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 protease, 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.

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.

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. Even today, amputation is the only effec- of haemoglobin into the serum. The most intensely tive life-saving treatment. studied channel-forming toxin is α-haemolysin which Not all clostridial toxins are as toxic as gas-gangrene. is a 243-residue monomer secreted by Staphylococcus Most of us will have had an unpleasant encounter with aureus. Seven monomers form a non-specific hepta- Clostridium perfringens enterotoxin7 which is responsi- meric pore (Figure 3) that makes a channel through the ble for over 20% of cases of food poisoning and is cur- membrane of the host cell, leading to osmotic imbalance rently being studied in our laboratory. The epithelial cells and cell death. which line our gut are separated by tight junctions that Some toxins specifically target domestic animals. are home to membrane proteins called claudins. These Young sheep and goats are susceptible to enterotox‑ proteins have specific extracellular loops that only allow aemia that results in neurological disorders, pulpy the paracellular (between cells) transport of certain small kidney disease and rapid death. The causative agent is ions and small molecules between the circulatory system produced by Clostridium perfringens and is known as

ε-toxin9. The bacterium first secretes a non-toxic prototoxin which closely resembles in structure the pore- forming toxin aerolysin from Aeromonas hydrophila (see Figure 4), but the latter is over a hundred times less toxic. The activated ε‑toxin can be produced by cleav- ing N- and C-terminal peptides from the prototoxin with an enzyme, produced by the bacterium, called λ-protease. Interestingly, although highly toxic, ε-toxin does not appear to be an enzyme. Figure 4 compares the highly toxic ε-toxin with the mildly toxic aerolysin. Oligomers of ε-toxin may bind to receptors, whose

identity is unknown, to form pores in the epithelial Downloaded from http://portlandpress.com/biochemist/article-pdf/32/4/4/5064/bio032040004.pdf by guest on 02 October 2021 cells of the gut.

Some toxins elicit inappropriate immune responses

Other toxins specifically target the immune system. Gram-negative bacteria have lipopolysaccharides (LPS) in their outer membrane. These molecules consist of a sugar chain and a lipid moiety, known as lipid A. These molecules are not secreted and are therefore known as endotoxins. Receptors that recognize LPS are found on Figure 5. Staphylococcus aureus

dendritic cells and other cells of the immune system. This recognition stimulates the release of signalling proteins such as cytokines and this ultimately leads to the bacterium being neutralized or destroyed. How- ever, serious adverse effects can also result from LPS. The presence of LPS in the serum can trigger signalling cascades in leucocytes that produce fever and lowering of blood pressure. Other bacteria such as Staphylococcus aureus (Fig- ure 5) and Streptococcus pyogenes produce another type of toxin, called a superantigen10. This is a protein that interferes with T-cell signalling and stimulates the release of cytokines. In an ordinary infection, only T‑cells that recognize foreign peptides, bound to MHC proteins, are stimulated to release cytokines. However, superantigens bind to MHC molecules and stimulate up to 20% of the T-cell population, leading to a storm of pro-inflammatory cytokines resulting in rashes and fever and sometimes coma and death.

Vaccines and antitoxins have been Figure 4. Comparison between the highly toxic ε-prototoxin developed to neutralize bacterial toxins from Clostridium perfringens on the left and the mildly toxic pore-forming aerolysin from Aeromonas hydrophila on the Bacterial toxins are often highly immunogenic. They right. The similarity between the two structures has enabled stimulate the adaptive immune system to produce high- us to identify residues in the prototoxin that may be involved affinity antibodies that bind to the toxin molecules and in membrane insertion9. Aerolysin has an extra domain that inactivate them. These are IgG antibodies that diffuse is coloured grey, but it is not known whether this contributes through the circulatory system and IgA antibodies that to the difference in toxicity. confer protection in the mucosal secretions. However,

the generation of high-affinity antibodies by the adap- Professor David Moss is Senior Research tive immune system is a long and complex process, tak- Fellow in the Department of Biological ing several days, which may be too long in the case of Sciences at Birkbeck College, London. He intoxication by the most virulent toxins. has a long-standing interest in biomo- Some toxin molecules can be modified by treatment lecular structure–function relationships with chemicals such as formaldehyde that impair the and has used X-ray crystallography, enzymatic function without removing the immuno- biophysical and simulation techniques to probe protein–ligand genicity. Such toxoids can be used as vaccines that lead interactions. His current research topics include bacterial to the production of memory B‑cells that can be quickly toxins and the immune response to antigenic molecules. stimulated to produce high-affinity antibodies when email: [email protected] later challenged with the toxin. This approach has been Dr Ajit Basak is a Senior Research As-

used to develop the protective vaccines against diph- Downloaded from http://portlandpress.com/biochemist/article-pdf/32/4/4/5064/bio032040004.pdf by guest on 02 October 2021 theria and tetanus toxins. Poisoning by bacterial toxins sociate in the Department of Biological can be treated with antitoxins that contain pre‑formed Sciences at Birkbeck College, London. antibodies. Antitoxins are produced by immunizing He leads the Birkbeck Bacterial Toxin Re- animals such as horses and then recovering the neutral- search Group and has been involved in izing antibodies that develop in the serum. Equine anti‑ structure–function studies of clostridial toxins developed in this way are used in the treatment of toxins for 20 years. He is currently working on the ε- and ente- botulism and diphtheria. ro-toxins and also on Listeria proteins associated with disease. There is an ongoing need for more effective vac- email: [email protected] cines against bacterial toxins. For example, better vaccines against ε-toxin will give newborn lambs extra Dr Claire Naylor is a research scien- protection against enterotoxaemia, whereas vaccines tist in the Birkbeck Bacterial Toxin against all strains of botulinum toxin would benefit Research Group with particular inter- anybody who might be exposed to their use as agents est in the use of computational and of bioterrorism. ■ biophysical techniques. She has solved many crystal structures of bacterial We thank the Medical Research Council and the Wellcome toxins and currently has MRC funding for working on the Trust for providing project grant support for toxin work in interactions of gas‑gangrene toxin with cell membranes. our laboratory. email: [email protected]

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