Upon binding as monomers, they oligomerize to form large pores with up to 30 nm internal diameter. These are the largest pores known, permitting permeation not only of ions and small metabolites but also of macromolecules. The latter property renders these toxins useful tools in cell biology.

While several of these cytolysins have been shown to be determinants of bacterial pathogenicity, their biological roles may vary, as do the lifestyles of the bacteria secreting them. A unique function is surely fulfilled by listeriolysin O, which helps the intracellular pathogen Listeria monocytogenes escape from phagolysosomes and then spread to adjacent host cells.

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.

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