Pore forming toxins

Pore forming toxins (PFTs) are protein toxins, typically, (but not exclusively) produced by bacteria, such as C.perfringens and S.aureus. They are frequently cytotoxic (i.e., they kill cells) as they create unregulated pores in the membrane of targeted cells.

Types of PFTs
PFTs can be divided into the following subcategories:
 * Beta-pore forming toxins
 * eg. α-Haemolysin (Fig 1).
 * Binary toxins
 * eg. Anthrax toxin
 * Cholesterol-dependent cytolysins (CDCs)
 * eg. Pneumolysin
 * Small pore-forming toxins
 * eg. Gramicidin A

Beta-pore forming toxins
β-PFTs are so-named because of their structural characteristics: they are composed mostly of β-strand-based domains. Whilst they frequently have divergent sequences, X-ray crystallographic structures have revealed some commonalities: α-haemolysin and Panton-Valentine leukocidin S are structurally related, as are aerolysin and Clostridial Epsilon-toxin.

Mode of action
β-PFTs are dimorphic proteins that exist as soluble monomers and then assemble to form multimeric assemblies that constitute the pore. Fig 1 shows the pore-form of α-Haemolysin, the only crystal structure of a β-PFT in its pore-form to-date. 7 α-Haemolysin 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. The Panton-Valentine Leucocidin S structure (PDB 1T5R) 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.

Assembly
The transition between soluble monomer and membrane associated heptamer is not a trivial one: it is believed that β-PFTs, follow as similar assembly pathway as the CDCs (see Cholesterol-dependant 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. I.E, they oligomerise, but do not penetrate the membrane. Following this, the large-scale conformational change occurs in which the membrane spanning section is formed and inserted into the membrane.

Specificity
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, 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 Cyto-lethal effects of the pore
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 signalling pathways to be spuriously activated or deactivated. The uncontrolled entry of water into a cell can cause the cell to swell up uncontrollably: initially, this causes a process called blebbing, where large parts of the cell membrane are distorted and come away under the mounting internal pressure. Ultimately this can cause the cell to burst.

Binary toxins
''See the main article for more information on Anthrax toxins.

Binary toxins, such as Anthrax lethal & edema toxins, C.perfringens Iota toxin and C.difficile cyto-lethal toxins consist of two components (hence binary):
 * an emzymatic component - A
 * a membrane-altering component - B

The B component facilitates the entry of the enzymatic 'payload' into the target cell, by forming homomeptameric pores, as shown above for βPFTs. The A component then enters the cytosol and inhibits normal cell functions by one of the following means:

Mono-ADP-Ribosylation of G-actin
ADP-Ribosylation is a common enzymatic methods used by various bacterial toxins from various species. These toxins (including C.perfringens Iota toxin & ''C.Botulinum C2 toxin) attach a ribosyl-ADP moiety to surface Arginine residue 117 of G-actin. This prevents G-actin assembling to form F-actin, and thus the cytoskeleton breaks down, resulting in cell death.

Proteolysis of Mitogen-activated protein kinase kinases (MAPKK)
The A component of Anthrax_toxin lethal toxin is zinc-metalloprotease which shows specificity for a conserved family of Mitogen-activated protein kinase kinases. The loss of these proteins results in a breakdown of cell signalling which in turn renders the cell insensitive to outside simuli - therefore no immune response is triggered.

Increasing intracellular levels of cAMP
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.

Cholesterol-dependant cytolysins
CDCs, such as pneumolysin, from S.pneumoniae, form pores as large as 260Å (26nm), containing between 30 and 44 monomer units. 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 utilise a common mechanism (Fig 4). Eukaryote MACPF proteins function in immune defence and are found in proteins such as perforin and complement C9.



Why bother?
Q. Bacteria can invest an awful lot of 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, so why bother?
 * A(1). Food. After the target cell has ruptured and released its contents, the bacteria can scavenge the remains for nutrients.
 * A(2). Environment. The mammalian immune response helps create the anaerobic environment that anaerobic bacteria require.