Bacterial toxins can be classified as exotoxins or endotoxins. Exotoxins are protein toxins secreted by bacteria, while endotoxins are structural components of the outer membrane of gram-negative bacteria. Exotoxins can be inactivated by heat or chemicals to form immunogenic toxoids, whereas endotoxins cannot. Exotoxins play an important role in several diseases by directly damaging host cells or tissues both locally and systemically.

1. Classification of Bacterial Toxins
Dr Ravi Kant Agrawal, MVSc, PhD,
Senior Scientist (Veterinary Microbiology)
Food Microbiology Laboratory
Division of Livestock Products Technology
ICAR-Indian Veterinary Research Institute
Izatnagar 243122 (UP) India

2. Chemical Poisons Vs Biological Toxins
• Poison: A chemical substance that can cause illness or death when it enters our
bodies.
• Toxin: A poison of biological origin, specifically a protein molecule produced by a
plant, animal or microbe.
• The terms are often used interchangeably.
• Toxins from bacteria are large protein molecules.
• Mycotoxins from molds are much smaller.
• Frequently toxins are the main pathogenic factor.
• Toxigenicity: Ability of a microbe to produce toxins.
• Toxemia: Presence of toxins in the blood.
• Toxin effects: May include fever, cardiovascular problems, diarrhea, shock,
destruction of red blood cells and blood vessels, and nervous system disruptions.
• Of 220 known bacterial toxins, 40% damage eucaryotic cell membranes.
• Two types of toxins:
• Exotoxins
• Endotoxins

3. What are toxins?
 A toxin (from Ancient Greek Ancient Greek: toxikon) is
a poisonous substance produced within living cells or
organisms). Synthetic toxicants created by artificial
processes are thus excluded.
 The term was first used by organic chemist Ludwig
Brieger.
 Toxins can be small molecules, peptides,
or proteins that are capable of causing disease on
contact with or absorption by body tissues interacting
with biological macromolecules such
as enzymes or cellular receptors.
 Toxins vary greatly in their severity, ranging from
usually minor (such as a bee sting) to almost
immediately deadly (such as Botulinum toxin).
 Toxins were the first bacterial virulence factors to be
identified (Diphtheria toxin isolated in 1888), and were
also the first link between bacteria and cell biology.
 Cellular microbiology was, in fact, naturally born a
long time ago with the study of toxins, and only
recently, thanks to the sophisticated new
technologies, has it expanded to include the study of
many other aspects of the interactions between
bacteria and host cells.

4.  There goes an ongoing terminological dispute between
NATO and the Warsaw pact over whether to call a toxin a
biological or chemical agent, in which NATO opted for
biological agent, and the Warsaw pact, for chemical agent.
 According to Title 18 of united states code, the term “toxin”
means toxic material or product of plants, animals,
microorganisms (including but not limited to bacteria,
viruses, fungi, or protozoa) or infectious substances,
whatever their origin and method of production.
Radioactive symbol Toxic hazard symbol Biohazard symbol

5. Types of toxicity
• There are generally three types of toxic entities; chemical,
biological, and physical:
• Chemical toxicants include inorganic substances such
as lead, mercury, hydrofluoric acid, and chlorine gas, and organic
compounds such as methyl alcohol, most medications, and poisons
from living things.
• Biological toxicants include bacteria and viruses that can induce
disease in living organisms. Biological toxicity can be difficult to
measure because the "threshold dose" may be a single organism.
Theoretically one virus, bacterium or worm can reproduce to cause
a serious infection.
• Physical toxicants are substances that, due to their physical nature,
interfere with biological processes. Examples
include coal dust, asbestos fibers or finely divided silicon dioxide,
all of which can ultimately be fatal if inhaled. Corrosive chemicals
possess physical toxicity because they destroy tissues, but they're
not directly poisonous unless they interfere directly with biological
activity.

6. Toxicity definition
• The degree to which a substance (a toxin or poison) can harm humans or animals.
• Toxicity is the degree to which a substance can damage an organism.
• Toxicity can refer to the effect on a whole organism, such as an animal , bacterium , or plant ,
as well as the effect on a sub-structure of the organism, such as a cell (cytotoxicity) or an organ
such as the liver (hepatotoxicity).
• By extension, the word may be metaphorically used to describe toxic effects on larger and
more complex groups, such as the family unit or society at large.
Types:
• Acute toxicity involves harmful effects in an organism through a single or short-
term exposure.
• Subchronic toxicity is the ability of a toxic substance to cause effects for more than
one year but less than the lifetime of the exposed organism.
• Chronic toxicity is the ability of a substance or mixture of substances to cause
harmful effects over an extended period, usually upon repeated or continuous
exposure, sometimes lasting for the entire life of the exposed organism.
• Toxicity depends on dose.
• A central concept of toxicology is that effects are dose-dependent; even water can
lead to water intoxication when taken in too high a dose, whereas for even a very
toxic substance such as snake venom there is a dose below which there is no
detectable toxic effect.
• Some substances are beneficial at low concentration and toxic at high conc.

7. Measuring toxicity
• Assessing all aspects of the toxicity of cancer-causing agents involves
additional issues, since it is not certain if there is a minimal effective dose
for carcinogens , or whether the risk is just too small to see.
• It is more difficult to determine the toxicity of chemical mixtures than a
pure chemical, because each component displays its own toxicity, and
components may interact to produce enhanced or diminished effects.
Common mixtures include gasoline, cigarette smoke, and industrial waste.
Even more complex are situations with more than one type of toxic entity,
such as the discharge from a malfunctioning sewage treatment plant, with
both chemical and biological agents.
• The preclinical toxicity testing on various biological systems reveals the
species-, organ- and dose- specific toxic effects of an investigational
product.
• The toxicity of substances can be observed by (a) studying the accidental
exposures to a substance (b) in vitro studies using cells/ cell lines (c) in vivo
exposure on experimental animals.
• Toxicity tests are mostly used to examine specific adverse events or
specific end points such as cancer, cardiotoxicity, and skin/eye irritation.
• Toxicity testing also helps calculate the “No Observed Adverse Effect
Level” (NOAEL) dose and is helpful for clinical studies.

8. Antitoxin
• An antitoxin is an antibody with the ability to neutralize a specific toxin.
• This procedure involves injecting an animal with a safe amount of a
particular toxin. Then, the animal’s body makes the antitoxin needed to
neutralize the toxin. Later, the blood is withdrawn from the animal. When
the antitoxin is obtained from the blood, it is purified and injected into a
human or other animal, inducing passive immunity.
• Antitoxin, antibody, formed in the body by the introduction of a bacterial
poison, or toxin, and capable of neutralizing the toxin. People who have
recovered from bacterial illnesses often develop specific antitoxins that
confer immunity against recurrence.
• To prevent serum sickness, it is often best to use antitoxin generated from
the same species . (e.g. use human antitoxin to treat humans).
• Antitoxins to diphtheria and tetanus toxins were produced by
Emil Adolf von Behring and his colleagues from 1890 onwards.
• The use of diphtheria antitoxin for the treatment of diphtheria was
regarded by the Lancet as the "most important advance of the [19th]
Century in the medical treatment of acute infectious disease".
• For medical use in treating human infectious diseases, antitoxins are
produced by injecting an animal with toxin; the animal, most commonly a
horse, is given repeated small doses of toxin until a high concentration of
the antitoxin builds up in the blood. The resulting highly concentrated
preparation of antitoxins is called an antiserum.

9. Two Main Categories of Toxins
• Exotoxin: Easily inactivated by formaldehyde, iodine, and other chemicals to form immunogenic
toxoids.

10. 10

11. Characteristic of endotoxin and exotoxin
Property Exotoxin Endotoxin
Chemical nature Protein (50-100kDa) Polysaccharide (10kDa)
Relationship to cell Extracelluler Part of outer membrane
Denatured by boiling Usually No
Form toxoid Yes No
Potency High (1 µg) Low (> 100 µg)
Antigenic Yes Yes
Specificity High degree Low degree
Pyrogenicity Occasionally Yes
Enzymatic activity Usually No

12. Exotoxins versus Endotoxins

13. Endotoxins

15. Role of bacterial exotoxins in disease
•In the first example, the exotoxin is produced by bacteria growing in food.
When food is consumed, the preformed exotoxin is also consumed. The
classical example is staphylococcal food poisoning caused solely by the
ingestion of preformed enterotoxin. Since the bacteria (Staphylococcus
aureus) cannot colonize the gut, they pass through the body without
producing any more exotoxin; thus, this type of bacterial disease is self-
limiting.
•In the second example, bacteria colonize a mucosal surface but do not invade
underlying tissue or enter the bloodstream. The toxin either causes disease
locally or enters the bloodstream and is distributed systemically where it can
cause disease at distant sites. The classical example here is the disease
cholera caused by Vibrio cholerae. Once the bacteria enter the body, they
adhere to the intestinal mucosa where they are not invasive but secrete the
cholera toxin, which is an AB exotoxin that catalyzes an ADP – ribosylation
similar to that of diphtheria exotoxin. As a result, cholera toxin stimulates
hypersecretion of water and chloride ions and the patient loses massive
quantities of water.
•The third example of exotoxins in disease pathogenesis occurs when bacteria
grow in a wound or abscess. The exotoxin causes local tissue damage or kills
phagocytes that enter the infected area. A disease of this type is gas
gangrene in which the exotoxin (-toxin) of Clostridium perfringens causes the
tissue destruction in the wound.

17. EXOTOXINS
Can be classified on the basis of
 Site of action
 Mode of action
 Biochemical target
 Structure

18. By Site of Action, cellular or anatomical
 Neurotoxins
 Enterotoxins
 Cytotoxins - Nephrotoxin, Hepatotoxin, Cardiotoxin
 Cytolytic toxins - haemolysins, Leucotoxins/leucocidins
 Dermonecrotic toxins
 Superantigens
Specific Host Site Exotoxins

19. By Mode of Action
 ADP-ribosyltransferase e.g. Cholera toxin
 Glucosyltransferase e.g. Clostridium difficile toxin A (TcdA)
 Zinc endopeptidases e.g. Clostridium tetani neurotoxin
 N-glycosidase e.g. Shigella dysenteriae Shiga toxin
 Deamidase e.g. Bordetella pertussis Dermonecrotic toxin
 Metalloprotease e.g. Bacillus anthracis Lethal factor
 Adenylate cyclase e.g. Bordetella pertussis and Bacillus anthracis (Oedema
factor)
 Phospholipases C (lecithinase, sphingomyelinase) e.g. Staphylococcus
aureus β-toxin
 Pore-forming toxins e.g. Staphylococcus aureus α-toxin
 Superantigens e.g. Staphylococcus aureus Toxic Shock Syndrome Toxin
(TSST-1)

20. By Biochemical Target Site
 Cholesterol e.g. streptolysin O (SLO) of Streptococcus pyogenes
 Elongation factor 2 e.g. diphtheria toxin (DT)
 Guanylate cyclase e.g. ST enterotoxin of Escherichia coli
 28 S rRNA e.g. Shiga toxin
 Rho family of small GTP-binding proteins e.g. Bordetella pertussis
dermonecrotic toxin
 Synaptobrevin e.g. Clostridium tetani neurotoxin
 G-actin e.g. C2 toxin of Clostridium botulinum

21. By structure of Bacterial Toxins
 Single chain polypeptides e.g. botulinum toxin, diphtheria toxin
 Oligomeric molecules – Multimeric complexes comprising two or more non-
covalently linked subunits, e.g., cholera toxin, shiga toxin
 Macromolecular complexes associated with non-toxic moieties, e.g.
botulinum neurotoxins (HA and non-HA proteins)
 Binary toxins – composed of two independent polypeptide chains e.g.
anthrax toxin (LF + PA or EF + PA)
 Protoxins – toxins secreted in inactive forms (proenzymes) that are converted
to active forms by proteolytic enzymes, e.g., iota toxin of Clostridium
perfringens, botulinum C2 toxin

22. three groups of bacterial toxins.
•Group 1 toxins act on cell membrane
act either by binding receptors => sending a signal to the cell
or by forming pores => perturbing the cell permeability barrier.
•Group 2 toxins are A/B toxins,
binding domain (B subunit), enzymatically active (A subunit).
•Group 3 toxins are injected directly from the bacterium into the cell by a
specialized secretion apparatus (type III or type IV secretion system).
• Group 4 toxins are toxins with unknown mechanism of action.
Classification by Entrance Mechanism

23. Acting on
intracellular
targets
Injected into
eukaryotic cells
Unknown
mechanism of
action
Acting on the
cell surface
Immune system
(Superantigens)
ClassTarget
Surface molecules
Cell membrane
Large pore- forming toxins
Small pore- forming toxins
Insecticidal toxins
Membrane-perturbing
toxins
Other pore- forming
toxins
RTX toxins
Protein synthesis Mediators of apoptosis
Signal transduction
Cytoskeleton structure
Intracellular trafficking
Inositol phosphate
metabolism
Cytoskeleton
Signal transduction

26. Toxins acting on the cell surface:
Immune system (Superantigens)
• Superantigens are bacterial and
viral proteins that share the ability
to activate a large fraction of T-
lymphocytes.
Acting on the
cell surface
Immune system
(Superantigens)
ClassTarget
Surface molecules
Cell membrane
Large pore- forming toxins
Small pore- forming toxins
Insecticidal toxins
Membrane-perturbing
toxins
Other pore- forming
toxins
RTX toxins

27. Toxins acting on the cell surface: Immune system
(Superantigens)
Toxin Organism Activity Consequence
SEA-SEI, TSST-1, SPEA,
SPEC, SPEL, SPEM, SSA,
and SMEZ
SPE-streptococcal
exotoxin; SMEZ,
streptococcal mitogenic
exotoxin z
SSA, streptococcal
superantigen
Staphylococcus
aureus and
Streptococcus
pyogenes
Binding to MHC class II
molecules and to Vβ or
Vγ of T cell receptor
T cell activation and
cytokines secretion
MAM (Mycoplasma
arthritidis mitogen)
Mycoplasma
arthritidis
Binding to MHC class II
molecules and to Vβ or
Vγ of T cell receptor
Chronic
inflammation
YPMa
Y. pseudotuberculosis-
derived mitogen
Yersinia
pseudotuberculosis
Binding to MHC class II
molecules and to Vβ or
Vγ of T cell receptor
Chronic
Inflammation
SPEB
streptococcal exotoxin S. pyogenes Cysteine protease
Alteration in
Immunoglobulin
binding properties
ETA, ETB, and ETD
exfoliative toxins S. aureus Trypsin-like serine
proteases
T-cell proliferation,
intraepidermal
layer separation

28. Toxins acting on the cell surface:
Surface molecules
 BFT enterotoxin: The pathogenicity of ETBF is ascribed to a
heat-labile 20-kDa toxin (∼ B. fragilis toxin [BFT], also called
fragilysin).
 This toxin binds to a specific intestinal epithelial cell receptor
and stimulates cell proliferation.

29. Toxins acting on the cell surface: Surface molecules
Toxin Organism Activity Consequence
BFT enterotoxin Bacteroides fragilis Metalloprotease,
cleavage of E-
cadherin
Alteration of
epithelial
permeability
AhyB Aeromonas
hydrophyla
E l a s t a s e ,
metalloprotease
Hydrolization of
casein and elastine
Aminopeptidase Pseudomonas
aeruginosa
E l a s t a s e ,
metalloprotease
Corneal infection,
inflammation and
ulceration
ColH
(collagenase)
Clostridium
histolyticum
Collagenase,
metalloprotease
Collagenolytic activity
Nhe
(nonhemolytic
entertoxin)
Bacillus cereus Metalloprotease and
collagenase
Collagenolytic activity

30. Toxins acting on the cell surface:
Pore-Forming
• Protein toxins forming pores in biological membranes occur
frequently in Gram-positive and Gram-negative bacteria.
• Pore-forming toxins, also known as “lytic factors”.
• Some of them which act on RBCs are also called “hemolysins”.

31. Toxins acting on the cell surface:
Large Pore-Forming Toxins
• Generally secreted by diverse species of Gram-positive
bacteria.
• Binding selectively to cholesterol on the eukaryotic cell
membrane.

32. Toxins acting on the cell surface: Large pore forming toxins
Toxin Organism Activity Consequence
PFO
perfringolysin
O
C. perfringens Thiol-activated cytolysin,
cholesterol Binding Gas gangrene
SLO
streptolysin O
S. pyogenes Thiol-activated cytolysin,
cholesterol Binding
Transfer of other toxins,
cell death
LLO
listeriolysin O
L.
monocytogenes
Induction of Lymphocyte
apoptosis
Membrane damage
Pneumolysin S. pneumoniae Induction of Lymphocyte
Apoptosis
Complement activation,
cytokine production,
apoptosis

33. Toxins acting on the cell surface:
Small pore forming toxins
• Creating very small pores 1-1.5 nm diameter.
• Selective permeabilization to solutes with a molecular mass
less than 2 kDa.

34. Toxins acting on the cell surface: Small pore forming toxins
Toxin Organism Activity Consequence
Alveolysin B. alveis Induction of
lymphocyte Apoptosis
Complement activation,
cytokine production, apoptosis
ALO
anthrolisin O
B. anthracis Induction of
lymphocyte apoptosis
Complement activation,
cytokine production, Apoptosis
α-Toxin S. aureus Binding of erythrocytes Release of cytokines, cell lysis,
apoptosis
PVL leukocidin
(LukS-LukF)
S. aureus Cell membrane
permeabilization
Necrotic enteritis, rapid shock-
like syndrome
γ-Hemolysins
(HlgA- HlgB and
HlgC- HlgB)
S. aureus Cell membrane
permeabilization
Necrotic enteritis, rapid shock-
like syndrome
β-Toxin C. perfringens Cell membrane
permeabilization
Necrotic enteritis, neurologic
effects

35. Toxins acting on the cell surface:
RTX toxins
• The RTX toxin family is a group of cytotoxins produced by
Gram-negative bacteria.
• There are over 1000 known members with a variety of
functions.
• The RTX family is defined by two common features:
characteristic repeats in the toxin protein sequences, and
extracellular secretion by the type I secretion system (T1SS).
• The name RTX (repeats in toxin) refers to the glycine and
aspartate-rich repeats located at the C-terminus of the toxin
proteins.

36. Genomic Structure
• The toxin is encoded by four genes, one of which, hlyA,
encodes the 110-kDa hemolysin. The other genes are required
for its post-translational modification (hlyC) and secretion
(hlyB and hlyD).

37. Toxins acting on the cell surface: RTX toxins
Toxin Organism Activity Consequence
Hemolysin II B. cereus Cell membrane permeabilization Hemolytic activity
CytK B. cereus Cell membrane Permeabilization Necrotic enteritis
HlyA E. coli Calcium-dependent formation of
transmembrane Pores
Cell permeabilization and
lysis

38. Toxins acting on the cell surface: Membrane
perturbing toxins
• Soap like structure.
• The toxin binds nonspecifically parallel to the surface of any
membrane without forming transmembrane channels.
• Cells first become permeable to small solutes and eventually
swell and lyse, releasing cell intracellular content.

39. Toxins acting on the cell surface: Membrane perturbing
toxins
Toxin Organism Activity Consequence
ApxI, ApxII, and
ApxIII
A.
pleuropneumoniae
Exotoxin I, II and III
A. pleuropneumoniae
Calcium-dependent
formation of
transmembrane
Pores
Lysis of
erythrocytes and
other nucleated
Cells
LtxA
Leucotoxin A
A.
actinomycetemcomitans
Calcium-dependent
formation of
transmembrane
Pores
Apoptosis
LtxA
Leucotoxin A
P. haemolytica
Calcium-dependent
formation of
transmembrane
Pores
Activity specific
versus ruminant
leukocytes

40. Toxins acting on the cell surface: Other pore forming
toxin
• Like other functionally related toxins, aerolysin changes its
topology in a multi-step process from a completely water-
soluble form to a membrane-soluble heptameric
transmembrane channel that destroys sensitive cells by
breaking their permeability barriers.

41. Toxins acting on the cell surface: Other pore forming
toxins
Toxin Organism Activity Consequence
δ-Hemolysin S. aureus
Perturbation of the lipid
bilayer
Cell permeabilization and lysis
Aerolysin A. hydrophila
Perturbation of the lipid
bilayer
Cell permeabilization and lysis
AT
α-toxin
C. septicum
Perturbation of the lipid
bilayer
Cellpermeabilization and lysis

42. Toxins acting on the cell surface: Insecticidal
toxins
 The class of insecticidal proteins, also known as δ-endotoxins, includes a
number of toxins produced by Bacillus thuringiensis.
 These exert their toxic activity by making pores in the epithelial cell
membrane of the insect midgut.
 δ-Endotoxins form two multigenic families, cry and cyt;
 members of the cry family are toxic to insects of Lepidoptera, Diptera
and Coleoptera orders (Hofmann et al., 1988),
 whereas members of the cyt family are lethal specifically to the larvae
of Dipteran insects (Koni and Ellar, 1994).
 Lepidoptera is a large order of insects that includes moths and butterflies.
 True flies are insects of the order Diptera.
 Coleoptera is an order of insects commonly called beetles.

43. Toxins acting on the cell surface: Insecticidal toxins
Toxin Organism Activity Consequence
PA B. anthracis Perturbation of the lipid
bilayer
Cell permeabilization and lysis
HlyE E. coli Perturbation of the lipid
bilayer
Osmotic lysis of cells lining the
Midgut
CryIA,
CryIIA,
CryIIIA, etc
Bacillus
thuringiensis
Destruction of the
transmembrane
Potential
Osmotic lysis of cells lining the
Midgut
CytA, CytB B. thuringiensis
Destruction of the
transmembrane
Potential
Osmotic lysis of cells lining the
Midgut
BT toxin B. thuringiensis
Destruction of the
transmembrane
Potential
Cytocidal activity on human cells

44. Toxins Acting on Intracellular Targets
• The group of toxins with an intracellular
target (A/B toxins) contains many toxins with
different structures that have only one
general feature in common: they are
composed of two domains generally
identified as “A” and “B”.
 The A domain is the active portion of the
toxin; it usually has enzymatic activity and
can recognize and modify a target molecule
within the cytosol of eukaryotic cells.
 The B domain is usually the carrier for the A
subunit; it bind the receptor on the cell
surface and facilitates the translocation of A
across the cytoplasmic membrane.
Actingon
intracellular
targets
Actingonthe
cell surface
Immune system
(Superantigens)
Class
Surface molecules
Cellmembrane
Largepore-forming toxins
Proteinsynthesis
Signal transduction
Cytoskeletonstructure
Intracellulartrafficking

45. Toxins acting on intracellular targets: Protein synthesis
• These toxins are able to cause rapid cell death at extremely low
concentrations.
• The reaction leads to the formation of a completely inactive EF2-ADP-
ribose complex.
• A very important step in the elucidation of the mechanism of
enzymatic activity has been the determination of the crystal
structure for the complex of diphtheria toxin with NAD.
• Upon the addition of NAD to nucleotide-free DT crystals, a
significant structural change occurs.
• This change lead to recognition and binding of the acceptor
substrate EF-2.
• This would explain why DT recognizes EF-2 only after NAD has
bound.

46. Toxins acting on intracellular targets:
Protein synthesis
Toxins acting on intracellular targets: Protein synthesis
Toxin Organism Activity Consequence
DT
Corynebacterium
diphtheriae
ADP-ribosylation of EF-2 Cell death
PAETA P. aeruginosa ADP-ribosylation of EF-2 Cell death
SHT S. dysenteriae N-glycosidase activity on 28S RNA Cell death, apoptosis

47. Toxins acting on intracellular targets: Signal
transduction
• Two types of transduction mechanism:
– Tyrosine phosphorylation
– Modification of a receptor-coupled GTP-binding protein
• cyclic AMP
• inositol triphosphate
• diacylglycerol

48. Pertussis toxin
• The A domain acts on eukaryotic cells by ADP-ribosylating their GTP-binding
proteins
• The B domain is a nontoxic oligomer that binds the receptors on the surface
of eukaryotic cells and allows the toxic subunit S1 to reach its intracellular
target proteins.
• Role of many residues of S1 has been tested by site-directed mutagenesis to
produce nontoxic mutants of the toxin to be used as vaccines.
PT Subunits
A B

49. Cholera toxin (CT) and E. coli heat-labile enterotoxins (LT-I and
LT-II)
• Cholera toxin (CT) and E. coli heat-labile enterotoxins (LT-I and LT-II) share
an identical mechanism of action and homologous primary and 3D
structures.
• While V. cholerae exports the CT toxin into the culture medium, LT
remains associated to the outer membrane bound to lipopolysaccharide.
• The corresponding genes of CT and LT are organized in a bicistronic
operon and are located on a filamentous bacteriophage and on a plasmid,
respectively.

50. Clostridium difficile Toxins
• Enterotoxin A (TcdA) and cytotoxin B (TcdB) of Clostridium difficile are the
two virulence factors responsible for the induction of antibiotic-associated
diarrhea.
• The toxin genes tcdA and tcdB together with three accessory genes (tcdC-
E) constitute the pathogenicity locus (PaLoc) of C. difficile.

51. Toxins acting on intracellular targets: Signal transduction
1
Toxin Organism Activity Consequence
PT Bordetella pertussis ADP-ribosylation of Gi cAMP increase
CT Vibrio cholerae ADP-ribosylation of Gi cAMP increase
LT E. coli ADP-ribosylation of Gi cAMP increase
α-Toxin (PLC) C. perfringens
Zinc-phospholipase C,
hydrolase
Gas gangrene
Toxins A and B
(TcdA and TcdB)
C. difficile
Monoglucosylation of Rho,
Rac, Cdc42
Breakdown of
cellular actin stress
fibers
Adenylate
cyclase (CyaA)
B. pertussis
Binding to calmodulin
ATP→cAMP conversion
cAMP increase

52. Anthrax Edema and Lethal Factors
• The EF and LF genes are located on a large plasmids.
• Cleavage of the N-terminal signal peptides yields mature EF and
LF proteins.
• LF, is able to cause apoptosis in human endothelial cells.

53. E. coli Cytotoxin Necrotizing Factors (CNF) and
Bordetella Dermonecrotic Toxin (DNT)
• CNF1 & CNF2: produced by a number of
uropathogenic and neonatal meningitis-
causing pathogenic E. coli strains.
• cnf1 is chromosomally encoded, cnf2 is
carried on a large transmissible F-like plasmid
called “Vir”.
• DNT is a transglutaminase, which causes
alteration of cell morphology, re-organization
of stress fibers, and focal adhesions on a
variety of animal models.

54. Cytolethal Distending Toxins
• HdCDT is a complex of three proteins (CdtA, CdtB and CdtC)
encoded by three genes that are part of an operon.
• Members of this family have been identified in E. coli, Shigella,
Salmonella, Campylobacter, Actinobacillus and Helicobacter
hepaticus.

55. Toxins acting on intracellular targets: Signal transduction 2
Toxin Organism Activity Consequence
Anthrax edema
factor (EF)
B. anthracis
Binding to calmodulin
ATP→cAMP conversion
cAMP increase
Anthrax lethal factor
(LF)
B. anthracis
Cleavage of MAPKK1 and
MAPKK2
Cell death, apoptosis
Cytotoxin necrotizing
factors 1 and 2
(CNF1, 2)
E. coli
Deamidation of Rho, Rac
and Cdc42
Ruffling, stress fiber
formation.
DNT
Bordetella
species
Transglutaminase,
deamidation or
polyamination of Rho
GTPase
Ruffling, stress fiber
formation
CDT
Several
species
DNA damage, formation of
actin stress fibers via
activation of RhoA
Cell-cycle arrest,
cytotoxicity,
apoptosis

56. Toxins acting on intracellular targets: Cytoskeleton
structure
• The cytoskeleton is a cellular structure that consists of a fiber
network composed of microfilaments, microtubules, and the
intermediate filaments.
• It controls a number of essential functions in the eukaryotic
cell:
 Exo- and endocytosis
 Vesicle transport
 Cell-cell contact and
 Mitosis
• Most of them do it by modifying the regulatory, small G
proteins, such as Ras, Rho, and Cdc42, which control cell shape.

57. Lymphostatin
• Lymphostatin is a very recently identified protein in enteropathogenic
strains of E. coli.
• Lymphostatin selectively block the production of IL-2, IL-4, IL-5 and γ-
interferon by human cells and inhibit proliferation of these cells, thus
interfering with the cellular immune response.

58. Toxins acting on intracellular targets: Cytoskeleton
structure
Toxin Organism Activity Consequence
Toxin C2 and
related proteins
C. botulinum
ADP-ribosylation of monomeric G
actin
Failure in actin
polymerization
Lymphostatin E. coli Block of interleukin production Chronic diarrhea
Iota toxin and
related proteins
C. perfringens Block of interleukin production Chronic diarrhea

59. Toxins acting on intracellular targets:
Intracellular trafficking
• Vesicle structures are essential in:
– receptor-mediated endocytosis
– and exocytosis
• One example of exocytic pathway is that involving the release
of neurotransmitters

60. Mechanism of action of clostridial neurotoxins (CNT)
Synaptosomal-associated protein 25 (SNAP-25)
Synaptosomal-associated protein 25
(SNAP-25)
SNARE: SNAP REceptor
Acetylcholine & neurotransmiters

61. Helicobacter pylori Vacuolating Cytotoxin Vac A
• This toxin is responsible for massive growth of
vacuoles within epithelial cells.
• VacA can insert into membranes forming
hexameric, anion-selective pores.

62. Toxins acting on intracellular targets: Intracellular
trafficking
Toxin Organism Activity Consequence
TeNT
tetanus neurotoxin
C. tetanii
Cleavage of VAMP/
synaptobrevin
Spastic paralysis
BoNT-B, D, G and F
neurotoxins
C.
botulinum
Cleavage of VAMP/
synaptobrevin
Flaccid paralysis
BoNT-A, E
neurotoxins
C.
botulinum
Cleavage of SNAP-25 Flaccid paralysis
BoNT-C neurotoxin
C.
botulinum
Cleavage of syntaxin, SNAP-25 Flaccid paralysis
Vacuolating
cytotoxin VacA
H. pylori
Alteration in the endocytic
pathway
Vacuole formation,
apoptosis
NAD glycohydrolase S. pyogenes Keratinocyte apoptosis
Enhancement of GAS
proliferation
Vesicle associated membrane proteins (VAMP) are a family of SNARE proteins with similar
structure, and are mostly involved in vesicle fusion.

63. Toxins injected into eukaryotic cells
• These bacteria intoxicate individual eukaryotic
cells by using a contact-dependent secretion
system to inject or deliver toxic proteins into the
cytoplasm of eukaryotic cells.
• This is done by using specialized secretion
systems that in Gram-negative bacteria are called
"type III" or "type IV,“.
Actingon
intracellular
targets
Injectedinto
eukaryoticcells
Actingonthe
cellsurface
Immunesystem
(Superantigens)
Class
Surfacemolecules
Cellmembrane
Largepore-formingtoxins
Proteinsynthesis Mediatorsofapoptosis
Signaltransduction
Cytoskeletonstructure
Intracellulartrafficking
Inositolphosphate
metabolism
Cytoskeleton
Signaltransduction

64. Toxins injected into eukaryotic cells: Mediators of
apoptosis: IpaB in Shigella
• Shigella invasion plasmid antigen (Ipa) proteins: IpaA, IpaB,
IpaC, IpaD.
• Only IpaB is required to initiate cell death.

65. Toxins injected into eukaryotic cells: Mediators of
apoptosis: SipB in Salmonella
• Salmonella invasion protein B (SipB) is an analog of Shigella
invasin IpaB.
• In contrast to Shigella, Salmonella does not escape from the
phagosome, but it survives and multiplies within the
macrophages.
• Salmonella virulence genes are encoded by a chromosomal
operon named sip containing five genes (sipEBCDA).

66. Toxins injected into eukaryotic cells: Mediators of
apoptosis
Toxin Organism Activity Consequence
IpaB Shigella
Binding to ICE
(interleukin-1β-converting enzyme)
Apoptosis
SipB Salmonella Cysteine proteases Apoptosis
YopP/YopJ
Yersinia outer
protein
Yersinia species
Cysteine protease, blocks MAPK
and NFkappaB pathways
Apoptosis
 ICE: Caspase 1/interleukin-1 converting enzyme is an enzyme that
proteolytically cleaves other proteins, such as the precursor forms of the
inflammatory cytokines interleukin 1-β and interleukin 18, into active mature
peptides.
 Mitogen-activated protein kinases are involved in directing cellular responses
to a diverse array of stimuli, such as mitogens, osmotic stress, heat shock.
 NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) is a
protein complex that controls the transcription of DNA.
 NF-κB is found in almost all animal cell types and is involved in cellular
responses to stimuli such as stress, cytokines, free radicals, ultraviolet
irradiation, oxidized LDL and bacterial or viral antigens

67. Toxins injected into eukaryotic cells: Inositol
phosphate metabolism
• Salmonella outer protein B (SopB): in Salmonella is homologous
to the Shigella flexneri invasion plasmid gene D (lpgD) virulence
factor.
• Both proteins contain two regions of sequence similarities with
human inositol polyphosphatases types I and II.

68. Toxins injected into eukaryotic cells: Inositol phosphate
metabolism
Toxin Organism Activity Consequence
SopB
Salmonella outer
protein B
Salmonella
species
Inositol phosphate phosphatase,
cytoskeleton rearrangements
Increased chloride
secretion (diarrhea)
IpgD
Invasion plasmid
gene D
S. flexneri
Inositol phosphate phosphatase,
cytoskeleton rearrangements
Increased chloride
secretion (diarrhea)

69. Toxins injected into eukaryotic cells: Signal transduction
Toxin Organism Activity Consequence
ExoS P. aeruginosa
ADP-ribosylation of Ras,
Rho GTPase
Collapse of cytoskeleton
C3 exotoxin C. botulinum ADP-ribosylation of Rho Breakdown of cellular actin stress fibers
EDIN-A, B
and C
S. aureus ADP-ribosylation of Rho Modification of actin cytoskeleton
SopE S. typhimurium Rac and Cdc42 activation
Membrane ruffling, cytoskeletal
reorganization, proinflammatory
cytokines production
SipA S. typhimurium Rac and Cdc42 activation
Membrane ruffling, cytoskeletal
reorganization, proinflammatory
cytokines production
IpaA Shigella species Vinculin binding Depolymerization of actin filaments
YopE Yersinia species
GAP activity towards
RhoA, Rac1 or Cdc42
Cytotoxicity, actin depolymerization
YopT Yersinia species
Cysteine protease,
cleaves RhoA, Rac, and
Cdc42 releasing them
from the membrane
Disruption of actin cytoskeleton
VirA Shigella flexneri
Inhibition of tubulin
polymerization
Microtubule destabilization and
membrane ruffling

70. Toxins injected into eukaryotic cells: Signal transduction
Toxin Organism Activity Consequence
YpkA
(Yersinia protein kinase
A)
Yersinia species
Protein
serine/threonine
kinase
Inhibition of
phagocytosis
YopH
Yersinia outer protein
Yersinia species Tyrosine
phosphatase
Inhibition of
phagocytosis
Tir
(Translocated intimin
receptor)
E. coli EPEC Receptor for intimin Actin nucleation and
pedestalformation
CagA
(Cytotoxin-associated
gene A)
H. pylori Tyrosine
phosphorylated
Cortactin
dephosphorylation
YopM
(Yersinia outer protein) Yersinia species
Interaction with
PRK2 and RSK1
kinases
Cytotoxicity
SptP
(Salmonella
typhimurium protein
tyrosine phosphatase)
S. typhimurium
Inhibition of the
MAP kinase
pathway
Enhancement of
Salmonella capacity to
induce TNF-alpha
secretion
ExoU
(Exoenzyme U)
P. aeruginosa Lysophospholipase
A activity
Lung injury

71. Toxins with unknown mechanism of action
Toxin Organism Activity Consequence
Zot
zonula
occludens
toxin
V. cholerae ?
Modification of intestinal tight
junction permeability
Hemolysin
BL (HBL)
B. cereus
Hemolytic,
dermonecrotic and
vascular permeability
activities
Food poisoning, fluid
accumulation and diarrhea
BSH
bile salt
hydrolase
L. monocytogenes ?
Increased bacterial survival
and intestinal colonization
Toxins with unknown mechanism of action

72. Toxins with enzymatic activities
Enzymatic activities
Glucosyl-transferases
Deamidases
ADP-ribosyltransferases
N-Glycosidases
Metalloproteases

73. ADP-ribosyltransferases
DT ElongationfactorEF-2 Celldeath
PAETA ElongationfactorEF-2 Celldeath
PT Gi,Go andtransducin
CT
Gs,Gt andGolf
cAMPincrease
E.coli LT
Clostridium botulinumC2 Actin Failureinactin
P.aeruginosaExoS Ras Collapseofcytoskeleton
Clostridium botulinumC3 Rho
Breakdownofcellularactin stress
fibers
ADP-ribosyltransferases Toxin Substrate Effect
Paeta: Pseudomonas aeroginosa exotoxin a

74. ClostridiumdifficiletoxinsAandB Rho/RasGTPases Breakdownofcytoskeletalstructure
Toxin Substrate Effect
Glucosyl-transferases
Deamidases
E.coliCNF1 Rho,RacandCdC42
BordetellaDNT Rho,
Stressfiberformation
Shigatoxin RibosomalRNA
Disruptionofnormalhomoeostatic
functions
N-Glycosidases
Metalloproteases
BacillusanthracisLF Macrophages
ClostridiumtetaniiTeNT VAMP/synaptobrevin Spasticparalysis
FlaccidparalysisC.botulinumBoNTs VAMP/synaptobrevin,SNAP-25
Stopofproteinsynthesis

75. Thanks
Acknowledgement: All the presentations available online on the
subject are duly acknowledged.
Disclaimer: The author bear no responsibility with regard to the
source and authenticity of the content.