Bacterial Toxins

By
Prof. Dr. Mohamed Refai
Department of Microbiology
Faculty of Veterinary Medicine, Cairo
University
 Bacterial toxins
Definition
Bacterial toxins are soluble substances that alter
the normal metabolism of host cells with
deleterious effect on the host

Since the isolation of diphtheria toxin by Roux and

Yersin in 1888 bacterial toxins have been
recognized as the primary virulence factors of a
variety of pathogenic bacteria
 Bacterial toxins

• A few cause clinical illness, e.g. botulinum

toxin in botulism
• The majority are important component of
virulence factors of pathogenic bacteria
Classification of bacterial toxins
1. Pattern of release
a. Endotoxins
b. Exotoxins
 Endotoxins
• Produced solely by Gram-negative
bacteria
• Released when cell wall disintegrates
• Less potent and less specific
• Antigenic and immunogenic
• Do not form toxoids
 BACTERIAL ENDOTOXINS
• Endotoxins are the lipopolysaccharide
complex associated with the outer membrane of
Gram-negative bacteria, whether the organisms
are pathogens or not.
• Both Lipid A (the toxic component of LPS) and
the polysaccharide side chains (the nontoxic but
immunogenic portion of LPS) act as
determinants of virulence in Gram-negative
bacteria.
Fig.1B: Structure of a Gram-Negative Cell Wall
 Endotoxin-producing bacteria
• Salmonella • Brucella
• Shigella • Pasteurella
• Plesiomonas • Yersinia
• E. coli • Vibrio
• Pseudomonas, • Aeromonas
• Haemophilus, • Campylobacter
• Bordetella • Neisseria,
 BACTERIAL ENDOTOXINS
• endotoxins remain associated with the cell
wall until disintegration of the bacteria.
• In vivo , this results from autolysis of the
bacteria, external lysis mediated by
complement and lysozyme, and
phagocytic digestion of bacterial cells.
 BACTERIAL ENDOTOXINS
as a virulence factor
• First, they act as a permeability barrier
that is permeable only to low molecular
weight, hydrophilic molecules
• This prevents penetration of the bacteria
by bile salts and other toxic molecules
from the GI tract. It is also a barrier to
lysozyme and many antimicrobial agents.
 BACTERIAL ENDOTOXINS
as a virulence factor
• Second, it impedes destruction of the
bacterial cells by serum components and
phagocytic cells.
• Third, LPS plays an important role as a
surface structure in the interaction of the
pathogen with its host.
 BACTERIAL ENDOTOXINS
as a virulence factor
The injection of living or killed Gram
negative cells, or purified LPS, into
experimental animals causes :
1. fever,
2. changes in white blood cell counts,
3. disseminated intravascular
coagulation,
4. hypotension, shock and death..
 BACTERIAL ENDOTOXINS
as a virulence factor
• The sequence of events follows a regular
pattern: (1) latent period; (2) physiological
distress (diarrhea, prostration, shock); (3)
death.
• How soon death occurs varies on the dose
of the endotoxin, route of administration,
and species of animal.
• Animals vary in their susceptibility to
endotoxin.
Mechanism of action of endotoxins
• It is thought that LPS released into the
bloodstream by lysing Gram-negative bacteria is
first bound by certain plasma proteins identified
as LPS-binding proteins.
• The LPS-binding protein complex interacts with
CD14 receptors on monocytes and
macrophages and other types of receptors on
endothelial cells. In monocytes and
macrophages three types of events are
triggered during their interaction with LPS:
Mechanism of action of endotoxins
• In monocytes and macrophages three
types of events are triggered during their
interaction with LPS:
• 1. Production of cytokines
• 2. Activation of the complement
• 3. Activation of the coagulation
cascade.
 1. Production of cytokines
• IL-1, IL-6, IL-8, tumor necrosis factor
(TNF) and platelet-activating factor.
• These in turn stimulate production of
prostaglandins and leukotrienes.
• These are powerful mediators of
inflammation and septic shock that
accompanies endotoxin toxemia.
2. Activation of the complement
• C3a and C5a cause histamine release
(leading to vasodilation) and effect
neutrophil chemotaxis and
accumulation.
• The result is inflammation.
 3. Activation of the coagulation
cascade.
a. coagulation: a blood clotting cascade that
leads to coagulation, thrombosis, acute
disseminated intravascular coagulation
b. plasmin activation which leads to
fibrinolysis and hemorrhage.
c. kinin activation releases vasoactive peptides
which cause hypotension.

• The net effect is to induce inflammation,

intravascular coagulation, hemorrhage and
shock.
 Exotoxins
• Extracellular proteins
• Highly toxic and specific in action
• Produced mainly by Gram positive
bacteria and some Gram negative bacteria
• Immunogenic, neutralizable by antibodies
• Heat-labile, except Staph enterotoxins
• Variable structure and pharmacol actions
• Inactivated by heat or chemicals (toxoids)
Classification of bacterial toxins
Mechanisms of action

a. Damage membranes (pore-forming)

b. Inhibit protein synthesis
c. Activate second mesenger pathway
d. Activate immune response
e. Protease
 a. Membrane damaging (pore-
forming) toxins (Cytolysins)
• Pore-forming toxins disrupt the
selective influx and efflux of ions
across the plama membrane by
• inserting transmembrane pore or by
enzymatic hydrolysis through
hyaluronidases, collagenases,
phospholipases and streptokinase
 Membrane damaging (pore-
forming) toxins (Cytolysins)
Organism Toxin Target
• A. hydrophila aerolysin: Glycoprotein
• C. perfringens perfringolysin: Cholestrol
• E. coli haemolysin: Plasma membrane
• S. aureus α-toxin: Plasma membrane
• S. pneumoniae pnemolysin: Cholestrol
• L. monocytogenes listeriolysin:Cholesterol
 2. Toxins causing inhibition of
protein synthesis
1. Act as ADP-ribosyltransferase on
elongation factor 2
a. C. diphtheriae diphtheria toxin
b. P aeruginosa exotoxin A
1. Act as N-glycosidase on 28S rRNA
a. E. coli
b. Sh. dysentriae
Toxins causing inhibition of protein
synthesis
• The modified EF2 is no longer able
function in protein synthesis
• The inactivation of the ribosomal RNA can
no longer interact with the elongation
factors
• The inhibition of protein synthesis by this
group of toxins ultimately results in death
of the target cells
 c. Toxins activating second
messenger pathway
1. Escherichia coli
. Cytotoxic necrotizing factor (CNF)
acts as deamidase, Target: Rho G-protein
. Heat-labile toxin (LT)
acts as ADP-ribosyltransferase, Target: G-
protein
. Heat-stable toxin (ST),
stimulates guanylate, Target: guanylate
cyclase receptor
 d. Toxins activating second
messenger pathway
2. Bacillus anthracis
Oedema factor: Adneylate cyclase: ATP
3. Bordetella pertussis
a. Dermonecrotic toxin: Deamidase: Rho
G-protein
b. Pertussis toxin: ADP-
ribosyltransferase: G-protein
 Toxins activating second
messenger pathway
• Deamidase,Target: Rho G-protein
a. E. coli Cytotoxic necrotizing factor
b. B. pertussis Dermonecrotic toxin
• ADP-ribosyltransferase, Target: G-protein
a. B. pertussis Pertusis toxin
b. V. cholerae Cholera toxin
• Adneylate cyclase, Target : ATP
a. B. anthracis Oedema factor
 Toxins activating second
messenger pathway
• The Rho is a subfamily of small GTP-
binding proteins that are regulators of the
actin cytoskeleton
• The modification of Rho renders it unable
to hydrolyze bound GTP
 e. Toxins activating immune
response
S. aurues enterotoxins, exfoliative toxins
and toxic-shock syndrome toxin
Act as
superantigen
On
TCR and MHC II
Food poisoning, Scalded skin syndrome
(SSS), Toxic-shock syndrome (TSS)
Toxins activating immune response
The binding of superantigen to T-cells
results in massive proliferation of up to
20% of peripheral T-cells and massive
release of cytokines (IL 1,2 and 6) which
serve as mediators of the hypotension,
high fever,and diffuse erythematous rash
Classification of bacterial toxins
Structural model
A-B subunit toxins
a. Enzymatic component (A)
b. Adhesion component (B)
 Exotoxins
A-B subunit toxins

• Subunit A, toxic component

. Originally a single polypeptide
. Proteolysed into :
. Polypeptide A1 (toxic part)
. Polypeptide A2 (moves A1 into cells)
• Subunit B, non-toxic component
associted with binding to host cell
 Exotoxins
A-B subunit toxins
• Diphtheria toxin
• Pseudomonas aeruginosa exotoxin A
• Botulinum toxin
• Tetanus toxin
• Shiga toxin
• Shiga-like toxin (verotoxin)
• Cholera toxin
 Diphtheria toxin
Corynebacterium diphthriae
• The sole pathogenic factor
• Produced in the primary site of infection in
the throat or nasopharynx
• Transported by blood to remote tissues
• Causes haemorrhagic and necrotic
damages, particularly in the heart and
nervous system
• Inhibit protein synthesis as a result of
inactivation of elongation factor 2 (EF2)
 Diphtheria toxin
62-63 kDa
• A1 fradment , 22 kDa
. Exceedingly stable to short boiling and
pH extremes
• A2 fragment , 44 kDa
. Very unstable in normal buffers
. Soluble in the presence of urea,
guanidine or sodium dodocylsulphate
 Botulinum toxin
Clostridium botulinum
• Toxic component, uniform , 150 kDa (7S)
• Non-toxic component, varible, 7S and
more, accordingly the complex may:
. Medium (M = 12 S)
. Large (L =16 S)
. Extra-large (LL= 19 S)
 Botulinum toxin
• Immunological types: A, B, C, D, E, F,G
. Type A: involves M, L and LL forms
. Types B, C, D involve M and L forms
• Toxic component is nicked by protease to:
. Light chain, 55 kDa
. Heavy chain, 100 kDa
• Neurotoxic, paralysis and death within 3-5
days from respiratory failure
 Botulism in man
• Usually food-born
. Caused by ingestion of already made
toxin in food ( canned food, fish, milk
whey)
. Most common types A, B and E
• Intestinal form in infants, spores germinate
in the intestine, multiply and produce toxin
• Wound botulism was reported in man
 Botulism in animals
• Caused mainly by types C and D
• Caused by:
. ingestion of already made toxin in feeds,
. ingestion of carcasses (cannibalism),
. ingestion of fly magots or
. driking water ( as in fish )
• Toxin can be produced in the intestine or
organs as the liver
 Tetanus toxin
Clostridium tetani
• Components A, B, and C, each 50 kDa
• The A chain is the toxic component
• The B chain forms the channel in the lipid
membrane of the host cell
• The C chain contains the ganglionic
binding site
• It acts on the central reflex apparatus of
CNS exciting motor neurons and hence
spastic paralysis, particularly in the jaw
 Shiga toxin
Shigella dysentriae type 1
• The A subunit, 30-32 kDa
Nicked by protease into:
. Enzymatic A1 fragment, 27 kDa and
. Caroxyl terminal A2 fragment, 3-5 kDa
• The B subunit, 50-65 kDa, pentameric
• It causes dysentry in man
 Shiga toxin
• Cytotoxic : death of tissue culture cells
• Enterotoxic: fluid accumulation in ileal loop
• Neurotoxic : paralysis in animals injected
• Neuronotoxic: cytotoxic to rat neuorons
• Inhibition of protein synthesis: cessation of
peptide chain elongation
 Shiga-like toxin (SLT), Verotoxin
Eshericha coli
• The A subunit, 33 kDa, 2 fragments:
. N-terminal, enzymatically active, 27 kDa
. C-terminal, 6 kDa
• The B subunit, pentameric, each 7.5 kDa
It binds SLT to glycolipid receptors
• SLT causes diarrhoea, haemorrhagic
colitis and haemolytic uraemic syndrome
in man, oedema in pigs
 Pertusis toxin
Boretella pertusis
• It is the main pathogenic factor
responsible for clinical manifestations of
whooping cough
• It has several names:
. Histamine-sensitizing factor
. Lymphocytosis promoting factor
. Islet-promoting protein
 Anthrax toxin
Bacillus anthracis
• Consists of 3 serologically distinct proteins
• The subunit A, 89 Kda (lethal factor, LF)
• The subunit A1, 83 kDa (edema factor,EF)
• The subunit B, 82.7 kDa (protective
antigen, PA)
• Causes sudden death of infected animals
• Acts on CNS leading to respiratory failure
Classification of bacterial toxins
3. Cellular / Tissue target
a. Enterotoxins
b. neurotoxins
c. leukotoxins
d. Haemolysins, etc.
4. Major biological effects
a. Dermonecrotic
b. Oedema-producing
c. Diarrhoe-producing
 Enterotoxins
* Act on the intestines causing diarrhoea
* Produced by:
• Aeromonas hydrophila
• Bacillus cereus
• Clostridium perfringens
• Escherichia coli
• Vibrio cholera
Aeromonas hydrophyla enterotoxin
• Type 1 enterotoxin, 15 kDa
. Cytotoxic
. Cholera-like, but unrelated serologically
. Heat-stable at 56C for 10 minutes
• Type 2 enterotoxin, 50 kDa
. Cytotoxic
. Heat unstable at 56 C for 10 minutes
 Bacillus cereus enterotoxin
• Type 1 enterotoxin, 39-46 kDa:
. Thermostable
. Inactivated by proteolytic enzymes
. Causes diarrhoea (within 17 h)
• Type 2 enterotoxin, 10 kDa:
. Thermostable
. Resistant to proteolytic enzymes
. Causes vomiting (within 4 h)
Clostridium perfringens enterotoxin
• Consists of a single polypeptide, 43 kDa
• Activated by trypsin
• Produced in the intestines within 8-24 h
after ingestion of foods contaminated with
C. perfringens
• Causes diarrhoea and abdominal pain
• Nausea may occur, vomiting uncommon
 Staphylococal enterotoxins
• A group of 6 serologically distinct types
. A (27.8 kDa), B (28.3 kDa), C1 (34.1
kDa), C2 (34 kDa), D and E (29.6 kDa)
. Heat-stable
. Water-soluble
. Resist trypsin and pepsin
• Sudden onset of symptoms, within 2-6 h
• Severe cramps, vomiting, diarrhoea
 Cholera toxin
Vibrio cholerae
• 1. The subunit A, 28 kDa
. Fragment A1, 21 kDa
. Fragment A2, 7 kDa
• 2. The subunits 2 are 5 identical
monovalently associated, each 11.5 kDa
 Cholera toxin
• Activates adenylate cyclase
• Increased intracellular cyclic adeosine
monophosphate
• Active secretion of chloride ions and
hydrogencarbonate ions from mucosal
cells into intestinal lumen
• Secretion into the lumen of large amount
of water resulting in watery diarrhoea
• Massive loss of body fluids, dehydration
 Escherichia coli enterotoxins
• 1. Heat-labile enterotoxins (LT)
. LT-I closely resembles cholera toxin in
structure, antigenicity and activity
. LT-II has similar activity but is distinct
from LT-1 antigenically
• 2. Heat-stable enterotoxins (ST)
. Resists heat at 100C for 15 min
. Resists proteolytic enzymes and acids
 Escherichia coli enterotoxis
• Produced by enterotoxigenic E.
coli
• Cause traveller’s diarrhoea in man
• Cause diarrhoea in infants and
young domestic animals
 Cytolysins
• Family: Thiol-activated
• Family: Not-Thiol activated
 Thiol-activated Cytolysins
• Streptolysin O • Septicolysin
• Cereolysin • Histolyticolysin
• Listeriolysin • Botulinolysin
• Pneumolysin • Tetanolysin
• Perfringolysin • Thuringolusin
 Thiol-activated Cytolysins
• Immunogenic
• Immunogenically closely related
• Differ in amino acid sequences
• Single chain
• Oxygen-labile
• Inactivated by a low level of cholestrol
Not-Thiol-activated Cytolysins
• Streptolysin S
• Haemolysin II of B. cereus
• Haemolysin of V. parahaemolyticus
• Haemolysin α, β,γ, δ of staphylococci
• Leucocidin of staphylococci
 Identification and determination of
exotoxins
• 1. Laboratory animals
• Monkeys in Staph enterotoxin (feeding)
• Mice in botulinum toxin (i.p.) with and
without antitoxin
• Guinea pigs in diphtheria toxin (i.c.), 5 h
later antitoxin is injected i.v.
• Rabbit ileal loop test in E. coli enterotoxins
• 2. Tissue cultures
 Identification and determination of
exotoxins
• 3. Serological methods as gel diffusion,
radioimmunoassay, reverse passive
haemagglutination, reverse passive latex
agglutination and ELISA
• 4. Molecular biological techniques
 Identification and determination of
endotoxins
• Schwarzman reaction
. Inject endotoxin i.d. into rabbits induces
mild inflammation
. Injection of endotoxin i.v. 24 h later
induces haemorrhaigic necrosis at the
original injection
• Limulus assay
Induction of clotting of horseshoe crab
(Limulus) ambeocyte lysates
 Therapeutic uses of toxins
• Vaccines prepared from toxins (toxoids)
are used in prevention of several diseases
e.g. diphtheria, tetanus, botulism, anthrax
• Antitoxins raised against diphtheria,
tetanus and botulism are used for passive
immunity (prophylaxis) or treatment of
seriously ill patients
 Therapeutic uses of toxins
Several potent cytotoxins are used as potential
therapies for certain cancers
• Stx binds to the cell surface glycolipid CD77,
expressed by B-cells in B-cell lymphoma
• Diphtheria toxin and P. aeruginosa exotoxin
A are engineered as the cell-killing
component of immunotoxins in the treatment
of B-cell lymphoma, leukemia etc.
• Application of streptokinase to clear blocked
arteries in patients with heart attacks
 Botulinum toxins
• 1973 type A was used to treat strabismus
in monkeys
• 1981 FDA approved type A toxin
(Oculinum, Botox) to treat strabismus
• 1999 type A toxin ( Dysport) was approved
to treat facial dystonias
• 2000 type B (Neuro Block) was approved
to treat cervical dystonia
 Application of biotechnology
• Protein engineering and vaccine design
Preparation of mutants with reduced
toxicity
• Receptor binding crystallography and drug
design leading to design receptor binding
inhibitors and blockers
 Application of biotechnology
• Receptor binding domain of all neurotoxins
is evaluated as a delivery vehicle for
therapeutic substances to the nervous
system and may be used to transport
substances across the blood-brain barrier