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).

Some pathogens produce pore-forming toxins (PFTs) that disrupt the plasma membrane (PM) integrity by forming transmembrane pores. High PFT concentrations cause massive damage leading to cell death and facilitating infection. Sub-lytic PFT doses activate repair mechanisms to restore PM integrity, support cell survival, and limit disease. Shedding of extracellular vesicles (EVs) is one mechanism to eliminate PFT pores and restore PM integrity. Alves et al. 2022 showed that cholesterol-dependent cytolysins (CDCs) are at least partially eliminated through EV release, and proteins important for PM repair are included in EVs shed by cells during repair. To identify new PM repair proteins, Alves et al. 2022 collected EVs released by cells challenged with sub-lytic doses of two different bacterial CDCs, listeriolysin O and pneumolysin, and they determined the EV proteomic repertoire by LC-MS/MS. Intoxicated cells release similar EVs irrespectively of the CDC used. They release more and larger EVs than non-intoxicated cells. A cluster of 70 proteins, including calcium-binding proteins, molecular chaperones, cytoskeletal scaffold proteins and membrane trafficking proteins, was detected, enriched in EVs collected from intoxicated cells. While some of these proteins have well-characterized roles in repair, the involvement of others is not knkown. Copine-1 and Copine-3 proteins, abundantly detected in EVs released by intoxicated cells, are required for efficient repair of CDC-induced PM damage. New proteins potentially involved in PM repair are described, and they give new insights into common mechanisms and machinery engaged by cells in response to PM damage (Alves et al. 2022).


The generalized transport reaction catalyzed by CDC family members is:

small and large molecules (in) → small and large molecules (out).



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TC#NameOrganismal TypeExample

Perfringolysin O, PFO. In the formation of the pore forming toxin, the elongated toxin monomer binds stably to the membrane in an "end-on" orientation, with its long axis approximately perpendicular to the plane of the membrane bilayer (Ramachandran et al. 2005). This orientation is largely retained, even after monomers associate to form an oligomeric prepore complex. The domain 3 (D3) polypeptide segments that ultimately form transmembrane beta-hairpins remain far above the membrane surface in both the membrane-bound monomer and prepore oligomer. Upon pore formation, these segments enter the bilayer, whereas D1 moves to a position that is substantially closer to the membrane. Therefore, the extended D2 beta-structure that connects D1 to membrane-bound D4 appears to bend or otherwise reconfigure during the prepore-to-pore transition of the perfringolysin O oligomer (Ramachandran et al. 2005). The prepore to pore transition has been visualized by electron microscopy (Dang et al. 2005). Phosphatidylcholine in the outer leaflet increases the cholesterol concentration required to induce PFO binding while phosphatidylethanolamine and phosphatidylserine in the inner leaflet of asymmetric vesicles stabilized the formation of a deeply inserted conformation that does not form pores, even though it contains transmembrane segments (Lin and London 2014). This conformation may represent an important intermediate stage in PFO pore formation.  Cholesterol recognition, oligomerization, and the conformational changes involved in pore formation have been reviewed (Johnson and Heuck 2014), and the involvement of the D1 domain in structural transitions leading to pore formation has been studied (Kacprzyk-Stokowiec et al. 2014). Interaction of PFO with cholesterol is sufficient to initiate an irreversible sequence of coupled conformational changes that extend throughout the toxin molecule and induce pore formation (Heuck et al. 2007).  Once this transmembrane beta-barrel protein is inserted, PFO assembles into pore-forming oligomers containing 30-50 PFO monomers. These form a pore of up to 300 Å, far exceeding the size of most other proteinaceous pores.  Decreasing the length of the β-strands causes the pore to shrink (Lin et al. 2015). Site-directed mutagenesis data combined with binding studies performed with different sterols, and molecular modeling are beginning to shed light on the interaction with cholesterol (Savinov and Heuck 2017). Fine-tuning of the stability of beta-strands by Y181 in perfringolysin O directs the prepore to pore transition (Kulma et al. 2019). 


Perfringolysin O of Clostridium perfringens (P0C2E9)


CDC family protein of 588 aas

CDC protein of Treponema medium


CDC homologue of 511 aas

CDC protein of Deinococcus deserti


Uncharacterized protein of 656 aas

UP of Streptomyces mobaraensis


Intermedilysin of 532 aas and 1 N-terminal TMS, ILY or Ply.  It binds to membranes containing the human protein CD59 but forms pores only if the membrane contains sufficient cholesterol (Heuck et al. 2007).  CD59 is required for the specific coordination of intermedilysin (ILY) monomers and for triggering collapse of an oligomeric prepore. Movement of Domain 2 with respect to Domain 3 of ILY is essential for forming a late prepore intermediate that releases CD59, while the role of cholesterol may be limited to insertion of the TMSs (Boyd et al. 2016). The pore-forming regions are initially folded up on the surfaces of the soluble precursors. To create the transmembrane pores, these regions must extend and refold into membrane-inserted beta-barrels (Tilley and Saibil 2006).

Intermedilysin of Streptococcus intermedius


Thiol-activated cytolysin of 500 aas and 1 N-terminal TMS.  It is a sulfhydryl-activated toxin that causes cytolysis by forming pores in cholesterol containing host membranes. After binding to target membranes, the protein undergoes a major conformation change, leading to its insertion in the host membrane and formation of an oligomeric pore complex.  Biomimetic nanosponges neutralize this cytolysin, protect the retina, preserve vision, and may provide an adjunct detoxification therapy for bacterial infections (LaGrow et al. 2017).

Cytotoxin of Enterococcus faecalis


Cholesterol-dependent cytolysin or thiol-activated cytolysin of 532 aas (Pleckaityte 2019).

CDC or TAC of Gemella bergeri


Vaginolysin (VLY) of 516 aas and 1 N-terminal TMS. It plays a role in bacterial vaginosis (BV), a vaginal anaerobic dysbiosis that affects women of reproductive age worldwide. BV is microbiologically characterized by the depletion of vaginal lactobacilli and the overgrowth of anaerobic bacterial species. Gardnerella spp. have a pivotal role among BV-associated bacteria in the initiation and development of BV (Pleckaityte 2019). Inerolysin (INY) (TC# 1.C.12.1.17)-induced damage of artificial membranes is directly dependent on the cholesterol concentration in the bilayer, whereas VLY-induced damage occurs only with high levels of membrane cholesterol (>40 mol%) (Ragaliauskas et al. 2019). VLY primarily forms membrane-embedded complete rings in the synthetic bilayer, whereas INY forms arciform structures with smaller pore sizes. VLY activity is high at elevated pH, which is characteristic of BV, whereas INY activity is high at more acidic pH, which is characteristic of a healthy vagina (Pleckaityte 2019).

Vaginolysin of Gardnerella vaginalis


Inerolysin (INY) or cholesterol-dependent cytolysin of 519 aas and one N-terminal TMS. Lactobacillus iners is a prevalent constituent of healthy vaginal microbiota, but it produces this cytotoxin (Pleckaityte 2019). INY-induced damage of artificial membranes is directly dependent on the cholesterol concentration in the bilayer, whereas VLY (TC# 1.C.12.1.16)-induced damage occurs only with high levels of membrane cholesterol (>40 mol%) (Ragaliauskas et al. 2019). VLY primarily forms membrane-embedded complete rings in the synthetic bilayer, whereas INY forms arciform structures with smaller pore sizes. VLY activity is high at elevated pH, which is characteristic of BV, whereas INY activity is high at more acidic pH, which is specific for a healthy vagina (Pleckaityte 2019).

INY of Lactobacillus iners


Pore-forming Alveolysin of 501 aas and one N-terminal TMS.

Gram-positive bacteria

Alveolysin of Bacillus alvei (P23564)


Cereolysin O (hemolysin I) (Ramarao and Sanchis 2013).

Gram-positive bacteria

Hemolysin I of Bacillus cereus (Q93LA9)


Streptolysin O, SLO or SpyM3, (transports NAD-glycohydrolase into the host cell) (Meehl and Caparon, 2004).  Injections into cells modulates cell metabolism which induces streptolysin synthesis and S. pyogenes growth (Baruch et al. 2014). This sulfhydryl-activated toxin causes cytolysis by forming pores in cholesterol containing host membranes. After binding to target membranes, the protein undergoes a major conformation change, leading to its insertion. The domino-like prepore-to-pore transition of Streptolysin O has been visualized (Ariyama 2022).


Gram-positive bacteria

Streptolysin O of Streptococcus pyogenes (P0C0I3)


Pneumolysin (PLS or PLY) or Intermedilysin (ILY), the shortest members of the CDC family (Gonzalez et al., 2008). It exhibits a broad range of conductances (El-Rachkidy et al., 2008) and localizes to the cell wall of S. pneumoniae (Price and Camilli, 2009). Binding of ILY to human CD59 initiates a series of conformational changes within the toxin that result in the conversion of the soluble monomer into an oligomeric membrane-embedded pore complex. The assembly intermediates increase the sensitivity of the host cell to lysis by its complement membrane attack complex, apparently by blocking the hCD59-binding site for complement proteins C8α and C9 (LaChapelle et al., 2009).  The herbal bioflavonoid, Apigenin, inhibits oligomerization of PLY and protects against pneumonia (Song et al. 2016).  Pneumolysin alters lysosomal integrity in epithelial cells, but not in macrophages, inducing lysosomal membrane permeabilization and release of lysosomal content (Malet et al. 2016). A four-step mechanism of membrane attachment and pore formation has been proosed (van Pee et al. 2016). Pneumolysin is both necessary and sufficient to promote inflammation, increasing shedding and causing transmission to others (Zafar et al. 2017). The release of pneumococcal autolysin, which promotes cell lysis and the release of pneumolysin, is inhibited by treatment with azithromycin and erythromycin, but recombinant autolysin restores the release of pneumolysin (Domon et al. 2018). Pneumolyin exhibits direct cardiotoxic and immunosuppressive activities, as well as indirect pro-inflammatory/pro-thrombotic activities (Anderson et al. 2018). The transmembrane beta-hairpins of the PLY pore are stable only for oligomers, forming a curtain-like membrane-spanning beta-sheet, and its hydrophilic inner face draws water into the protein-lipid interface, forcing lipids to recede (Vögele et al. 2019). Formation of pre-pore complexes of pneumolysin is accompanied by a decrease in short-range order of lipid molecules throughout vesicle bilayers (Faraj et al. 2020). Although pneumolysin-induced inflammation drives person-to-person transmission from the nasopharynx, the primary reservoir for pneumococcus, it also contributes to high mortality rates, creating a bottleneck that hampers widespread bacterial dissemination, thus acting as a double-edged sword (Badgujar et al. 2020). Serotype 1 ST306, a widespread pneumococcal clone, harbours a non-hemolytic variant of pneumolysin (Ply-NH). Crystal structural analyses of Ply-NH led to the identification of Y150H and T172I as key substitutions responsible for loss of its pore-forming activity. A novel inter-molecular cation-pi interaction governs formation of the transmembrane beta-hairpins (TMH) in the pore state of Ply, which can be applied to other CDCs. H150 in Ply-NH disrupts this interaction, while I172 provides structural rigidity to domain-3 through hydrophobic interactions, inhibiting TMH formation. Loss of pore-forming activity enables improved cellular invasion and autophagy evasion, promoting an atypical intracellular lifestyle for pneumococcus, a finding that was corroborated in in vivo infection models. Attenuation of inflammatory responses and tissue damage promoted tolerance of Ply-NH-expressing pneumococcus in the lower respiratory tract. Adoption of this altered lifestyle may be necessary for ST306 due to its limited nasopharyngeal carriage with Ply-NH, aided partly by loss of its pore forming ability, facilitating a benign association of SPN in an alternative, intracellular host niche (Badgujar et al. 2020). Apigenin protects mice from pneumococcal pneumonia by inhibiting the cytolytic activity of pneumolysin (Song et al. 2016). PLY can disrupt plasma membrane integrity, deregulating cellular homeostasis. At lytic concentrations, PLY causes cell death, but at sub-lytic concentrations, PLY triggers host cell survival pathways that cooperate to reseal the damaged plasma membrane and restore cell homeostasis (Pereira et al. 2022). While PLY is generally considered a pivotal factor promoting S. pneumoniae colonization and survival, it is also a powerful trigger of the innate and adaptive host immune response against bacterial infection. The dichotomy of PLY as both a key bacterial virulence factor and a trigger for host immune modulation allows the toxin to display both "Yin" and "Yang" properties during infection, promoting disease by membrane perforation and activating inflammatory pathways, while also mitigating damage by triggering host cell repair and initiating anti-inflammatory responses. Due to its cytolytic activity and diverse immunomodulatory properties, PLY is integral to every stage of S. pneumoniae pathogenesis and may tip the balance towards either the pathogen or the host depending on the context of infection (Pereira et al. 2022).

Gram-positive bacteria

Pneumolysin of Streptococcus pneumoniae (P0C2J9)

1.C.12.1.6Ivanolysin Gram-positive bacteria Ivanolysin of Listeria ivanovii (P31831)

Listeriolysin O, Listeriolysin-O, LLO, Hly, HlyA, Lis of 507 aas and 1 N-terminal TMS (Viala et al., 2008). CFTR transiently increases phagosomal chloride concentrations after infection, potentiating pore formation and vacuole lysis. Thus, Listeria exploits mechanisms of cellular ion homeostasis to escape the phagosome (Radtke et al., 2011).  LLO is an example of a large beta-barrel pore that exhibits plasticity, controlled by environmental cues like pH (Podobnik et al. 2015).  Pore formation is a multistep process involving the sequential formation of arcs, slits, small rings and larger rings before formation of transmembrane pores (Mulvihill et al. 2015).  LLO promotes nanoscale membrane reorganization (Sarangi et al. 2016). It alters lysosomal integrity in epithelial cells, but not in macrophages, inducing lysosomal membrane permeabilization and release of lysosomal content (Malet et al. 2016). LLO pore activity is active at acidic pH (<6), but not at neutral pH because pore-formation is controlled by rapid, irreversible denaturation of its structure at neutral pH at temperatures >30 degrees C. Denaturation is triggered at neutral pH by the premature unfolding of the domain 3 transmembrane beta-hairpins, structures that normally form the transmembrane beta-barrel. A triad of acidic residues within domain 3 functions as the pH sensor (Schuerch et al. 2005). Kisovec et al. 2017 have made a mutant variant with hemolytic activity that is pH-dependent. LLO does not form pores of regular shape or size, but rather forms membrane inserted arcs that propagate and damage lipid membranes as lineactants (Jiao et al. 2021). At low PFT concentrations, a regime of increased lipid diffusivity is attributed to lipid ejection events because of a preponderance of ring-like pore states (Ilangumaran Ponmalar et al. 2021). At higher protein concentrations in which membrane-inserted arc-like pores dominate, lipid ejection is less efficient and the ensuing crowding results in a lowering of lipid diffusion.


Listeriolysin O of Listeria monocytogenes (P13128)


Suilysin (SLY, a hemolysin) of 497 aas is a pore-forming cholesterol-dependent cytolysin of S. suis and a true virulence factor (Tenenbaum et al. 2016). It plays a role during the development of S. suis meningitis in pigs and humans, and is a potential vaccine candidate. Amentoflavone, a natural biflavonoid compound isolated from Chinese herbs is a potent antagonist of suilysin (SLY)-mediated hemolysis without interfering with its expression. Amentoflavone effectively inhibited SLY oligomerization, which is critical for its pore-forming activity. Treatment with amentoflavone reduced S. suis-induced cytotoxicity in macrophages, and S. suis-infected mice that received amentoflavone exhibited lower mortality and bacterial burden (Shen et al. 2018).

Gram-positive bacteria

Hemolysin of Streptococcus suis (O85102)


The cholesterol-dependent pore-forming cytoslysin, Pyolysin of 534 aas with one N-terminal TMS.  The pathology of Trueperella pyogenes and this pyolysin have been described and reviewed (Rzewuska et al. 2019). Liu et al. 2022 located and mutated two different highly conserved structural sites in the primary sequence of the protein that are critical for PLO structure and function.

Gram-positive bacteria

Pyolysin of Arcanobacterium pyogenes (Trueperella pyogenes) (O31241)


TC#NameOrganismal TypeExample

TC#NameOrganismal TypeExample
1.C.12.2.1FlavomodulinBacteriaFlavomodulin of Flavobacterium psychrophilum (A6GVU3)

Uncharacterized protein of 373 aas

UP of Prevotella micans


Tetanolysin O of 369 aas.  A three dimensional model of the toxin is availalbe (Skariyachan et al. 2012).

Tetanolysin O of Capnocytophaga canimorsus


CDC homologue of 489 aas

CDC homologue of Chryseobacterium indologenes


TC#NameOrganismal TypeExample
1.C.12.3.1Hypothetical Protein, HPBacteriaHP of Nostoc sp. PCC7120 (Q8YX86)

Cytolysin, a secreted calcineurin-like phosphatase of 361 aas

Cytolysin of Mesorhizobium loti


Cytolysin, a secreted calcineurin-like phosphatase of 458 aas

Cytolysin of Candidatus Liberibacter americanus


TC#NameOrganismal TypeExample