Cholesterol-dependent cytolysin
Encyclopedia
Cholesterol-Dependent Cytolysins (CDC) are a family of β-barrel
pore-forming exotoxin
s that are secreted by Gram-positive bacteria. CDC are secreted as water-soluble monomer
s of 50-70 kDa, that once bound to the target cell, will form a circular homo-oligomeric complex containing up to 50 monomers. Through multiple conformational changes, the β-barrel transmembrane structure (~250Ǻ in diameter depending on the toxin) is formed and inserted into the target cell membrane. The presence of cholesterol
in the target membrane is required for pore formation, though presence of cholesterol is not required by all CDC for binding. Intermedilysin (ILY) secreted by Streptococcus intermedius
will bind only to target membranes containing a specific protein receptor, but cholesterol is required by intermedilysin (ILY) for pore formation. The distribution of cholesterol in the membrane can affect toxin binding, though the exact molecular mechanism that cholesterol regulates the cytolytic activity of the CDC is not totally understood.
environment and what enters and leaves the cell is lost. The pore being ~250Ǻ in diameter is large enough to allow the loss of amino acids, nucleotides, small proteins as well as ions (Ca2+,Na+, K+, etc.). The loss of calcium in particular, which is involved in multiple molecular pathways will have a large impact on cell survival. The pore will also lead to an influx of water, which may lead to blebbing and cell death.
s. By targeting immune cells such as macrophage
s the bacteria will be protected against phagocytosis and destruction by respiratory burst
.
ize once bound to the target cell membrane forming a β-barrel structure which will be inserted into the target cell membrane.
The core section of amino acids, which are required for pore formation are more conserved between CDC, which allows all CDC to exhibit similar three-dimensional structures and function. The structurally conserved domain 4 of CDC contains four conserve loops L1-L3 and an undecapeptide region, which is believed to be involved in cholesterol dependent recognition. Single amino acid modifications in these loops prevented Perfingolysin O (PFO), which is a CDC secreted by Clostridium perfringens
from binding to cholesterol rich liposome
s.
that are located on both sides of the central β-sheet on domain 3 are each folded as three short α-helices
to minimize the exposure of hydrophobic residues. The α-helices are inserted into the target cell membrane bilayer and a conformational change takes place into amphipathic β-hairpins. A concerted mechanism of insertion is required so that the hydrophilic surfaces of the β-hairpins remain exposed to the aqueous medium, and not the hydrophobic membrane core. Six short α-helices in D3 unfold to form two transmembrane β-hairpin (TMH), TMH1 (red) and TMH2 (green).
CDC are sensitive to both oxygen and cholesterol. Toxins isolated-form culture supernatants were inactivated once exposed to oxygen after being pre-incubated with cholesterol. CDC are also pH-sensitive. A change of pH in a medium from 7.4 to 6.0 caused a conformational change in perfringolysin O, leading to the exposure of tryptophan
residues to the aqueous solvent and altering the minimum cholesterol threshold required for binding. Another CDC, listeriolysin O (LLO), which optimally functions at an acidic pH, will lose its function at a neutral pH. Listeriolysin O function can be restored if the cholesterol concentration within the target membrane is increased.
, and glycerol backbones closer to the membrane surface. Even with the 3-β-OH group near the membrane surface, it is not very exposed compared to the phospholipid head groups.
The availability of cholesterol at the membrane surface is dependent upon its interaction with other membrane components such as phospholipids and proteins; and the more cholesterol interacts with these components the less available it is to interact with extramembranous molecules. Some factors that affect cholesterols availability are size of the polar head groups and the ability of the phospholipid
to hydrogen bond
with the 3-β-OH group of cholesterol. Cholesterol associates with phospholipids, forming a stoichiometric complex, though, increasing the cholesterol beyond a certain point, free cholesterol will begin to precipitate out of the membrane. The binding and pore formation of CDC will occur when the concentration of cholesterol exceeds the association capacity of the phospholipids, allowing the excess cholesterol to associate with the toxin.
The presence of cholesterol aggregates in an aqueous solution were sufficient to initiate a conformation change and oligomerization of perfringolysin O (PFO), while no changes were seen by perfingolysin O with epicholesterol aggregates in solution. Epicholesterol is a sterol
that differs from cholesterol by the orientation of the 3-β-OH group, which is axial in epicholesterol and equatorial in cholesterol. Since the orientation of the hydroxyl group has such an effect on the bind/pore-formation of CDC, the equatorial conformation may be required for docking of the sterol to the binding pocket in domain 4, or to be properly exposed at the surface of lipid structures.
. Lipids having a conical molecular shape alter the energetic state of membrane cholesterol, augmenting the interaction of the sterol with the cholesterol-specific cytolysin
. Since high cholesterol concentrations are required for CDC binding/pore-formation, it was thought that CDC would associate with lipid raft
s. A later study showed that sphingomyelin
, a necessary component of lipid raft formation, inhibited rather than promoted the binding of perfringolysin O to the target membrane.
, which cleave the head groups of phospholipids increasing the exposure of cholesterol. This has been seen in two organisms, perfingolysin O (CDC) and α-toxin
secreted by clostridial myonecrosis. and listeriolysin O (CDC) and phospholipases C released by Listeria monocytogenes leading to the virulence of these bacteria.
Beta barrel
A beta barrel is a large beta-sheet that twists and coils to form a closed structure in which the first strand is hydrogen bonded to the last.Beta-strands in beta-barrels are typically arranged in an antiparallel fashion...
pore-forming exotoxin
Exotoxin
An exotoxin is a toxin excreted by a microorganism, like bacteria, fungi, algae, and protozoa. An exotoxin can cause damage to the host by destroying cells or disrupting normal cellular metabolism. They are highly potent and can cause major damage to the host...
s that are secreted by Gram-positive bacteria. CDC are secreted as water-soluble monomer
Monomer
A monomer is an atom or a small molecule that may bind chemically to other monomers to form a polymer; the term "monomeric protein" may also be used to describe one of the proteins making up a multiprotein complex...
s of 50-70 kDa, that once bound to the target cell, will form a circular homo-oligomeric complex containing up to 50 monomers. Through multiple conformational changes, the β-barrel transmembrane structure (~250Ǻ in diameter depending on the toxin) is formed and inserted into the target cell membrane. The presence of cholesterol
Cholesterol
Cholesterol is a complex isoprenoid. Specifically, it is a waxy steroid of fat that is produced in the liver or intestines. It is used to produce hormones and cell membranes and is transported in the blood plasma of all mammals. It is an essential structural component of mammalian cell membranes...
in the target membrane is required for pore formation, though presence of cholesterol is not required by all CDC for binding. Intermedilysin (ILY) secreted by Streptococcus intermedius
Streptococcus intermedius
Streptococcus intermedius is a commensal bacterium and a member of the Streptococcus anginosus group. The S. anginosus group, occasionally termed “Streptococcus milleri group” display hemolytic and serologic diversity, yet share core physiological traits. Despite being commensal organisms, members...
will bind only to target membranes containing a specific protein receptor, but cholesterol is required by intermedilysin (ILY) for pore formation. The distribution of cholesterol in the membrane can affect toxin binding, though the exact molecular mechanism that cholesterol regulates the cytolytic activity of the CDC is not totally understood.
Cyto-lethal effects
Once the pore is formed within the target cell membrane, the regulation of the intracellularIntracellular
Not to be confused with intercellular, meaning "between cells".In cell biology, molecular biology and related fields, the word intracellular means "inside the cell".It is used in contrast to extracellular...
environment and what enters and leaves the cell is lost. The pore being ~250Ǻ in diameter is large enough to allow the loss of amino acids, nucleotides, small proteins as well as ions (Ca2+,Na+, K+, etc.). The loss of calcium in particular, which is involved in multiple molecular pathways will have a large impact on cell survival. The pore will also lead to an influx of water, which may lead to blebbing and cell death.
Purpose
Bacteria invest energy into creating these toxins because they act as virulence factorVirulence factor
Virulence factors are molecules expressed and secreted by pathogens that enable them to achieve the following:* colonization of a niche in the host...
s. By targeting immune cells such as macrophage
Macrophage
Macrophages are cells produced by the differentiation of monocytes in tissues. Human macrophages are about in diameter. Monocytes and macrophages are phagocytes. Macrophages function in both non-specific defense as well as help initiate specific defense mechanisms of vertebrate animals...
s the bacteria will be protected against phagocytosis and destruction by respiratory burst
Respiratory burst
Respiratory burst is the rapid release of reactive oxygen species from different types of cells....
.
Structure
The 28 CDC family members are known to vary in amino acid identity from 28.1 – 99.6%, with amino acid sequences ranging from 471 to 665 amino acids. Within the primary structure of the proteins there is a low degree of conservation at the N-terminus, it is presumed because some CDC contain additional domains at this region and different species use different signal sequences for secretion. The CDC monomer consist of 4 structural domains, with domain 4 (D4) being involved with membrane binding. Multiple CDC monomers will oligomerOligomer
In chemistry, an oligomer is a molecule that consists of a few monomer units , in contrast to a polymer that, at least in principle, consists of an unlimited number of monomers. Dimers, trimers, and tetramers are oligomers. Many oils are oligomeric, such as liquid paraffin...
ize once bound to the target cell membrane forming a β-barrel structure which will be inserted into the target cell membrane.
The core section of amino acids, which are required for pore formation are more conserved between CDC, which allows all CDC to exhibit similar three-dimensional structures and function. The structurally conserved domain 4 of CDC contains four conserve loops L1-L3 and an undecapeptide region, which is believed to be involved in cholesterol dependent recognition. Single amino acid modifications in these loops prevented Perfingolysin O (PFO), which is a CDC secreted by Clostridium perfringens
Clostridium perfringens
Clostridium perfringens is a Gram-positive, rod-shaped, anaerobic, spore-forming bacterium of the genus Clostridium. C. perfringens is ever present in nature and can be found as a normal component of decaying vegetation, marine sediment, the intestinal tract of humans and other vertebrates,...
from binding to cholesterol rich liposome
Liposome
Liposomes are artificially prepared vesicles made of lipid bilayer. Liposomes can be filled with drugs, and used to deliver drugs for cancer and other diseases. Liposomes are composite structures made of phospholipids and may contain small amounts of other molecules...
s.
Assembly/Translocation
The mechanism of pore formation of perfingolysin O (PFO), which is secreted by Clostridium perfringens, begins with encountering and binding to cholesterol on the target membrane. The C-terminus of PFO domain 4 (D4) encounters the membrane first. The binding of D4 triggers a structural rearrangement in which the PFO monomers oligomerize forming the pre-pore complex. The binding of CDC to the target membrane is required for oligmerization. The oligomerization of the CDC requires the exposure of hidden polypeptide regions that are triggered by conformational changes induced by protein-lipid interactions or protein-protein interactions. The water-soluble form of the toxins is prevented from oligomerizing by having the access of one edge of a core β-sheet in the monomer blocked. To be specific, β5, a short polypeptide loop, hydrogen-bonds to β4, preventing β4 interaction with β1 on the adjacent monomer. The binding of D4 to the membrane surface triggers a conformational change in domain 3, which rotates β5 away from β4, exposing β4 allowing it to interact with the β1 strand of another PFO molecule, initiating oligomerization. Unlike most of the exposed surface residues of CDC that are not conserved the residues at the surface of the D4 tip, which are involved in membrane interactions, are highly conserved. Pore formation begins once two amphipathic transmembrane β-hairpins from ~35 PFO monomers are concertedly insert, which then create a large β-barrel that perforates the membrane. The toxin gets around the energy barrier of inserting the CDC into the membrane by the formation of the β-barrel, which will lower the energy requirements compared to what would be required for the insertion of single β-hairpins. In the water-soluble monomeric form of CDC, the transmembrane β-hairpinsBeta hairpin
The beta hairpin structural motif is the simplest protein motif involving two beta strands that look like a hairpin. The motif consists of two strands that are adjacent in primary structure oriented in an antiparallel arrangement and linked by a short loop of two to five amino acids...
that are located on both sides of the central β-sheet on domain 3 are each folded as three short α-helices
Alpha helix
A common motif in the secondary structure of proteins, the alpha helix is a right-handed coiled or spiral conformation, in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier...
to minimize the exposure of hydrophobic residues. The α-helices are inserted into the target cell membrane bilayer and a conformational change takes place into amphipathic β-hairpins. A concerted mechanism of insertion is required so that the hydrophilic surfaces of the β-hairpins remain exposed to the aqueous medium, and not the hydrophobic membrane core. Six short α-helices in D3 unfold to form two transmembrane β-hairpin (TMH), TMH1 (red) and TMH2 (green).
Specificity
The binding of CDC to its target membrane requires the recognition of cholesterol or in the case of intermedilysin (ILY), the recognition of CD59 membrane-anchored protein. The recognition of cholesterol provides specificity for eukaryotic cells and the specificity for the glycosylphosphatidylinositol-anchored protein CD59 provides specificity for human cells. Even though cholesterol is not required for intermedilysin (ILY) to bind to a target cell, the presence of cholesterol is required for pore formation by all CDC.CDC are sensitive to both oxygen and cholesterol. Toxins isolated-form culture supernatants were inactivated once exposed to oxygen after being pre-incubated with cholesterol. CDC are also pH-sensitive. A change of pH in a medium from 7.4 to 6.0 caused a conformational change in perfringolysin O, leading to the exposure of tryptophan
Tryptophan
Tryptophan is one of the 20 standard amino acids, as well as an essential amino acid in the human diet. It is encoded in the standard genetic code as the codon UGG...
residues to the aqueous solvent and altering the minimum cholesterol threshold required for binding. Another CDC, listeriolysin O (LLO), which optimally functions at an acidic pH, will lose its function at a neutral pH. Listeriolysin O function can be restored if the cholesterol concentration within the target membrane is increased.
Role of cholesterol
The presents of cholesterol in the membrane of the target cell is required for CDC pore formation. The arrangement of cholesterol molecules in the bilayer is important for successful binding. The non-polar hydrocarbon tail of cholesterol orients itself toward the polar center of the membrane lipid bilayer, while the 3-β-OH group is oriented closer to the ester bonds formed by the fatty acid chainsFatty acid
In chemistry, especially biochemistry, a fatty acid is a carboxylic acid with a long unbranched aliphatic tail , which is either saturated or unsaturated. Most naturally occurring fatty acids have a chain of an even number of carbon atoms, from 4 to 28. Fatty acids are usually derived from...
, and glycerol backbones closer to the membrane surface. Even with the 3-β-OH group near the membrane surface, it is not very exposed compared to the phospholipid head groups.
The availability of cholesterol at the membrane surface is dependent upon its interaction with other membrane components such as phospholipids and proteins; and the more cholesterol interacts with these components the less available it is to interact with extramembranous molecules. Some factors that affect cholesterols availability are size of the polar head groups and the ability of the phospholipid
Phospholipid
Phospholipids are a class of lipids that are a major component of all cell membranes as they can form lipid bilayers. Most phospholipids contain a diglyceride, a phosphate group, and a simple organic molecule such as choline; one exception to this rule is sphingomyelin, which is derived from...
to hydrogen bond
Hydrogen bond
A hydrogen bond is the attractive interaction of a hydrogen atom with an electronegative atom, such as nitrogen, oxygen or fluorine, that comes from another molecule or chemical group. The hydrogen must be covalently bonded to another electronegative atom to create the bond...
with the 3-β-OH group of cholesterol. Cholesterol associates with phospholipids, forming a stoichiometric complex, though, increasing the cholesterol beyond a certain point, free cholesterol will begin to precipitate out of the membrane. The binding and pore formation of CDC will occur when the concentration of cholesterol exceeds the association capacity of the phospholipids, allowing the excess cholesterol to associate with the toxin.
The presence of cholesterol aggregates in an aqueous solution were sufficient to initiate a conformation change and oligomerization of perfringolysin O (PFO), while no changes were seen by perfingolysin O with epicholesterol aggregates in solution. Epicholesterol is a sterol
Sterol
Sterols, also known as steroid alcohols, are a subgroup of the steroids and an important class of organic molecules. They occur naturally in plants, animals, and fungi, with the most familiar type of animal sterol being cholesterol...
that differs from cholesterol by the orientation of the 3-β-OH group, which is axial in epicholesterol and equatorial in cholesterol. Since the orientation of the hydroxyl group has such an effect on the bind/pore-formation of CDC, the equatorial conformation may be required for docking of the sterol to the binding pocket in domain 4, or to be properly exposed at the surface of lipid structures.
Effects of other membrane lipids
The phospholipid composition of a cell membrane affects the arrangement of cholesterol within the membrane and the ability for CDC to bind and initiate pore-formation. For example, perfringolysin O will preferentially bind to cholesterol-rich membranes composed mainly of phospholipids containing 18-carbon acyl chainsFatty acid
In chemistry, especially biochemistry, a fatty acid is a carboxylic acid with a long unbranched aliphatic tail , which is either saturated or unsaturated. Most naturally occurring fatty acids have a chain of an even number of carbon atoms, from 4 to 28. Fatty acids are usually derived from...
. Lipids having a conical molecular shape alter the energetic state of membrane cholesterol, augmenting the interaction of the sterol with the cholesterol-specific cytolysin
Cytolysin
Cytolysin refers to the substance or antibody elaborated by microorganisms, plants or animals that is specifically toxic to individual cells, in many cases causing their dissolution through lysis. Cytolysins that have a specific action for certain cells are named accordingly...
. Since high cholesterol concentrations are required for CDC binding/pore-formation, it was thought that CDC would associate with lipid raft
Lipid raft
The plasma membrane of cells is made of a combination of glycosphingolipids and protein receptors organized in glycolipoprotein microdomains termed lipid rafts...
s. A later study showed that sphingomyelin
Sphingomyelin
Sphingomyelin is a type of sphingolipid found in animal cell membranes, especially in the membranous myelin sheath that surrounds some nerve cell axons. It usually consists of phosphorylcholine and ceramide...
, a necessary component of lipid raft formation, inhibited rather than promoted the binding of perfringolysin O to the target membrane.
Possible Coordination With Other Toxins
It is possible that the exposure of cholesterol at the membrane surface might be facilitated by other membrane-damaging toxins secreted such as phospholipase CPhospholipase C
Phosphoinositide phospholipase C is a family of eukaryotic intracellular enzymes that play an important role in signal transduction processes. In general, this enzyme is denoted as Phospholipase C, although three other families of phospholipase C enzymes have been identified in bacteria and in...
, which cleave the head groups of phospholipids increasing the exposure of cholesterol. This has been seen in two organisms, perfingolysin O (CDC) and α-toxin
Clostridium perfringens alpha toxin
Clostridium perfringens alpha toxin is a toxin produced by the bacterium Clostridium perfringens and is responsible for gas gangrene and myonecrosis in infected tissues. The toxin also possesses hemolytic activity....
secreted by clostridial myonecrosis. and listeriolysin O (CDC) and phospholipases C released by Listeria monocytogenes leading to the virulence of these bacteria.