Pore-forming proteins (PFTs, also known as pore-forming toxins) are usually produced by bacteria, and include a number of protein exotoxins but may also be produced by other organisms such as apple snails that produce perivitellin-2 [1] [2] or earthworms, who produce lysenin. They are frequently cytotoxic (i.e., they kill cells), as they create unregulated pores in the membrane of targeted cells.
PFTs can be divided into two categories, depending on the alpha-helical or beta-barrel architecture of their transmembrane channel [3] that can consist either of
Other categories:
According to TCDB, there are following families of pore-forming toxins:
| Leukocidin | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Identifiers | |||||||||
| Symbol | Leukocidin | ||||||||
| Pfam | PF07968 | ||||||||
| InterPro | IPR001340 | ||||||||
| TCDB | 1.C.3 | ||||||||
| OPM superfamily | 35 | ||||||||
| OPM protein | 7ahl | ||||||||
| |||||||||
β-PFTs are so-named because of their structural characteristics: they are composed mostly of β-strand-based domains. They have divergent sequences, and are classified by Pfam into a number of families including Leukocidins, Etx-Mtx2, Toxin-10, and aegerolysin. X-ray crystallographic structures have revealed some commonalities: α-hemolysin [6] and Panton-Valentine leukocidin S [7] are structurally related. Similarly, aerolysin [8] and Clostridial Epsilon-toxin. [9] and Mtx2 are linked in the Etx/Mtx2 family. [10]
The ß-PFTs include a number of toxins of commercial interest for the control of pest insects. These toxins are potent but also highly specific to a limited range of target insects, making them safe biological control agents.
Insecticidal members of the Etx/Mtx2 family include Mtx2 [10] and Mtx3 [11] from Lysinibacillus sphaericus that can control mosquito vectors of human diseases and also Cry15, Cry23, Cry33, Cry38, Cry45, Cry51, Cry60, Cry64 and Cry74 from Bacillus thuringiensis [12] that control a range of insect pests that can cause great losses to agriculture.
Insecticidal toxins in the Toxin–10 family show an overall similarity to the aerolysin and Etx/Mtx2 toxin structures but differ in two notable features. While all of these toxins feature a head domain and a larger, extended beta-sheet tail domain, in the Toxin_10 family, the head is formed exclusively from the N-terminal region of the primary amino acid sequence whereas regions from throughout the protein sequence contribute to the head domain in Etx/Mtx2 toxins. In addition, the head domains of the Toxin_10 proteins show lectin-like features of carbohydrate binding domains. The only reported natural targets of Toxin_10 proteins are insects. With the exception of Cry36 [13] and Cry78, [12] the Toxin_10 toxins appear to act as two-part, binary toxins. The partner proteins in these combinations may belong to different structural groups, depending on the individual toxin: two Toxin_10 proteins (BinA and BinB) act together in the Bin mosquitocidal toxin of Lysinibacillus sphaericus; [14] the Toxin_10 Cry49 is co-dependent on the 3-domain toxin family member Cry48 for its activity against Culex mosquito larvae; [15] and the Bacillus thuringiensis Toxin_10 protein Cry35 interacts with the aegerolysin family Cry34 to kill Western Corn Rootworm. [16] This toxin pair has been included in insect resistant plants such as SmartStax corn.
β-PFTs are dimorphic proteins that exist as soluble monomers and then assemble to form multimeric assemblies that constitute the pore. Figure 1 shows the pore-form of α-Hemolysin, the first crystal structure of a β-PFT in its pore-form. 7 α-Hemolysin monomers come together to create the mushroom-shaped pore. The 'cap' of the mushroom sits on the surface of the cell, and the 'stalk' of the mushroom penetrates the cell membrane, rendering it permeable (see later). The 'stalk' is composed of a 14-strand β-barrel, with two strands donated from each monomer.
A structure of the Vibrio cholerae cytolysin [17] in the pore form is also heptameric; however, Staphylococcus aureus gamma-hemolysin [18] reveals an octomeric pore, consequently with a 16-strand 'stalk'.
The Panton-Valentine leucocidin S structure [19] shows a highly related structure, but in its soluble monomeric state. This shows that the strands involved in forming the 'stalk' are in a very different conformation – shown in Fig 2.
Structural comparison of pore-form α-Hemolysin (pink/red) and soluble-form PVL (pale green/green). It is postulated that the green section in PVL 'flips out' to the 'red' conformation as seen in α-Haemolysin. (PDB: 7AHL, 1T5R) β-PFTs are dimorphic proteins that exist as soluble monomers and then assemble to form multimeric assemblies that constitute the pore. Figure 1 shows the pore-form of α-Hemolysin, the first crystal structure of a β-PFT in its pore-form. 7 α-Hemolysin monomers come together to create the mushroom-shaped pore. The 'cap' of the mushroom sits on the surface of the cell, and the 'stalk' of the mushroom penetrates the cell membrane, rendering it permeable (see later). The 'stalk' is composed of a 14-strand β-barrel, with two strands donated from each monomer. A structure of the Vibrio cholerae cytolysin PDB:3O44 [20] in the pore form is also heptameric; however, Staphylococcus aureus gamma-hemolysin (PDB:3B07) [21] reveals an octomeric pore, consequently with a 16-strand 'stalk'. The Panton-Valentine leucocidin S structure (PDB: 1T5R) [7] shows a highly related structure, but in its soluble monomeric state. This shows that the strands involved in forming the 'stalk' are in a very different conformation – shown in Fig 2. While the Bin toxin of Lysinibacillus sphaericus is able to form pores in artificial membranes [22] and mosquito cells in culture, [23] it also causes a series of other cellular changes including the uptake of toxin in recycling endosomes and the production of large, autophagic vesicles [24] and the ultimate cause of cell death may be apoptotic. [25] Similar effects on cell biology are also seen with other Toxin_10 activities [26] [27] but the roles of these events in toxicity remain to be established.
The transition between soluble monomer and membrane-associated protomer to oligomer is not a trivial one: It is believed that β-PFTs, follow as similar assembly pathway as the CDCs (see Cholesterol-dependent cytolysins later), in that they must first assemble on the cell-surface (in a receptor-mediated fashion in some cases) in a pre-pore state. Following this, the large-scale conformational change occurs in which the membrane spanning section is formed and inserted into the membrane. The portion entering the membrane, referred to as the head, is usually apolar and hydrophobic, this produces an energetically favorable insertion of the pore-forming toxin. [3]
Some β-PFTs such as clostridial ε-toxin and Clostridium perfringens enterotoxin (CPE) bind to the cell membrane via specific receptors – possibly certain claudins for CPE, [28] possibly GPI anchors or other sugars for ε-toxin – these receptors help raise the local concentration of the toxins, allowing oligomerisation and pore formation.
The BinB Toxin_10 component of the Lysinibacillus sphaericus Bin toxin specifically recognises a GPI anchored alpha glycosidase in the midgut of Culex [29] and Anopheles mosquitoes but not the related protein found in Aedes mosquitoes, [30] hence conferring specificity on the toxin.
When the pore is formed, the tight regulation of what can and cannot enter/leave a cell is disrupted. Ions and small molecules, such as amino acids and nucleotides within the cell, flow out, and water from the surrounding tissue enters. The loss of important small molecules to the cell can disrupt protein synthesis and other crucial cellular reactions. The loss of ions, especially calcium, can cause cell signaling pathways to be spuriously activated or deactivated. The uncontrolled entry of water into a cell can cause the cell to swell up uncontrollably: this causes a process called blebbing, wherein large parts of the cell membrane are distorted and give way under the mounting internal pressure. In the end, this can cause the cell to burst. In particular, nuclear - free erythrocytes under the influence of alpha-staphylotoxin undergo hemolysis with the loss of a large protein hemoglobin.
There are many different types of binary toxins. The term binary toxin simply implies a two part toxin where both components are necessary for toxic activity. Several β-PFTs form binary toxins.
As discussed above, the majority of the Toxin_10 family proteins act as part of binary toxins with partner proteins that may belong to the Toxin_10 or other structural families. The interplay of the individual components has not been well studied to date. Other beta sheet toxins of commercial importance are also binary. These include the Cry23/Cry37 toxin from Bacillus thuringiensis. [31] These toxins have some structural similarity to the Cry34/Cry35 binary toxin but neither component shows a match to established Pfam families and the features of the larger Cry23 protein have more in common with the Etx/Mtx2 family than the Toxin_10 family to which Cry35 belongs.
Some binary toxins are composed of an enzymatic component and a component that is involved in membrane interactions and entry of the enzymatic component into the cell. The membrane interacting component may have structural domains that are rich in beta sheets. Binary toxins, such as anthrax lethal and edema toxins (Main article: Anthrax toxin), C. perfringens iota toxin and C. difficile cyto-lethal toxins consist of two components (hence binary):
In these enzymatic binary toxins, the B component facilitates the entry of the enzymatic 'payload' (A subunit) into the target cell, by forming homooligomeric pores, as shown above for βPFTs. The A component then enters the cytosol and inhibits normal cell functions by one of the following means:
ADP-ribosylation is a common enzymatic method used by different bacterial toxins from various species. Toxins such as C. perfringens iota toxin and C. botulinum C2 toxin, attach a ribosyl-ADP moiety to surface arginine residue 177 of G-actin. This prevents G-actin assembling to form F-actin, and, thus, the cytoskeleton breaks down, resulting in cell death. Insecticidal members of the ADP-ribosyltransferase family of toxins include the Mtx1 toxin of Lysinibacillus sphaericus [32] and the Vip1/Vip2 toxin of Bacillus thuringiensis and some members of the toxin complex (Tc) toxins from gram negative bacteria such as Photorhabdus and Xenorhabdus species. The beta sheet-rich regions of the Mtx1 protein are lectin-like sequences that may be involved in glycolipid interactions. [33]
The A component of anthrax toxin lethal toxin is zinc-metalloprotease, which shows specificity for a conserved family of mitogen-activated protein kinases. The loss of these proteins results in a breakdown of cell signaling, which, in turn, renders the cell insensitive to outside stimuli – therefore no immune response is triggered.
Anthrax toxin edema toxin triggers a calcium ion influx into the target cell. This subsequently elevates intracellular cAMP levels. This can profoundly alter any sort of immune response, by inhibiting leucocyte proliferation, phagocytosis, and proinflammatory cytokine release.
CDCs, such as pneumolysin, from S. pneumoniae , form pores as large as 260Å (26 nm), containing between 30 and 44 monomer units. [36] Electron microscopy studies of pneumolysin show that it assembles into large multimeric peripheral membrane complexes before undergoing a conformational change in which a group of α-helices in each monomer change into extended, amphipathic β-hairpins that span the membrane, in a manner reminiscent of α-haemolysin, albeit on a much larger scale (Fig 3). CDCs are homologous to the MACPF family of pore-forming toxins, and it is suggested that both families use a common mechanism (Fig 4). [35] Eukaryote MACPF proteins function in immune defence and are found in proteins such as perforin and complement C9 [37] though perivitellin-2 is a MACPF attached to a delivery lectin that has enterotoxic and neurotoxic properties toward mice. [1] [2] [38]
A family of highly conserved cholesterol-dependent cytolysins, closely related to perfringolysin from Clostridium perfringens are produced by bacteria from across the order Bacillales and include anthrolysin, alveolysin and sphaericolysin. [29] Sphaericolysin has been shown to exhibit toxicity to a limited range of insects injected with the purified protein. [39]
Bacteria may invest much time and energy in making these toxins: CPE can account for up to 15% of the dry mass of C. perfringens at the time of sporulation. [ citation needed ] The purpose of toxins is thought to be one of the following:
An exotoxin is a toxin secreted by bacteria. 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. Exotoxins may be secreted, or, similar to endotoxins, may be released during lysis of the cell. Gram negative pathogens may secrete outer membrane vesicles containing lipopolysaccharide endotoxin and some virulence proteins in the bounding membrane along with some other toxins as intra-vesicular contents, thus adding a previously unforeseen dimension to the well-known eukaryote process of membrane vesicle trafficking, which is quite active at the host–pathogen interface.
An enterotoxin is a protein exotoxin released by a microorganism that targets the intestines. They can be chromosomally or plasmid encoded. They are heat labile (>60⁰), of low molecular weight and water-soluble. Enterotoxins are frequently cytotoxic and kill cells by altering the apical membrane permeability of the mucosal (epithelial) cells of the intestinal wall. They are mostly pore-forming toxins, secreted by bacteria, that assemble to form pores in cell membranes. This causes the cells to die.
A latrotoxin is a high-molecular mass neurotoxin found in the venom of spiders of the genus Latrodectus as well as at least one species of another genus in the same family, Steatoda nobilis. Latrotoxins are the main active components of the venom and are responsible for the symptoms of latrodectism.
Anthrax toxin is a three-protein exotoxin secreted by virulent strains of the bacterium, Bacillus anthracis—the causative agent of anthrax. The toxin was first discovered by Harry Smith in 1954. Anthrax toxin is composed of a cell-binding protein, known as protective antigen (PA), and two enzyme components, called edema factor (EF) and lethal factor (LF). These three protein components act together to impart their physiological effects. Assembled complexes containing the toxin components are endocytosed. In the endosome, the enzymatic components of the toxin translocate into the cytoplasm of a target cell. Once in the cytosol, the enzymatic components of the toxin disrupts various immune cell functions, namely cellular signaling and cell migration. The toxin may even induce cell lysis, as is observed for macrophage cells. Anthrax toxin allows the bacteria to evade the immune system, proliferate, and ultimately kill the host animal. Research on anthrax toxin also provides insight into the generation of macromolecular assemblies, and on protein translocation, pore formation, endocytosis, and other biochemical processes.
Tetanolysin is a toxin produced by Clostridium tetani bacteria. Its function is unknown, but it is believed to contribute to the pathogenesis of tetanus. The other C. tetani toxin, tetanospasmin, is more definitively linked to tetanus. It is sensitive to oxygen.
Cytolysin refers to the substance secreted 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. For instance, the cytolysins responsible for the destruction of red blood cells, thereby liberating hemoglobins, are named hemolysins, and so on. Cytolysins may be involved in immunity as well as in venoms.
Hemolysins or haemolysins are lipids and proteins that cause lysis of red blood cells by disrupting the cell membrane. Although the lytic activity of some microbe-derived hemolysins on red blood cells may be of great importance for nutrient acquisition, many hemolysins produced by pathogens do not cause significant destruction of red blood cells during infection. However, hemolysins are often capable of lysing red blood cells in vitro.
Lysinibacillus sphaericus is a Gram-positive, mesophilic, rod-shaped bacterium commonly found on soil. It can form resistant endospores that are tolerant to high temperatures, chemicals and ultraviolet light and can remain viable for long periods of time. It is of particular interest to the World Health Organization due to the larvicide effect of some strains against two mosquito genera, more effective than Bacillus thuringiensis, frequently used as a biological pest control. L. sphaericus cells in a vegetative state are also effective against Aedes aegypti larvae, an important vector of yellow fever and dengue viruses.
The Membrane Attack Complex/Perforin (MACPF) superfamily, sometimes referred to as the MACPF/CDC superfamily, is named after a domain that is common to the membrane attack complex (MAC) proteins of the complement system and perforin (PF). Members of this protein family are pore-forming toxins (PFTs). In eukaryotes, MACPF proteins play a role in immunity and development.
Alpha-toxin, also known as alpha-hemolysin (Hla), is the major cytotoxic agent released by bacterium Staphylococcus aureus and the first identified member of the pore forming beta-barrel toxin family. This toxin consists mostly of beta-sheets (68%) with only about 10% alpha-helices. The hly gene on the S. aureus chromosome encodes the 293 residue protein monomer, which forms heptameric units on the cellular membrane to form a complete beta-barrel pore. This structure allows the toxin to perform its major function, development of pores in the cellular membrane, eventually causing cell death.
Delta endotoxins (δ-endotoxins) are a family of pore-forming toxins produced by Bacillus thuringiensis species of bacteria. They are useful for their insecticidal action and are the primary toxin produced by the genetically modified (GM) Bt maize/corn and other GM crops. During spore formation the bacteria produce crystals of such proteins that are also known as parasporal bodies, next to the endospores; as a result some members are known as a parasporin. The Cyt (cytolytic) toxin group is another group of delta-endotoxins formed in the cytoplasm. VIP toxins are formed at other stages of the life cycle.
Streptolysins are two hemolytic exotoxins from Streptococcus pyogenes. Types include streptolysin O, which is oxygen-labile, and streptolysin S, which is oxygen-stable.
The AB toxins are two-component protein complexes secreted by a number of pathogenic bacteria, though there is a pore-forming AB toxin found in the eggs of a snail. They can be classified as Type III toxins because they interfere with internal cell function. They are named AB toxins due to their components: the "A" component is usually the "active" portion, and the "B" component is usually the "binding" portion. The "A" subunit possesses enzyme activity, and is transferred to the host cell following a conformational change in the membrane-bound transport "B" subunit. These proteins consist of two independent polypeptides, which correspond to the A/B subunit moieties. The enzyme component (A) enters the cell through endosomes produced by the oligomeric binding/translocation protein (B), and prevents actin polymerisation through ADP-ribosylation of monomeric G-actin.
The RTX toxin superfamily is a group of cytolysins and cytotoxins produced by bacteria. There are over 1000 known members with a variety of functions. The RTX family is defined by two common features: characteristic repeats in the toxin protein sequences, and extracellular secretion by the type I secretion systems (T1SS). The name RTX refers to the glycine and aspartate-rich repeats located at the C-terminus of the toxin proteins, which facilitate export by a dedicated T1SS encoded within the rtx operon.
The thiol-activated Cholesterol-dependent Cytolysin(CDC) family is a member of the MACPF superfamily. Cholesterol dependent cytolysins are a family of β-barrel pore-forming exotoxins that are secreted by gram-positive bacteria. CDCs are secreted as water-soluble monomers of 50-70 kDa, that when bound to the target cell, form a circular homo-oligomeric complex containing as many as 40 monomers. Through multiple conformational changes, the β-barrel transmembrane structure is formed and inserted into the target cell membrane. The presence of cholesterol in the target membrane is required for pore formation, though the presence of cholesterol is not required by all CDCs for binding. For example, intermedilysin secreted by Streptococcus intermedius will bind only to target membranes containing a specific protein receptor, independent of the presence of cholesterol, but cholesterol is required by intermedilysin for pore formation. While the lipid environment of cholesterol in the membrane can affect toxin binding, the exact molecular mechanism that cholesterol regulates the cytolytic activity of the CDC is not fully understood.
Cry6Aa is a toxic crystal protein generated by the bacterial family Bacillus thuringiensis during sporulation. This protein is a member of the alpha pore forming toxins family, which gives it insecticidal qualities advantageous in agricultural pest control. Each Cry protein has some level of target specificity; Cry6Aa has specific toxic action against coleopteran insects and nematodes. The corresponding B. thuringiensis gene, cry6aa, is located on bacterial plasmids. Along with several other Cry protein genes, cry6aa can be genetically recombined in Bt corn and Bt cotton so the plants produce specific toxins. Insects are developing resistance to the most commonly inserted proteins like Cry1Ac. Since Cry6Aa proteins function differently than other Cry proteins, they are combined with other proteins to decrease the development of pest resistance. Recent studies suggest this protein functions better in combination with other virulence factors such as other Cry proteins and metalloproteinases.>
Cry34Ab1 is one member of a binary Bacillus thuringiensis (Bt) crystal protein set isolated from Bt strain PS149B1. The protein exists as a 14 kDa aegerolysin that, in presence of Cry35Ab1, exhibits insecticidal activity towards Western Corn Rootworm. The protein has been transformed into maize plants under the commercialized events 4114 (DP-ØØ4114-3) by Pioneer Hi-Bred and 59122 (DAS-59122-7) by Dow AgroSciences. These events have, in turn, been bred into multiple trait stacks in additional products.
Kausik Chattopadhyay is an Indian structural biologist, protein biologist, and a professor at the Department of Biological Sciences. He was the Dean of R&D at the Indian Institute of Science Education and Research, Mohali until May 2021. He is known for his studies on the Pore-forming protein toxins and T-cell costimulatory molecules. The Department of Biotechnology of the Government of India awarded him the National Bioscience Award for Career Development, one of the highest Indian science awards, for his contributions to biosciences, in 2014.
Lysenin is a pore-forming toxin (PFT) present in the coelomic fluid of the earthworm Eisenia fetida. Pore-forming toxins are a group of proteins that act as virulence factors of several pathogenic bacteria. Lysenin proteins are chiefly involved in the defense against cellular pathogens. Following the general mechanism of action of PFTs lysenin is segregated as a soluble monomer that binds specifically to a membrane receptor, sphingomyelin in the case of lysenin. After attaching to the membrane, the oligomerization begins, resulting in a nonamer on top of membrane, known as a prepore. After a conformational change, which could be triggered by a decrease of pH, the oligomer is inserted into the membrane in the so-called pore state.
Cytotoxin-K (CytK) is a protein toxin produced by the gram-positive bacteria Bacillus cereus. It was first discovered in a certain Bacillus cereus strain which was isolated from a food poisoning epidemic that occurred in a French nursing home in 1998. There were six cases of bloody diarrhea, three of which were fatal. None of the known enterotoxins from B. cereus could be detected at this time. Later, this B. cereus strain and its relatives were classified as a brand-new species called Bacillus cytotoxicus, which is the thermo-tolerant member of the B. cereus genus. The cytotoxin-K gene is present in approximately 50% of Bacillus cereus isolates, and its expression is regulated by several factors, including temperature and nutrient availability.
