Introduction

Unlike endotoxins that are a structural component of the outer membrane of gram-negative bacteria, exotoxins can be produced by any bacteria. Exotoxins are either actively secreted by the bacterial cells or released from the protoplasm on death of the bacterial cells. Some of the most potent exotoxins known are produced by bacteria. Knowledge of how the different types of exotoxins work assists us in counteracting them and we can also use this knowledge in the manufacture of novel drugs.

Reference (not essential)

Pathogenesis of Bacterial Infections in Animals, edited by Carlton L. Gyles, John F. Prescott, Glenn Songer, Charles O. Thoen – more related to specific bacteria rather than principals. (Available on-line via JCU library)

classification of exotoxins

Exotoxins are generally proteins produced by both gram-positive and gram-negative bacteria that cause disease. They either produced in the host by infecting bacteria or by the ingestion of bacterial toxins (toxicoses) that have been produced by environmental bacteria. The severity of disease due to exotoxins is usually dose-dependent with each toxin having its own lethal dose (LD). For example the 1 g of type D botox (produced by Clostridium botulinum) is enough to kill 60 000 head of cattle. Exotoxins often adhere to specific cell membrane receptors and enter cells via the cell’s transport mechanisms or by bacterial manufactured porins.  Many exotoxins act as enzymes within their target host cells. (Refer to the Table comparing endotoxins and exotoxins in the previous chapter).

Although exotoxins are highly variable in their activity and effect, they can often be classified into the following broad categories.

They can be defined by their mode of action:

1.  Type I: Act directly on host cell plasma membrane receptors
2. Type II: Damage cell membranes
3. Type III: Enter cells and affect cell processes = intracellular toxins

They can be defined by the tissues they affect:

1. Tissue toxins = histiotoxins/cytotoxins
2. Intestinal toxins = enterotoxins
3. Peripheral and central nervous system toxins = neurotoxin

type i membrane active exotoxins

Toxins that bind to the surface of host cell plasma membrane receptors and modify host cell physiology by triggering intracellular signaling. An example is that of Enterotoxigenic Escherichia coli causing watery diarrhoea in neonatal animals. This is also an example of an enterotoxin – one that is active in the gastrointestinal tract. Another example is superantigen production by some bacteria including Staphylococcus aureus.

1. Sucking one dry: the story of enterotoxigenic Escherichia coli

White scours is a common contagious disease of colostrum-deprived calves, lambs, kids, foals and piglets caused by  enterotoxigenic Escherichia coli. When provided with lots of lactose (in milk), this enterotoxigenic Escherichia coli will grow rapidly, adhere to the brush borders of intestinal epithelium using F 4 (K88) fimbriae in pigs and F5, F6 and F41 fimbriae in calves and pigs. They then produce heat-labile or heat-stable enterotoxins. These toxins are able to cross into the cell cytoplasm using a A-B transporter system (see later). Heat-labile enterotoxin activates adenyl cyclase raising the cAMP levels which stimulate the transmembrane chloride transporter to secrete chloride ions and water.  Heat stable toxin binds to membrane guanylate cyclase stimulating the production of cGMP. cGMP stimulates transmembrane channels to secrete chloride and bicarbonate ions and bring water with them. This fluid loss can result in rapid dehydration which can be fatal in neonatal animals.

2. Superantigens: A shocking situation

Superantigens bridge MHCII class receptors and T-cell antigen receptors resulting in at least a fifth of the T-cells proliferating in a non-specific way. Activated T-cells release huge amounts of proinflammatory cytokines such as gamma-IFN, Il-1, IL-6 and TNF leading to “inflammatory storms”.  In other words, superantigens are potent T-cell mitogens. This can result in life-threatening shock. TSST-1 and enterotoxins, both exotoxins produced by Staphylococcus aureus can act as superantigens.

Shock due to superantigens is mainly recognised in human disease, however, there is an increasing body of evidence to show that it is also happening in animals. It is involved in the pathogenesis of deep pyodermas in dogs caused by either Staphylococcus pseudintermedius or S. aureus and mastitis caused by S. aureus in dairy cows.

type II membrane damaging exotoxins

Toxins that damage membranes often cause haemolysis when their source bacteria are cultured on blood agar and are thus known as haemolysins. These toxins create channels in cell membranes or produce enzymes that alter the characteristics of the cell membrane

A. Large Porins

These exotoxins insert in a lipid raft or cholesterol-rich portion of the target cell membrane and form large pores. An example of a cholesterol-dependent cytolysin (porin) is an enterotoxin and endotheliotoxin known as Epsilon toxin. It is produced by Clostridium perfringens toxin type D.

The story of Pulpy kidney caused by a cholesterol-dependent Porin

Epsilon toxin of Clostridium perfringens type D the cause of Pulpy kidney in lambs and kids is a typical porin. This disease is also called enterotoxaemia (the presence of toxins in the blood originating from the intestines), giving an indication that Epsilon toxin is an enterotoxin (targets intestinal epithelium) as well as a endothelial toxin (targets the endothelium of blood vessels).

Clostridium perfringens, gram-positive rods normally present in the large intestine, will accelerate their growth when provided with more nutrients than normal e.g. when animals overeat after a period of fasting. This allows them to become the predominant bacterium in the large intestine. As the bacteria use up all the nutrients and die, they release toxins. Clostridium perfringens toxin type D will release a lot of Epsilon toxin. This toxin is released as a protoxin that will be active when it is cleaved by intestinal enzymes such as trypsin. This toxin attaches to lipid rafts within the cell membranes and then expands to form a hepatomer (7 sided) channel. This channel is large enough to affect the sodium-potassium balance of the cell as well as destroy the structural integrity of the cell. Thus fluid is lost from the cell and the cells can rupture. The effect is greatest in the capillary beds of the brain and kidney with oedema and haemorrhage resulting.

B. RTX (repeats in toxin) porins

These porin toxins have repeat sequences and are usually produced by gram-negative bacteria. They are cytolysins,  like leukotoxins (white blood cell destructive) or haemolysins (red blood cell destructive).   At low concentrations they cause increased intracellular calcium, cell apoptosis and degranulation and at high concentrations they cause cell lysis.  The toxins insert into host cell membranes forming channels. The leukotoxin (leucocidan) of Mannheimia haemolytica, the primary bacterial cause of transport or feedlot pneumonia in cattle, especially targets and damages alveolar macrophages of cattle which promotes bacterial proliferation and hyperinflammation.

C. ENZYMATICALLY active toxins

One example is the α toxin of Clostridium perfringens, which causes gas gangrene; α toxin has lecithinase (phospholipase C) activity.

TYPE III INTRACELLULAR ACTIVE TOXINS

These toxins enter the cytoplasm of cells and disrupt cellular activities.

They can do this in several ways:

1. AB toxins.
2. Using a molecular injector

1. AB Toxins

The B subunit attaches to target regions on the cell membrane are then ingested by the cell in a phagosome. Acidification of the phagosome allows the toxic portion “A” to detach from “B” and enter the cytoplasm of the cell where it exerts an effect. A short you-tube video shows an animation of this.

NEUROTOXINS

Neurotoxins exert their effect on the central and peripheral nervous systems. Botulism and tetanus toxin are similar in mode of action but target different cells and hence animals show very different clinical signs.

Botulism

Botulism toxin is the most potent toxin known. After an animal ingests botulism toxin, it enters via the intestinal tract and circulates in the lymphatics and bloodstream to the muscles. Once at the muscle it binds to specific proteins on the presynaptic membrane on motor-end plates where it is taken up by the axon. The A subunit of the toxin cleaves the vesicle associated membrane proteins (VAMP) or SNARE proteins preventing vesicles from fusing with the motor-end plate membrane and releasing the acetylcholine into the synaptic junction. Thus, the muscle cannot contract in response to a nerve stimulus. The DNA encoding for all types of botoxin are found in prophages (See Introduction).

Tetanus

Once the spores of Clostridium tetani found in faecally contaminated soil enters wounds, it germinates and divides producing tetanus toxin (TeNT). It is the second most potent toxin known. TeNT circulates in the lymphatics and bloodstream and binds to the presynaptic junction. Once it has entered the axon it moves retrograde up nerves to the central nervous system. There it cleaves to inhibitory interneurons, is endocytosed and binds to synaptobrevin, a vesicle membrane protein and prevents the neuroinhibitors GABA and glycine from being released. Thus, no signal returns to the motor-end plates to initiate muscle relaxation. Thus muscle contractions continue. Genes encoding this toxin are transferred between bacteria by conjugation.

systemic toxin

Anthrax toxin: a killer of macrophages and other cells

Bacillus anthracis, a gram-positive rod, produces an AB toxin where the B subunit is known as Protective antigen (PA). It is antigenic and inserts into the cell membrane of target cells, is activated to form a heptamer, to which lethal factor (LF) and oedema factor (EF) attach. These are then phagocytosed. The PA inserts itself in the membrane of the phagocytic vesicle and expands to form a narrow channel. This provides a gateway for one of the A subunits, LF and EF, to enter the cell. See this YouTube animation to understand the process: Mechanism of Anthrax Toxins. Lethal factor affects snips off a portion of the mitogen activated protein kinases leading to cell apoptosis and Oedema factor increases cAMP in the cell affecting water homeostasis.

ENTEROTOXIN 

Killing cells: Stopping intracellular protein manufacture by the Shigatoxin of enterohaemorrhagic E. coli

Once enterohaemorrhagic strains of Escherichia coli adhere to the brush border of intestinal cells they produce shigatoxin (STx) which enters cells via receptor-mediated endocytosis and is transported to the ribosomes on the endoplasmic reticulum. There it cleaves an adenosine molecule from the ribosome preventing aminoacyl-tRNA binding and thus the initiation of protein manufacture resulting in cell death.

DERMOTOXIN: A MITOGEN

Pasteurella multocida, the cause of atropic rhinitis in pigs, produces a dermotoxin (skin necrosis toxin) which is also considered to be a mitogen. In other words it induces cell mitosis and proliferation.

This is an AB toxin that enters the cell via receptor-mediated endocytosis where it has effects of the manufacture of G-proteins. It inhibits apoptosis and stimulates the proliferation of osteoclasts and increases cell death in osteoblasts. This results in nasal turbinate and septum loss and remodeling (see picture below).

Using a molecular injector

Some gram-negative bacteria use a type III secretion system (T3SS) to inject toxins into the cell. An example is Salmonella species where it uses an injector to stimulate endocytosis of itself (refer to earlier section on intracellular lifestyle = trigger mechanism). An animation of the injector is shown in this YouTube video: Salmonella Entering the Intestinal Tract.

Enteropathogenic Escherichia coli uses an injector to inject intimin into cells which stimulate cell membrane actin polymerisation, creating a pedestal providing E. coli with a more secure attachment to intestinal epithelium.

sharing of bacterial genes encoding for exotoxin production

Many of the exotoxins produced by bacteria are not essential for life and reproduction. They do, however, provide them with a competitive advantage in polymicrobial environments and in the presence of a hostile immune response by their hosts. Thus, many of the genes encoding toxin proteins are found in bacterial plasmids or in transposons. This means that bacteria are able to share these genes with bacteria of the same species and even of the same genus and family. They do this by transduction, conjugation and transformation. Transformation is by the acquiring of naked DNA from ruptured bacterial cells. This happens in biofilms and in bacteria-rich environments.

Transduction

Viruses known as bacteriophages (phages) have iscohedral symmetry with a long tail. The tail attaches specifically to bacterial cells and inject their DNA into the bacterial cell using a pumping action. The DNA then forms a circle and encodes the bacteria to replicate the phage DNA to manufacture proteins and new phages. The bacterium then ruptures releasing the new phage virions. This is known as the lytic cycle and phages that do this are known as virulent phages. Since virulent phages are destructive to bacteria, they can be used to control bacteria and treat bacterial infections. Their specific nature also allows us to use phage to identify specific bacterial species and even strains. This is observed as plaques or bacterial killing zones on a lawn of bacteria grown on the surface of an agar plate as they will crease circular areas of bacterial lysis or plaques in a lawn of bacterial colonies on an agar plate. You have seen this in a virology practical in tv2102.

However, it is the lysogenic phage that is of concern. Phage DNA integrate into the plasmid DNA as a prophage, becoming part of the bacterial DNA. If DNA coding for bacterial toxins or other virulence factors are part of the prophage, then an essentially harmless bacterium can become pathogenic. This is also one of the ways in which bacteria acquire DNA that encodes for antibiotic resistance. Bacteriophages can also provide other bacteria with a portion of bacterial DNA when they under stress, such as UV light or nutritional deprivation, escape from the bacterial DNA and start the virulent cycle. Together with their own DNA they can take a portion of bacterial DNA with them. The botoxin of botulism is an example of a bacterium where the DNA that encodes for botulinum toxin (botoxin) is found in a prophage. The example when a non-pathogenic bacterium Bacillus cereus becomes as toxic as Bacillus anthracis is a case of bacterial DNA encoding for exotoxins has been included in phage DNA during the conversion from the lysogenic to the virulent cycle.

Proof that this happens : a story of a Chimpanzee and a soil bacterium with new tools – Don’t need to know

Veterinarians in the Cote de Ivoire, Africa, found a chimpanzee that had died from what appeared to be Bacillus anthracis (anthrax) infection. However, when they grew the bacterium they discovered that it was a Bacillus cereus a normal soil inhabitant that is usually not very virulent. On investigation of the genetics of this bacterium, they discovered that the bacterium had acquired a prophage that now encoded for the anthrax toxin.

CONJUGATION

Conjugation is when one bacterium transfers plasmid DNA to another bacterium of the same species or related species via sexual pili. The process is described in the diagram below. Note that some bacteria can also transfer chromosomal DNA. Tetanospasmin produced by Clostridium tetani is encoded on a plasmid that is transferred by conjugation.

Tissue damaging molecules

There are a number of bacterial enzymes  that are not toxins but act synergistically to aid bacteria in spreading and tissue damage. Many of these enzymes are also used to assist in tissue invasion and spread. Thus refer to the chapter on bacterial invasion. However, there are some that have not been described previously.

1. Siderophore. Whilst not an enzyme, these molecules are produced by bacteria to assist them in scavenging, trapping and transporting iron. Iron is an important micro-nutrient for bacteria as it is needed for the growth and metabolism of all bacteria.

2. DNAses. These are hydrolysing enzymes produced by bacteria that allow them to break down DNA in cells and other microbes.

3. Urease. Some bacteria especially those that colonise the urogenital tract are able to produce the enzyme urease. This allows bacteria to utilise urea as a nutritive source. The breakdown of urea leads to alkalinisation of the urine with struvite crystal formation.

Toxic cyanobacteria (so-called “Blue-green algae”, “pond scum”)

Up to 80 species of blue-green bacteria in blooms including Dolichospermum spp., Cylindrosporum sp., Microcystis sp., and Nodularia spp are toxic. It is not always the same species that blooms in a given water body, and the dominant species can change over the course of the season. As the bacteria die-off and their outer membranes rupture and some species may release exotoxins. Animals and people who ingest these toxins via the drinking of contaminated water can become ill.

See this poster that the Florida Sea Grant distributed in 2020 to warn dog owners about algal blooms: Cyanobacteria and Dogs: By the Numbers

Microcystin is usually produced by Microcystis aeruginosa and nodularin by Nodularia spumigena.   Since the liver is damaged, typical clinical signs in dogs ingesting microcystin include vomiting and diarrhoea. People may develop a skin rash and blistering or the lips when in contact with microcystin. Saxitoxin is produced by Dolichospermum circinale – it causes muscle paralysis and death due to respiratory muscle failure.

Control of blooms

Good water management aimed at reducing overgrowth by cyanobacteria in drinking supplies reduces the risk. Measures include the use of riparian plants, proper treatment of sewage and stormwater run-off, and mixing of water to prevent an upper warm layer forming. The DPI usually provides a “Red Alert” warning when a bloom happens. Unacceptable levels in freshwater: Criteria >4mm³ M. aeruginosa/1000mL or > 10mm³ cyanobacteria /1000mL. Thus it is advisable to avoid those water bodies or at least shower after being in them and not to drink untreated water. Note that boiling does not destroy the toxin. In fact, the destruction of the bacteria by boiling will release even more protoplasmic-bound toxin.  Water treatment with chlorine and algaecides can reduce the bacterial load and coagulation and filtration of the water will cause the bacteria to clump and not pass through filters.

Advice to animal owners is the following:

• Provide plentiful clean, clear, fresh water for your animals. Keep water bowls, buckets, and troughs clean and well-maintained.
• Never let your pets (or children) swim in, play in, or drink discoloured, slimy, scummy, or otherwise suspicious water. Assume any bloom is toxic.
• Pay attention to local health and water advisories and respect any water body closures. Water that appears clean can still contain high concentrations of toxins.
• Fence off farm ponds, creeks, and other natural water sources to prevent livestock from contaminating them as well as drinking from them.
• Barley straw added in onion bags to at-risk water, early in summer, can prevent the proliferation of Microcystis. When decomposing the barley straw uses up nitrogen in the water and produce flavonolignins that inhibit the growth of Microcystis.
• Ferric alum will remove phosphorus from the water. It is a preventative so use early in summer.
• Simazine is a registered triazine herbicide that can be used to destroy the cyanobacteria. Be careful as the amount of toxin in the water will increase soon after the bacteria are destroyed. So don’t let animals drink from the water until it is clear.
• Fence off backyard ponds and other natural water sources to keep pets from accessing them.
• Prevent fertilizer and/or manure from running off into water sources.
• If your pet does access suspicious water, wash him thoroughly with clean, fresh water, and prevent him from licking his fur. Wash your own hands and arms after washing your pet, as exposure to blue-green algae can cause skin, eye, nose, and throat irritations in humans.
• If animals become ill after exposure to a pond, lake, or other natural water source, seek immediate veterinary care – even if the water appears clean, toxins can still be present. Tell your veterinarian if your animal might have been exposed to blue-green algae. This can help direct treatment, as many other illnesses can have similar signs.