Bacterial Exotoxins
• First bacterial virulence factors identified
• Diphtheria toxin isolated in 1888
• Enterocyte intracellular signaling leading to intestinal
secretion. Four main pathways seem to be involved in
intestinal secretion of water and electrolytes: cAMP,
cGMP, Ca, and cytoskeleton. These pathways are
activated by several enteric pathogens, either directly or
through elaboration of enterotoxic products. CT, cholera
toxin; LT, heat-labile enterotoxin; TDH, thermostable
direct hemolysin; CD, Clostridium difficile; EAST1,
enteroaggregative E. coli heat-stable toxin 1; STa, heatstable toxin a; AC, adenylate cyclase; GC, guanylate
cyclase; CM, calmodulin; PKC, protein kinase C; ZOT,
zonula occludens toxin; EGF-R, epidermal growth factor
receptor; ECM, extracellular matrix; MAPK, mitogenactivated protein kinase.
• Mode of action of CT. A: adenylate
cyclase, located in basolateral membrane
of intestinal epithelial cells, is regulated by
G proteins. CT binds via the B subunit
pentamer to GM1 ganglioside receptor
inserted in lipid bilayer.
• the A subunit enters the cell, perhaps via endosomes, and is
proteolitically cleaved into A1 and A2 peptides. A1 is activated and
transfers an ADP-ribose moiety (ADPR) and NAD to the -subunit of
Gs protein. ADP-ribosylated -subunit dissociates from other
subunits of Gs and activates adenylate cyclase, thereby increasing
intracellular cAMP concentration. Three possible scenarios have
been proposed to explain entry of the toxin and activation of
adenylate cyclase: 1) A1 subunit translocates through apical
membrane, leaving B pentamer on apical membrane; 2) A1 peptide
ADP-ribosylates an -subunit in apical membrane, and the ADPribosylated -subunit traverses the cell to attach to adenylate cyclase
located in basolateral membrane; 3) entire toxin enters cell via
endosomes, and A subunit translocates through endosomal
membrane, then A1 peptide ADP-ribosylates Gs located in
basolateral membrane, perhaps after endosome-plasma membrane
fusion. ARF, adenosine rybosil factor.
• increased cAMP activates protein kinase
A, leading to protein phosphorylation.
Protein phosphorylation leads to increased
Cl ion secretion in crypt cells and
decreased NaCl-coupled absorption in
villus cells.
Exotoxins
• They are toxic proteins secreted from a living
bacterium. They alter the normal metabolism of host
cells and have deleterious effects on the host.
• They are different from ENDOTOXINS (LPS or LOS).
• Exotoxins are diverse i.e., they have different
structures and different modes of action.
– Some will form pores in the host cell plasma membrane, others will bind
receptors, and others will be internalized by the host cell.
• One pathogen may produce one toxin or one
pathogen may produce several toxins
– S. aureus produces 25 or 30 different toxins.
Toxin Classification
• Type I toxins: superantigens
• They bind to the host cell surface, but they
are not translocated into the cell
– They will modulate the immune response
Toxin Classification
• Type II toxins: pore forming toxins
• They act on the host cell membrane.
– Host cells will leak then die
Toxin Classification
• Type III toxins: A-B toxins
• They bind to the host cell one specific
receptor and are translocated into the cell.
– They will become active and modify some proteins or other
components of the host cell.
A-B toxins
Cell surface
Active
Binding
A
B
Toxin Classification
• Effector proteins that are translocated
into host cells through a type III
secretion system.
– This type is present in many gram negative bacteria.
– No binding, direct injection.
Toxin effects
• Toxins that lyse cells (hemolysins, leukocidins)
• Toxins that elevate cyclic AMP (cholera toxin)
• Toxins that block protein synthesis (diphtheria
toxin)
• Toxins that block nerve function (botulinum,
tetanus)
Type III Toxins
Domain Organization
• A domain; catalytic domain.
• B domain; receptor-binding domain.
• AB5 toxins; consist of 6 non-covalently
bound proteins.
Domain Organization
• AB toxins; one single protein.
– Diphtheria has an N-terminal catalytic domain and a
C-terminal domain receptor-binding domain.
• A-B toxins; proteins that are not
associated in solution, but associate
upon binding to the host cell.
– Anthrax toxins (edema toxin and lethal toxin).
– This type is synthesized as two different proteins.
Examples of AB Toxins
What is the Role of a Toxin In
Disease?
– Toxins may have several roles (these are not mutually exclusive)
– Directly toxic to host
• Aid in the establishment of the disease
• help in the acquisition of nutrients
– interfere with the normal function of immune, or other cell types,
cells during the infection
– The contribution of toxins to disease remains unclear or
incompletely understood
Diphtheria
• Diphtheria is normally an infection of
the throat caused by a bacterium,
Corynebacterium diphtheriae.
• Gram-positive
• Non-spore forming
• Non-motile
• Aerobic rod
• The tox gene codes for the AB toxin.
Corynebacteria diphtheriae
Pathogenesis of Diphtheria
• Encounter – Corynebacterium diphtheriae
encountered only from other people (carriers)
• Entry – respiratory droplets; organism colonizes
pharynx
• Spread
• Multiplication – iron likely a factor
• Evasion of host immune response – adhesins;
toxin may kill phagocytes contributing to
pseudomembrane
• Damage – inflammation; circulating toxin
• Transmission – aerosolized droplets;
HPR10
Diphtheria: infection of upper respiratory tract by Corynebacterium diphtheriae
• bacteria grow on throat tissues
• characteristic formation of pseudomembrane (greyish membrane of bacteria,
damaged host cells) as a result of host’s inflammatory response
• systemic exotoxin release is responsible for tissue damage
• tox gene carried by lysogenic bacteriophage → only lysogenized bacteria cause
serious disease
• formerly major childhood disease; now rare due to DTP vaccine
• “D” component of vaccine is formalin-treated diphtheria toxin (= toxoid)
• a toxoid is nontoxic but remains immunogenic
pseudomembrane
other A-B toxins include:
• the neurotoxin of Clostridium botulinum
• tetanus toxin (neurotoxin produced by
Clostridium tetani)
• pertussis toxin (whooping cough, Bordetella
pertussis)
• Shiga toxin (bacterial dysentery, Shigella
dysenteriae)
tox Gene
• There are toxigenic and nontoxigenic
strains of Corynebacterium diphtheriae.
• The tox gene encoding DT is carried by
a family of corynebacteriophages
• Toxigenic strains of Corynebacterium
diphtheriae are lysogenized by these
phages.
Diphtheria tox
Gene in Beta
Bacteriophage
and Prophage
tox Gene
Molecular Structure of Diphtheria Toxin
Catalytic Region
A Subunit
B Subunit
Translocation Region
Receptor-Binding Region
Structure/function of Dt
Domain Structure of Dt
• R domain or C-terminal receptor-binding (R) domain:
It binds to cell surface receptor, allowing the toxin to
enter the cell by receptor-mediated endocytosis.
• C domain or N-terminal catalytic (C) domain: It
blocks protein synthesis by transfer of ADP-ribose
from NAD to a diphthamide residue of EF-2.
• T domain or central domain of diphtheria toxin is the
translocation (T) domain: pH-induced conformational
change of this domain triggers insertion into the
endosomal membrane and facilitates the transfer of
the catalytic domain into the cytoplasm.
Diphtheria Toxin
Binding to the host-cell receptor:
• The R-domain, which is part of the B-chain,
binds to a specific receptor on the host cell
surface.
• This receptor is the heparin-binding
epidermal growth factor (HB-EGF) precursor.
– This is a membrane protein expressed on the host
cell.
A Model for DT binding to the
HB-EGF Receptor
Endocytosis and Translocation
• Endocytosis of the toxin-receptor complex.
• Acidic pH in the endosome promotes a
conformational change (T-domain) that
inserts the toxin into the vesicle membrane.
• The A-chain is translocated into the
cytoplasm.
• Reduction of the disulfide releases the Achain into the cytoplasm.
Endocytosis & Translocation
Enzymatic activity
Enzymatic activity
•
In the cytoplasm, the A-chain catalyzes the ADP-ribosylation of EF-2.
– ADP-ribosylation uses NAD as a substrate.
•
•
In eukaryotes, EF-2 participates in the elongation step of translation.
ADP-ribosylation renders EF-2 inactive and blocks protein synthesis.
– Causes cell to die.
•
ADP-ribosylation occurs at an unusual derivative of histidine, called
diphtamide (post-translational modification).
•
Physiological function of diphtamide is unknown.
•
A single molecule of A-chain is enough to kill one eukaryotic cell.
– Very Powerful.
Diphtheria Vaccine
• Toxoid: the toxin is rendered nontoxic but
still immunogenic by chemical modification
or heat treatment.
• Diphtheria toxoid = Toxin treated with
formaldehyde is nontoxic but immunogenic.
• Formaldehyde reacts with Lys amino groups
and forms internal cross-links [prot]-NH-CH2NH-[prot]
Summary
Diagnostic Schick Skin Test
Immune Status to C. diphtheriae and
Sensitivity to Diphtheria Toxoid
TOXIN
TOXOID
In vivo Detection
of Diphtheria Exotoxin
toxin
receptor
Anthrax toxin is an AB-type toxin
•
•
•
•
“A” moiety is the effector molecule
“B” is the receptor-binding domain which
facilitates the “A” moiety’s entry into the host cell
Two different types of “A” moieties -- EF and LF
“B” moiety is PA
EF and LF moieties cannot produce pathogenic
effect on their own -- they must be present with
PA moiety
Edema factor
– Edema factor (EF) is called what it is due to
its effect in producing edema in animals
injected with it
– EF is a calmodulin-dependent adenyl cyclase
enzyme -- increases the concentration of
cAMP in the cells
– This increase in cAMP upsets water
homeostasis in cells -- this may be the cause
of the edema observed
– 84 kDa
Lethal Factor
– Lethal factor (LF) is called this because it causes
death through an unclear mechanism when injected
into animals
– Inhibitis neutrophil function in vitro; neutrophil function
is inhibited in patients with cutaneous anthrax
– Acts as a Zn2+ - dependent metalloprotease, and
may inactivate MAP-kinase kinase
– Stimulates macrophages to release TNF-a and IL-1b,
which may cause sudden death
– 90 kDa
Protective Antigen
• Protective antigen is so named due to its
use as an anthrax vaccine (commonly
used, including by the US Army -- a
source of some controversy!) Acts as the
“B” moiety of the AB toxin
• Can interact with both EF and LF
• Forms a channel through the host cell
membrane (shown using voltage-gating
studies)
Protective Antigen
• Best-characterized of the three toxins
– Cleaved to become active
– Precursor binds to cells as an 83 kDa
structure
– Host cell furin cleaves PA83 into two subunits
-- PA20, which dissociates, and PA63 which
forms into heptamers and binds to the
membrane to form the pore
Protective Antigen
Anthrax toxin
Trends Immunol. 2006 Sep;27(9):434-40.
Epub 2006 Jul 24.
• Anthrax toxins: A paradigm of bacterial
immune suppression.
• Baldari CT, Tonello F, Paccani SR,
• Montecucco C.
• Department of Evolutionary Biology,
University of Siena, 53100, Siena, Italy.