What Makes Some Bacterial Toxins So Dangerous? the Most Poisonous Substances Known

Features Poisons and antidotes What makes some bacterial toxins so dangerous? The most poisonous substances known David Moss, Ajit Basak We are used to thinking of proteins as beneficial, so it is surprising to realize that the most toxic sub- and Claire Naylor stances known to man are also protein molecules. These are the bacterial exotoxins, proteins secreted (Birkbeck College, London) by pathogenic bacteria. Toxicity is measured by the median lethal dose (LD50). An LD50 value is defined as the mass of toxin per kg of body weight required to wipe out half of an animal population. Whereas Downloaded from http://portlandpress.com/biochemist/article-pdf/32/4/4/5064/bio032040004.pdf by guest on 02 October 2021 classic poisons, such as potassium cyanide or arsenic trioxide, have LD50 values in the range 5–15 mg/ kg, the causative agent of botulism, botulinum toxin, has an LD50 in the range 1–3 ng/kg, a million times more toxic! Highly pathogenic toxins have a catalytic ing. Treatment has to be repeated every few months. and a binding domain or subunit In the early 19th Century, it was thought that cholera was caused by ‘bad air’. It was then discovered that Nearly all of the most potent toxins have two compo- cholera was the result of ingesting faecal-contaminated nents, A and B, that are either separate polypeptide water and we now know that the pathogenic agent is an chains or are separate domains. Component A is an 87 kDa toxin secreted by the bacterium Vibrio cholerae3,4. enzyme which modifies an important protein inside the This bacterium is the cause of many deaths in the de- host cell, whereas B is a protein that enables the toxin veloping world in the aftermath of flooding. When -in to bind to specific receptors that are abundant on target gested, the bacterium adheres to the intestinal mucosa. cell-surface membranes and enables A to be taken up into the cell. Enzymes can act at very low concentrations, and the hallmark of the most potent toxins is the posses- sion of enzymes that target vital cellular processes1. Botulinum toxin provides an example. It is an AB toxin secreted by Clostridium botulinum, an anaerobic bacterium that is sometimes found in improperly canned food and was originally known as ‘sausage poison’ (Latin botulus=sausage)2. The intoxication, called botulism, arises from the consumption of pre-formed botulinum toxin. Each molecule of this toxin is synthesized as a 150 kDa single polypeptide chain which is cleaved into a heavy polypeptide chain (100 kDa) and a light chain (50 kDa) that are joined by a disulfide bridge. The heavy chain is responsible for binding specific gangliosides and protein receptors on the plasma membrane of neurons (nerve cells) and enables the light chain, which is a pro- tease, to enter the cell by endocytosis. This enzyme in- activates a SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) protein that is responsible for the fusion of acetylcholine-containing vesicles with the cell membrane. This prevents the re- Figure 1. A space-filling model of cholera toxin from Vibrio lease of the acetylcholine, a neurotransmitter, into the cholerae viewed along the plane of the membrane. The A synapse (the junction between neurons), preventing the subunit is coloured red and is the catalytic subunit, part of transmission of nerve pulses. Flaccid paralysis of muscles which escapes into the cytoplasm of the host cell. Below occurs and, once respiratory muscles are involved, death this subunit is the B subunit that has 5-fold symmetry and Key words: AB toxin, can result. In medical applications, under the name of interacts with ganglioside receptors on the host cell surface, Clostridium, enterotoxin, BOTOX, this toxin is used for treating wrinkles and enabling the A subunit to be taken up into the host cell by exotoxin, superantigen other conditions such as excessive sweating and blink- receptor-mediated endocytosis. 4 August 2010 © 2010 The Biochemical Society Poisons and antidotes Features The most poisonous substances known The five identical B subunits (103 residues each) form a ring with 5-fold symmetry (see Figure 1) that binds to GM1 gangliosides of the epithelial cells of the gut. The A subunit (240 residues) is then taken into the host cell Downloaded from http://portlandpress.com/biochemist/article-pdf/32/4/4/5064/bio032040004.pdf by guest on 02 October 2021 by receptor-mediated endocytosis. Part of the A subunit ends up in the cytoplasm of the cell where it catalyses the attachment of an ADP-ribose moiety to a G-protein that in turn leads to the activation of adenylate cyclase, causing excessive production of intracellular cAMP. This causes huge quantities of water and electrolytes to move into the lumen of the gut, causing severe diarrhoea and possible death from dehydration. Another well-studied AB toxin causes diphtheria5. This toxin is secreted by the bacteriumCorynebacterium Figure 2. The gas-gangrene toxin from Clostridium perfrin- diphtheriae, when it is infected by the bacteriophage co- gens, hydrolysing a phospholipid from a cell membrane. The rynephage β. The toxin is a phage-encoded monomeric α-helical N-terminal domain on the left is a zinc-containing protein (535 residues) whose B fragment binds to a spe- phospholipase and the C-terminal domain on the right ena- cific receptor protein on the cell surface, leading to the bles the toxin to attach itself to cell membranes. Hydrophobic toxin being taken up into clathrin-coated vesicles. The tryptophan and phenylalanine residues can be seen penetrat- A fragment escapes from the vesicle into the cytoplasm ing the membrane on the extreme left and right of the toxin. where it causes the ADP-ribosylation of the eukaryo- tic elongation factor eEF2. This factor is responsible for elongation of the nascent polypeptide chain during protein synthesis and this is inhibited by the covalent modification. Enzymes can work effectively at very low concentrations and just one molecule of diphtheria toxin can ADP-ribosylate all of the eEF2 molecules in a cell, stopping protein production and leading to cell death. Other toxins do not enter the host cell At the siege of Châlus in 1199, King Richard I received a wound to his shoulder from a crossbow bolt. When the wound became gangrenous, he immediately knew that he only had a few days to live. More recently, gas-gangrene has been responsible for many thousands of deaths in tsunamis. The main causative agent of gas-gangrene is a toxin (Figure 2) produced by the anaerobic bacterium Clostridium perfringens. This bacterium is said to produce more toxins than any other. Like all bacteria, it has a number of different strains and not all strains produce Figure 3. The α-haemolysin from Staphylococcus aureus which the same toxins. It can also survive as spores, and, when forms a heptameric transmembrane pore. This mushroom- the right anaerobic conditions arise, it can secrete potent shaped molecule has 7-fold symmetry. A channel of length exotoxins. Gas-gangrene toxin6 (also known as α-toxin) 100 Å (1 Å=0.1 nm) runs through the stem of the heptamer is the main causative agent of gas-gangrene, a disease which penetrates the host cell membrane allowing the efflux that associates anaerobic conditions in wounds with of ions and small molecules. August 2010 © 2010 The Biochemical Society 5 Features Poisons and antidotes Table 1. Some important toxins and their effects in animal hosts. Disease Bacterium Toxin LD50 (μg/kg) Effect Botulism Clostridium botulinum Botulinum toxin 0.001–0.003 Blocks the release of acetylcholine from storage vesicles, causing flaccid muscle paralysis Tetanus Clostridium tetani Tetanus toxin 0.001 Blocks the release of acetylcholine from storage vesicles, causing spastic muscle paralysis Anthrax Bacillus anthracis Oedema toxin and lethal toxin Inhalation of 8000–50000 Invades and kills macrophages, spores leading to internal bleeding Downloaded from http://portlandpress.com/biochemist/article-pdf/32/4/4/5064/bio032040004.pdf by guest on 02 October 2021 and septic shock Cholera Vibrio cholerae Cholera toxin 250 Increases cAMP in epithelial cells of gut, causing ion imbalance Gas-gangrene Clostridium perfringens α-Toxin 3–5 Hydrolyses phospholipids of host cells, causing cell death Food poisoning Clostridium perfringens Enterotoxin 80–100 Disrupts epithelial cells of the gut, causing diarrhoea Diphtheria Corynebacterium diphtheriae Diphtheria toxin 0.1 Inhibits protein synthesis in the ribosome Enterotoxaemia Clostridium perfringens ε-Toxin 0.1 Interferes with neurological functions in sheep and goats Toxic-shock Syndrome Staphylococcus aureus Toxic-shock syndrome toxin 20–50 Over-activates T-cells in the immune system vascular damage, surgical interventions and poor cir- and the gut. The enterotoxin binds to claudin to form culation in the extremities of limbs. The toxin molecule a large complex that disrupts the control of paracellular has two domains. The C-terminal domain is responsible transport and may disturb cell signalling in the epithelial for binding the membrane surface of the host cell and cells. These perturbations lead to the efflux of water into the N-terminal domain cleaves the phospholipids (phos- the gut, resulting in diarrhoea. phatidylcholine and sphingomyelin) of the membrane to Some toxins do not have enzymatic activity. Among yield diacylglycerol which acts as a second messenger, these are the haemolysins and leucocidins which, as their interfering with signalling pathways, leading to local cell names suggest, cause lysis of erythrocytes or leucocytes death and systemic effects from vasoconstriction and which are phagocytic cells of the immune systems8. The muscle spasm to shock and organ failure. Once the toxin bacterium Streptococcus pyogenes, the causative agent enters the circulation, it rapidly kills muscle cells and the of scarlet fever, produces a haemolysin, streptolysin-O, black putrefying flesh emits hydrogen, hence the name which forms pores in erythrocytes leading to the release gas-gangrene.

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