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EXOTOXIN
DEFINATION: Exotoxin is a protein excreted by a microorganism including bacteria, fungi and
protozoa.They produce exotoxin as a part of their growth and multiplication.
Toxin are of two types:
a. Exotoxin
b. Endotoxin
CHARACTERISTICS:
1. Exotoxins are protein in nature(Polypeptide) with molecular weight of 10,000-900,000.
Produced by both gram positive and negative bacteria.
Example:
Gram-positive: Staphylococcus, Streptococcus, Clostridium, Bacillus, C. diphtherae.
Gram-negetive: Vibrio Cholerae, E.Coli, Bordatella pertusis, Pseudomonas.
2. Relatively unstable, toxicity often destroyed rapidly by heating at temperatue above 60C
exceptional in case of staph enterotoxin.
3. Highly antigenic.Stimulate formation of high-titer of antibodies called antitoxin.
4. Highly toxic fatal dose may be one micro-gram such as Tetanus toxin.
5. Usually bind to specific receptors on cell surface.
6. Are encoded by genes eg plasmids, B. phage and chromosomal.
7. Medical importance: it can be consisted to antigenic, nontoxic toxoids by formalin,acid,
heat etc. toxoids are used to immunity e.g. tetanus toxoid
8. Exotoxins are most toxic substances known eg.Botulinum toxin.
STRUCTURE
Most exotoxins have two structural domains:


A subunit (active domain): That causes cell cytotoxicity.
B subunit (binding domain):That binds the “A” domain to specific cellular receptor. A
subunit is transferred to the interior of the cell, where the cell ingury is induced.
Isolated A subunits are enzymatically active but lack binding ability and cell entry capability.
Isolated B subunits bind to target cells but they are nontoxic and biologically inactive.
Exotoxin that have an A-B subunit structure-diptheria toxin, tetanus toxin, botulium toxin,
cholera toxin, entero toxin of E.coli. Pseudomonus exotoxin A.
The binding of a B subunit determines the specificity of the action of the exotoxin. E.g.
botulinum toxic acts on the neuromascular junction because B subunits bind to the specific
receptors on the surface of the motor nuron at the junction.
Activaion of toxin: activation occurred due to proteolytic cleavage & reduction of disulphide
bond as the folloing figure…
Figure: Diphtheria exotoxin. Intact extracellular toxin binds to aeukaryotic cell by its B region
(dark fragment). After proteolytic cleavage and reduction of the disulfide bond, the A region
(light fragment) containing the ribosylating enzyme is activated.
SECRETION SYSTEM
1.Exotoxins are released from bacteria by specialized structures called secretion systems. Some
secretion systems transport the exotoxins into the extracellular space, but others transport the
exotoxins directly into the mammalian cell. Those that transport the exotoxins directly into the
mammalian cell are especially effective because the exotoxin is not exposed to antibodies in the
extracellular space. Six types of secretion systems have been identified, but the type III
secretionsystem (also called an injectosome) is particularly important in virulence. This secretion
system is mediated by a needlelike projection (sometimes called a “molecular syringe”) and by
transport pumps in the bacterial cell membrane. The importance of the type III secretion system
is illustrated by the finding that the strains of Pseudomonas aeruginosa that have this secretion
system are significantly more virulent than those that do not. Other medically important gramnegative rods that utilize injectosomes include Shigella species, Salmonella species, E. coli, and
Y. pestis.
2.Another secration system is the following pic….
3.Some bacteria are released by autilusis e.g Cl. Tetani,Cl.botulinim.
CLASSIFICATION
A. According to genetics:
1. Toxin is encoded on a plasmid e.g.
- LT and St toxins of entropathogenic E. Coli
- Tenus toxintanus of Cl. Tetani.
2. toxin is encoded on a lysogenic phage e.g.
-C. diphtheria
- C. botulium
- Staph. Aureus
-Strepto. Pyogyn
-Shiga like toxin of E.Coli
- Cholera toxin
3. Chromosomal encoded e.g
- Partussis toxin.
B. According to function:
1. Enterotoxin: that affects GIT and include cholera toxin, E.Coli enterotoxin. Shiga
toxin.
2. Neurotoxin: that affects cells of nervous tissue e.g. botulinum toxin, tetanus toxin.
3. Cytotoxin: that affects cells of various tissue e.g. diphtheria toxin, peudomonus toxin.
C. According to mode of action:
Common exotoxin producing Bacteria:
ANTITOXIN:
Exotoxin polypeptide are good antigens and induce the synthesis of protective antibodies called
antitoxin, some of which are useful in prevention of diseases e.g. botulism, tetanus.
TOXOID:
When exotoxin treated with formaldehyde,acid or heat the exotoxin polypeptides are converted
into toxoids, which are used in protective vaccines bcause they retains their antigenicity but have
lost their toxicity.
A. ADP-RIBOSYLATION
DIPHTHERIA TOXIN:
Produced by Corynebacterium diphtheriae, inhibits protein synthesis by ADP-ribosylation of
EF-2. The consequent death of the cells leads to two prominent symptoms of diphtheria e.g
pseudomembrane formation in the throat and myocarditis.
The exotoxin binds to cell membrane receptors via a region near its carboxyl end. The toxin is
transported across the membrane, and the proteolytic nick and reduction of the disulfide bonds
occur. This releases the active fragment A, which inactivates EF-2. The enzymatic activity is
specific for EF-2; no other protein is ADP-ribosylated. The specificity is due to the presence in
EF-2 of a unique amino acid, a modified histidine called diphthamide. The reaction occurs in all
eukaryotic cells; there is no tissue or organ specificity. Prokaryotic and mitochondrial protein
synthesis is not affected because a different, nonsusceptible elongation factor is involved. The
reaction is as follows:
EF-2 + NAD → EF-2–ADP-ribose + Nicotinamide
HEAT-LABILE ENTEROTOXIN
Produced by E. coli causes watery, nonbloody diarrhea by stimulating adenylate cyclase
activity in cells in the small intestine. The resulting increase in the concentration of cyclic AMP
causes excretion of the chloride ion, inhibition of sodium ion absorption, and significant fluid
and electrolyte loss into the lumen of the gut. The heat-labile toxin, which is inactivated at 65°C
for 30 minutes, is composed of two subunits: a B subunit, which binds to a ganglioside receptor
in the cell membrane, and an A subunit, which enters the cell and mediates the transfer of ADPribose from NAD to a stimulatory coupling protein (Gs protein). This locks the Gs protein in the
“on” position, thereby continually stimulating adenylate cyclase to synthesize cyclic AMP. This
activates cyclic AMP–dependent protein kinase, an enzyme that phosphorylates ion transporters
in the cell membrane, resulting in the loss of water and ions from the cell.
In addition to the labile toxin, there is a heat-stable toxin, which is a polypeptide that is not
inactivated by boiling for 30 minutes. The heat-stable toxin affects cyclic guanosine
monophosphate (GMP) rather than cyclic AMP. It stimulates guanylate cyclase and thus
increases the concentration of cyclic GMP, which inhibits the reabsorption of sodium ions and
causes diarrhea.
SHIGA TOXIN:
Also known as verotoxin and Shiga-like toxin.It is an exotoxin
produced primarily by strains of E. coli with the O157:H7 serotype. These
enterohemorrhagic strains cause bloody diarrhea and are the cause of outbreaks
associated with eating undercooked meat, especially hamburger in fast-food
restaurants. The toxin is named for a very similar toxin produced by Shigella
dysenteriae. The toxin inactivates protein synthesis by removing adenine from a
specific site on the 28S rRNA in the large subunit of the human ribosome. The term
verotoxin refers to its cytopathic effect on Vero (monkey) cells in culture.
Shiga toxin is encoded by a temperate (lysogenic) bacteriophage. When Shiga
toxin enters the bloodstream, it can cause hemolytic–uremic syndrome (HUS).
Shiga toxin binds to receptors on the kidney and on the endothelium of small blood
vessels. Inhibition of protein synthesis results in death of those cells, leading to renal
failure and microangiopathic hemolytic anemia. Certain antibiotics, such as
ciprofloxacin, can increase the amounts of Shiga toxin produced by E. coli O157,
which predisposes to HUS.
Cholera Toxin:
The enterotoxin produced by V. cholerae, the agent of cholera and Bacillus cereus, a cause of
diarrhea, act in a manner similar to that of the heat-labile toxin of E. coli.
Pertussis toxin:
produced by Bor. pertussis, the cause of whooping cough, is an exotoxin that catalyzes the
transfer of ADP-ribose from NAD to an inhibitory G protein. Inactivation of this inhibitory
regulator has two effects: one is in the stimulation of adenylate cyclase activity and a consequent
increase in the amount of cyclic AMP within the affected cells. This results in edema and other
changes in the respiratory tract, leading to the cough of whooping cough. The second
effect is the inhibition of the signal transduction pathway used by chemokine
receptors. This causes the marked lymphocytosis seen in patients with pertussis.
The toxin inhibits signal transduction by all chemokine receptors, resulting in an
inability of lymphocytes to migrate to and enter lymphoid tissue (spleen, lymph
nodes). Because they do not enter tissue, there is an increase in their number in the
blood.
Difficile Toxin:
Two exotoxins are produced by Clostridium difficile, both of which are involved in the
pathogenesis of pseudomembranous colitis. Exotoxin A is an enterotoxin that causes watery
diarrhea. Exotoxin B is a cytotoxin that damages the colonic mucosa and causes
pseudomembranes to form. Exotoxins A and B are glucosyl transferases that glucosylate signal
transduction proteins called Rho GTPases—a process that inhibits these GTPases from
performing their signal transduction function. Glucosylation by exotoxin B causes
disaggregation of actin filaments in the cytoskeleton, leading to apoptosis and cell death.
B. SUPERANTIGEN
TOXIC SHOCK SYNDROME TOXIN:
Superantigen produced primarily by certain strains of Sta. aureus but also by certain strains of
Str. pyogenes. TSST binds directly to class II major histocompatibility (MHC) proteins on the
surface of antigen-presenting cells (macrophages) without intracellular processing. This complex
interacts with the β-chain of the T-cell receptor of many helper T cells. This causes the release of
large amounts of interleukins, especially interleukin-1 and interleukin-2. These cytokines
produce many of the signs and symptoms of toxic shock. TSST is also a T-cell “mitogen”
(i.e., it induces T cells to multiply), which contributes to the overproduction of cytokines.
STAPHYLOCOCCAL ENTEROTOXIN:
It is also a superantigen, but because of it’s ingestion, it acts locally on the lymphoid cells lining
the small intestine. The enterotoxin is produced by Sta. aureus in the contaminated food and
causes food poisoning, usually within 1 to 6 hours after ingestion. The main symptoms are
vomiting and watery diarrhea.The prominent vomiting seen in food poisoning is thought to be
caused by cytokines released from the lymphoid cells stimulating the enteric nervous system that
is vagus nerve which activates the vomiting center in the brain.
ERYTHROGENIC TOXIN:
produced by Str. pyogenes, causes the rash characteristic of scarlet fever. Its mechanism of
action is similar to that of TSST (it acts as a superantigen). The DNA that codes for the toxin
resides on a temperate bacteriophage. Nonlysogenic bacteria do not cause scarlet fever, although
they can cause pharyngitis.
C. PROTEASE
TETANUS TOXIN:
C.Tetani is a anaerobic gm (+ve) rod that causes tetanus. spores of C. Tetani germinate in the
anaerobic environment of the devitalized tissue. Vegetative forms of it produce the toxin
tetanospasmin (150,000), that is cleaved by a bacterial protease in to two peptides linked by
disaccharide bond.
Toxin is produced around the peripheral wond.
Toxin binds to the presynaptic membrane of motor neuron & migrates by retrograde axonal
transport system to anterior horn cell of spinal cord & brain.Toxin degrades synaptobrevin ( a
protein required for docking of neurotransmitter vesicels on the prynaptic membrane).
Blocking the release of inhabitory neurotransmitter glycine and Gama-aminobutyric acid -motor
neuron are not inhibited-spastic paralysis occurs-Spasm of Jaw & neck muscle called lock Jaw.
BOTULINUM TOXIN:
Botulinum toxin is absorbed from the gut binds to receptors of presynaptic membrane of motor
neurons of peripheral nervous system and cranial nerves.
Proteolysis of target protein in the neurons causing inhibition of release of the
acetylcholine(excitatory neurotransmitter) at the synapse.
-
Lack of muscle contraction and flaccid paralysis occurs.
It is one of the most toxic substance known.
Approximately one microgram is lethal for human.
ANTHRAX TOXIN:
Three exotoxins are produced by Bacillus anthracis, the agent of anthrax: edema factor, lethal
factor, and protective antigen. The three exotoxins associate with each other, but each component
has a distinct function. Edema factor is an adenylate cyclase that raises the cyclic AMP
concentration within the cell, resulting in loss of chloride ions and water and consequent edema
formation in the tissue. Lethal factor is a protease that cleaves a phosphokinase required for the
signal transduction pathway that controls cell growth. Loss of the phosphokinase results in the
failure of cell growth and consequent cell death. Protective antigen binds to a cell surface
receptor and forms pores in the human cell membrane that allow edema factor and lethal factor
to enter the cell. The name protective antigen is based on the finding that antibody against this
protein protects against disease. The antibody blocks the binding of protective antigen, thereby
preventing edema factor and lethal factor from entering the cell.
EXFOLIATIN TOXIN: is a protease produced by Sta. aureus that causes scalded skin syndrome.
Exfoliatin cleaves desmoglein, a protein in the desmosomes of the skin, resulting in the
detachment of the superficial layers of the skin. Exfoliatin is also called epidermolytic toxin.
PANTON-VALENTINE (PV) LEUKOCIDIN:
is a pore-forming exotoxin produced by methicillin-resistant strains of Sta. aureus (MRSA). It
destroys white blood cells, skin, and subcutaneous tissue. The two subunits of the toxin assemble
in the cell membrane to form a pore through which cell contents exit into the extracellular space.
D. LECITHINASE
Perfringens Toxin:
Multiple toxins are produced by Clostridium perfringens and other species of clostridia that
cause gas gangrene. A total of 7 lethal factors and 5 enzymes have been characterized, but no
species of Clostridium makes all 12 products. The best characterized is the alpha toxin, which is
a lecithinase that hydrolyzes lecithin in the cell membrane, resulting in destruction of the
membrane and widespread cell death. The other four enzymes are collagenase, protease,
hyaluronidase, and deoxyribonuclease (DNase). The seven lethal toxins are a heterogeneous
group with hemolytic and necrotizing activity. Certain strains of Clo. perfringens produce an
enterotoxin that causes watery diarrhea. This enterotoxin acts as a superantigen similar to the
enterotoxin of Sta. aureus.
FUNGAL TOXIN:
Some fungus produce exotoxins. As follows;
1. Aflatoxin- Aspergillous flavous produce toxins in spoiled grains and peanut-ingestionmetabolised by liver & Epoxide (potent carcinogen) is formed - mutation of p53 gene
which is tumor suppressor gene - loss of p53 protein - loss of growth control of hepatocyte
- hepatic carcinoma is formed.
2. Amanaitin and phalloidin (hepatotoxin) – produced by Amanita mushroom – inhibit
protein synthesis—inhibit RNA polymerase-hepatotoxicity.
3. Claviceps purpurea (Mold) infect grains and produces alkaloids (e.g. ergotamine and
lysergic acid diethylamide) that cause pronounced vascular and neurologic effects.
PROTOZOAL TOXIN
Dinoflagella are marine protozoa. They produce various toxins. When we eat undercoocked seafood such as shell fish, they produce various effects on GIT, skin and respiratory tract.
IDENTIFICATION OF EXOTOXIN:
Toxin production by pathogens can be studied by several techniques.
1.
2.
3.
4.
5.
6.
Illeal loop test
Cell culture test
Serological test ( immunoassay)
Molecular method for detection of DNA of toxin producing organism in specimen.
Precipitation – Elick test (Diphtheria)
EIESA
APPLICATION:
1. They used as toxoid for vaccine.
2. Ig which is produced against them is used for prevention of some diseases.
3. Botox produced from botulinum toxin used in cosmetic purpose.
Prepeard by
Dr. kakali Halder
Resident, 5th batch,
Microbiology & Immunology, BSMMU.