1.C.12 The Thiol-activated Cholesterol-dependent Cytolysin (CDC) Family
Cholesterol-binding 'sulfhydryl-activated' toxins bind to cholesterol containing animal cell membranes and can be reversibly inactivated by oxidation. The prototype of the family, perfringolysin O (PFO), can lyse cholesterol-containing membranes of eukaryotic host cells. Cholesterol is the receptor for toxin binding, and following binding, the proteolytically processed subunits oligomerize to form the integral membrane, pore-forming, ring-shaped structure. Structural data for perfringolysin O are available, and a model for its membrane-associated form has been proposed. The oligomeric state of Pneumolysin involves 30-50 monomers complexed with lipid (Boney et al., 2001). Christie et al. 2018 presented an overview of the known features of the structures and functions of the CDCs, including the structure of the secreted monomers, the process of interaction with target membranes, and the transition from bound monomers to complete pores.
One CDC family member, Listeriolysin O (1.C.12.1.7), is produced by the intracellular parasite, Listeria monocytogenes. This pore-forming toxin contains a Pro-Glu-Ser-Thr (PEST) sequence that is essential for virulence and intracellular compartmentalization to the cytosol. Without the PEST sequence, the host cell is killed. Listerolysin O is probably targeted for degradation due to the presence of the PEST sequence. Thus, the PEST sequence converts the toxic cytolysin into a nontoxic derivative that allows intracelluar growth (Decatur and Portnoy, 2000). Listeriolysin O mediates lysis of L. monoctogenes-containing phagosomes and also facilitates cell-to-cell spreading (Dancz et al., 2002). Thus, it is bifunctional, but both functions probably depend on its pore-forming activity.
Pore-forming toxins of the CDC family allow delivery of macromolecules of up to 100 kDa to the host cell cytoplasm in a fully folded native conformation (Gonzalez et al., 2008). The large pores formed may contain up to 50 subunits. The animal cell receptors of many bacterial toxins have been tabulated by Gonzelez et al. (Gonzelez et al. 2008). Streptolysin O-permeabilized cells can be resealed by the action of Ca2+-calmodulin plus microtubules. CDC family toxins may thus serve to deliver proteins to the host cell cytoplasm, and they can be useful for artificial delivery of macromolecules to animal cells in general (Walev et al., 2001).
At the level of the primary structure, cholesterol dependent cytolysins (CDCs) display a high degree of sequence similarity ranging from 40% to 80%. This is mainly reflected in the conserved core of about 471 amino acids shared by all CDCs, which essentially corresponds to the sequence of pneumolysin, the shortest member of the family (Gonzalez et al., 2008). CDCs with longer sequences usually display variations in the N terminus, the functions of which are unknown for many members. Furthermore, all CDCs contain a highly conserved undecapeptide, which is thought to be critical for cholesterol-mediated membrane recognition. CDCs indeed all share a strict cholesterol dependency for oligomerization, which gave them their name. Most CDCs seem to use choletserol directly as a receptor. Intermedilysin (ILY) from Streptococcus intermedius, however, was shown to have a proteinaceous receptor, i.e., the GPI-anchored protein CD59 (Gonzalez et al., 2008). Interestingly, ILY shows a lower degree of conservation in the conserved undecapeptide important for cholesterol binding. As for all CDCs, pore formation by ILY requires the presence of choletserol for the membrane insertion step.
As noted above, the water-soluble monomeric cytolysin, perfringolysin O (PFO), secreted by Clostridium perfringens, oligomerizes and forms large pores upon encountering cholestrol-containing membranes. These pores, composed of 40-80 monomers, are large enough (15-30 nm diameter) to allow passage of macromolecules. Cysteine-scanning mutagenesis and multiple independent fluorescence techniques have suggested that each PFO monomer containing four domains, one of which is primarily involved in pore formation and has two amphipathic β-hairpins that span the membrane. In the soluble monomer, these transmembrane segments are folded into six α-helices (Shatursky et al., 1999; Billington et al., 2000). The insertion of two transmembrane hairpins per toxin monomer and a major change in secondary structure (vertical collapse) define a novel paradigm for the mechanism of membrane insertion by a cytolytic toxin (Czajkowsky et al., 2004).
The structural basis of Pneumolysin (1.C.12.1.5) has been presented (Tilley et al., 2005). As for other members of the CDC family, it is released from the bacterial cell as a monomer and assembles into large oligomeric rings in the target cell plasma membrane. Using cryoelectron microscopy and image processing, Tilley et al. have determined the structures of membrane-surface bound (prepore) and inserted-pore oligomer forms, providing a direct observation of the conformational transition into the pore form of a cholesterol-dependent cytolysin. In the pore structure, the domains of the monomer separate and double over into an arch, forming a wall sealing the bilayer around the pore. This transformation is accomplished by substantial refolding of two of the four protein domains along with deformation of the membrane. Extension of protein density into the bilayer supports earlier predictions that the protein inserts β-hairpins into the membrane. With an oligomer size of up to 44 subunits in the pore, this assembly creates a transmembrane channel 260 Å in diameter lined by 176 β-strands.
Despite their designation as 'thiol-activated' cytolysins, thiol activation does not appear to be a physiologically important property of these toxins. These proteins have therefore been renamed 'cholesterol-dependent cytolysins' (CDC). A detailed analysis of membrane interactive structures at the tip of perfringolysin O (PFO) domain 4 reveals that a threonine-leucine pair mediates CDC recognition of and binding to membrane cholesterol. This motif is conserved in all known CDCs, and conservative changes in its sequence or order are not well tolerated. Thus, the Thr-Leu pair mediates CDC-cholesterol recognition and binding (Farrand et al., 2010).
CDCs form large β-barrel pore complexes that are assembled from 35 to 40 soluble CDC monomers. Pore formation is dependent on the presence of membrane cholesterol, which functions as the receptor for most CDCs. Cholesterol binding initiates significant secondary and tertiary structural changes in the monomers, which lead to the assembly of a large membrane embedded β-barrel pore complex. The molecular mechanism of assembly of the CDC membrane pore complex has been reviewed (Hotze and Tweten, 2011).
As noted above, membrane-bound oligomers assemble into a prepore oligomeric form, following which the prepore assembly collapses towards the membrane surface, with concomitant release and insertion of the membrane spanning subunits (Reboul et al. 2014). During this rearrangement it is proposed that Domain 2, a region comprising three β-strands that links the pore forming region (Domains 1 and 3) and the Ig domain, must undergo a significant conformational change. Simple rigid body rotations may account for the observed collapse of the prepore towards the membrane surface. Domains 1, 2 and 4 are able to undergo significant rotational movements with respect to each other (Reboul et al. 2014).
The generalized transport reaction catalyzed by CDC family members is:
small and large molecules (in) → small and large molecules (out).