Table of contents
Summary
List of abbreviations
Preface: Clostridium botulinum: botulinum neurotoxin (BoNT)
Discovery
Clostridium botulinum bacteria
Structure
Mechanism of action
Disease symptoms
Clinical applications
Toxicity
Research question
Chapter 1: Introduction to bacteria and toxins
1.1 Gram classification
1.2 The immune [system and] response to bacterial pathogens
1.3 Endo- and exotoxins
Chapter 2: Bacterial protein toxin classification
2.1 Membrane damaging toxins (cytolysins)
2.2 Receptor-targeting toxins
2.3 Internalized toxins
Chapter 3: Membrane damaging toxins (cytolysins)
3.1 Pore-forming toxins (PFTs)
3.2 Enzymatic cytolysins
3.3 Detergent-like cytolysins
Chapter 4: Receptor-targeting toxins
4.1 E. coli heat-stable enterotoxin
4.2 Superantigens
Chapter 5: Internalized toxins
5.1 Shiga toxin (Shigella dysenteria)
5.2 Cholera toxin (Vibrios cholera)
5.3 Diphtheria toxin (Corynebacterium diphtheria)
5.4 Tetanus toxin (Clostridium tetani)
5.5 Anthrax toxin (Bacillus anthracis)
Chapter 6: Application in therapies
6.1 Medical toxins for diseases (hypothetical)
6.2 Botox-like medical toxins
Chapter 7: Discussion
References
Appendix
3
4
5
5
6
7
8
11
12
13
13
14
14
15
18
19
19
19
19
20
20
27
29
30
30
32
34
34
36
38
39
40
43
43
44
45
46
61
2|Page
Summary
For several years, even decades, people have under gone cosmetic surgeries to approach a better
looking skin without the signs of aging. Botox has been the ingredient that many people let inject to
treat the symptom of getting older, the wrinkles it brings along. Even other syndromes, like migraine,
or involuntary twitches are targeted for treatment with botox. But where does this agent come from
that has been able to create wonders in peoples minds. It is produced by Clostridium botulinum, a
relative from Clostridium tetani, the causative bacterium of tetanus. Clostridium botulinum was first
known to cause the so-called ‘sausage-disease’, as it was often hidden in meat and canned food. The
discoverer of the toxin now used for botox has predicted that eventually we would know enough
about the toxin for us to find a beneficial aspect for the toxin. And indeed, it is now incorporated in
daily use and procedures. But how safe is it really, and are their possibly other bacterial toxins that
can help us ameliorate diseases or treat them as a whole? To dig deeper in the existence of bacterial
toxins, three categories will be discussed in their method of toxicity, namely the membranedamaging toxins, receptor-targeting toxins and the internalised toxins, spread over several chapters.
The final chapters are dedicated to the hypothetical design of toxins that can be used in treatments.
3|Page
List of abbreviations
Atrial natriuretic peptide
Anthrax toxin receptor 1/2
Antigen-presenting cell
ANP
ANTXR1/2
APC
Bone marrow derived lymphocyte
B-type natriuretic peptide
Botulinum neurotoxin
Cyclic AMP
Cytosolic translocation factor
Cystic fibrosis transmembrane
conductance regulator
Cyclic GMP
B cell
BNP
BoNT
cAMP
CFT
CFTR
C-type natriuretic peptide
Coat protein-I
C-reactive protein
Diacylglycerol
Deoxyribonucleic acid
Death receptor 5
Elongation factor 2
Enterohaemorragic E. Coli
Endoplasmic reticulum
Enterotoxigenic E.coli
Globotriosylceramide
Guanylyl cyclase-C
CNP
COPI
CRP
DAG
DNA
DR5
EF-2
EHEC
ER
ETEC
Gb3
GC-C
Glycolipid monosialoganglioside
GM1
Glycophosphatidylinositolanchored protein
GPIanchored
protein
H
hb-EGF
Heavy chain
Heparin binding epidermal growth
factor-like precursor
C terminal of heavy chain
C-subdomain of HC
N-subdomain of HC
cGMP
HC
HCC
HCN
α-haemolysin
γ-haemolysin
N terminal of heavy chain
Hla
Hlg
HN
Immunoglobulins
Ket-deoxyoctulosonate
Ig
Kdo
N-terminal of light chain
Lipopolysaccharide
Low-lipoprotein receptor-related
protein 6
Heat-labile enterotoxin
Mitogen-associated protein kinase
MapK Kinase
Major histocompatibility complex
Nicotinamide adenine dinucleotide
N-ethylmaleimide-sensitive factor
LC
LPS
LRP6
Pathogen associated molecular
pattern
Protein disulphide isomerase
Pore-forming toxins
Phosphatidylinositol phosphate
cAMP-dependent kinase
Protein kinase A
Protein kinase C
cGMP-dependent protein kinase II
Pattern recognition receptor
Ribonucleic acid
Ribosomal RNA
Superantigen
Synaptosomal-associated protein of
25kDA
Soluble NSF-attachment protein
receptor
Heat-stable enterotoxin
PAMP
Synaptic vesicle
Thymus derived lymphocyte
SV
T cell
T cell receptor
Toll-like receptor
TNF-related apoptosis-inducing
ligand
Amino Acyl transfer RNA
Unfolded protein response
Vescile-associated membrane
protein
TCR
TLR
TRAIL
LT
MAPK
MAPKK
MHC
NAD
NSF
PDI
PFT
PIP
PKA
PKA
PKC
PKGII
PRR
RNA
rRNA
SAg
SNAP-25
SNARE
ST
tRNA
UPR
VAMP
4|Page
Preface: Clostridium Botulinum: Botulinum neurotoxin
Botulinum toxin (BoNT) is most commonly known for its cosmetic uses by having smoothening
effects on the facial skin of numerous women. Its commercial name, Botox, is therefore often used in
advertisements on anti-aging methods. Botulinum toxin however has multiple other uses, from the
prevention of involuntary muscle contractions to amelioration of migraine. Unfortunately, even
though the toxin is used in the clinic in combination with substances that will prevent the onset of
botulism, Botox stays a dangerous and deadly substance, with the ability to kill a million people with
only a single gram. Even today, there is hardly any substantial proof that treatment with Botox is
completely harmless. Nevertheless, thousands of people continue risking their lives in some way by
using Botox. Is there an alternative to Botox, a toxin that has the same mechanism as Botulinum
toxin but without the possible deadly consequences? By comparing the mechanism of Botulinum
toxin including its way of entry, with other bacterial toxins, I attempt to give an answer to this
question.
Discovery
The correlation between botulism and ingesting of contaminated food was not recognised in
ancient times. Reports on paralysis due to botulism are therefore rare and sometimes even
attributed to a different organism [Erbguth et al, 2004]. At the end of the 18th century, there were
several outbreaks in the Kingdom of Wurttemberg in Germany [Grusser et al, 1986]. In 1802, the
government set out a warning for the consumption of ‘bloody sausages’, recognising this may be the
cause of the disease. Nine years later, the department of internal affairs readdressed the poisonous
sausages, however contributing it to the occurrence of hydrocyanic acid, in those days also known as
prussic acid [Erbguth et al, 2000]. A german poet and physician, Justinus Kerner did not think such a
disease would be caused by an inorganic substance like hydrocyanic acid. Instead, like the Medical
institute in Tübingen, he suspected a biological poison [Erbguth et al, 2004]. In 1820, he published a
report, a so-called monograph, on 77 cases, and gave a full and accurate description and definition of
botulism like we know it nowadays [Erbguth et al, 2004; Kerner, 1820 (German)]. 5 years later, he
published a second monograph on a further 155 cases and described the effects of the ‘sausage
poison’ on the muscles. He also did extensive experiments on animals and on himself, and he
deduced from the results that the toxin has effect on the signal transduction from neuron to muscle
[Erbguth, 1998]. One of his conclusions in his monograph was: “The nerve conduction is brought by
the toxin into a condition in which its influence on the chemical process of life is interrupted. The
capacity of nerve conduction is interrupted by the toxin in the same way as in an electrical conductor
by rust’’ [Kerner, 1822 (German)]. He even made speculations of the use of the toxin in conditions of
hyperexcitability of the nervous system [Erbguth et al, 2000]. After his publications, no other scientist
had made substantial contributions to the knowledge on ‘sausage poisoning’. Kerner became quite
known for his work on botulism that the disease also adopted the name ‘Kerner’s disease’. Later it
got the name botulism, from ‘botulus’, which means sausage in Latin [Torrens, 1998].
5|Page
Clostridium Botulinum bacteria
With the discovery that botulism was not caused by an inorganic reagent but merely a biological
toxin, the causative organism was soon to be discovered as well. After a minor outbreak in Belgium
at a funeral lead to the correlation of the ‘sausage poisoning’ with bacteria found in the pickled and
smoked ham by Emile Pierre Marie van Ermengem [Kreyden, 2002]. He called it ‘Bacillus botulinus’
which was later renamed as ‘Clostridium botulinum’ [Erbguth et al, 2008]. Clostridium botulinum is a
rod-shaped, Gram-positive bacterium that can be found ubiquitously in soil and water. Under
stressful conditions, these bacteria can form spores that can survive extreme conditions. These
spores are destroyed when heated to 121°Celsius, whereas the toxin is destructed when heated to
85°C. Insufficiently heated food, like home-canned food or traditional meals in Alaska, may therefore
be a source of botulism. The optimal conditions for Clostridium botulinum to germinate and survive
in are anaerobic, a nonacidic pH (above 4,5) and low salt and sugar levels [Sobel et al, 2005]. Within
these conditions, the bacterium will grow and produce its toxin, resulting in disease when ingested
[Johnson et al, 2001].
Botulism can occur in multiple forms, of which 4 are most prevalent. These include food-borne,
wound-borne, infant and adult intestinal botulism. Botulism caused by inhalation of the bacterium or
spores is also reported but only in laboratorists working with the bacteria [Sobel, 2005; Holzer et al,
1962 (German)]. Food-borne botulism is caused by inappropriate heating of the food, causing the
bacteria to survive and allowing it to produce the botulinum toxin, which when ingested can lead to
botulism. The ingestion of spores rarely leads to germination of the bacteria due to suboptimal
conditions in adult intestines. Nowadays, disease prevalence by this type of botulism is low due to
modern heating and canning techniques. Home canned foods or the traditional Alaskan meals are
still sources of food-borne botulism owing to insufficient heating. Wound-borne botulism is caused
by contamination of wounds by botulinum spores. This occurs mostly in drug users who apply drugs
by means of injections [Werner et al, 2000]. Especially the users of black tar heroin are at risk for
development of wound-borne botulism [Passaro et al, 1998]. When the injection site gets
contaminated with the spores, the subsequent formation of an abscess establishes an optimal
environment for the spores to germinate into the full bacterium again, which will start producing the
toxin. Black tar heroin users are more at risk as they inject the drug subcutaneously rather than
intravenously, creating a more suitable environment for the bacteria to grow [Passaro et al, 1998].
Infant botulism is caused by ingestion of spores that are able to germinate in the intestines of infants.
The normal bacterial flora of the infant has not yet been fully developed, giving the Clostridium
botulinum bacteria the opportunity to colonize the intestines. On top of this, the intestines of
formula-fed infants have a nearly neutral pH, making it a suitable environment for the bacteria to
grow in [Mills et al, 1987]. A similar variant of botulism can occur in adults but only rarely. These
patients often have abnormalities in the anatomy or function of the intestines, or are on
antimicrobial medication, which interrupts the protection by the normal flora which allows the
bacteria to colonize the intestines [Arnon, 1995].
6|Page
Structure
There are seven serologically distinct botulinum neurotoxins (BoNTs) defined, each produced by
single serotype of Clostridium botulinum, and given the addition of a letter A-G [chronology of
discovery described in Erbguth et al, 2008]. All have a similar, conserved amino acid sequence. Only
four of these serotypes are found to be able to cause botulism in humans, namely A, B, E and F.
Botulinum toxin is synthesized as a single polypeptide chain which is cleaved into two chains, the
light (L) and heavy (H) chain. The
cleavage is conducted by either
clostridial or tissue proteases. These
chains become connected to each other
by means of and essential disulphide
bridge and a loop deriving from the
heavy chain that is wrapped around the
light chain, the so-called belt [Lacy et al,
1999]. The active polypeptide consists
of
three
domains,
the
zinc
metalloprotease domain, located on the
N terminal of the light chain (LC), the
translocation domain located at the N
terminal of the heavy chain (HN), and
the receptor binding domain, located on
the C terminal of the heavy chain (HC)
[Lacy et al, 1999]. Even though the
serotypes bear a highly similar amino
acid sequence, this can lead to
structural differences, as is seen for
BoNT/A-B and E. Figure 1a shows the
architecture
of
BoNT/A,
which
resembles the structure of BoNT/B as
well. The N terminal of the heavy chain
(dark blue) is situated between the light
chain (light blue) and the C terminal of
the heavy chain (green). The belt (red),
deriving from the heavy chain is shown
to ‘hug’ the light chain [Montal et al,
2010; Lacy et al, 1999; Lacy et al, 1998;
Swaminathan et al, 2000]. Figure 1b
shows the structure of BoNT/E, where Figure 1: Structure of BoNT/A and E.
Structure of BoNT/A (a) and BoNT/E (b) are shown with different colours
the LC and HC are situated on top of depicting the domains. The light chain (LC) is shown in light blue, the heavy
chain (HC) in green and dark blue. The dark blue depicts the N terminal of the
each other with the N terminal of the heavy chain (Hn), and the green domain presents the C terminal (Hc). The
heavy chain located on the side. In this latter is subdivided in two subdomains, the Hcn and the Hcc. In red, the belt is
presented looping around the LC. For BoNT/A, the Hn is located between the
structure, the belt also embraces the Hc and the LC. In BoNT/E, the Hc is located beneath the LC with the Hn at the
light chain [Montal et al, 2010; Kumaran side. [Structural presentations adopted from Montal et al, 2010]
7|Page
et al, 2009]. The HC is depicted in two different shades of green, to differentiate between a beta
sheet jelly roll fold (dark green, HCN) and a beta tree foil fold carboxy subdomain (light green, HCC)
[Lacy et al, 1999]. These two particular subdomains have their specific role in the binding, entry and
translocation steps of the toxin and will be discussed in the next section.
Mechanism of action
All serotypes of BoNT work in a four step
manner. They bind to the target cell, get
internalized, translocated the catalytic domain
(light chain) to the cytosol where the toxin
exerts its effects (figure 2). Each stage will be
discussed in more detail below.
Step i and ii: the binding of the toxin to neurons
at the peripheral neuromuscular junction and
subsequent internalization.
Figure 2: overview of chronological events of botulinum
The carboxy subdomain (HCC) of the HC binds to toxin action.
different molecules specific for neurons. The Botulinum neurotoxin binds to surface receptors and
polysialogangliosides on the neuron cell surface (i) and
initial association of the domain to the neuron is trigger receptor-mediated endocytosis (ii). When the toxin is
established
by
binding
with in the endosomal compartment, the toxin forms a channel
in the endosomal membrane and translocates the LC (iii)
polysialogangliosides present on the plasma which then cleaves a protein of the SNARE complex and
membrane [Simpson et al, 1971; Kazoki et al, inhibits neurotransmitter release (iv). [Schematic
1998]. These lipid molecules have a role in representation adopted from Breidenbach et al, 2005]
signal transduction and seem to be important in the development of neurons. The only BoNT that
does not bind to polysialogangliosides is BoNT/D, which has been shown to lack the
polysialoganglioside binding site, found in the other serotypes, and instead binds to
phosphatidylethanolamine [Tsukamota et al, 2005]. The polysialonganglioside molecules are not the
only molecules the toxin binds to enter the cell as they would not explain the binding affinity of
BoNTs to the cell membrane. It was found they also bind protein receptors playing a role in synaptic
vesicle recycling [Simpson et al, 2004]. The protein synaptotagmin, a Ca2+ sensor for triggering
synaptic vesicle fusion, has been found to be the protein receptor for BoNT/B and BoNT/G as well
[Nishiki et al, 1996; Binz et al, 2009]. Later, members another family of synaptic vesicle proteins (SV2)
have been identified to act as the protein receptors for the serotypes A, E and F [Dong et al, 2006,
Dong et al, 2008 and Fu et al, 2009 respectively]. The receptor specificity is interestingly correlated to
the amino acid sequence similarity between the serotypes [Binz et al, 2009]. The protein receptors
for BoNT/C and D remain to be identified. Binding to these two receptors, the protein receptor and
the ganglioside receptor, triggers receptor-mediated endocytosis, through which the toxin is
enveloped by a synaptic vesicle and transported to an endosome [Simpson et al, 2004].
Step iii: translocation of the protease domain to the cytosol.
The C terminal domain of the heavy chain contains 2 subdomains, the HCC and HCN. The HCC, as
described before, is involved in receptor binding and triggering receptor-mediated endocytosis. The
HCN however, seems to be involved in the translocation [Montal et al, 2010]. Within this subdomain,
8|Page
a phosphatidylinositol phosphate (PIP)-binding motif was identified [Muraro et al, 2009]. Not only
are PIPs described to be involved in intracellular signalling regarding vesicular trafficking, another
bacterial toxin, diphtheria toxin, has been shown to bind to PIPs, promoting channel formation
[Donovan et al, 1982]. This suggests a role for the HCN for bringing the HN domain in close proximity
to the membrane, possibly thereby promoting the onset of translocation when endosomal conditions
are optimal. The HN domain is responsible for translocating the catalytic domain. Upon acidification
of the endosome, a conformational change is triggered in the HC domain, uncovering the HN domain
to bind and insert itself in the endosomal membrane. When the HC domain is absent, the HN domain
can still insert itself, even when the pH in the endosome is kept neutral (pH ~7)[Fisher et al, 2008].
The catalytic light chain remains in a folded state in neutral pH, and unfolds when the pH is lowered,
and is chaperoned by the HC for translocation through the HN [Fisher, 2013; Koriazova et al, 2003].
Presumably, the LC is kept inactive in the endosome by the belt, as this may act as a pseudosubstrate
[Brunger et al, 2007]. This will advance translocation over the endosomal domain. The channel
formed by the HN domain, is a cation conductive channel. In an open state, the channel conducts
sodium ions over the membrane. Membrane potential assays have shown that the channel seems
occluded in the beginning stages of translocation (step 2, figure 3 [fully described by Fisher et al,
2007a]). Presumably, the further the LC domain has unfolded and translocated through the channel,
the more Na+ ions are conducted and the higher the conductance is (step 3-4, figure 3). When the LC
domain has fully translocated to the cytosol, the reducing environment of the cytosol reduces the
disulphide bridge which releases the LC domain (step 5, figure 3), an important step in translocation
as described by Fischer et al [Fischer et al, 2007b]. The fate of the HN channel has not been fully
elucidated yet, but it presumable stays closed until it is somehow degraded. This likely prevents
cellular detection of the presence of a pathogenic protein in the cytosol [Fischer et al, 2007a].
Figure 3: Sequence of events during LC translocation
The sequence of translocation events is shown through steps 1-5. These events are deduced from the
conductance of the channel formed by HC. The cis side of the membrane represents the endosomal compartment,
while the trans side depicts the cytosol. The characteristics important for translocation are depicted on the left,
considering the pH and reducing properties of the compartments. In (1), the BoNT/A toxin is present as a folded
tertiary structure. Due to the lower pH, structural changes occur and HN is incorporated in the membrane while LC
is unfolded and begins to translocate through the channel (2). This is accompanied by limited conductance levels.
As soon as the LC becomes more unfolded and occupies the channel (3-4), the conductance increase until in (5),
the conductance is at its maximum. Due to the reducing properties of the cytosol, the sulphide bond is broken and
the LC is released. [Schematic representation is adopted from Montal et al, 2010 and described in Fisher et al,
2007a]
9|Page
Step iv: proteolytic activity of the LC domain in the cytosol.
Depending on the serotype, the catalytic domain of the LC domain has various proteins as target,
however similar in function. They target so-called SNARE proteins (soluble NSF attachment protein
receptor, where NSF stands for N-ethylmaleimide-sensitive factor). These proteins play an important
role in the fusion of synaptic vesicles (SV) containing neurotransmitters [reviewed in Brunger et al,
2005 and Jahn et al, 2003]. These vesicles fuse completely with the neuronal cell membrane to
release their neurotransmitting content upon the arrival of an action potential. This fusion is
mediated by SNARE proteins, proteins that are located either on the synaptic vesicle membrane
(synaptobrovin or Vesicle-associated Membrane Protein (VAMP)) or on the inner side of the plasma
membrane (Synaptosomal-associated Protein of 25 kDA (SNAP-25) and syntaxin)(figure 4). When
unbound to a partner, the SNARE proteins find themselves with an unstructured core domain, but
once bound to a partner, the core domains adopt a fully helical, coiled-coil formation which can
interact with the coiled-coil core domain of binding partner [Fasshauer et al, 1997; Hazzard et al,
1999]. A particular membrane protein Munc18 is bound to Syntaxin, keeping it in its closed
conformation and stabilizing the protein (figure 4)[Dulubova et al, 1999]. Presumably, following the
arrival of an action potential, this binding is somehow disrupted, either by conformational change of
Munc18 or by an interaction with Munc13, which releases syntaxin [Dulubova et al, 1999]. Syntaxin is
then able to change into its open formation and bind to SNAP-25 (figure 4). This pre-assembly of the
SNARE protein complex can bind to synaptobrevin (figure 4). However, the exact chronology of
events has still yet to be elucidated [Bowen et al, 2005]. Munc18 is thought to stabilize and assist the
formation of the SNARE protein complex in a yet to be elucidated way [Dulubova et al, 2006]. The
helices of the SNARE proteins continue to interact with each other, extending the complex towards
the C terminal. This eventually brings the synaptic vesicle in close proximity to the plasma
membrane. Because the phospholipid make-up of the synaptic vesicle and plasma membrane repulse
each other, fusion becomes an energetically favoured option [Simpson, 2004]. There are several
Figure 4: Schematic representation of the events during synaptic vesicle fusion.
The SNARE proteins are depicted in the events occurring prior to synaptic vesicle fusion. Upon a still to be elucidated
trigger, Syntaxin (red) is released from Munc18 (grey) and can bind with SNAP-25 (green). This pre-formation of the SNARE
complex is then able to bind to synaptobrevin (blue) and continues to form a complex in a zipper formation, pulling the
synaptic vesicle in proximity of the plasma membrane. This ultimately leads to fusion of the synaptic vesicle with the plasma
membrane, releasing the contents in to synapse (not shown in figure). [Figure is adopted from Fasshauer, 2003].
10 | P a g e
different types of SNAREs and each BoNT has its SNARE specificity. BoNT/A and E recognise SNAP-25,
BoNT/B, D, F and G cleave synaptobrevin, and BoNT/C targets syntaxin, but has been shown to be
able to target SNAP-25 as well [Williamson et al, 1996]. This target specificity of the BoNTs to their
particular SNARE protein is established by an extended SNARE binding region. The extension of
protease-protein binding region works as both
specification of target recognition but also to
position the target in a way that scissile peptide
bond (general name for the bond which is cleaved
by the protease) is brought close to the catalytic
site [Breidenbach et al, 2004]. In figure 5, it is
shown that the LC of BoNT/A(gray) embraces the
target protein almost completely and thereby
bringing a particular site at the C terminal of SNAP25 near the zinc atom (purple). Even though the
protease cleaves only 9 residues, it binds to
approximately sixty. These residues, both in
respect to cleavage site and binding site, differ
between the 7 serotypes, attributing to specificity
and differential cleavage site [Breidenbach et al,
2004; Breidenbach et al, 2005]. Any mutations in
the C terminal, or binding of the target SNARE Figure 5: binding region of SNAP-25 to BoNT/A
protein to a partner reduces binding efficiency of The structure of the complex formed by BoNT/A with
SNAP-25 has shined some light on how specificity of
proteolysis by the toxin, therefore inhibiting BoNTs are mediated. The long stretch of SNAP-25
protease reactivity [Breidenbach et al, 2005]. After protein (green) (approximately 30 amino acids in length)
brings the scissile bond near the Zinc atom (purple)
cleavage of the SNARE proteins, the SNARE where the bond is hydrolysed by the toxin. The region
complex cannot be formed any longer, restricting bound by the various BoNTs on the SNARE proteins
synaptic vesicle fusion with the plasma membrane, differs between, depicting their specificity of hydrolysis
site and target protein. [Structure adopted from
and the vesicles are therefore unable to release Breidenbach et al, 2005]
their neurotransmitting content.
Disease symptoms
Through the inhibition of synaptic vesicle fusion with the neuronal cell membrane, the neuron does
not release its neurotransmitters to the receiving muscle. The neurotransmitters are important in
transmitting any signals from the brain to the muscles, which in response will either contract or relax
the muscle fibres. The first signs of foodborne botulism are digestive complaints, like stomach
cramps, nausea and diarrhea [Zembek et al, 2007]. The first neurological signs occurring after BoNT
intoxication are located around the eyes, caused blurred and double vision and drooping eye lids.
This is followed by difficulties to swallow and talk, indicating that other neuromuscular junctions are
affected by the toxin [Zembek et al, 2007]. Subsequently, the patient is unable to move its head, its
upper extremities and has trouble breathing and only later will the lower extremities become
affected. When respiratory muscle failure appears, the patient is likely to die without proper
treatment. The success of treatment decreases with time, and therefore it is important that BoNT
11 | P a g e
intoxication is diagnosed within a few hours [Zembek et al, 2007]. In addition, BoNTs can also have
more effects on the activity of the autonomous nervous system, the nervous system that controls the
involuntary movements) leading to decreases in heart rate, hypothermia and retention of urine
among many other symptoms [Zembek et al, 2007]. Severity and incubation time depends on
serotype and the amount of toxin ingested. BoNT/A causes more severe disease with effects on
skeletal and respiratory muscles while BoNT/B and E are more frequently associated with the
autonomous nerve dysfunction. BoNT/E has been shown to have a more rapid onset of clinical
symptoms than BoNT/A or B. In general, symptoms can occur within a few hours up to a few days,
with a median of 24 hours (1 day) [Zembek et al, 2007].
Clinical applications
Justinus Kerner had speculated in his monograph in 1822 that when “The fatty acid or zoonic acid
administered in such doses, that its action could be restricted to the sphere of the sympathetic
nervous system only, it could be of benefit in the many diseases which originate from
hyperexcitation of this system’’ and that “by analogy it can be expected that in outbreaks of sweat,
perhaps also in mucous hypersecretion, the fatty acid will be of therapeutic value” [Kerner, 1822;
Erbguth et al, 2004]. However, he did mention that his speculation “about the fatty acid as a
therapeutic drug belongs to the realm of hypothesis and may be confirmed or disproved by
observations in the future” [Kerner, 1822; Erbguth et al, 2004]. His speculations in 1822 have long
become reality today. Even though its use as wrinkle reducer is one of the commonly known
purposes, it has been used in many other clinical applications. In the early nineties, purifications of
BoNT/A were developed by American scientists to be used in clinical applications, more precisely for
strabismus. Strabismus is a condition where the eyes are not perfectly aligned, which can have many
causes including a disorder in the strength of one of the muscles aimed for eye motion. Late nineties,
BoNT/A was approved to treat blepharospasm (twitching of the eyelid) by the American Food and
Drug Association, only to be followed by the approval for Botox (small amounts of BoNT/A) to be
used to reduce wrinkles as a cosmetic operation [Dhaked et al, 2010]. Nowadays, several
preparations of BoNT/A and B are sold on the market for clinical purposes (a partial overview of
these clinical applications is shown in table 1, full table by Dhaked et al, 2010). Most of them contain
bacterial and humoral proteins to stabilise the toxin and to facilitate its injection. Even though
injecting a potent bacterial toxin into a patient’s body seems like a risky procedure, medical scientists
believe it is safe. The toxin does not spread more than 2 cm from where it has been injected, limiting
the affected area to the injection site.
Table 1: Uses of botulinum toxin in clinic
Furthermore, the amount of toxin
Example
used in the procedures is far less than Indication
Frown lines
will be deadly to a human being Cosmetology
Migraine
[Dhaked et al, 2010; Anderson, 2004]. Pain syndromes
Tic, tremors
Nevertheless, a potential threat Movement disorders
Strabismus
continues to exist by exposing the Opthalmology
Exocrine gland hyperactivity
Crocodile tears syndrom
body to a highly lethal toxin.
12 | P a g e
Toxicity
As has been mentioned before, botulinum neurotoxins are highly potent and lethal toxins. 30 ng of
the toxin can kill an adult when ingested, while 900 ng and 150 ng is enough when inhaling or
injecting intravenously respectively. When each person on this earth weighs 70 kg, less than 40 gram
will be sufficient to eradicate humankind. Because of this potency, it is often feared that BoNTs will
be used as a weapon for bioterrorism. And indeed, several cases of the use of botulinum neurotoxins
are used as a means of killing people. In the 1930s, a Japanese biological warfare group has been
feeding isolated and purified BoNT/A to prisoners [Arnon et al, 2001]. In the 1990s, an international
arms control team found warheads filled up with BoNT/A, in total 19.000 litres, when they swept Iraq
during the Gulf War [Cohen et al, 2001]. The fear that more bioterrorists will be using the toxin as
their weapon is because the toxin is easy to isolate, stable under various conditions and exceptionally
lethal. The toxin can be spread by deliberate contamination of food and drinks or by dispersing it in
aerosols. Such an attack would be noticed too late as symptoms occur late during incubation, when
the chance of successful treatment for every victim is low.
Research question
As is discussed through this preface, botulinum toxin is a lethal and dangerous toxin. Its actions on
neurons seem to be straight forward and therefore other bacteria can possibly produce a toxin which
has similar clinical applications but without the lethality. Therefore the question has been raised to
be answered in this thesis: Are there other less toxic bacterial toxins applicable in the clinic?
13 | P a g e
Chapter 1: Introduction to bacteria and toxins
Bacteria have always been part of our everyday life. Teeth are brushed to prevent the formation of
cavities; hands are washed by surgeons before continuing to the next patient et cetera. However, the
human species has not always been aware of these small organisms. In 1676, almost 340 years ago,
the Dutch scientist Antoni van Leeuwenhoek discovered small living creatures in water enriched with
pepper, now known as bacteria [van Leeuwenhoek, 1941]. 200 years later, a student of Dr Robert
Koch described the link between microbes and infectious diseases in the case of diphteria. He also
described what is now known as Koch’s postulates, stating a method to proof microbe involvement
[Loeffler, 1884; Brock, 1999]. Robert Koch himself described the causative agent of tuberculosis
[Koch, 1882]. Fortunately, not all bacterial species are pathogenic and dangerous. For example, the
human body is inhabited by a variety of bacterial species, forming a complex microflora. Studies have
shown that the gut microflora is maintained by the immune system and health is preserved by the
microflora [Willing et al, 2010].
1.1 Gram classification
The most regularly used bacterial classification is the Gram classification. This classification is
established by Hans Christian Gram in 1884. It was originally not used to distinguish bacterial species
from each other, but rather to stain the bacteria in order to recognise them in tissue sections. The
staining techniques used in those days also coloured nuclei and fibrin, making it difficult to
distinguish the bacteria from tissue. By decolorizing aniline-gentian violet stained tissue cells by
means of applying an iodine and potassium iodide solution and alcohol to the sections, bacteria
Figure 6: Composition of gram-positive versus gram-negative bacterial cell walls
Left is shown the composition of the gram-positive bacterial cell wall and on the right the gram-negative wall. The
peptidoglycan layer is thicker in the gram-positive wall whereas the gram-negative bacteria have an outer envelope, with
the presence of lipopolysaccharides. Adapted from http://biology-forums.com/gallery/33_15_07_11_12_13_35.jpeg accessed 13-11-2013
14 | P a g e
maintained their stain, making them easy to recognise. Dr Gram reported 20 cases, of which 19 cases
showed retained bacterial colorization. However, one case showed a decreased staining intensity. He
described in addition that the bacteria that retained their staining did not show capsule formation
[Austrian, 1960; Gram, 1884]. Nowadays, the gram staining is an important aspect of bacterial
classification. The staining renders bacterial species either gram-positive or gram-negative. This gram
staining principle is based on the composition of the bacterial cell wall (figure 6). Both gram negative
and positive bacterial cell walls contain a peptidoglycan layer. For gram negative bacteria however,
this peptidoglycan layer is thinner in contrast to the peptidoglycan layer of gram positive bacteria
and is protected by a second membrane. This outer membrane is composed out of proteins and
lipopolysaccharides (LPS), the latter being a defence molecule, structural compound as well as target
of the immune system. These structural differences in membrane between gram-positive and –
negative bacteria are basis of whether the bacteria retain their stain or not. The staining is composed
out of crystal violet and iodine, which forms a complex inside the cell. By treating the bacteria
afterward with alcohol, causes the peptidoglycan-layers of the gram-positive bacteria to close its
pores, making it impossible for the crystal violet-iodine complex to escape from the cells, whereas
the lipid-rich outer membrane of gram-negative bacteria is easily penetrated by alcohol, releasing
the stain-complex [described more extensively in review by Beveridge TJ, 2001]. Gram classification
may be important for deciding which antibiotics to use during a bacterial infection. Gram-positive
bacteria can be more easily targeted with antibiotics that attack the peptidoglycan layer, such as
penicillin, whereas gram-negative bacteria are rendered unharmed. Antibiotics are divided into
classes based on whether they are applicable for gram-positive, or –negative infections, or during
both. However, physicians often use antibiotics that have the narrowest spectrum to prevent
development of antibiotic resistance.
1.2 The immune [system and] response to bacterial pathogens.
The human body is provoked daily by several bacterial invasions. However, this rarely leads to the
development of an infectious disease. This is partially realised by the hosts defence, the immune
system. The immune system is a corporation of two types of elements, the innate immunity and
acquired (or adaptive) immune system. The innate immune system is the first barrier for pathogens
to defeat and is activated early during infection. It contains the epithelial tissues (e.g. the skin) and a
diversity of cells and proteins. These cellular and humoral components of the innate system
recognise molecular patterns specific to bacteria and is present since birth. The adaptive immune
system develops with age and pathogen encounter. It contains different humoral and cellular
components than the innate immune system and is able to develop memory for pathogens. This
enables the adaptive immune system to respond and activate faster during a recurring pathogen
invasion. Below these two immune systems will be discussed further in more detail [reviewed in
Medzhitov, 2007].
1.2.1 The innate immune system
As previously mentioned, the innate immune system is activated early during bacterial encounter. It
consists of mechanical barriers (e.g. epithelial tissues), which is the first barrier for bacteria to
overcome, and a variety of immune cells and proteins. These immune cells and proteins work closely
together and also form a corporation with the acquired immune system. Among these immune cells
15 | P a g e
are dendritic cells, natural killer cells, macrophages and neutrophils. These cells are early on site of
infection and can induce inflammatory immune responses, phagocytise the pathogen in order to
eliminate the pathogen and activate the acquired immune system (figure 7). The principle of
recognition by these cells is the distinction between self- and non-self-antigens. These can be
distinguished by patterns associated with microorganisms (also known as PAMPs, Pathogen
associated molecular patterns) which do are not present on mammalian cells. These patterns are
recognised by pattern recognition receptors (PRRs) present on the innate immune cells, activating
immune responses to eliminate the invading organisms. One particular PRR group is the Toll-like
receptors family. This receptor family was first discovered in the Drosophila melanogaster in the late
20th century, named after the discoverer, Toll. The mammalian homologue was discovered one year
later, when a receptor was shown to activate the expression of genes involved in inflammatory
responses. This receptor was then named Toll-like receptor (TLR). Nowadays, at least 11 members of
the TLR family are described. Each member recognises a different PAMP and is located on the cell
membrane or inside the cell dependent on the ligand. For example, TLR4 is located on the cell
membrane as it recognises the endotoxin LPS whereas TLR3 is located on an intracellular cell
membrane due to its recognition of double stranded RNA. Recognition of pathogens through these
receptors causes the cell to phagocytise the pathogen and produce inflammatory cytokines,
recruiting more immune cells to the site of infection. Antigen-presenting cells (APCs), dendritic cells
in particular but to some extent also macrophages, can present parts of the pathogen on major
histocompatibility complexes (MHC) class II to cells of the acquired immune system, activating this
immune component and thereby forming a bridge between innate and acquired immunity. In
addition to the immune cells, the innate immune system also comprises proteins that are important
in the initial immune response. These
proteins are part of the acute phase
response proteins as they are produced
upon signals of cell damage or infection
released into the blood stream. Among
these are the C-reactive protein (CRP)
and the complement system. CRP binds
to
polysaccharide
and
phosphorylcholine, located on microbial
membranes, in a calcium-dependent
manner. This attachment of CRP to the
microbial
membranes
promotes
phagocytosis by the innate immune cells
as they have receptors recognising this
protein. In addition, CRP can activate
the complement system to attack
microbial
membranes.
Some
components of the complement system
work the same as CRP, as it binds the
microbial membrane and phagocytes Figure 7: Pathogen stimulated immune cell responses
thereby inducing phagocytosis. In The pathogen is recognised by pathogen recognising receptors (PRRs)
and activate innate immune cells. These innate immune cells attempt
addition, the complement system can to clear the infection and in addition induce an adaptive immune
form pores in the bacterial membrane response. Adapted from Medzhitov, 2007
16 | P a g e
through a cascade of complement component cleavage. This pore formation leads to microbial lysis.
Furthermore, the complement system can corporate with proteins produced by acquired immune
cells, promoting phagocytosis to a higher extent. This will be discussed in the next paragraph.
1.2.2 Acquired immune system
The acquired immune system is activated later than the innate immune system. As the name of this
immune system already indicates, the specificity of the acquired immune cells and proteins is
acquired with infections by pathogens. When the acquired immune system is activated to generate
an immune response, simultaneously a memory is developed for the particular pathogen. This
enables to generate a faster activation during a recurring infection. The acquired immune system
consists of two different immune cells, the B and the T cells. The B of B cells stands for bone marrow
and the T of T cells for thymus, therefore referring to the location where one thought that the cells
originate from. The B cells are formed in the bone marrow but translocate to the lymph nodes
throughout the body where they mature upon stimulation. This stimulation is mediated by the T cells
and the innate dendritic cell. Upon stimulation, the B cell starts to produce antibodies, also known as
immunoglobulins (Ig). Immunoglobulins consist of 2 heavy chains and 2 light chains, forming a small
Y-shaped protein. The base of the immunoglobulin, the constant portion, can be recognised by
receptors (Fc receptors) on other immune cells, inducing among others the process of phagocytosis.
The two branches on top of the immunoglobulin are variable, thereby creating a large variety of
specificities. During B cell maturation, the B cell receptor (having the specificity as the variable parts
of the immunoglobulins it produces) is generated by Ig-gene segment rearrangement. Binding of
antibodies to pathogens has multiple consequences. In addition to induction of phagocytosis,
immunoregulatory signals are generated which can negatively and positively affect the immune
response. The T cell on the other hand secretes other products than antibodies, they are a huge
factory for the production of cytokines. Within the T cell population, two subpopulations are to be
distinguished. The alpha beta (αβ) T cells are dependent on MHC mediated antigen presentation and
are activated by the presentation of pathogenic antigens on infected cells or APCs. These MHCpeptide complexes are recognised by their T cell receptor (TCR) consisting out of an α and β chain.
The other subpopulation is called the gamma delta (γδ) T cell subpopulation. They do not recognise
MHC-peptide complexes but recognise the transformation of cells due to expressed of stress
molecules on the cell membrane. In addition, γδ T cells possess many of the innate immune
receptors, e.g. TLRs and natural killer receptors that are viable in recognising pathogens. Αβ T cells
are activated by dendritic cells upon infection and either target the extracellular pathogen, infected
cells or stimulate B cells to start producing antibodies. On the other hand, T cells produce cytokines
that attract other immune cells, mainly innate immune cells, to the site of infection. The various
aspects of the immune system provide a complex but strong protection of the body against
pathogens, however this system is a logical target for the bacteria to aim for to promote bacterial
survival.
17 | P a g e
1.3 Endo- and exotoxins.
Two different groups can be distinguished in bacterial toxins: endotoxins and exotoxins. Endotoxins
are molecules attached to the membrane of gram-negative bacteria and are also known as
lipopolysaccharide (LPS). Exotoxins are soluble proteins that are secreted by bacteria to facilitate
invasion of the host tissues and spreading. This paragraph will describe the endotoxins to some
extent as the remaining chapters are dedicated to discussing mechanisms and toxicity of exotoxins.
1.3.1 Endotoxins
As is mentioned before, endotoxins are exclusively present on the majority of gram-negative
bacterial membranes. The endotoxic moiety of endotoxins is also known as lipopolysaccharide,
consisting of a lipid bound to polysaccharide. Endotoxins were postulated to be the cause of fever by
Richard Pfeiffer, back in late 19th century [Hitchcock et al, 1986]. The molecule itself was isolated and
described by three research groups under the lead of Boivin and Messrobeanu (1935), Goebel et al
(1945) and Morgan (1937)[Hitchcock et al, 1986]. They described the endotoxin to be a complex of
lipid, protein and polysaccharides [Hitchcock et al, 1986]. Only two decades later, the endotoxin was
purified and extracted from its proteins leaving LPS as protein free endotoxin, by Westphal et al
[Hitchcock et al, 1986]. This lipid bound polysaccharide molecule had all endotoxic properties of the
endotoxin. But even the polysaccharide moiety of LPS has been shown to be redundant [Hitchcock et
al, 1986]. Subsequently, the lipid part (lipid A) of LPS was isolated and organically synthesized. This
synthetic lipid showed similar toxicity as its biological relative [Hitchcock et al, 1986; Galanos et al,
1984].
Lipopolysaccharide consists of three portions, the core polysaccharide, the O polysaccharide
and lipid A (figure 8a). The precise composition of each portion varies among the gram-negative
bacterial species. The O polysaccharide is a strain of repetitive subunits made out of sugar units. Each
O polysaccharide chain can comprise of up to 50 subunits [Caroff et al, 2003]. There are some gramnegative
bacterial
a
b
species that possess
lipopolysaccharides that
lack
the
O
polysaccharide
chain.
These species have are
also called rough-type
bacteria, the presence
of LPS with the O
polysaccharide chain is
characteristic of the
smooth type bacteria
Figure 8: Structure of bacterial LPS
with as intermediate
Structure of LPS, consisting of lipid A and polysaccharide. A) Within the polysaccharide, 2
different parts can be distinguished, the O polysaccharideand the core saccharide having
semi-rough-type,
an inner and outer core. B) Among the LPS expressing bacteria, there are different types.
containing only one ORough type bacteria do not have the O polysaccharide chain (consisting of multiple units)
whereas smooth-type do. An intermediate is the semi-rough type, having a single O
chain unit (figure 8b)
polysaccharide unit. Adapted from Caroff et al, 2003
[Schmidt et al, 1968].
18 | P a g e
The function of these saccharide chains is protection against several antibiotics and to the
complement system [Nikaido, 1976]. O polysaccharide chains are also used by researchers to classify
serotypes among various bacteria families, determining the specificity of antibodies directed against
these bacteria [Caroff et al, 2003]. Linked to the O polysaccharide chains (if present) is the core
polysaccharide. Even though the core polysaccharide is less variable between bacterial species, the
only common feature between all gram-negative species is the keto-deoxyoctulosonate (Kdo)
residue [Cipolla et al, 2010]. This residue links lipid A to the core polysaccharide and is an important
sugar residue for maintaining the structure of LPS. The core polysaccharide is in general composed of
several sugar unites and negatively charged due to the substitution of phosphate or carboxylic
moieties to the carbohydrates, probably contributing to the integrity of the outer membrane [Cipolla
et al, 2010]. The anchor of the LPS molecule consists of the final domain, the lipid A domain. This lipid
anchor has a backbone consisting of glucosamine disaccharides, which have fatty acids on specific
positions. The number of fatty acids can vary between 4 and 7. Both the fatty acids and the backbone
can differ between bacterial species [described in various reviews, including Alexander et al, 2001
and Caroff et al, 2003].
The toxicity of LPS is mediated through the extensive immune response it induces.
Mononuclear cells have a high sensitivity to the molecule as they express TLR4 and CD14 on the cell
membrane and respond to detection of LPS by producing and releasing a broad spectrum of proinflammatory cytokines and reduced oxygen species [Rossol M et al, 2011; Rietschel et al, 1996]. In
addition, the complement system is activated, and facilitates clearance of the gram-negative bacteria
by opsonisation of the bacteria or by direct killing through the creation of pores in the bacterial
membrane [Freudenberg MA et al, 1978]. Activation of the first line of immune cells leads to the
activation of the adaptive immune cells, inducing a specific and complex response to the pathogen or
LPS. As has been described before, activation of the adaptive immune system also triggers memory
formation. When this immune response is balanced and moderate, it leads to the clearance of the
bacteria with occasional occurrence of mild fever. However, in patients where the immune system
has been suppressed for a period of time and reacts to strong to the LPS bearing pathogen, the
immune response gets too strong. This leads to a so-called cytokine storm, through stimulation and
activation of innate immune cells; the formation of blood clots in the vascular system by the
complement system and reparation of damaged vessel walls causing organs to dysfunction and
failure. Ultimately, this immune response can lead to septic shock with often lethal outcome
[Rietschel et al, 1996; Woltmann et al, 1998].
19 | P a g e
Chapter 2: Bacterial protein toxin classification
There are many possible methods to classify bacterial protein toxins, but there is no universally
accepted taxonomy. One method uses the location, another method focuses on the bacterial species
that produces them, and others use the mechanism of action. Here, the location of target has been
used to provide an order of discussing the exotoxin mechanisms of action. The locations consist of
the plasma membrane, receptors and intracellular targets.
2.1 Membrane damaging toxins (cytolysins)
It is vital for a cell to maintain its interior milieu, through the influx of water and efflux of ions. The
membrane damaging toxins (cytolysins) discussed in this chapter target the cellular membrane to
disturb its permeability by the formation of pores, degradation of membrane lipids or through a
surfactant activity. This disturbance leads to an alteration of the interior milieu, causing the cell to
burst. In addition, even sublytic doses of membrane damaging toxins can have extended
consequences, especially on immune cells. For example, it induces the release of cytokines but also
inhibits chemotaxis of neutrophils. In the following chapter, the different types of membrane
targeting toxins are discussed, as well as their mechanism of action and the most familiar bacterial
toxins that belong to this class of exotoxins.
2.2 Receptor-targeting toxins
A cell relies on signals and stimuli from outside to survive. These signals and stimuli are received by
receptors, located extracellular on the plasma membrane. For example, epithelial cells are stimulated
to replicate when the epithelial growth factor (EGF) binds to the epithelial growth factor receptor
(EGFR). This binding induces all kinds of changes to intracellular targets, for instance the
phosphorylation or acetylation of proteins or DNA, leading to DNA replication and mitosis.
Overactivation of receptors can have profound consequences. There are some exotoxins that target
receptors, affecting the intracellular pathways and physiological state of the cell and body systems.
These exotoxins and their mechanisms of action are discussed in chapter 4.
2.3 Internalized toxins
The interior milieu of the cell is protected by the plasma membrane, keeping proteins in and out,
allowing molecules to pass the membrane only when the cell makes this possible. Toxins targeting
intracellular targets must first be translocated through the plasma membrane before they can reach
their target. The main mechanism for the translocation is internalisation, induced through binding to
receptors. All internalized toxins therefore have a similar mechanism to bind to the cell and get
internalized, as the general structure of these toxins is alike. The most prominent difference between
internalized toxins is the intracellular target. By modulating the intracellular target, the exotoxins
modulate cellular pathways from within the inside, having profound effects on the cellular
physiological state. These toxins are discussed in chapter 5, giving a detailed overview on the targets,
effects and mechanisms on internalisation using specific toxins as examples.
20 | P a g e
Chapter 3: Membrane damaging toxins (cytolysins)
Although cytolysins are discussed here as solely products of bacterial species, mammalian and
arthropod species produces similar proteins with cytolytic activity. For example, the perforins
secreted by mammalian immune cells, components of the complement system of mammalian
organisms and other membrane damaging agents have been described to execute a similar function
as bacterial cytolysins. An enzymatic cytolysin, phospholipase D produced by multiple bacterial
species among which Corynebacterium pseudotuberculosis, has been shown also to be produced by
the brown recluse spider (Loxosceles reclusa) [Bernheimer, 1996; Kurpiewski et al, 1981]. These two
phospholipases were indistinguishable based on their activity, but are different in antigenicity. In
addition, a similar parallel is described between a toxin produced by a sea anemone and streptylysin
O. Here too, both toxins are alike considering their toxicity, but differ in antigenicity [Bernheimer,
1996; Bernheimer et al, 1979]. One of the first membrane-damaging toxin was studied by P. Ehrlich
in 1898 [Bernheimer, 1996], and was termed tetanolysin. Tetanolysin is a pore-forming toxin
produced by Clostridium tetani. Another membrane-active toxin described in the early 20th century
was perfringolysin O, however it is not certain whether Herter, the researcher on the studies, had not
been working on the alpha-toxin of Clostridium perfringens [Bernheimer, 1996]. It was long thought
that most toxins had a narrow specificity when it comes to cell types. For example, hemolysins were
thought to only target erythrocytes, however it is since 1940 known that other cell types were
vulnerable to these proteins. Ever since, more research has been conducted on the function and
structure of membrane-damaging toxins, and more details on how the toxins operate on cell
membranes are discovered. Here, cytolysins are divided in three classes, pore-forming toxins,
enzymatic cytolysins and detergent-like toxins. Of all three classes, the structure, mechanism and
prototype toxins will be described.
3.1 Pore-forming toxins (PFTs)
The largest group representing the cytolysins and known bacterial toxins are the pore-forming
toxins (PFTs) [Alouf et al, 2001]. These toxins are water-soluble but are able to penetrate the cell
membrane by protein conformation changes. These changes allow the proteins to transform from a
hydrophilic state to amphiphilic [Balfanz et al, 1996]. Most PFTs require oligomerization before they
are able to penetrate the plasma membrane [Balfanz et al, 1996; Gonzalez et al, 2008]. Pore-forming
toxins can be divided in many types based on their membrane-binding mechanism or pore size.
However, the structure of PFTs as classification criteria is a more preferred criterion, used in many
reviews [Gonzalez et al, 2008; Parker et al, 2005]. Based on this structure, two classes can be
distinguished, the α-PFTs and the β-PFTs. The Greek letter refers to secondary protein structures,
namely alpha-helices and beta-barrels. The α-PFTs are shown to have an alpha-helical region for
spanning the plasma membrane (figure 9a), β-PFTs use beta-barrels (figure 9b). Both PFT classes are
discussed below, concerning structure, mechanism of pore formation and toxin prototypes.
21 | P a g e
a
b
Figure 9: Structure of colicin N and
aerolysin.
a) The α helical structure is shown for
colicin N. The black helices are shown
to be inserted into the membrane for
the pore formation. b) The toxin
aerolysin consists mainly of β-strands
structured in β-sheets. The dark grey
domain, domain 4 is inserted into the
cell membrane, including the black
loop from domain three. These two
toxins are shown as example for αand β-PFTs. Adapted from Parker et al,
2005
3.1.1 α-PFTs
Alpha pore forming toxins contain α-helices that facilitate the insertion into the plasma membrane.
Some toxins contain a three-layered structure consisting of up to 10 α-helices, enclosing a
hydrophobic hairpin that presumably facilitates membrane insertion [Parker et al, 2005]. Some
familiar toxins that belong to this family are the pore-forming colicins produced by various strains of
Escherichia Coli, diphtheria toxin produced by Corynebacterium diphtheria, exotoxin A secreted by
Pseudomonas aeruginosa and the Cry toxins produced by Bacillus thuringiensis [Parker et al, 2005;
Iacovache et al, 2008]. The colicins by E. coli have received considerable attention on its structure
and mechanism of action during its insertion into the cell membrane and are therefore the best
described α pore forming toxin. These toxins are produced by E. coli in order to attack other E. coli
species during infection and invasion. Naturally, these bacterial species have created a defence
mechanism against their own toxin [Parker et al, 2005; Weaver et al, 1981]. The toxin is released
upon lysis of the producing cells, facilitated by the colicin lysis protein (also known as bacteriocidin
release protein or as the killing (kil) protein [Van der Wal et al, 1995]. Upon secretion, the toxin binds
to its target protein on the target cell and gets transported over the outer membrane by one of two
bacterial protein transport systems, Ton or Tol [Lloubès et al, 2001; Postle et al, 2003]. The transport
system used for the toxin divides the known bacterial colicins into two groups, colicin A, N, E1-E9 and
K use the Tol pathway and colicins B, Ia, Ib, D, M, V, 10 and 5 use the Ton pathway [Lazdunski et al,
1998]. Colicins are structurally divided in three domains, each involved in specific phases of invasion,
namely receptor binding, translocation and cell death [Parker et al, 2005]. The translocation domain
facilitates the transportation by the protein transport system. The domain involved in the facilitation
of cell death can have multiple functions within this aspect. Only five colicins (A, B, N, E1 and Ia) are
known to kill the target cell by forming ion-channels and thereby disrupting the regular processes
inside the bacterial cell [Elkins et al, 1997; Hilsenbeck et al, 2004; Vetter et al, 1998; Wiener et al,
1997; Parker et al, 1989]. The other colicins kill the target cell via different mechanisms, e.g. by
functioning as DNase or RNase or by inhibiting protein synthesis [Cascales et al, 2007]. The poreforming domain of colicins A, B, N, E1 and Ia consists of 10 tightly packed alpha helices, consisting of
22 | P a g e
two hydrophobic helices surrounded by mostly amphipatic helices [Elkins et al, 1997; Hilsenbeck et
al, 2004; Vetter et al, 1998; Wiener et al, 1997; Parker et al, 1989]. Without the presence of a
membrane potential, the hydrophobic helices penetrate the inner membrane, separating the
periplasm from the cytoplasm, via an umbrella like configuration, leaving the other helices
embedded in the membrane surface [Muga et al, 1993], shown in figure 10b. This insertion creates a
pore in its so called closed state. There are studies showing that the hydrophobic helices, at least for
colicin A and B, undergo transition to a molten-globule state prior to interaction with the membrane
[Evans et al, 1996; Van der Goot et al, 1991;
Muga et al, 1993]. In this state, the tertiary
structure is lost but the secondary structure
remains intact. For colicin E1 and N, there is
evidence that this molten-globule state does
not occur [Schendel et al, 1994; Zakharov et al,
2002, Evans et al, 1996]. Upon application of
positive voltage, the alpha helices of the domain
insert themselves in the membrane as well,
forming an ion channel, the pore in its open
state (figure 10c). The size of the pore is
approximately 10 Å, but studies have shown
that larger molecules can pass the channel. The
smallest reported pore size was shown to be 7
Å, formed by colicin Ia [Henry et al, 2004]. Not
only ions pass the colicin channel, far larger
molecules are also shown to be transported
through the channel. Colicin Ia was presented
with a range of proteins with varying sizes that
had the potential to be transported by the pore.
Proteins as large as 26 Å were transported by
the channel, even though the protein
maintained its tertiary structure through
disulphide bonds [Kienker et al, 2003]. These
proteins are presumable transported by the
conformation changes when the pore
transitions from closed to an open state. The
presumed pore size of the colicin channel is still
under investigation, as the protein available for
the formation of the channel is likely to be
inadequate to be able to form the reported size. Figure 10: Umbrella-like insertion of colicins and other αPFTs
The combination of multiple monomers would a) Toxin structure in its completely folded form prior to
explain the pore size, however studies suggest binding to the target cell. B) Binding to the target cell
that the pores are formed by single proteins. induces a conformational change through which the
hydrophobic hairpin, here shown with helices 8 and 9,
Another explanation would be that the forming a closed pore, while the rest of the pores are
transmembrane helices recruit lipids located in embedded in the cell membrane. C) Through the application
of positive voltage or other cues, the other helices insert
the membrane to participate in the formation of themselves as well, forming the open pore. Adapted from
the pore, as is used for multiple other proteins Parker et al, 2005
23 | P a g e
forming multimeric pores [Lin et al. 2000; Matsuzaki, 1998; Matsuzaki et al, 1996]. Some evidence
has reported on this mechanism for colicins [Sobko et al, 2004; Sobko et al, 2006]. A similar pore
forming structure is found in by the cry toxins secreted by Bacillus thuringiensis. This group of toxins
apply a different mechanism of pore formation as the monomeric toxins oligomerize into pre-pore
complexes before membrane insertion [Gomez et al, 2002]. Nevertheless, the insertion of the prepore complex into the membrane has a similar series of events. The oligomerisation of the toxin
monomer leads to a conformational change into a molten-globule state, revealing the hydrophobic
hairpins of the oligomer inducing the insertion. Unlike most toxins, the molten-globule state is
induced by alkaline pH, whereas for numerous toxins that undergo such a conformation change, it is
induced by acidic pH [Raussell et al, 2004]. While the hydrophobic core inserts itself into the
membrane, the other helices are embedded in the membrane surface [Pardo-López et al, 2006]. The
interaction of the pre-pore complex with its receptor has been shown to be important for the
insertion process [Pardo-López et al, 2006]. Another toxin that has been studied in the relevance of
pore formation is diphtheria toxin produced by Corynebacterium diphtheria. This toxin does not
entirely belong to the α-PFT family as its pore formation is likely involved in the translocation process
for the catalytically active domain of the toxin. However, studies have shown that the toxin is capable
of forming ion-channels [Papini et al, 1998; Kagan et al, 1981; Donovan et al, 1981]. It is still unclear
whether these channels are involved in the translocation of the cell-killing domain into the
cytoplasm. Like colicins, diphtheria translocation domain consists of 10 helices and contains a
hydrophobic hairpin that is masked by the other helices [Parker et al, 2005]. Upon arrival in the
endosome, the pH is lowered, inducing changes in amino acid charges, initiating insertion of the
hydrophobic hairpin [Choe et al, 1992]. It has been suggested that the pore-forming, translocation
domain undergoes the molten-globule conformation change, facilitating the membrane insertion
[Chenal et al, 2002]. When comparing all the mechanisms of the pore-forming domains of α-PFTS and
their structures, many differences are apparent. Nevertheless, despite these differences, they are
more similar than appears. For one, they all have a helical secondary structure, as they belong to the
α-PFTs. Furthermore, most pore-forming toxins consist of three domains, each exhibiting a specific
function attributed to the toxin. Most α-PFTs create a pore as a monomer, except for Cry toxins and
E. coli haemolysin E. The majority of the α-PFTs bind to a receptor before the membrane-insertion is
initiated, except for colicins [Schein et al, 1978]. Perhaps, the receptor binding positions the
translocation domain optimally for insertion, or the binding reveals the hydrophobic core, as for Cry
toxins. Structure wise, there are many similarities as well. The pore-forming domains that consist out
of a large bundle of helices assembled into a three-layer structure. This three-layer structure consists
of two outer layers of amphipathic helices, masking the inner hydrophobic core. The three-layer
conformation allows the protein to be water-soluble and diffuse through intracellular spaces. For
none of the α-PFTs, the exact mechanism is known through which the pore-forming domain changes
its conformation to allow the hydrophobic helices to insert themselves into the target membrane,
however for most a model has been designed that may predict the mechanism. The formation of
pores in the target cells may have several benefits for the bacteria. Due to the induced pores in the
membrane, the cellular osmotic balance is disrupted, causing the cell to rupture and release its
nutrients, beneficial for the bacterial growth and spreading [Cascales et al, 2007; Knowles et al,
1987], with the exception of diphtheria toxin.
24 | P a g e
3.1.2 β-PFTs
The β pore forming toxins, or the β-barrel pore forming toxins create pores with the use of β-strands.
Unlike the α-PFTs, β-PFT monomers are required to oligomerise to form a pre-pore before they are
able to penetrate the target membrane, with only a few exceptions. The general order of events is
Figure 11: General process of β-PFT pore formation
General steps of the pore formation for β-PFTs are shown. The bacterial cell secretes the toxins that bind to the target cell
and oligomerise to form a pre-pore complex. Conformational changes induce the insertion of the pre-pore complex into the
cell membrane, sometimes after prior internalisation.
the secretion of the pro-toxin, cleavage for activation, binding to the target receptor, oligomerisation
and formation of a pre-pore complex and finally insertion (figure 11). The first toxin in this category
whose structure was identified is aerolysin. This toxin is produced by three Aeromonas species,
namely A. hydrophila, A. trota and A. salmonicida [Rossjohn et al, 1998a]. Unlike most β-PFTs,
aerolysin does not show apparent hydrophobic stretches and appears to be hydrophilic [Parker et al,
1994]. The toxin is secreted as protoxin, after being folded and dimerised in the periplasm [Howard
et al, 1996]. After binding to its cell receptor, it is cleaved by gut proteases or furin family members
[Howard et al, 1985; Abrami et al, 1998]. Cleavage of the toxin activates it and promotes
oligomerisation [Van der Goot et al, 1992]. If the concentration of aerolysin at the cell membrane is
high enough, they oligomerize in a heptameric pre-pore complex [Wilmsen et al, 1992; Moniatte et
al, 1996]. When the pre-pore has been formed, insertion in the target membrane is initiated as the
activating cleavage has induced conformational changes, exposing a hydrophobic β-sheet [Rossjohn
et al, 1998a]. Aerolysin consists of two protein lobes linked together, a small and a large one. The
small one operates as domain I, while the large lobe consists of three domains, domain II-IV, even
though the lobe constitutes out of long stretches of uninterrupted β-strands [Rossjohn et al, 1998a].
The first domain, the small lobe, is involved in the dimerization as proaerolysin and receptor binding.
Domain II also operates in the receptor binding phase and together with domain III are engaged
during oligomerisation. Domain IV gets inserted into the target membrane, possibly partially
together with domain III [Rossjohn et al, 1998a; Rossjohn et al, 1998b]. The final pore resembles a
mushroom, similar construction is shown in figure 12a, with domain III and IV as stem and domain I
and II as the head [Rossjohn et al, 1998a; Parker et al, 2005]. There are a few β-PFTs that form
channels in a similar fashion as aerolysin. Among these homologues are the alpha toxin of
Clostridium septicum (high sequence homology), the enterolobin from the seeds of Enterolobium
contortisiliquum, a brazillian tree, and ε toxin of Clostridium perfringens (structurally identical)
[Ballard et al, 1993; Cole et al, 2004; Bittencourt et al, 2003; Sousa et al, 1994]. Interesting to note is
25 | P a g e
that aerolysin or homologues are synthesised by a gram-negative, -positive bacteria and a plant.
Another toxin that is a paradigm to the channel formation by β-PFTs is α-haemolysin (Hla) produced
by Staphylococcus aureus. Like aerolysin, the appearance of the final pore looks like a mushroom,
with the cap at the membrane surface and the stem inside the membrane, functioning as channel
(figure 12a) [Song et al, 1996]. The toxin is released as a monomer (figure 12b) and binds to among
others, human platelets and endothelial cells [Gouaux et al, 1998; Bhakdi et al, 1988; Suttorp et al,
1988]. When bound to its, still unidentified, receptor, it oligomerises into a heptameric structure,
ready to get inserted [Gouaux et al, 1994, Gouaux et al, 1998]. The event of oligomerisation is a
cooperative process whereas the insertion of the pre-pore is an independent action of the
incorporated monomers. So if it happens that one of the monomers is unable to insert itself into the
membrane due to mutations, only the defective monomer does not continue to insert, leading to the
formation of a hexamer channel [Valeva et al,
1997]. The exact cue for pre-pore insertion is
unknown, but it is known that through the
oligomerisation, the glycine rich region in the
stem is rearranged, exposing other residues that
are likely involved in the insertion [Gouaux et al.
1998]. Α-haemolysin homologues are produced by
the same bacteria, namely the leukocidins and γhaemolysin [Parker et al, 2005]. Leukocidins are
bimeric toxins, consisting of a slow (S) and fast (F)
eluting unit. The slow eluting unit binds to the
target cell, allowing the fast eluting unit to bind to
the S unit and insert itself into the membrane
[Colin et al, 1994]. The formation of oligomers
consists of the combination of 4 S units and four F
units, forming a β-barrel pore [Miles et al, 2002],
but the formation of hexameric and even
heptameric with uneven distribution of the units
has been proposed [Sugawara et al, 1999;
Sugawara-Tomito et al, 2002]. This would suggest
that unlike α-haemolysin, leukocidin pores
probably consist of an even number of monomers.
The insertion of the leukocidin pore complex is
likely to follow the same process as α-haemolysin;
nevertheless the exact order of events for
insertion is still unknown. A familiar leukocidin
belonging to the α-haemolysin homologues is the
Panton-Valentine leukocidin [Löffler et al, 2010]. Figure 12: Structure of a mushroom-like pore formed by
several β-PFTs.
Similar to leukocidins and homologue to α- a) The pore formed by some β-PFTs appears like a
haemolysin is γ-haemolysin (Hlg) [Parker et al, mushroom. It consists of a cap, rim and stem, the latter
2005; Rossjohn et al, 1998]. The gene for this functioning as the pore spanning the cell membrane. 7
monomeric toxins are used to form this pore. b) The
toxin encodes three different proteins, HlgA, HlgB monomeric toxin α-haemolysin. The stem domain (black)
and HlgC, forming a bimeric toxin [Menestrina et inserts in the cell membrane while the cap domain (dark
grey) and the rim domain (light grey) stay on the outside.
al, 2003]. The HlgB protein pairs up with either Adapted from Parker et al, 2005
26 | P a g e
HlgA or HlgC, similar to the fast and slow eluting units, where HlgB would suffice as fast and HlgA and
C as slow eluting [Menestrina et al, 2003]. They oligomerise and insert itself in a similar fashion as for
the leukocidins. Moving on to other β-PFTs is the anthrax protective antigen produced by Bacillus
anthracis. The complete anthrax toxin consists of three exotoxins secreted as singular proteins. The
translocating part of the toxin, protective antigen (PA), binds to the cell surface and gets cleaved by
furin [Collier et al, 2003]. Cleavage of the protein initiates the formation of a pre-pore by
oligomerisation and its stabilisation by binding of the catalytic domains that are part of the anthrax
toxin [Wigelsworth et al, 2004]. The binding and oligomerisation of the PA protein initiates the
internalisation of the receptor-pre-pore-catalytic domain complex [Abrami et al, 2003; Beauregard et
al, 2000; Liu et al, 2003]. The pre-pore complex with the enzymatic unit attached is trafficked to an
endosome, in which the low pH triggers the pore insertion by conformational changes of the PA unit
[Milne et al, 1993] and translocation of the enzymatic factors. This translocation process is described
in chapter 5.5. A complete different category in the class of β-PFTs is the PFTs that are dependent on
cholesterol-binding. These toxins only bind to membranes that are rich in cholesterol in order to be
able to penetrate. However, whether the cholesterol is relevant for membrane binding or membrane
insertion remains a central dogma in the field [Giddings et al, 2003; Ramachandran et al, 2002]. In
addition, it is known that the cholesterol-dependent toxins oligomerize. An extensively studied toxin
in this category is perfringolysin O, secreted by Clostridium perfringens. This toxin binds to the cell
surface and penetrates the membrane with a hydrophobic dagger and thereby anchoring itself
[Ramachandran et al, 2002 Parker]. Another domain of the toxin is brought closer to the surface,
making the toxin accessible for oligomerisation [Abdel Ghani et al, 1999]. The process of creating an
oligomer induces conformational changes in each monomer, likely exposing and providing space for
β-hairpins to penetrate the membrane and insertion of the pre-pore [Hotze et al, 2001; Heuck et al,
2001]. From the previous discussed toxins, there are two aspects that bring similarity between them
all, namely the presence of β-sheets and the prerequisite to form oligomers in order to create a pore.
This prerequisite is most likely because the toxin needs to create a channel that is hydrophilic
channel whereas the exterior has to be hydrophobic in order to insert. However, a pore-inserting
aspect that unites the β-PFTs is harder to identify. This lies perhaps in a 3 stranded β-sheet that
transforms to a hydrophobic β-hairpin that at least for α-haemolysin, cholesterol-dependent
cytolysins and anthrax PA is shown to exist and play an important role in their conformational
changes. The primary function of the pore formation by β-PFTs is like α-PFTs, the disruption of
osmotic balance and cell rupture, in the bacteria’s benefit. However, these pores also play a role in
the translocation of enzymatic domains into the cytosol.
3.2 Enzymatic cytolysins
The eukaryotic cell membrane consists mainly of phospholipids (including sphingomyelin) and
cholesterol. These lipids form a bilayer, keeping the cell in osmotic balance while being selective in
what goes into and out of the cell. The cellular enzymes that maintain the integrity of the lipid bilayer
are phospholipases, sphingomyelinases and cholesterol oxidases. In addition to taking care of the
lipid turnover, these enzymes also contribute to the signal transduction by providing precursors of
second messengers [Schmiel et al, 1999]. Among the phospholipases, different classes can be
distinguished (A-D), based on the hydrolysis site. These different phospholipases are responsible for
the formation of different second messenger precursers, dependent on the hydrolysis site. For
27 | P a g e
example, hydrolysis by phospholipase C leads to the release of choline phosphate, inositol
phosphate, inositol triphosphate and diacylglycerol, while phospholipase A cleaves of the fatty acids
from the glycerol [Schmiel et al, 1999; Goni et al, 2012]. Moreover, sphingomyelinase cleaves
spinghomyelin into phosphorycholine and ceramide [Goni et al, 2012]. In contrast to the eukaryotic
phospholipases and cholesterol oxidases, some bacteria species are known to produce similar
enzymes, but instead of keeping the cell membrane intact, they disrupt its integrity. The majority of
these cytolysins damage the membrane by hydrolysing phospholipids, while the minority targets
either sphingomyelin or cholesterol [Menestrina et al, 1994]. The process of membrane damaging
consists of two steps, the first being the damaging of the outer layer of the lipid bilayer, while the
second step is lytic [Menestrina et al, 1994]. Some of the enzymatic cytolysins are able to lyse the cell
within the first step of membrane damage. But for the majority of the toxins, the second step is
performed by other enzymatic cytolysins or even by by-products of the hydrolysis process. A well
described phospholipase is phospholipase C produced by Clostridium perfringens, also known as αtoxin. This is one of the toxins that lyses the cell during the first step of the lytic process. As a zinc
metalloprotease, it hydrolyses phospholipids with high efficiency, however has also been shown to
cleave sphingomyelin to some extent [Oda et al, 2008]. These fatal hydrolysing effects are only
induced by high concentrations of the toxin, in low concentrations, it is limited in its hydrolysis
activities, leading to the activation of diacylglycerol and ceramide [Oda et al, 2008, Ochi et al, 2004].
Diacylglycerol (DAG) is a second messenger in eukaryotic cells and, among other effects, activates
protein kinase C (PKC). PKC in its turn activates eukaryotic phospholipase A and C, inducing even
more phospholipid degradation [Goni et al, 2012; Nishizuka, 1992; Nishizuka, 1995]. In addition, its
sphingomyelinase activities lead to the production of ceramide, a second messenger involved in
apoptosis, cell differentiation and cell proliferation [Okazaki et al, 1989; Kim et al, 1991; Wiegman et
al, 1994; Gomez-Muñoz et al, 1994; Kuy et ak, 1995; Obeid et al, 1993]. Thus, low and high
concentrations of phospholipase C induce the death of the target cell. The phospholipases (A, C or D),
the sphingomyelinases and cholesterol oxidases produced by the bacteria are very similar in their
mechanisms of action. The toxins disrupt the membrane integrity by cleaving its components and
with or without the help of other enzymes or second messengers, to either escape their own
degradation or lyse the cell in order to release nutrients that promote bacterial growth and spread
[Songer et al, 1997; Schmiel et al, 1999].
28 | P a g e
3.3 Detergent-like toxins
Another category of toxins are the detergent-like toxins. These toxins have not been studied
extensively in the exact mode of action of cytolysis. However, the best characterized, although this is
an overstatement, is the δ-lysin produced by Staphylococcus aureus. This toxin binds to its target
cells unspecifically and is therefore thought to bind to the lipids of the cellular bilayer. Through
experiments, it has been shown that in low concentrations, the δ-toxin binds to the cell membrane
but does not penetrate it, although through its binding shifts the lipids slightly aside, causing an
increased permeability of the cell membrane (figure 13a) [Talbot et al, 2001]. During this phase, it
may also initiate several processes in the cell, including the activation of cellular phospholipase A. A
higher concentration of toxin causes the toxin, which is highly amphiphatic, to insert itself
perpendicularly into the cell membrane and mingle with other inserted toxin monomers (figure 13b).
As soon as the toxin monomers adopt a certain conformation in which they expose their hydrophilic
surface, an ion channel can open, causing an osmotic imbalance [Mellor et al, 1988; Raghunathan et
al, 1990]. An even higher concentration of δ-toxin, it may even behave as a detergent, enclosing
parts of the bilayer membrane and solubilizing it (figure 13c), creating holes in the cell membrane
causing the cell to erupt and die [Bernheimer, 1974; Lohner et al, 1999; Thelestam et al, 1975].
Another toxin that by some papers gets categorized in the detergent-like toxins is streptolysin S,
produced by Streptococcus pyogenes [Balfanz et al, 1996; Menestrina et al, 1994]. To date, despite
the years of research, the structure nor functions and mode of action have been conclusively defined
and described. It has
been reported that
the toxin creates
pores in a similar way
as the complement
system; however, the
complement system
has
not
been
suggested to form
such pores in a
detergent-like manner
[Carr et al, 2001]. The
only toxin that has
been described to be
detergent-like,
in
addition
to
Staphylococcus
Figure 13: The membrane damaging mechanism of detergent-like toxins.
aureus’ δ-toxin is a) Low concentrations of detergent-like toxin δ-lysin bind to the cell membrane
unspecifically and push the lipids in the lipid bilayer slightly apart. This creates a weak
melittin,
a
non- permeability. B) Higher concentrations will induce the dimerization of monomers and the
bacterial toxin found perpendicular insertion in to the cell membrane, forming ion channels. C) Even higher
concentrations will form micelles around parts of the cell membrane, solubilising them and
in bee-venom [Pott et creating holes in the cell membrane. Adapted from Verdon et al, 2009.
al, 1995; Dufourq et
al, 1986; Suttorp et al, 1985].
29 | P a g e
Chapter 4: Receptor-targeting toxins
Previous chapter has discussed the toxins that target the membrane and make it permeable for
ions, molecules and water. This allows the toxin to kill the cell, either by the lysis through osmotic
imbalance or through the allowance of its cell-killing subunit to enter the cell and initiate cell death.
This chapter will discuss the few toxins that are known to bind to receptors and induce signalling
pathways, leading to disease symptoms like diarrhea and weak immune system through initial
overstimulation of the immune system.
4.1 E. Coli heat-stable enterotoxin
Escherichia
coli
are
extensively studied bacteria,
especially
regarding
its
pathogenicity and its toxins.
Within this bacterial species
there are several strains, some
harmless and others highly
virulent,
including
enterohaemorragic (EHEC) and
enterotoxigenic (ETEC) strains
[Giannella et al, 2003]. The
latter
produces
several
enterotoxins and is the most
common pathogenic E. coli Figure 14: Mechanism of heat-stable enterotoxin of CFTR activation and NHE3
strain for humans [Giannella et inhibition.
Besides the heat-stable toxin, guanylin and uroguanylin bind to the GC-C
al, 2003]. These enterotoxins receptor, inducing the dimerization and activation of the catalytic domain. The
include heat-labile enterotoxin catalytic domain synthesises high levels of cGMP, which in its turn activates
PKGII and PKA. PKA and PKGII induce the translocation of CFTRs to the cell
(LT), Shiga-like toxin (STEC) and surface, while PKA inhibits the Na+/H+ exchanger NHE3, inhibiting the absorption
heat-stable enterotoxin (ST). of NA+. Furthermore, the high levels of cGMP accumulating inhibits PDE3,
Besides the difference in therefore inhibiting the degradation of cAMP, activating PKA even more.
Adapted from Weiglmeier et al, 2010
structure, these toxins also
differ in their mechanisms of action. The LT activates adenylyl cyclase, increasing cyclic AMP (cAMP)
levels in the cell and activating cAMP-dependent kinase (PKA) leading to Cl- secretion and inhibition
of Na+ absoption [Spangler et al, 1992]. These electrolyte balance alterations lead to the secretion of
water into the lumen, causing diarrhea. The mechanism of action is similar to cholera toxin, the
enterotoxin produced by Vibrio cholerae and causative agent of diarrhea in Vibrio cholerae infected
patients (discussed in chapter 5.2) [Spangler et al, 1992]. Despite its activation of adenylyl cyclase,
this happens through intracellular interactions with the cyclase and not through the binding of an
extracellular binding domain, leading to activation [Spangler et al, 1992]. The heat-stable enterotoxin
produced by ETEC strains does bind to a receptor leading to direct activation of a different cyclase,
namely membrane-bound guanylyl cyclase C (GC-C) [Schulz et al, 1990; Field et al, 1978; Vaandrager
et al, 1993]. The guanylyl cyclase C receptor is a transmembrane receptor, passing the membrane
30 | P a g e
once, with an extracellular domain that orchestrates binding of its ligands and two intracellular
subdomains, a kinase-homology domain and a guanylyl cyclase catalytic domain (figure 14)
[Vaandrager, 2003]. There are seven members of the GC family known (A-G), of which three its
ligands are known. GC-A is bound by atrial natriuretic peptide (ANP) and GC-B is the receptor for
brain and C-type natriuretic peptide (BNP and CNP respectively) [Wong et al, 1992; Wedel et al,
2001; Potter et al, 2001; Schulz et al, 1999; Drewett et al, 1994]. Besides ST produced by E. coli, the
GC-C also gets activated by guanylin and uroguanylin, two polypeptides that are released by body
cells in the intestine, binding to the same binding domain on the extracellular part of GC-C and
inducing the same cellular processes as ST [24-26 Hasegawa]. The activation of gyanylyl cyclase leads
to the accumulation and increase of cyclic GMP (cGMP) [Vaandrager et al, 1997; Vaandrager et al,
1998]. The accumulation of cGMP leads to the activation of cGMP-dependent protein kinase II (PKG),
with a subsequent translocation of cystic fibrosis transmembrane conductance regulator (CFTR) to
the cell membrane, regulation the secretion of Cl- , HCO3- and water as a result (figure 14)
[Vaandrager et al, 1997; Vaandrager et al, 1998;Kleizen et al, 2000; Golin-Bisello et al, 2005]. In
addition, the accumulating levels of cGMP are able to inhibit phosphodiesterase 3 (PDE3). This
enzyme hydrolyses cAMP, therefore its inhibition leads to increase levels of cAMP, initiating the
activation of protein kinase A (PKA) [Chao et al, 1994]. PKA in its turn inhibits the absorption of Na+
and induces CFTR targeting to the cell membrane as well as inhibition of NHE3, a Na+/H+-exchanger
(figure 14) [Chao et al, 1994; Tousson et al, 1996; He et al, 2010]. Like with the cholera toxin and LT,
Cl- secretion leads to the occurrence of diarrhea as the water secretion in the intestinal lumen
exceeds the reabsorbance capacity of the colon. The difference between the binding of ST and
guanylin/uroguanylin is that ST overactivates guanylyl cyclase C, whereas binding of guanylin and
uroguanylin is a normal bodily process, regulating the balance of electrolytes in intestinal cells.
Besides this regulatory function in the intestine, GC-C has been reported to be expressed in a variety
of other organs, including in the kidney, liver and lungs [Wong et al, 1992; Wedel et al, 2001; Laney
et al, 1992; Carrithers et al, 2000; Sindice et al, 2002]. ST/guanylin/uroguanylin has been shown to
bind to different receptors as well [Sindice et al, 2002; Carrithers et al, 1999]. First of all, high and low
binding affinities by these polypeptides have been reported in the intestine [Crane et al, 1992].
Furthermore, when GC-C is absent, the polypeptides are still able to bind to the intestinal cells,
without guanylyl cyclase activity [Charney et al, 2001; Albano et al, 2001; Mann et al, 1993]. Thirdly, a
research group has reported that urogyanylin is able to stimulate two different signalling pathways in
the kidney [30 Giannella]. Besides the cGMP dependent signalling pathway, another pathway,
independent from cGMP and GC-C, involves a pertussis toxin-sensitive G protein [Sindice et al, 2002].
The function of this alternative receptor for ST/guanylin/urogyanylin has not been defined yet,
however it is almost certain that it does not take part in maintaining the Cl- levels, but might regulate
HCO3- transport in the intestines [Childs et al, 2001]. The heat-stable enterotoxin that binds to GC-C is
also produced by a variety of other bacteria, including both O1 and non-O1 Vibrio cholera, Yersinia
enterocolitica and Citrobacter freundii [Guarino et al, 1989; Takao et al, 1985a; Yoshino K et al, 1994;
Takao et al, 1985b; Yoshino K et al, 1993] sharing homologous sequences in the receptor binding
domain. It is therefore likely that these enterotoxins cause similar consequences as E. coli’s ST. The
benefit of causing diarrhea for the causative bacteria is unclear. The secretion of Cl- into the
intestinal lumen can be lethal for bacteria, and diarrhea is known to completely empty the intestines,
and therefore getting rid as well of the bacteria. A possible benefit may however be in the further
spreading of the bacteria amongst individuals, through contact with bacteria contaminated faeces.
31 | P a g e
4.2 Superantigens
As has been explained and discussed in chapter 1, the human body has evolved an intricate immune
system that is very potent in targeting pathogens, transformed and stressed cells. Therefore, it is to
be expected that pathogens take advantage of this system for their own benefit. Indeed, several
bacterial species use the immune system to weaken the host to positively influence bacterial
flourishing. These toxins are called superantigens (SAgs) and are produced by several viruses and
bacteria. The best characterized bacterial superantigens are produced by Staphylococcus aureus and
Streptococcus pyogenes [Llewelyn et al, 2002]. They both express a large panel of SAgs which can be
subdivided in four groups depending on their sequence identity. The mechanism in which SAgs can
weaken the host is by binding to MHC class II molecules on antigen-presenting cells and TCRs on T
cells, but in a way that does not resemble the convential peptide presentation [Marrack et al, 1990].
SAgs bind to the invariant regions of MHC class II molecules and not in the antigen-presenting
groove. In addition, it also binds solely to the β chain of TCRs, avoiding the specificity-determining
region (figure 15). The binding of SAgs to these two molecules brings them in close proximity, close
enough for the co-stimulatory proteins, expressed on both cells, to activate both the antigenpresenting cell and the T cell [Florquin et al, 1997]. This activation initiates the production and
secretion of a range of inflammatory cytokines, including monokines and lymphokines, each
stimulating lymphocytes and APCs even more. This overstimulation results in an inflammatory
cytokine cascade and replication of inflammatory T cells, overwhelming the regulatory systems of the
host immune system and damaging organs and tissue [Uchiyama et al, 1994; Ulrich et al, 1995; Kotb,
1997; Müller-Alouf et al, 1996; Müller-Alouf et al, 1997; Florquin et al, 1997]. Besides these
destructive effects, under certain conditions, anergy and even apoptosis of T cells is elicitated,
through extensive T cell replication and
inhibition of IL-2 production [O’Hehir et al,
1990; Miethke et al, 1993; Kawabe et al,
1991], leading to an enhanced susceptibility
of the host for other pathogens and their
toxic effects. Both streptococcal and
staphylococcal SAgs have been suggested to
contribute to the onset of toxic shock
syndromes, with the Streptococcal toxic
shock syndrome having a higher rate of
mortality [McCormick et al, 2001; Stevens,
1996]. The shock syndrome is likely to be
caused by the high levels of TNFα secreted
during the cytokine cascade. Ultimately, if
the conditions caused by SAgs are not Figure 15: Binding region of superantigens
treated early or at all, they can ultimately Superantigens bind to the Vβ chain of the TCR, while avoiding
lead to death. Besides other bacteria shown the specificity determing region, and invariant region of the
MHC class II molecule on the antigen presenting cell. This
to produce toxins that are able to initiate binding is different from the covalent binding of antigens in the
extensive T cell replication without MHC antigen-presentation groove in the middle. However,
class II presentation, there has been superantigens do not bind simultaneously with antigens.
evidence reported that the human genome Adopted from Müller-Alouf et al, 2001.
32 | P a g e
encodes a superantigen as well. This SAg is likely to be inserted by the human endogenous retrovirus
(HERV), coded for by the env gene [Sutkowski et al, 2001]. This has been suggested as infection of
cells by Epstein-Barr virus (EBV) induces the expression of SAg with specificity for a TCR β chain, even
though the SAg gene has not been found to be encoded by the EBV genome [Sutkowski et al, 2001].
A possible function of the SAg during EBV infection is the expansion of host T cells, providing cells in
which EBV can establish latency and persist in the host organism [Sutkowski et al, 2001]. This
mechanism of induction of SAg expression might explain the absence of SAgs encoded in several
other viruses, including cytomegalovirus and Human immune-deficiency virus (HIV), despite the SAglike T cell proliferation occurrence during infection [Llewelyn et al, 2002]. All together, the SAgs
provide a way for bacteria to modulate the immune system of its host, by causing an extensive
cascade of cytokines, leading to T cell replication, leading to IL2 depletion and subsequent T cell
anergy. Due to T cell anergy, the host is unable to elicit an adequate T cell or humoral immune
response towards neither the causative agents nor other pathogens that invade the body in its
immunologically weak condition.
33 | P a g e
Chapter 5: Internalized toxins
As the final chapter, the internalized toxins will be discussed here. These toxins are required to
enter the cell before they are able to execute their toxic effect. It is to this category that BoNT
belongs to, as it targets an
intracellular protein. The general
mechanism of action for these
toxins can be subdivided into
three steps, the target cell
binding,
induction
of
internalisation and its entry into
the cytosol, and ultimately the
modification of target molecules.
The general structure of such
toxins consists out of at least two
domains, one to provide the
catalytic activity (A domain, the
light chain of the toxin) and the Figure 16: Summarising overview of the toxins that will be discussed.
other domain for cell entry (B Schematic overview is shown for the 5 toxins that will be discussed in further
detail in chapter 5. The route of internalisation and location of translocation is
domain, heavy chain). The shown for anthrax toxin, diphtheria toxin, clostridial neurotoxins, Shiga toxin
specificity of the toxins to its and cholera toxin. The mechanisms of cell entry, internalisation, translocation
and intoxication will be described. Adopted from Falnes et al, 2000
target cell is depicted by its
binding domain, which binds to a cell specific receptor. There are too many toxins that belong to this
group and have a slightly different process of toxicity to discuss each and every one, therefore, only a
few will be discussed regarding their target cell binding and receptor specificity, the process of
internalisation and their target molecule modification. The toxins that will be discussed are: Shiga
toxin (Shigella dysenteria), Cholera toxin (Vibrio cholerae), Diphtheria toxin (Corynebacterium
diphtheria), Tetanus toxin (Clostridium tetani) and Antrax toxin (Bacillus anthracis) (figure 16). Each
of these five toxins will be discussed separately, going through all the steps of its mechanism of
action to avoid overcomplicating these toxins. Similar toxins to the ones that will be discussed can be
found in the appendix.
5.1 Shiga toxin (Shigella dysenteria)
Shiga toxin is secreted by Shigella dysenteria as a polypeptide, consisting of the two domains
discussed before. The A domain, containing the catalytic active region, is inactive upon cell binding
and will need to be cleaved by furin before the A1 subdomain can exert its toxic function [Sandvig et
al, 1996]. The B domain consists out of 5 B subunits, forming a pentameric ring structure around the
C terminal of the A domain [Paton et al, 1998]. Each B subunit contains three binding domains for its
receptor, globotriaosylceramide (Gb3) [Jacewicz et al., 1986; Lindberg et al., 1987; Lingwood et al.,
1987], thus the complete B domain is able to bind to up to 15 Gb3 molecules. This extensive binding
moiety contributes to the high binding affinity to the target cell [Windschiegl et al, 2009]. Gb3
molecules are expressed on a variety of cells, including kidney epithelial and endothelial cells,
34 | P a g e
microvascular endothelium cells in the lamina propria of the intestine and neurons of the central
nervous system [Engedal et al, 2011; Ren et al, 1999; Obata et al, 2008]. The extensive range of
target cells explains the severe effects of Shiga toxin on the body and its processes. The binding of
Shiga toxin to Gb3 induces the activation of multiple protein kinases, inducing the endocytosis in a
clatherin-coated vesicle manner [Sandvig et al, 1989; Nichols et al., 2001; Lauvrak et al., 2004; SaintPol et al., 2004; Sandvig et al, 1996], but also in a clatherin-independent manner [Lauvrak et al,
2004]. When the toxin is endocytosed, the lowering of the pH induces cleavage of the A domain into
A1 and A2 by furin [Garred et al, 1997, Garred et al, 1995]. The A1 subdomain stays bound to A2 by a
disulphide bridge [Garred et al, 1997; Olsnes et al, 1981]. These vesicles, containing the nicked toxin
bound to its receptor, are then targeted to the trans-Golgi network [Donta et al, 1995]. This sorting
process is likely to be dependent on the presence of Gb3 within lipid rafts, enriched with cholesterol,
specific membrane proteins and sphingolipids [Takenouchi et al, 2004; Falguières et al, 2001; Smith
et al, 2006; Tam et al, 2008], as well as the activation of
protein kinases involved in the p38 mitogen-associated
protein kinase (MapK) pathway [Wälchli et al., 2008;
Skånland et al, 2009] and actin cytoskeleton remodelling
[Hehnley et al, 2006; Takenouchi et al, 2004]. In addition,
several other proteins are shown to be involved in the
retrograde transport of the vesicle to the Golgi-apparatus
[Bujny et al, 2007; Popoff et al., 2007; Utskarpen et al.,
2007]. Upon arrival in the Golgi-organelle, it is unclear
whether the toxin is transported through each part (trans,
mediate and cis) of the Golgi-apparatus or whether the
toxin has a way to circumvent this system and is
transported directly to the endoplasmic reticulum (ER)
[Sandvig et al, 2010; Starr et al, 2010]. The Shiga toxin is
transported to the ER in transport carriers lacking Coat
protein I (COP-I), but is reported to be dependent on
transport via the actin network, mediated by myosin II, a
motor protein [Duran et al, 2003]. One of the proteins that
has been shown to specifically regulate Shiga toxin
retrograde transport to the ER is Rab6a, a retrograde
transport regulating protein, consistent with its function in
Shiga toxin transport [Girod et al, 1999; White et al, 1999;
Del Nery et al, 2006]. Depletion or interrupting with any of
Figure 17: Cell entry mechanism of Shiga
the proteins involved in the transportation process
toxin.
abrogates Shiga toxin retrograde transport or targets the
Shiga toxin binds to the Gb3 molecule on the
cell membrane, inducing the endocytosis and
toxin to the lysosomes to be degraded [Bergan et al, 2012].
transportation in clathrin-coated vesicles to
Therefore, this process has to be secure and precise for the
the early endosome. Here the toxin is targeted
toxin to arrive in the ER. Upon arrival in the ER, the
towards the Golgi in a retrograde manner, and
subsequently transported to the ER where the
disulphide bridge between A1 and the A2-B-domain is
catalytic domain is translocated into the
reduced, releasing A1 into the ER lumen. This release
cytosol where it can execute its enzymatic
function. Whether the toxin goes through all
exposes the hydrophobic C terminal of the A1 domain and
the parts of the Golgi or whether it has a way
is recognised by the ER export system to be an improperly
around it has not yet been elucidated.
Adapted from O’Loughlin et al, 2001
folded protein [LaPointe et al, 2005]. As a consequence,
35 | P a g e
the A1 peptide is translocated to the cytosol to be degraded. However, this translocation allows the
toxic domain to remove one adenine from adenosine in the 5’ terminal of the 28S ribosomal RNA
(rRNA) [Endo et al, 1988]. As a consequence, the amino acyl transfer RNA (tRNA) is unable to bind to
the ribosome, leading to the abrogation of protein synthesis [Reisbig et al, 1981, Thompson et al,
1976, Brown et al, 1980]. The lack of protein synthesis has several consequences. First of all, the
ribotoxic stress due to the abrogated protein synthesis activates the mapK pathways and triggers
caspase 3 activation leading to the induction of apoptosis [Iordanov et al, 1997, Smith et al, 2003].
Second of all, the cell starts to secrete cytokines, portraying its cellular stress and increasing Gb3
expression on other cells, increasing their susceptibility to Shiga toxin [Eisenhauer et al, 2001]. Lastly,
the abolished protein synthesis leaves truncated proteins in the ER lumen, as well as the unfolding of
the Shiga toxin A1 domain induces the alarm response of three sensors involved in unfolded protein
response (UPR) [Tabas et al, 2011]. This response leads to the expression of chaperones to mend the
proteins. Prolonged UPR signalling however induces the expression of death receptor 5 (DR5) on the
cell membrane and secretion of TNF-related apoptosis-inducing ligand (TRAIL), inducing apoptosis
through the caspase cascade [Tabas et al, 2011]. This apoptosis process damages the endo- and
epithelial lining of many organs and tissues, leading to severe damage and eventually to death when
left untreated.
5.2 Cholera toxin (Vibrio cholerae)
The cholera toxin is produced by Vibrio cholerae. This toxin has been shown to be the causative
agent for the severe diarrhea patients have after infection with Vibrio cholerae. The disease cholera
was most prevalent when villages and cities did not have a sufficient sewer system, therefore the
bacteria was easily transmitted between individuals through contact with the faeces of one another
[Finkelstein et al, 1963]. Like the Shiga toxin, cholera toxin consists of an A and B domain, the B
domain consisting of 5 subdomains [Chinnapen et al, 2007]. When secreted, the cholera toxin B
domain binds to its receptor, glycolipid monosialoganglioside (GM1) [Merritt et al, 1994]. The
importance of its location within lipid rafts has been reported to initiate the correct intracellular
transport pathway for the toxin to become active, likely due to cholesterol signalling [Orlandi and
Fishman, 1998; Wolf et al, 1998; Wolf et al, 2002]. The toxin is, after binding and endocytosis,
trafficked to the early endosome in preferentially non-coated vesicles [Chinnapen et al, 2007; Nichols
et al, 2001; Shogomori et al, 2001]. However, the transport of cholera toxin in clatherin-coated
vesicles has been reported as well [Hansen et al, 2005; Nichols et al, 2001; Shogomori et al, 2001].
Within the early endosome, the low pH induces furin to cleave the proteolytic bond between A1 and
A2 subdomains, after which A1 is attached to the A2-B-domain complex via a disulphide bond in a
similar fashion as is seen in Shiga toxin. From the early endosome, the toxin is transported to the
Golgi-apparatus [Fujinaga et al, 2003]. The manner in which the toxin is dislocated is still under
debate. Some studies have reported the importance of Rab7 and Rab9 proteins in this process, while
others say that the toxin is carried in COP-I coated vesicles [Chen et al, 2002; Richards et al, 2002].
One way or another, the toxin is transported to the Golgi, after which it gets transported to the ER.
The manner of Golgi-ER transportation has been thought to be brought about by COP-I coated
vesicles, consistent to the presence of the KDEL motif in the A2 domain [Bastiaens et al, 1996; Majoul
et al, 1998; Majoul et al, 2001]. However, again other studies propose the involvement of Rab
proteins, namely Rab6 [Chen et al, 2002; Girod et al, 1999; White et al, 1999]. Another aspect that is
36 | P a g e
still under discussion is whether the B
domain is transported to the ER as
well. Some state that the KDEL motif in
the A2 domain, necessary for
transportation to the ER, has to be
exposed by separation from the B
domain [Bastiaens et al, 1996; Majoul
et al, 1998; Majoul et al, 2001]. Others
report the unnessacity of the KDEL
motif for toxicity as well as the
passage of the B domain through the
Golgi as well as towards the ER [Lencer
et al, 1995; Fujinaga et al, 2003]. Once
arrived in the ER, the A1 domain is
recognised by protein disulphide
isomerase (PDI) which reduces the
disulphide bond between A1 and A2
and is probably also involved in the
unfolding of A1 [De Haan et al, 2004;
Tsai et al, 2002; Tsai et al, 2001]. The
unfolded protein is subsequenctly
recognised by the ER export
mechanism and targets it for Figure 18: Cell entry and intracellular transportation of cholera toxin.
translocation to the cytosol to be Cholera toxin binds to the target cell and is endocytosed in smooth nondegraded, similar to the translocation coated or clatherin-coated vesicles. There are transported to the early
endosome and in COPI-coated vesicles transported to the Golgi in a
by the active domain of Shiga toxin retrograde manner. Subsequently, the toxin is transported to the ER,
[Sandvig et al, 2002; Rodighiero et al, where the catalytic domain is released and unfolded with the help of
PDI. Translocation into the cytosol allows the catalytic domain to
2002]. Once in the cytosol, the active modify the Gα protein of adenylyl cyclase, continuously activating it and
domain targets one of the subunits of inducing via PKA the expression of CFTRs on the cell membrane.
the G protein involved in the signalling Adapted from Vanden Broeck et al, 2007
by adenylyl cyclase, namely the G protein alpha unit (Gα) [Sanchez et al, 2005]. This unit is rendered
active by mono-ADP-ribosylation by A1 domain, and therefore the adenylyl cyclase is continuously
active as well [Spangler et al, 1992]. Adenylyl cyclase increases the cAMP levels in the cell, leading to
the overactivation of protein kinase A (PKA) and overstimulated secretion of Cl- and H2O by CFTR,
indirectly targeted by heat-stabile enterotoxin produced by E. coli as has been described in chapter 4
[81, 82 Weiglmeier]. As a consequence of the secretion of Cl- and H2O is the onset of severe diarrhea,
as the reabsorbant capacity of the colon is overwhelmed [Sanchez et al, 2005]. Likely, this
consequence is used for the bacteria to spread amongst more individuals, inducing more illness
among them.
37 | P a g e
5.3 Diphtheria toxin (Corynebacterium diphtheria)
Diphtheria toxin has been previously discussed in
the section of α-PFTs where the ability to form
pores by this toxin is mediated by α-helices. The
diphtheria toxin is produced by Corynebacterium
diphtheria and is secreted as a single chain protein.
The toxin consists of two domains, the A and B
domain. Domain A is the catalytic domain that gets
translocated into the cytosol once inside the cell,
the B domain, consisting of two subdomains, is
responsible for cell binding (R domain) and
translocation (T domain). After secretion of the
toxin, the toxin binds to the target cell with its R
subdomain to heparin binding epidermal growth
factor-like precursor (hb-EGF) [Choe et al, 1992;
Naglich et al, 1992]. Another receptor has been
shown to be associated with diphtheria toxin
binding, namely CD9 [Iwamoto et al, 1994]. Once
bound to the receptors, the toxin induces
internalisation into clathrin coated vesicles,
transporting the toxin to the early endosome
[Moya et al, 1985]. Once in the endosome, the
toxin is proteotically cleaved by furin, transforming
the toxin into 2 domains, A and B, connected by a
disulphide bond [Collier et al, 1971; Choe et al.
1992; Gordan et al, 1995]. This cleavage renders Figure 19: Cell entry and cellular translocation pathway
the A domain active, therefore it will be able to of diphtheria toxin.
exert its toxic function once it gets translocated Diphtheria toxin binds to the target cell receptor with its
binding domain and induces endocytosis of the toxin.
over the endosomal membrane into the cytosol. As The toxin is transported to the early endosome where
has already been described, the lowering of the pH the acidification of the lumen leads to the insertion of
the translocation domain in the endosomal membrane.
in the early endosome induces conformational It is still unclear whether the catalytic domain is
changes in the T subdomain of the B domain [Choe translocated through the pore formed by the
domain or through another process. Upon
et al, 1992; Boquet et al, 1976a]. These changes translocation
arrival in the cytosol, the enzymatic domain targets EFexpose the hydrophobic α-hairpin that inserts itself 2, inhibiting protein synthesis. Adapted from
into the membrane [Bouquet et al, 1976b; Boquet http://brainboxes2.wikispaces.com/file/view/diphtheria
_toxin_mechanism.gif/279097328/502x827/diphtheria_
et al, 1976a]. The subsequent translocation process toxin_mechanism.gif - accessed on 13-11-2013
of the A domain remains uncertain and has yet to
be fully elucidated. For example, it remains elusive whether the formation of the pore by the T
subdomain is the structure through which the A domain gets translocated. It has however been
shown that without this pore formation, the A domain still ends up in the cytosol, therefore
possesses a doubtful function in the translocation process [Lanzrein et al, 1996], while others have
shown it is not [Trujilo et al, 2010; Vander Spek et al, 1994]. There are several hypothesis about the
manner the A domain is translocated over the membrane. Some studies suggest that a cytosolic
38 | P a g e
translocation factor (CTF) complex is involved, while others propose that the T domain in its partially
unfolded state can function as a chaperone in the translocation [Ratts et al, 2003; Ren et al, 1999; Oh
et al, 1999]. One way or another, when the A domain reaches the cytosol, the disulphide bond is
reduced and releases the catalytic domain into the cytosol, where it refolds and targets its substrate
[Lemichez et al, 1997]. Diphtheria toxin A domain targets elongation factor 2 (EF-2) by ADPribosylating the protein, dependent on the presence of nicotinamide adenine dinucleotide (NAD)
[Kochi et al, 1993]. EF-2 is an important protein involved in the process of protein synthesis. During
protein synthesis, EF-1 is necessary of escorting the tRNA, bound to its corresponding amino acid, to
the correct position, while EF-2 translocates the ribosome one step with regard to the mRNA and
synthesized protein. Therefore, the ADP-ribosylation of EF-2 renders the protein incapable of its
function and abrogates protein synthesis. Similar to the consequences of Shiga toxin mediated
protein synthesis abrogation, the cell undergoes ribotoxic stress leading to apoptosis and in addition,
the truncated proteins in the ER initiate apoptosis as well [Kochi et al, 1993]. This leads to the death
of many cells, damaging the tissue where Corynebacterium diphtheria resides, namely in the upper
respiratory tract. As a consequence, the pharynx and nasopharynx are severely damaged and can
lead to suffocation of the patient.
5.4 Tetanus toxin (Clostridium tetani)
Tetanus toxin is produced and secreted by Clostridium
tetani. This bacterial species is highly related to
Clostridium botulinum, the producer of BoNTs. Even
though the tetanus toxin and BoNT are similar in
structure and function, they differ in the consequences
of their toxicity. The essence of BoNT toxicity is the
deprival of stimulatory signals to the muscles, leading to
paralysis, whereas tetanus toxin abrogates the signalling
by
inhibitory
neurotransmitters,
leading
to
overstimulation of the muscles and the complete
contraction of body muscles. The toxin is produced as a
single peptide chain, often getting proteolytically
cleaved by bacterial proteases to give rise to an A
domain and a B domain [Lacy et al, 1998]. Like
diphtheria toxin, the B domain consists of a
translocation and receptor binding domain (T and R
respectively) while the A (or C) domain is the
catalytically active one, linked together by a disulphide
bond [Montecucco et al, 1994]. The toxin binds to
polysialoganglioside and another protein of which the
identity is not yet clear, but is possibly
glycophosphatidylinositol
(GPI)-anchored
protein
[Humeau et al, 2000; Munro et al, 2001; Herreros et al,
2000; Herreros et al, 2001]. These molecules are located
on motor neurons and, as with the other toxins, within
Figure 20: Schematic overview of the neuronal
cell entry and translocation to the inhibitory
interneuron of the tetanus toxin.
Tetanus toxin is internalised by the motor neuron
and translocated towards the neuron soma, where
it is released in the synapse between the
inhibitory interneuron and the motorneuron.
There the toxin binds again and is internalised
again, however is transported to the early
endosome where the enzymatic domain is
translocated over the endosomal membrane into
the cytosol where it can cleave VAMP protein.
Adapted from Rosetto et al, 2013
39 | P a g e
lipid rafts, involved in the targeting of the endocytosed toxin to the correct sorting region [Herreros
et al, 2001; Munro et al, 2001; Herreros et al, 2002; Lalli et al, 1999]. After the endocytosis, tetanus
toxin ends up in special vesicles, where acidification does not occur, and gets sorted to the
retrograde transport route [Bohnert et al, 2005; Deinhardt et al, 2006]. The vesicle containing
tetanus toxin is transported to the soma of the neuron, the cell body region where the Golgi and
nucleus are located, and is released in the synapse between the motor neuron and inhibitory
interneurons by fusion of the vesicle with the membrane [von Bartheld, 2004; Rind et al, 2005]. The
tetanus toxin then binds again to the polysialogangliosides on the inhibitory neuron, but also to GPIanchored protein [Herreros et al, 2000; Herreros et al, 2001; Munro et al, 2001]. This time, the toxin
undergoes endocytosis in recycled synaptic vesicles and is transported to the early endosome
[Matteoli et al, 1996]. Like with diphtheria toxin and BoNT, the acidification of the lumen alters the
conformation of the T domain of tetanus toxin, initiating the formation of a pore and translocation of
the A domain through the pore [Simpson, 1983, Simpson et al, 1994, Williamson et al, 1997;
Koriazova et al, 2003]. Once in the cytosol, the enzymatically active A domain cleaves vesicleassociated membrane protein (VAMP, also known as synaptobrevin to define its location in neuronal
location), disrupting its function in the cytosol [Schiavo et al, 1992a; Schiavo et al, 1992b; Schiavo et
al, 2000]. VAMP is part of the formation of the SNARE complex, bringing the synaptic vesicle in close
proximity to the membrane to induce fusion of the vesicle and release of its content. Therefore,
tetanus toxin, like BoNTs, inhibits the membrane fusion of the synaptic vesicles. In the case of
tetanus toxin, the content of the synaptic vesicles are neurotransmitters that transmit an inhibitory
signal to the motor neuron which the inhibitory neuron controls [Schiavo et al, 2000]. Therefore, the
motor neuron does not receive inhibitory signals that would prevent the secretion of stimulatory
neurotransmitters, therefore overstimulation the muscles by the continuous stimulatory signals,
leading to the contraction of the muscles. Without proper and fast treatment, this disease leads to
death, as the patient is not able to breath or for its heart to beat [Bleck, 1989].
5.5 Anthrax toxin (Bacillus anthracis)
The final bacterial toxin to be discussed is anthrax toxin, produced by Bacillus anthracis. This toxin
consists out of three proteins, but synthesised and secreted as single proteins which assemble at the
target cell membrane. The three proteins are protective antigen (PA), edema factor (EF, not to be
mistake with elongation factor) and lethal factor (LF) [Brossier et al, 2001]. Like with the other
internalised toxins, PA is the receptor binding and translocation domain, whereas EF and LF are
catalytically active and cause the intoxication. PA binds to Anthrax toxin receptor 1 or 2 (ANTXR1/2,
also known as tumor endothelial marker-8 and capillary morphogenesis protein 2 respectively) and
low-density lipoprotein receptor-related protein 6 (LRP6) [Bradley et al, 2001; Scobie et al, 2003). The
latter is involved in WNT signalling and acts as coreceptor for PA binding [Tamai et al, 2000; Abrami
et al, 2008]. Interesting to note, is that the receptor PA binds to has been discovered in the anthrax
toxin binding aspect and was likely not identified before the anthrax toxin research. The binding of
PA to its receptors induces the proteotically cleavage of a part of PA by furin, this enables the PA
domain to oligomerize with other receptor bound PA molecules into a heptameric prepore [6, 36
Abrami]. This prepore complex is then able to bind three molecules of the catalytic domains,
independent on the combination (e.g. 2 EFs and 1 LF molecule) [Mogridge et al, 2002]. This complex
of pre-pore and bound catalytic proteins is endocytosed and transported in clatherin-coated vesicles
40 | P a g e
to the early endosome [Abrami et al, 2003; Abrami et al, 2004]. The acidification of the endosomal
lumen triggers conformational changes in the pre-pore subunits and insertion of the PA pre-pore is
initiated [Collier et al, 2003; Nassi et al, 2002]. In addition, the EF and LF molecules are unfolded
because of the low pH, allowing them to get translocated through the PA formed pore [Krantz et al,
2004]. This translocation is possibly driven by a transmembrane proton gradient, delivering the
catalytic domains into the cytosol. There is evidence that suggests that the pre-pore inserts itself in
the membrane of intraluminal vesicles, translocating the domains into the lumen of the intraluminal
vesicles where the enzymatic domains are refolded again [Abrami et al, 2004; Haug et al, 2003]. The
vesicles travel to the late endosome where they are protected from degradation. A so called back
fusion events captures the enzymatic domains again and delivers them to the perinuclear region [45,
46]. Once in the cytosol, the LF and EF domains can exert their distinct toxic functions. LF is a zincdependent metalloprotease and cleaves the kinase that activates mitogen-activated protein kinase
(MapK), namely MapK kinase (MapKK) [Bardwell et al, 2004; Chopra et al, 2003]. These kinases are
involved in an important kinase cascade, regulating several cellular processes [Pearson et al, 2001;
Tanoue et al, 2003]. Because the receptors for anthrax toxin are ubiquitously expressed, the toxin is
able to affect a large variety of cells and tissues, disrupting multiple bodily processes. One of these
processes, or better said systems, that is affected is the immune system. The abrogation of the MapK
pathway in immune cells has multiple serious effects. First of all, the TLRs (Toll like receptors,
explained in chapter 1) signal through this pathway, and therefore the recognition by dendritic cells
or macrophages of bacterial or viral antigens does not result in the expression of cytokines.
Therefore, these cells are unable to activate
innate and adaptive immune cells [Agrawal et al, 2003; Tournier et al, 2005] In addition, LF inhibits
the expression of costimulatory molecules on its cell membrane, rendering the DC incapable of
activating T cells [Agrawal et al, 2003]. Second of all, the MapKK in the p38 pathway (one of the
MapK cascade pathways) is also cleaved by LT. Disruption of the pathway induces apoptosis in the
LPS activated macrophages [Hsu et al, 2004; Park et al, 2002]. Another pathway that induces
apoptosis is the TLR 4 signalling in macrophages. This signalling pathway induces an anti-apoptotic
pathway and therefore the disruption of this pathway renders the cell apoptotic [Hsu et al, 2004;
Park et al, 2002]. The effects on the immune system are likely to prevent the targeting and
elimination of the bacteria, and do not account for the lethality of the infection for the host [Pezerd
et al, 1991]. The symptoms that are found in anthrax disease, causing lethality, are pleural oedema,
liver necrosis and hepatic dysfunction. Other cells that are targeted by anthrax toxin and which are
possibly involved in the lethality of the disease are endothelial cells of the circulatory system [Kirby,
2004]. The cleavage of MapKK in the extracellular signal-regulated kinase (ERK) pathway in
endothelial cells induces apoptosis , despite the disruption in the c-Jun N-terminal kinase (JNK) and
p38 pathways which promote survival of the endothelial cell [Kirby, 2004]. The high extent of
endothelial cell apoptosis leads to vascular permeability and the onset of tissue hemorrhages and
gastraointestinal bleeding among other vascular damage mediated consequences. The other catalytic
domain of anthrax toxin, EF, is an adenylyl cyclase [Drum et al, 2002]. Like the cellular adenylyl
cyclase, it converts ATP to cAMP, increasing the cAMP levels in the cell. This has multiple effects, but
the best described is the inhibition of TNFα production by macrophages and nitric oxides by
phagocytes, inhibiting phagocytosis and degradation of the bacterium, providing the bacteria with an
advantage for bacterial growth and survival [O’Brien et al, 1985; Crawford MA et al, 2006]. The
infected patient will die from the severe consequences from the infection and intoxication by the
anthrax toxin.
41 | P a g e
Figure 21: The complex mechanism of cell entry, internalisation, translocation and the action of the LF and EF
domains in the cell during anthrax toxin intoxication.
The protective antigen binds to the cell membrane to its cell receptor and gets cleaved by furin, inducing the
formation of the pre-pore complex. The pre-pore complex binds three catalytic domains, any combination of LF and
EF. The pre-pore complex is internalised and transported to the early endosome. The low pH induces the insertion
of the pre-pore complex, allowing the EF and LF domains, perhaps not directly, translocate to the cytosol. There EF
and
LF
exerts
different
functions,
altering
many
body
systems.
Adapted
from
http://www.qiagen.com/geneglobe/static/images/Pathways/Mechanism%20of%20Anthrax%20Toxins.jpg
–
accessed on 13-11-2013
These toxin examples show that even though the toxins appear to be quite similar, they are unique in
the overall process from cell binding to intoxication and these differences appear in the clinical
picture belonging to each toxin and associated disease. Despite the differences, these toxins can
provide us with the possibility of designing vaccines, as each toxin may reveal a particular mechanism
another might use for its intoxication steps.
42 | P a g e
Chapter 6: Application in therapies
Until now, Clostridial botulinum neurotoxin is the only toxin that is often used in the clinic to treat a
variety of syndromes, from reducing wrinkles in the face to ameliorating migraines, and has been
effective and safe. Even though BoNT is far more poisonous than other toxins, other toxins are rarely
used to treat syndromes or in cosmetic surgeries. Therefore, in this chapter, the potencies for
bacterial toxins will be explored, within the boundaries of imagination. Import to note is that these
‘medical toxins’ are not derived from literature or ongoing studies, even though there may be studies
going on right now exploring bacterial toxins regarding medical treatments as well. In respect of the
imaginary toxins, some aspects are not as detailed, but the general idea will be stated.
6.1 Medical toxins for diseases
Years and years of research have been dedicated to cancer and its treatment. One of the aspects of
the transformation of cells into cancers cells has been shown to be the overexpression of certain
receptors on the cell surface. The overexpression of these receptors can be used to target ‘medical
toxins (MTs)’ to the cancer cells. By using a cancer-associated receptor binding domain and
combining it with a translocation domain similar to the one of diphtheria toxin or anthrax toxin, with
an EF-2 targeting or rRNA ADP-ribosylating catalytic domain, ribotoxic stress can be initiated leading
to the induction of apoptosis. Similarly, the binding domain of Shiga toxin can be altered or
exchanged for a cancer-associated receptor binding domain, to induce similar apoptosis inducing
events as in the target cells of the wild type Shiga toxin. In a similar fashion, cytomegalovirus infected
cells can be targeted, or any virus that is known to express infection-associated receptors or proteins
on the cell surface for that manner. Another way of targeting virally infected cells is by α-PFTs,
targeted to the same infection-associated receptors. The ion channels that are formed will cause
osmotic imbalance causing the cell to rupture. A cosmetic approach for a medical toxin is an
alternative for liposuction. At the moment, a particular machine is available to freeze adipocytes in
areas of the body. This machine cools down the adipocytes to an extent that they die by necrotic
processes. This approach of degrading fat creates a big mess in these areas, which takes longer to be
removed from the area and in addition may have tissue damaging effects as well. A medical
medicine, targeting adipocytes and inducing apoptosis through the inhibition of protein synthesis or
MapKKs will have a cleaner result, as the cells will be destroyed by apoptosis rather than by necrosis.
Another hypothetical idea for a medical toxin is a toxin that can be used in rheumatoid arthritis. This
disease is likely to be influenced by TNFα and other cytokines, produced by B and T cells. By
combining PA of anthrax toxin together with the EF, and alter the binding domain of PA to target
specific receptors on T and B cells, these cells are inhibited to produce cytokines. In addition, the
inclusion of wild type PA together with LF and EF will target the macrophages and dendritic cells at
the site of inflammation in the joints, leading to the inability of these cells to promote inflammation.
This approach however will only ameliorate the symptoms of rheumatoid arthritis instead of curing
the patient. The medical toxin cocktail will have to be injected continuously to get a long lasting
effect, of which the side-effects are to be established as well. Other autoimmune diseases might also
possibly be treated with this medical toxin, however, the site of inflammation will conduct whether
43 | P a g e
the toxin can be used. It is impossible to inject the toxin into the circulatory system, as then the
effects will be hard to control and the patient might become immune-compromised.
6.2 Botox-like medical toxins
Botox is used to limit or inhibit the secretion of acetylcholine in the synapse between motor
neurons and muscles, thereby inhibiting muscle contract to some extent. Similar approaches of
limiting the induction of muscle contraction might be the increase of inhibitory signals to the motor
neuron or the enhancement of degradation of acetylcholine. The first approach might be succeeded
by using modified tetanus toxin. By maintaining its binding domain to get internalised by the motor
neuron and trafficking to the synapse between inhibitory neuron and motor neuron, but altering the
mechanism of uptake by the inhibitory interneuron, leading to the binding of the toxin to the
membrane but not the endocytosis of it, the toxin can deliver its catalytic mechanism to the
interneuronal synapse. The binding to the receptor on the inhibitory interneuron might expose a
domain that can be cleaved and lead to the release of the catalytic domain. This catalytic domain
inhibits the enzyme that degrades the inhibitory neurotransmitters, creating an inhibitory signal that
lasts longer, increasing the threshold for the motor neuron to secrete its synaptic vesicles with
acetylcholine. This will inhibit the secretion induced by spontaneous activation of the neuron,
whereas signals from the brain will overpower the inhibitory signals and initiate secretion of its
vesicles, leading to voluntary muscle movements. However, the mechanisms this toxin must possess
to succeed in this inhibition of involuntary muscle contractions may be farfetched to be designed.
Another mechanism of inhibiting such muscle contractions is by altering the binding domain of
BoNTs to create a binding domain that does bind to the motor neuron but does not get internalised.
The bound BoNTs will get cleaved by an enzyme that is injected simultaneously, to release the
catalytic domain, consisting out of acetylcholinesterase. This enzyme degrades acetylcholine, and
therefore the increase of such enzymes in the synapse will lead to the degradation of more
neurotransmitters, allowing only a few to bind to the accepting muscle receptors and activate the
muscle. During voluntary muscle inducing contractions, the level of acetylcholine will increase,
overwhelming the capacity of the enzymes to degrade the transmitters and thus leading to muscle
contraction.
44 | P a g e
Chapter 7: Discussion
The whole of this thesis has been discussing the various bacterial toxins, starting with botulinum
neurotoxin, a very potent neurotoxin produced by Clostridium botulinum. This neurotoxin is used in
various therapeutic approaches to ameliorate a variety of hyperactive muscle-associated syndromes
as well as the appearance of wrinkles on the skin. Subsequently, three categories of bacterial toxins
have been described, stating their mechanism of action as well as the toxins that belong in the
category. Membrane-damaging toxins damage the membrane to make the cell susceptible for cell
rupture, death of the cell has advantages for the bacteria as this goes paired with the release of
nutrients. Receptor-targeting toxins target specific receptors on the cell, inducing the activation of a
signal within the cell without being internalised itself. These intracellular signals will eventually lead
to electrolyte imbalance and the onset of diarrhea or the initiation of an extreme inflammatory
response, damaging various tissues and organs, and the weakening of the host. Internalised toxins
are the category with the most diverse range of toxins. They have different target cells and their
catalytic domains execute a different enzymatic process, having consequences that range from
apoptosis of the intoxicated cell to abrogation of inhibitory signals, leading to extensive muscle
contraction. Even though most toxins have been studied extensively, any therapeutic benefits have
not yet been defined or described, except for the botulinum neurotoxin. This is probably because the
effects of the bacterial toxins will cause more harm than is desirable. In the previous chapter,
hypothetical medical toxins are described, combining domains of toxins or altering the receptorbinding domain to control the range of target cells. Possible targets can be virally infected or
transformed cells. Alternative toxins to botulinum toxin, the initial objective of this thesis, have
proven to be more difficult to design or imagine.
Funnily enough, I started this thesis with the thought that there would have to be a better
way to treat the botox-treated syndromes other than by botox itself. However, through the whole
journey through the world of botox and other bacterial toxins, botox has proven itself to be the best
candidate for these therapeutic approaches to ameliorate certain situations. Nevertheless, the idea
of using botox for amelioration of wrinkles on the face remains insane to me. There are a few
alternative remedies for this symptom of the aging disease, either by leaving your life before the
aging syndrome can occur and the wrinkles can set in, or remove all mirrors in order to not see them.
However, the best, most efficient remedy is by accepting this symptom and looking at it as the
beautiful aspect of aging.
45 | P a g e
References
1066.
33–48.
Abdel Ghani EM ,Weis S, Walev I et al. Streptolysin O: Inhibition of the conformational
change during membrane binding of the monomer prevents oligomerization and pore formation.
Biochemistry. 1999;38:15204-11
Abrami L, Fivax M, Decroly E et al. The pore-forming toxin proaerolysin is processed by furin.
J Biol Chem. 1998;273:32656-61
Abrami L, Kunz B, Deuquet J et al. Functional interactions between anthrax toxin receptos
and the WNT signaling protein LRP6. Cell Microbiol. 2008;10:2509-19
Abrami L, Lindsay M, Parton RG et al. Membrane insertion of anthrax protective antigen and
cytoplasmic delivery of lethal factor occur at different stages of the endocytic pathway. J Cell Biol.
2004;166:645-51
Abrami L, Liu S, Cosson P et al. Anthrax toxin triggers endocytosis of its receptor via a lipid
raft-mediated clathrin-dependent process. J Cell Biol. 2003;160:321-8
Abrami L, Liu S, Cosson P et al. Anthrax toxin triggers endocytosis of its receptor via a lipid
raft-mediated clathrin-dependent process. J Cell Biol. 2003;160:321-8
Agrawal A, Lingappa J, Leppla SH et al. Impairment of dendritic cells and adaptive immunity
by anthrax lethal toxin. Nature. 2003;424:329-34
Alan W, Bernheimer AW. Some aspects of the history of membrane-damaging toxins. Med
Microbiol Immunol. 1996;185:59-63
Albano F, Brasitus T, Mann EA et al. Colonocyte basolateral membranes contain E. coli heatstable enterotoxin receptors. Biochem Biophys Res Comm. 2001;284:331-4.
Alexander C, Rietschel ET. Bacterial lipopolysaccharides and innate immunity. J Endotoxin
Res. 2001;7:167-202.
Alouf JE. Pore-forming bacterial protein toxins: an overview. Curr Top Microbiol Immunol.
2001;257:1-14
Anderson ER Jr. Proper dose, preparation, and storage of botulinum neurotoxin serotype A.
Am J Health Syst Pharm. 2004;61:S24-9
Arnon S. Botulism as an intestinal toxemia. In: Blaser MJ, Smith PD, Ravdin JI, Greenberg HB,
Guerrant RL (ed). Infections of the gastrointestinal tract. New York: Raven Press. 1995:257–71.
Arnon SS, Schechter R, Inglesby TV et al. Botulinum Toxin as Biological weapon. JAMA.
2001;285:1059–1070.
Austrian R. The Gram stain and the etiology of lobar pneumonia, an historical note. Bacteriol
Rev. 1960;24:261-5
Balfanz J, Rautenberg P, Ullmann U. Molecular mechanisms of action of bacterial exotoxins.
Zbl Bakt. 1996;284:170-206
Ballard J, Sokolov Y, Yuan WL et al. Activation and mechanism of Clostridium septicum alpha
toxin. Mol Microbiol. 1993;10:627-34
Bardwell AJ, Abdollahi M, Bardwell L. Anthrax lethal factor-cleavage products of MAPK
(mitogen-activated protein kinase) kinases exhibit reduced binding to their cognate MAPKs. Biochem
J. 2004;378:569-77
Bastiaens PI, Majoul IV, Verveer PJ et al. Imaging the intracellular trafficking and state of the
AB5 quaternary structure of cholera toxin. EMBO J. 1996;15:4246-53.
Beauregard KE, Collier RJ, Swanson JA. Proteolytic activation of receptor-bound anthrax
protective antigen on macrophages promotes its internalization. Cell Microbiol. 2000;2:251-8
Bergan J, Dyve AB, Simm R et al, Shiga toxins. Toxicon. 2012;60:1085-107
46 | P a g e
Bernheimer AW, Avigad LS, Kim K. Comparison fo metridiolysin from the sea anemone with
thiol-activated cytolysins from bacteria. Toxicon. 1979;17(1):69-75.
Bernheimer AW. Interactions between membranes and cytolytic bacterial toxins. Biochim
Biophys Acta. 1974;465:378–90.
Beveridge TJ. Use of gram stain in microbiology. Biotech Histochem. 2001;76:111-8
Bhakdi S, Muhly M, Mannhardt U et al. Staphylococcal α-toxin promotes blood coagulation
via attack on human platelets. J Exp Med. 1988;168;527-42
Binz T, Rummel A. Cell entry strategy of clostridial neurotoxins. J Neurochem.
2009;109:1584–95
Bittencourt SE, Silva LP, Azevedo RB et al. The plat cytolytic protein enterolobin assumes a
dimeric structure in solution. FEBS Lett. 2003;549:47-51
Bleck TP. Clinical aspects of tetanus. In: Botulinum neurotoxins and tetanus toxin (ed.
Simpsom LL). 1989; San Diego, CA:379-98
Bohnert S, Schiavo G. Tetanus toxin is transported in a novel neuronal compartment
characterized by a specialized pH regulation. J Biol Chem. 2005;280:42336-44
Boquet P, Pappenheimer AMJ. Interaction of diphtheria toxin with mammalian cell
membranes. J Biol Chem. 1976b;251:5770-8
Boquet P, Silverman MS, Pappenheimer AM et al. Binding of triton X-100 to diptheria toxin,
crossreacting material 45, and their fragments. PNAS USA. 1976a;73:4449-53
Bowen ME, Weninger K, Ernst J et al. Single-molecule studies of synaptotagmin and
complexin binding to the SNARE complex. Biophys J. 2005;89:690-702
Bradley KA, Mogridge J, Mourez M et al. Identification of the cellular receptor for anthrax
toxin. Nature. 2001;414:225-9
Breidenbach MA, Brunger AT. New insights into clostridial neurotoxin-SNARE interactions.
Trends Mol Med. 2005;11:377-81
Breidenbach MA, Brunger AT. Substrate recognition strategy for botulinum neurotoxin
serotype A. Nature. 2004;432:925-9
Brock TD. Robert Koch: a life in medicine and bacteriology. American Society of Microbiology
Press, Washington. 1999
Brossier F, Mock M. Toxins of Bacillus anthracis. Toxicon. 2001;39:1747-55
Brown JE, Rothman SW, Doctor BP. Inhibition of protein synthesis in intact HeLa cells by
Shigella dysenteriae 1 toxin. Infect Immun. 1980;29:98-107
Brunger AT, Breidenbach MA, Jin R et al. Botulinum neurotoxin heavy chain belt as an
intramolecular chaperone for the light chain. PLoS Pathog. 2007;3:1191-4
Brunger AT. Structure and function of SNARE and SNARE-interacting proteins. Q Rev Biophys.
2005;38:1–47
Bujny MW, Popoff V, Johannes L et al. The retromer component sorting nexin-1 is required
for efficient retrograde transport of Shiga toxin from early endosome to the trans Golgi network. J
Cell Sci. 2007;120:2010-21
Caroff M, Karibian D. Structure of bacterial lipopolysaccharides. Carbohydr Res.
2003;338:2431-47
Carr A, Sledjeski DD, Podbielski A et al. Similarities between complement-mediated and
streptolysin S-mediated hemolysis. J Biol Chem. 2001;276:41790-6
Carrithers SL, Hill MJ, Johnson BR et al. Renal effects of uroguanylin and guanylin in vivo.
Braz J Med Biol Res. 1999;32:1337-44.
Carrithers SL, Taylor B, Cai WY et al. Guanylate cyclase C mRNA distribution along rat
nephron. Regulatory Peptides. 2000;95:65-74.
Cascales E, Buchanan SK, Duché D et al. Colicin biology. Microbiol Mol Biol Rev. 2007;71:158229
Chao AC, de Sauvage FJ, Dong YJ et al. Activation of intestinal CFTR Cl- channel by heat-stable
enterotoxin and guanylin via cAMP-dependent protein kinase. EMBO J. 1994;12:1065-72
47 | P a g e
Charney AN, Egnor RW, Alexander-Chacko JT et al. Effect of E. coli heat-stable enterotoxin on
colonic electrolyte transport in guanylyl cyclase-C receptor-deficient mice. Amer J Physiol.
2001;280:G216-21
Chen A, Hu T, Mikoryak C et al. Retrograde transport of protein toxins under conditions of
COPI dysfunction. Biochim Biophys Acta. 2002;1589:124-39
Chenal A, Savarin P, Nizard P et al. Membrane protein insertion regulated by bringing
electrostatic and hydrophobic interactions into play. J Biol Chem. 2002;277:43425-32
Childs D, Crombe DL, Pratha VSK et al. Heat-stable toxin of E. coli stimulates duodenal
mucosal bicarbonate secretion in cystic fibrosis knock out mice. Gastroenterology. 2001;120:A137.
Chinnapen D, Chinnapen JF, Saslowski H et al. Rafting with cholera toxin: endocytosis and
trafficking from plasma membrane to ER. FEMS Microbiol Lett. 2007;266:129-37
Choe S, Bennett MJ, Fugii G et al. The crystal structure of diphtheria toxin. Nature. 1992;357:
216-22
Choe S, Bennett MJ, Fujii G et al. The crystal structure of diphtheria toxin. Nature.
1992;357:216-22
Chopra AP, Boone SA, Liang X et al. Anthrax lethal factor proteolysis and inactivation of
MAPK kinase. J Biol Chem. 2003;278:9402-6
Cohen J, Marshall E. Vaccines for Biodefense: A system in Distress. Science. 2001;294:498–
501.
Cole AR, Gibert M, Popoff M et al. Clostridium perfringens epsilon-toxin shows structural
similarity to the pore-forming toxin aerolysin. Nat Struct Mol Biol. 2004;11:797-8
Colin DA, Mazurier I, Sire S et al. Interation of the two components of leukocidin from
Staphylococcus aureus with human polymorphonuclear leukocyte membranes: Sequential binding
and subsequent activation. Infect Immun. 1994;62:3184-8
Collier RJ, Kandel J. J. Structure and activity of diphtheria toxin. I. Thiol-dependent
dissociation of a fraction of toxin into enzymically active and inactive fragments. J Biol Chem.
1971;246:1496-503
Collier RJ, Young JA. Anthrax toxin. Annu Rev Cell Dev Biol. 2003;19:45-70
Collier RJ, Young JA. Anthrax toxin. Annu Rev Cell Dev Biol. 2003;19:45-70
Collier RJ. Understanding the mode of action of diphtheria toxin: a perspective on progress
during the 20th century. Toxicon. 2001;39:1793-803
Crane M, Hugues M, O'Hanley P et al. Identification of two affinity states of low affinity
receptors for E. coli heat-stable enterotoxin: correlation of occupation of lower affinity state with
guanylate cyclase activation. Molec Pharmacol. 1992;41:1073-80.
Crawford MA, Aylott CV, Bourdeau RW et al. Bacillus anthracis toxins inhibit human
neutrophil NADPH oxidase activity. J Immunol. 2006;176:7557-65
De Haan L, Hirst TR. Cholera toxin: A paradigm for multifunctional engagement of cellular
mechniasms. Mol Membr Biol. 2004;21:77-92
Deinhardt K, Salinas S, Verastegui C et al. Rab5 and Rab7 control endocytic sorting along the
axonal retrograde transport pathway. Neuron. 2006;52:293-305
Del Nery E, Miserey-Lenkei S, Falguières T et al. Rab6A and Rab6A’ GTPases play nonoverlapping roles in membrane trafficking. Traffic. 2006;7:394-407
Dembek ZF, Smith LA, Rusnak JM. Botulism: cause, effects, diagnosis, clinical and laboratory
identification, and treatment modalities. Disaster Med Public Health Prep. 2007;1:122-34
Dinges MM, Orwin PM, Schlievert PM. Exotoxins of Staphylococcus aureus. Clin Microbiol
Rev. 2000;13:16-34
Dong M, Liu H, Tepp WH, et al. Glycosylated SV2A and SV2B mediate the entry of botulinum
neurotoxin E into neurons. Mol. Biol. Cell. 2008;19:5226–37
Dong M, Yeh F, Tepp WH et al. SV2 is the protein receptor for botulinum neurotoxin A.
Science. 2006;312:592–96
Donovan JJ, Simon MI, Draper RK et al. Diphtheria toxin forms transmembrane channels in
planar lipid bilayers. PNAS USA. 1981;78:172-176
48 | P a g e
Donovan JJ, Simon MI, Montal M. Insertion of diphtheria toxin into and across membranes:
role of phosphoinositide asymmetry. Nature. 1982;298:669–72
Donta ST, Tomicic Tk, Donohue-Rolfe A. Inhibition of Shiga-like toxins by brefeldin A. J Infect
Dis. 1995;171:721-4
Drewett JG, Garbers DL. The family of guanylyl cyclase receptors and their ligands. Endocr
Rev. 1994;15:135–62.
Drum CL, Yan SZ, Bard J et al. Structural basis for the activation of anthrax adenylyl cyclase
exotoxin by calmodulin. Nature. 2002;415:396-402
Dufourcq J, Faucon JF, Fourche G et al. Morphological changes of phosphatidylcholine
bilayers induced by melittin: vesicularization, fusion, discoidal particles. Biochim Biophys Acta.
1986;859:
Dulubova I, Khvotchev M, Liu S et al. Munc18-1 binds directly to the neuronal SNARE
complex. PNAS. 2007;104:2697-702
Dulubova I, Sugita S, Hill S et al. A conformational switch in syntaxin during exocytosis: role
of munc18. EMBO J. 1999;18:4372-82
Durán JM, Valderrama F, Castel S et al. Myosin motors and not actin comets are mediators of
the actin-based Golgi-to-endoplasmic reticulum protein transport. Mol Biol Cell. 2003;14:445-59
Eisenhauer PB, Chaturvedi P, Fine RE et al. Tumor necrosis factor alpha increases human
cerebral endothelial cell Gb3 and sensitivity to Shiga toxin. Infect Immun. 2001;69:1889-1894
Elkins P, Bunker A, Cramer WA et al. A mechanism for toxin insertion into membranes is
suggested by the crystal structure of the channel-forming domain of colicin E1. Strucure. 1997;5:44358
Endo Y, Tsurugi K, Yutsudo T et al. Site of action of a Vero toxin (VT2) from Escherichia coli
O157: H7 and of Shiga toxin on eukaryotic ribosoms. RNA N-glycosidase activity of the toxins. Eur J
Biochem. 1988;171:45-50
Engedal N, Skotland T, Torgersen ML et al. Shiga toxin and its use in targeted cancer therapy
and imaging. Microb Biotechnol. 2011;4:32-46
Erbguth F, Naumann M. Historical aspects of botulinum toxin: Justinus Kerner (1786–1862)
and the ‘‘sausage poison’’. Neurology. 2000;53:1850–3
Erbguth F. Botulinum toxin, a historical note. Lancet. 1998;351:1280
Erbguth FJ. From poison to remedy: the chequered history of botulinum toxin. J Neural
Transm. 2008;115:559-65
Erbguth FJ. Historical notes on botulism, Clostridium botulinum, botulinum toxin, and the
idea of the therapeutic use of the toxin. Mov Disord. 2004;19:S2–S6
Evans LJ, Goble ML, Hales KA et al. Different sensitivities to acid denaturation within a family
of proteins: implications for acid unfolding and membrane translocation. Biochemistry.
1996;35:13180-5
Falguières T, Mallard F, Baron C et al. Targeting of Shiga toxin B-subunit to retrograde
transport route in association with detergent-resistant membranes. Mol Biol Cell. 2001;12:2453-68
Falnes PØ, Sandvig K. Penetration of protein toxins into cells. Curr Opin Cell Biol.
2000;12:407-13
Fasshauer D, Bruns D, Shen B et al. A structural change occurs upon binding of syntaxin to
SNAP-25. J Biol Chem. 1997;7:4582-90
Field M, Graf LH, Smith PL. Heat-stable toxin of E. coli: in vitro effects on guanylate cyclase,
cGMP, and ion transport in small intestine. PNAS. 1978;75:2800-4.
Finkelstein RA, Mukerjee S, Rudra BC. Demonstration and quantitation of antigen in cholera
stool filtates. J Infect Dis. 1963;113:99-104
Fischer A, Montal M. Crucial role of the disulfide bridge between botulinum neurotoxin light
and heavy chains in protease translocation across membranes. J Biol Chem. 2007b;282:29604–11
Fischer A, Montal M. Single molecule detection of intermediates during botulinum
neurotoxin translocation across membranes. PNAS. 2007a;104:10447–52
49 | P a g e
Fischer A, Mushrush DJ, Lacy DB et al. Botulinum neurotoxin devoid of receptor binding
domain translocates active protease. PLoS Pathog. 2008;4:published online
Fischer A. Synchronized chaperone function of botulinum neurotoxin domains mediates light
chain translocation into neurons. Curr Top Microbiol Immunol. 2013;364:115-37
Florquin S, Aaldering L. Superantigens: a tool to gain new insight into cellular immunity. Res
Immunol. 1997;148:373-386
Freudenberg MA, Galanos C. Interaction of lipopolysaccharides and lipid A with complement
in rats and its relation to endotoxicity. Infect Immun. 1978;19:875–882.
Fu Z, Chen C, Barbieri JT et al. Glycosylated SV2 and gangliosides as dual receptors for
botulinum neurotoxin serotype F. Biochemistry. 2009;48:5631–41
Fujinaga Y, Wolf AA, Rodighiero C et al. Gangliosides that associate with lipid rafts mediate
transport of cholera and related toxins from the plasma membrane to endoplasmic reticulum. Mol
Biol Cell. 2003;14:4783-3
Galanos C, Lehmann V, Luderitz O et al. Endotoxic properties of chemically synthesized lipid
A part structures. Comparison of synthetic lipid A precursor and synthetic analogues with
biosynthetic lipid A precursor and free lipid A. Eur J Biochem.1984;140:221-7
Garred O, Dubinina E, Polesskaya A et al. Role of the disulphide bond in Shiga toxin A-chain
for toxin entry into cells. J Biol Chem. 1997;272:11414-9
Garred O, Van Deurs B, Sandvig K. Furin-induced cleavage and activation of Shiga toxin. J Biol
Chem. 1995;270:10817-21
Giannella RA, Mann EA. E. coli heat-stable enterotoxin and guanylyl cyclase C: new functions
and unsuspected actions. Trans Am Clin Climatol Assoc. 2003;114:67-85
Giddings KS, Johnson Ae, Tweten RK. Redefining cholesterol’s role in the mechanism of the
cholesterol-dependent cytolysins. PNAS USA. 2003;100:11315-20
Girod A, Storry B, Simpsom JC et al. Evidence for A COP-I-independent transport rout from
the Golgi complex to the endoplasmic reticulum. Nat Cell Biol. 1999;1:423-30
Golin-Bisello F, Bradbury N, Ameen N. Sta and cGMP stimulate CFTR translocation to the
surface of villus enterocytes in rat jejunum and is regulated by protein kinase G. Am J Physiol Cell
Physiol. 2005;289:C708-16
Gómez I, Sánchez J, Miranda R et al. Cadherin-like receptor binding facilities proteolytic
cleavage of helix α-1 in domain I and oligomer pre-pore formation of Bacillus thuringiensis Cry1Ab
toxin. FEBS Lett. 2002;513:242-6
Gomez-Muñoz A, Marin A, O’Brien L et al. Cell-permeable ceramides inhibit the stimulation
of DNA synthesis and phospholipase D activity by phosphatidate and lysophosphatidate in rat
fibroblasts. J Biol Chem. 1994;269:8937-43
Goñi FL, Montes LR, Alonso A. Phospholipases C and sphingomyelinases: Lipids as substrates
and modulators of enzyme activity. Prog Lipid Res. 2012;51:238-66
Gonzalez MR, Bischofberger M, Pernot L et al. Bacterial pore-forming toxins: the (w)hole
story? Cell Mol Life Sci. 2008;65:493-507
Gordan VM, KLimpel KR, Arora N et al. Proteolytic activation of bacterial toxins by eukaryotic
cells is performed by furin and by additional cellular proteases. Infect Immun, 1995;63:82-7
Gouaux E. α-Hemolysin from Staphylococcus aureus: An archetype of β-barrel, channelforming toxins. J Struc Biol. 1998;121:110-22
Gouaux JE, Braha O, Hobaugh MR et al. Subunit stoichiometry of staphylococcal α-hemolysin
in crystals and on membranes: A heptameric transmembrane pore. PNAS USA. 1994;91:12828-31
Gram C. Ueber die isolierte Färbung der Schizomyceten der Pneumonie in Schnitt- und
Trockenpräparaten. Fortschr Med. 1884;2:185-9
Grüsser OJ. Die ersten systematischen Beschreibungen und tierexperimentellen
Untersuchungen des Botulismus. Zum 200. Geburtstag von Justinus Kerner am 18. September 1986.
Sudhoffs Arch. 1986;10: 167–87.
50 | P a g e
Guarino A, Giannella R, Thompson MR. (Citrobacter freundii produces an 18-amino acid heatstable enterotoxin identical to the 18-amino acid Escherichia coli heat-stable enterotoxin (STIa).
Infect Immun. 1989;57:649-52.
Hansen GH, Dalskov SM, Rasmussen CR et al.Cholera toxin entry into pig enterocytes occurs
via a lipid raft- and clathrin-dependent mechanism. Biochemistry. 2005;44:873-82
Haug G, Leemhuis J, Tiemann D et al. The host cell chaperone Hsp90 is essential for
translocation of the binary Clostridium botulinum C2 toxin into the cytosol. J Biol Chem.
2003;278:32266-74
Hazzard J, Sudhof TC, Rizo J. NMR analysis of the structure of synaptobrevin and of its
interaction with syntaxin, J. Biomol. 1999;14:203-7
He P, Yun CC. Mechanisms of the regulation of the intestinal Na+/H+ exchanger NHE3. J
Biomed Biotechnol. 2010:238080
Hehnly H, Longhini KM, Chen JL et al. Retrograde Shiga toxin trafficking is regulated by
ARHGAP21 and Cdc42. Mol Biol Cell. 2009;20:4303-12
Henry T, Pommier S, Journet L et al. Improved methods for producing outer membrane
vesicles in gram-negative bacteria. Res Microbiol. 2004;155:437-46
Herreros J, Lalli G, Montecucco C, Schiavo G. Tetanus toxin fragment C binds to a protein
present in neuronal cell lines and motoneurons. J Neurochem. 2000;74:1941–50.
Herreros J, Ng T, Schiavo G. Lipid rafts act as specialized domains for tetanus toxin binding
and internalization into neurons. Mol Biol Cell. 2001;12:2947–60.
Herreros J, Schiavo G. Lipid microdomains are involved in neurospecific binding and
internalisation of clostridial neurotoxins. Int J Med Microbiol. 2002;294:447-53
Heuck AP, Hotze EM, Tweten RK et al. Mechanism of membrane insertion of a multimeric
beta-barrel protein: perfringolysin O creates a pore using ordered and coupled conformational
changes. Mol Cell. 2000;6:1233-42
Hilsenbeck JL, Park H, Chen G, et al. Crystal structure of the cytotoxic bacterial protein colicin
B at 2.5 A resolution. Mol Microbiol. 2004;51:711-20
Hitchcock PJ, Leive L, Mäkelä PH et al. Lipopolysaccharide nomenclature – Past, present and
future. J Bacter. 1986;166:699-705
Holzer, VE. Botulismus durch inhalation. Med Klin. 1962;57:1735-1738.
Hotze EM, Heck AP, Czajkowski DM et al. Monomer-monomer interactions drive the prepore
to pore conversion of a beta-barrel-forming cholesterol-dependent cytolysin. J Biol Chem.
2002;277:11597-605
Howard SP, Buckley JT. Activation of the hole forming toxin aerolysin by extracellular
processing. J Bacteriol. 1985;163:336-40
Howard SP, Meiklejohn HG, Shivak D et al. A TonB-like protein and a novel membrane
protein encoding an ATP-binding cassette function together in exotoxin secretion. Mol Microbiol.
1996;22:595-604
Hsu LC, Park JM, Zhang K et al. The protein kinase PKR is required for macrophage apoptosis
after activation of Toll-like receptor 4. Nature. 2004;428:341-5
Humeau Y, Doussau F, Grant NJ et al. How botulinum and tetanus neurotoxins block
neurotransmitter release. Biochimie. 2000;85:427-46
Iacovache I, van der Goot FG, Pernot L. Pore formation: An ancient yet complex form of
attack. Biochim Biophys Acta. 2008;1778:1611-23
Iordanov MS, Pribnow D, Magun JL et al. Ribotoxic stress response: activation of the stressactivated protein kinase JNK1 by inhibitors of the peptidyl transferase reaction and by sequencespecific RNA damage to the alpha-sarcin/ricin loop in the 28S rRNA. Mol Cell Biol. 1997;17:3373-81
Iwamoto R, Higashiyama S, Mitamura T et al. Heparin-binding EGF-like growth factor, which
acts as the diphtheria toxin receptor, forms a complex with membrane protein DRAP27/CD9, which
up-regulates functional receptors and diphtheria toxin sensitivity. EMBO J. 1994;13:2322-30.
Jacewicz M, Clausen H, Nudelman E et al. Pathogenesis of Shigella diarrhea. XI. Isolation of a
Shigella
51 | P a g e
Jahn R, Lang T, Südhof TC. Membrane fusion. Cell. 2003;112:519–33
Johnson EA, Bradshaw M. Clostridium botulinum and its neurotoxins: A metabolic and cellular
perspective. Toxicon. 2001;39:1703–22.
Kagan BL, Finkelstein A, Colombini M. Diphtheria toxin fragment forms large pores in
phospholipid bilayer membranes. PNAS USA. 1981;4950-4
Kawabe Y, Ochi A. Programmed cell death and extrathymic reduction of Vbeta8+ CD4+ T cells
in mice tolerant to Staphylococcus aureus enterotoxin B. Nature. 1991;349:245-8.
Kerner J. Das Fettgift oder die Fettsäure und ihreWirkungen auf den thierischen Organismus,
ein Beytrag zur Untersuchung des in verdorbenen Würsten giftig wirkenden Stoffes. Cotta, Stuttgart.
1822
Kerner J. Neue Beobachtungen über die in Württemberg so häufig vorfallenden tödlichen
Vergiftungen durch den Genuss geräucherter Würste. Osiander, Tübingen. 1820
Kienker PK, Jakes KS, Blaustein RO et al. Sizing the protein translocation pathway of colicin Ia
channels. J Gen Physiol. 2003;122:161-76
Kim MY, Linardic C, Obeid L. Identification of sphingomyelin turnover as an effector
mechanism for the action of tumor necrosis factor alpha and gamma-interferon. Specific role in cell
differentiation. J Biol Chem. 1991;266:484-9
Kirby JE. Anthrax lethal toxin induces human endothelial cell apoptosis. Infect Immun.
2004;72:430-9
Kleizen B, Braakman I, de Jonge HR. Regulated trafficking of the CFTR chloride channel. Eur J
Cell Biol. 2000;79:544-56
Knowles BH, Ellar DJ. Colloid-osmotic lysis is a general feature of the mechanism of action of
Bacillus thuringiensis delta-endotoxins with different insect specificity. Biochim Biophys Acta.
1987;924:509-18
Koch R. Die aetiologie der tuberculose. Berliner Klinische Wochenschrift. 1882;19:221-30
Kochi SK, Collier RJ. DNA fragmentation and cytolysis in U937 cells treated with diphtheria
toxin or other inhibitors of protein synthesis. Exp Cell Res. 1993;208:296-302
Koriazova LK, Montal M. Translocation of botulinum neurotoxin light chain protease through
the heavy chain channel. Nat Struct Biol. 2003;10:13–18
Koriazova LK, Montal M. Translocation of botulinum neurotoxin ligh chain protease through
the heavy chain channel. Nat Struct Biol. 2003;10:13-8
Kotb M. Superantigens in human diseases. Clin Microbiol Newslett. 1997;19:145-52
Kozaki S, Kamata Y, Watarai S et al. Ganglioside GT1b as a complementary receptor
component for Clostridium botulinum neurotoxins. Microb Pathog. 1998;25:91–99
Krantz BA, Trivedi AD, Cunningham K et al. Acid-induced unfolding of the amino0terminal
domains of the lethal and edema factors of anthrax toxin. J Mol Biol. 2004;344:739-56
Kreyden OP. Botulinum toxin: from poison to pharmaceutical. In: Kreyden OP, Böne R, Burg
G (eds). Hyperhidrosis and botulinum toxin in dermatology. Curr Probl Dermatol, Karger, Basel,
2002;30:94–100
Kumaran D, Eswaramoorthy S, Furey W et al. Domain organization in Clostridium botulinum
neurotoxin type E is unique: its implication in faster translocation. J Mol Biol. 2009;386:233–45
Kurpiewski G, Forrester LJ, Barrett JT et al. Platelet aggregation and sphingomyelinase D
activity of a purified toxin from the venom of Loxosceles reclusa. Biochim Biophys Acta.
1981;678:467-76.
Lacy DB, Stevens RC. Sequence homology and structural analysis of the clostridial
neurotoxins. J Mol Biol. 1999;291:1091-104
Lacy DB, Tepp W, Cohen AC et al. Crystal structure of botulinum neurotoxin type A and
implications for toxicity. Nat Struct Biol. 1998;5:898-902
Lacy DB, Tepp W, Cohen AC et al. Crystal structure of botulinum neurotoxin type A and
implications for toxicity. Nat Struct Biol. 1998;5:898-902
Lalli G, Herreros J, Osborne SL et al. Functional characterisation of tetanus and botulinum
neurotoxins binding domains. J Cell Sci. 1999;112:2715-24
52 | P a g e
Laney DW, Mann EA, Dellon SC et al. Novel sites for expression of a E. coli heat-stable
enterotoxin receptor in developing rat. Amer J Physiol. 1992;263:G816-21.
Lanzrein M, Sand O, Olsnes S. GPI-anchored diphtheria toxin receptor allows membrane
translocation of the toxin without detectable ion channel activity. EMBO J. 1996;15:725-34.
LaPointe P, Wei X, Gariépy J. A role for the protease-sensitive loop region of Shiga-like toxin
1 in the retrotranslocation of its A1 domain from the endoplasmic reticulum lumen. J Biol Chem.
2005;280:23310-8
Lauvrak SU, Torgersen ML, Sandvig K. Efficient endosome-to-Golgi transport of Shiga toxin is
dependent on dynamin and clathrin. J Cell Sci. 2004;117:2321-31
Lazdunski C, Bouveret E, Rigal A et al. Colicin import into Escherichia coli cells. J Bacteriol.
1998;180:4993-5002
Lemichez E, Bomsel M, Devilliers G et al. Membrane translocation of diphtheria toxin
fragment A exploits early to late endosome trafficking machinery. Mole Microbiol. 1997;23:445-57
Lencer WI, Constable C, Moe S et al. Targeting of cholera toxin and Escherichia coli heat
labile toxin in polarized epithelia: role of COOH-terminal KDEL. J Cell Biol. 1995;131:951-62.
Lin JH, Baumgaertner A. Stability of a melittin pore in a lipid bilayer: a molecular dynamics
study. Biophys J. 2000;78:1714-24
Lindberg AA, Brown JE, Strömberg N et al. Identification of the carbohydrate receptor for
Shiga toxin produced by Shigella dysenteriae type 1. J Biol Chem. 1987;262:1179-85
Lingwood CA, Law H, Richarddson S et al. Glycolipid binding of purified and recombinant
Escherichia coli produced verotoxin in vitro. J Biol Chem. 1987;262:8834-9
Liu P, Anderson RG. Compartmentalized production of ceramide at the cell surface. J Biol
Chem. 1995;270:27179-85
Liu S, Leppla SH. Cell surface tumor endothelium marker 8 cytoplasmic tail-independent
anthrax toxin binding, proteolytic processing, oligomer formation, and internalization. J Biol Chem.
2003;278:5227-34
Llewelyn M, Cohen J. Superantigens: microbial agents that corrupt immunity. Lancet Infect
Dis. 2002;2:156-62
Lloubès R, Cascales E, Walburger A et al. The Tol-Pal proteins of Escherichia coli cell
envelope: an energized system required for outer membrane integrity? Res Microbiol. 2001;152:5239
Loeffler F. Untersuchungen über die Bedeutung der Mikroorganismen für die Entstehung der
Diptherie beim Menschen, bei der Taube und beim Kalbe. Mitth a.d. keiserl Gesundheitsampte;2:42199
Löffler B, Hussain M, Grundmeier M et al. Staphylococcus aureus Panton-Valentine
leukocidin is a very potent cytotoxic factor for human neutrophils. PLOS Path. 2010;6:e1000715
Lohner K, Staudegger E, Prenner EJ et al. Effect of staphylococcal delta-lysin on the
thermotropic phase behavior and vesicle morphology of dimyristoylphosphatidylcholine lipid bilayer
model membranes. Differential scanning calorimetric, 31P nuclear magnetic resonance and Fourier
transform infrared spectroscopic, and X-ray diffraction studies. Biochemistry. 1999;38:16514–28.
Majoul I, Sohn K, Wieland FT, et al. KDEL receptor (Erd2p)-mediated retrograde transport of
the cholera toxin A-subunit from the Golgi involves COPI, p23, and the COOH terminus of Erd2p. J Cell
Biol. 1998;143:601-12.
Majoul I, Straub M, Hell SW et al. KDEL-cargo regulates interactions between proteins
involved in COPI vesicle traffic: measurements in living cells using FRET. Dev Cell. 2001;1:139-53.
Mann EA, Cohen MB, Giannella RA. Comparison of receptors for E. coli heat-stable
enterotoxin: Novel receptor present in IEC-6 cells. Amer J Physiol. 1993;264:G172-G178.
Marrack P, Kappler J. The staphylococcal enterotoxins and their relatives. Science. 1990;248:
Matsuzaki K, Murase O, Fujii N et al. An antimicrobial peptide, magainin 2, induced rapid flipflop of phospholipids couples with pore formation and peptide translocation. Biochemistry.
1996;35:11361-8
53 | P a g e
Matsuzaki K. Magainins as paradigm for the mode of action of pore forming polypeptides.
Biochim Biophys Acta. 1998:1376:391-400
Matteoli M, Verderio C, Rossetto O et al. Synaptic vesicle endocytosis mediates the entry of
tetanus neurotoxin into hippocampal neurons. PNAS USA. 1996;93:13310–5.
McCormick JK, Yarwood JM, Schlievert PM. Toxic shock syndrome and bacterial
superantigens: an update. Annu Rev Microbiol. 2001;55:77–104.
Mellor IR, Thomas DH, Sansom MS. Properties of ion channels formed by Staphylococcus
aureus delta-toxin. Biochim Biophys Acta. 1988;942:280-94.
Menestrina G, Dalla Serra M, Comai M et al. Ion channels and bacterial infection: The case of
β-barrel pore-forming protein toxins of Staphylococcus aureus. FEBS Lett. 2003;552:54-60
Menestrina G, Schiavo G, Montecucco C. Molecular mechanisms of action of bacterial
protein toxins. Molec Aspects Med. 1994;15:79-193
Merritt EA, Sarfaty S, Van der Akker F et al. Crystal structure of cholera toxinB-pentamer
bound to receptor GM1 pentasaccharide. Protein Sci. 1994;3:166-75
Miethke T, Wahl C, Heeg K et al. Acquired resistance to superantigen-induced T cell shock.
Vbeta selective T cell unresponsiveness unfolds directly from a transient state of hyperreactivity. J
Immunol. 1993;150:3776–84.
Miles G, Movileanu L, Bayley H. Subunit composition of a bicomponent toxin: staphylococcal
leukocidin forms an octameric transmembrane pore. Protein Sci. 2002;11:894-902
Mills DC, Arnon SS. The large intestine as the site of Clostridium botulinum colonization in
human infant botulism. J Infect Dis. 1987;156:997–8
Milne JC, Collier RJ. pH-dependent permeabilization of the plasma membrane of mammalian
cells by anthrax protective antigen. Mol Microbiol. 1993;10:647-53
Mogridge J, Cunningham K, Collier RJ. Stoichiometry of anthrax toxin complexes.
Biochemistry. 2002;41:1079-82
Moniatte M, Van der Goot FG, Buckley JT et al. Characterization of the heptameric poreforming complex of the Aeromonas toxin aerolysin using MALDI-TOF mass spectrometry. FEBS Lett.
1996;384:269-72
Montal M. Botulinum neurotoxin: a marvel of protein design. Annu Rev Biochem.
2010;79:591-617
Montecucco C, Schiavo G. Mechanism of action of tetanus and botulinum neurotoxins. Mol
Microbiol. 1994;13:1-8
Moya M, Dautry-Varsat A, Goud B et al. Inhibition of coated pit formation in Hep2 cells
blocks the cytotoxicity of diphtheria toxin but not that of ricin toxin. J Cell Biol. 1985;101:548-59.
Muga A, González-Mañas JM, Lakey JH et al. pH-dependent stability and membrane
interaction of the pore-forming domain of colicin A. J Biol Chem. 1993;268:1553-7
Müller-Alouf H, Alouf JE, Gerlach D et al. Human pro- and anti-inflammatory cytokine
patterns induced by Streptococcus pyogenes erythrogenic (pyrogenic) exotoxins A and C
superantigens. Infect Immunol. 1996;64:1450-3
Müller-Alouf H, Carnoy C, Simonet M et al. Superantigen bacterial toxins: state of the art.
Toxicon. 2001;39:1691-1701
Müller-Alouf H, Gerlach D, Desreumaux P et al. Streptococcal pyrogenic exotoxin A (SPEA)
superantigen induced production of hematopoietic cytokines, IL-12 and IL-13 by human peripheral
blood mononuclear cells. Microb Pathogen. 1997;23:265-72
Munro P, Kojima H, Dupont JL et al. High sensitivity of mouse neuronal cells to tetanus toxin
requires a GPI-anchored protein. Biochem Biophys Res Commun. 2001;289:623-9
Muraro L,Tosatto S, Motterlini L et al. The N-terminal half of the receptor domain of
botulinum neurotoxin A binds to microdomains of the plasma membrane. Biochem Biophys Res
Commun. 2009;380:76–80
Naglich JG, Metheral JE, Russell DW et al. Expression cloning of a diphtheria toxin receptor:
identity with a heparin-binding EGF-like growth factor precursor. Cell. 1992;69:1051-61.
54 | P a g e
Nassi S, Collier RJ, Finkelstein A. PA63 channel of anthrax toxin: an extended beta-barrel.
Biochemistry. 2002;41:1445-50
Nichols BJ, Kenworthy AK, Polishchuk et al. Rapid cycling of lipid raft markers between the
cell surface and Golgi complex. J Cell Biol. 2001;153:529-41
Nikaido H. Outer membrane of Salmonella typhimurium. Transmembrane diffusion of some
hydrophobic substances. Biochim Biophys Acta. 1976;433:118-32
Nishiki T, Tokuyama Y, Kamata Y et al. The high-affinity binding of Clostridium botulinum type
B neurotoxin to synaptotagmin II associated with gangliosides GT1b/GD1a. FEBS Lett. 1996;378:253–
7
Nishizuka Y. Intracellular signalling by hydrolysis of phospholipids and activation of protein
kinase C. Science. 1992;259:607-14
Nishizuka Y. Protein kinase C and lipid signalling for sustained cellular response. FASEB J.
1995;9:484:96
O’Brien J, Friedlander A, Dreier T et al. Effects of anthrax toxic components on human
neutrophils. Infect Immun. 1985;47:306-10
O’Hehir RE, Lamb JR. Induction of specific clonal anergy in human T lymphocytes by
Staphylococcus aureus enterotoxins. PNAS USA. 1990;87:8884–8.
O’Loughlin EV, Robins-Browne RM. Effect of Shiga toxin and Shiga-like toxins on eukaryotic
cells. Microbes Infect. 2001;3:493-507
Obata F, Tohyama K, Bonev et al. Shiga toxin 2 effects the central nervous system through
receptor globotriaosylceramide localized to neurons. J Infect Dis. 2008;198:1398-1406
Obeid LM, Linardic CM, Karolak LA et al. Prgrammed cell death induced by ceramide. Science.
1993;259:1769-71
Ochi S, Oda M, Matsuda H et al. Clostridium perfringens alphatoxin activates the
sphingomyelin metabolism system in sheep erythrocytes. J Biol Chem. 2004;279:12181-9
Oda M, Matsuno T, Shiihara R et al. The relationship between the metabolism of
sphingomyelin species and the hemolysis of sheep erythrocytes induced by Clostridium perfringens
alphatoxin. J Lipid Res. 2008;49:1039-47
Oh, KJ, Senzel L, Collier RJ et al. Translocation of the catalytic domain of diphtheria toxin
across planar phospholipid bilayers by its own T domain. PNAS USA. 1999;96:8467-70
Okazaki T, Bell RM, Hannun YA. Spingomyelin turnover induced by vitamin D3 in HL-60 cells.
Role in cell differentiation. J Biol Chem. 1989;264:19076-80
Olsnes S, Reisbig R, Eiklid K. Subunit structure of Shigella cytotoxin. J Biol Chem.
1981;256:8732-8
Orlandi PA, Fishman PH. Filipin0dependent inhibition of cholera toxin: evidence for toxin
internalization and ac tvation through caveolae-like domains. J Cell Biol. 1998;141:905-15
Papini E, Sandoná D, Rappuoli R et al. On the membrane translocation of diphtheria toxin: at
low pH the toxin induces ion channels on cells. EMBO J. 1988;7:3353-9
Pardo-López L, Gómez I, Rausell C et al. Structural changes of the Cry1Ac oligomeric pre-pore
from Bacillus thuringiensis induced by N-acetylgalactosamine facilitates toxin membrane insertion.
Biochemistry. 2006;45:10329-36
Park JM, Greten FR, Li ZW et al. Macrophage apoptosis by anthrax lethal factor through p38
MAP kinase inhibition. Science. 2002;297:2048-51
Parker MW, Buckley JT, Postma JPM et al. Structure of the Aeromonas toxin proaerolysin in
its water-soluble and membrane-channel states. Nature. 1994;367:292-5
Parker MW, Feil SC. Pore-forming protein toxins: from structure to function. Prog Biophys
Mol Biol. 2005;88:91-142
Parker MW, Pattus F, Tucker AD et al. Structure of the membrane pore-forming fragments of
colicin A. Nature. 1989;337:93-6
Passaro DJ, Werner SB, McGee J et al. Wound botulism associated with black tar heroin
among injecting drug users. JAMA. 1998;279:859–63.
55 | P a g e
Paton JC, Paton AW. Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli
infections. Clin Microbiol Rev. 1998;11:450-79
Pearson G, Robinson F, Beers Gibson T et al. Mitogen-activated proteim (MAP) kinase
pathways: regulation and physiological functions. Endocr Rev. 2001;22:153-83
Pezerd C, Berche P, Mock M. Contribution of individual toxin components to virulence of
Bacillus anthracis. Infect Immun. 1991;59:3472-7
Popoff V, Mardones GA, Tenza D et al. The retromer complex and clathrin defin an early
endosomal retrograde exit site. J Cell Sci. 2007l120:2022-31
Postle K, Kadner RJ. Touch and go: tying TonB to transport. Mol Microbiol. 2003;49:869-882
Pott T, Dufourc EJ. Action of melittin on the DPPC-cholesterol liquid-ordered phase: a solid
state 2H- and 31P-NMR study. Biophys J. 1995;68:965–77.
Potter LR, Hunter T. Guanylyl cyclase-linked natriuretic peptide receptors: structure and
regulation. J Biol Chem. 2001;276:6057-60.
Raghunathan G, Seetharamulu P, Brooks Bret al. Models of delta-hemolysin membrane
channels and crystal structures. Proteins. 1990;8:213–25.
Ramachandran R, Heuck AP, Tweten RK et al. Structural insights into the membraneanchoring mechanism of a cholesterol-dependent cytolysin. Nature Struct Biol. 2002;11:823-7
Ratts R, Zeng H, Berg EA et al. The cytosolic entry of diphtheria toxin catalytic domain
requires a host cell cytosolic translocation factor complex. J Cell Biol. 2003;160:1139-50
Rausell C, Sánchez J, Muñoz-Garay C et al. Unfolding events in the water-soluble monomeric
Cry1Ab toxin during transition to oligomeric pre-pore and membrane inserted pore channel. J Biol
Chem. 2004;279:55168-75
Reisbig R, Olsnes S, Eiklid K. The cytotoxic activity of Shigella toxin. Evidence for catalytic
inactivation of the 60S ribosomal subunit. J Biol Chem. 1981;256:8739-44
Ren J, Kachel K, Kim H et al. Interaction of diphtheria toxin T domain with molten globule-like
proteins and its implications for translocation. Science. 1999;284:955-7
Ren J, Utsonomiya I, Taguchi K et al. Localization of verotoxin receptors in nervous system.
Brain Res. 1999;825:183-8
Richards AA, Stang E, Pepperkok R et al. Inhibitors of COP-mediated transport and cholera
toxin action inhibit Simian Virus 40 infection. Mol Biol Cell. 2002;13:1750-64
Rietschel ET, Brade H, Holst O et al. Bacterial endotoxin: chemical constitution, biological
recognition, host response, and immunological detoxification. Curr Top Microbiol Immunol.
1996;216:39–81.
Rind HB, Butowt R, von Bartheld CS. Synaptic targeting of retrograde transported trophic
factors in motoneurons: comparison of glial cell line-derived neurotrophic factor, brain-derived
neurotrophic factor, and cardiotrophin-1 with tetanus toxin. J Neurosci. 2005;25:539-549
Rodighiero C, Tsai B, Rapoport TA et al. Role of ubiquitination in retro-translocation of
cholera toxin and escape of cytosolic degradation. EMBO Rep. 2002;3:1222-7
Rosetto O, Scorzeto M, Megighian A et al. Tetanus neurotoxin. Toxicon. 2013;66:59-63
Rossjohn J, Feil SC, McKinstry WJ. Aerolysin – A paradigm for membrane insertion of betasheet protein toxins? J Struc Biol. 1998a;121:92-100
Rossjohn J, Raja SM, Nelsom KL et al. Movement of a loop in domain 3 of aerolysin is
required for channel formation. Biochemistry. 1998b;37:741-46
Rossol M, Heine H, Meusch U et al. LPS-induced cytokine production in human monocytes
and macrophages. Crit Rev Immunol. 2011;31:379-446
Saint-Pol A, Yélamos B, Amessou M et al. Clathrin adaptor epsinR is required for retrograde
sorting on early endosomal membranes. Dev Cell. 2004;6:525-38
Sanchez J, Holmgren J. Virulence factors, pathogenesis and vaccine protection in cholera and
ETEC diarrhea. Curr Opin Immunol. 2005;17:388-98
Sandvig K, Bergan J, Dyve AB et al. Endocytosis and retrograde transport of Shiga toxin.
Toxicon. 2010;56:1181-5
56 | P a g e
Sandvig K, Olsnes S, Brown JE et al. Endocytosis from coated pits of Shiga toxin: A glycolipidbinding protein from Shigella dysenteriae 1. J Cell Biol. 1989;108:1331-43
Sandvig K, Van Deurs B. Endocytosis, intracellular transport and cytotoxic action of Shiga
toxin and ricin. Physiol Rev. 1996;76;949-66
Sandvig K, Van Deurs B. Transport of protein toxins into cells: pathways used by ricin, cholera
toxin and Shiga toxin. FEBS Lett. 2002;529:49-53
Schein SJ, Kagan BL, Finkelstein A. Colicin K acts by forming voltage-dependent channels in
phospholipid bilayer membranes. Nature. 1978;276:159-63
Schendel SL, Cramer WA. On the nature of the unfolded intermediate in the in vitro
transition of the colicin E1 channel domain from the aqueous to the membrane phase. Protein Sci.
1994;3:2272-9
Schiavo G, Benfenati F, Poulain B et al. Tetanus and botulinum-B neurotoxins block
neurotransmitter release by a proteolytic cleavage of synaptobrevin. Nature. 1992b;359:832-5
Schiavo G, Matteoli M, Montecucco C. Neurotoxins affecting neuroexocytosis. Physiol Rev.
2000;80:1-50
Schiavo G, Poulain B, Rosetto O et al. Tetanus toxin is a zinc protein and its inhibition of
neurotransmitter release and protease activity depend on zinc. EMBO J. 1992a;11:3577-83
Schmiel DH, Miller VL. Bacterial phospholipases and pathogenesis. Micr Inf. 1999;1:1103-12
Schulz S, Green CK, Yuen PST et al. Guanylyl cyclase is a heat-stable enterotoxin receptor.
Cell. 1990;63:941–8
Schulz S, Waldman SA. (1999) The guanylyl cyclase family of natriuretic peptide receptors.
Vitam Horm. 1999;57:123–51.
Scobie HM, Rainey GJ, Bradley KA et al. Human capillary porphogenesis protein 2 functions
as an anthrax toxin receptor. PNAS USA. 2003;100:5170-4
Shogomori H, Futerman AH. Cholera toxin is found in detergent-insoluble rafts/domains at
the cell surface of hippocampal neurons but is internalized via a raft-independent mechanism. J Biol
Chem. 2001;276:9182-8
Simpson LL, Coffield JA, Bakry N. Inhibition of vacuolar adenosine triphosphatase
antagonizes the effects of clostridial neurotoxins but not phospholipase A2 neurotoxins. J Pharmacol
Exp Ther. 1994;269:256–62.
Simpson LL, Rapport MM. Ganglioside inactivation of botulinum toxin. J Neurochem.
1971;18:1341-3
Simpson LL, Rapport MM. The binding of botulinum toxin to membrane lipids: sphingolipids,
steroids and fatty acids. J. Neurochem. 1971;18:1751–59
Simpson LL. Ammonium chloride and methylamine hydrochloride antagonize clostridial
neurotoxins. J Pharmacol Exp Ther. 1983;225:546–52.
Simpson LL. Identification of the major steps in botulinum toxin action. Annu Rev Pharmacol
Toxicol. 2004;44:167–93
Sindice A, Basoglu C, Cerci A et al. Guanylin, uroguanylin and heat-stable enterotoxin activate
guanylate cyclase C and/or a pertussis toxin-sensitive G protein in human proximal tubular cells. J
Biol Chem. 2002;277:17758-64.
Skånland SS, Wälchli S, Sandvig K. beta-Arrestins attenuate p38 mediated endosome to Golgi
transport. Cell Microbiol. 2009;11:796-807
Smith DC, Sillence DJ, Falguières T et al. The association of Shiga-like toxin with detergentresistant membranes is modulated by glucosylceramide and is an essential requirement in the
endoplasmic reticulum for a cytotoxic effect. Mol Biol Cell. 2006;17:1375-87
Smith WE, Kane AV, Campbell St et al. Shiga toxin 1 triggers a ribotoxic stress response
leading to p38 and JNK activation and induction of apoptosis in intestinal epithelial cells. Infect
Immun. 2003;71:1497-1504
Sobel J. Botulism. Clin Infect Dis. 2005;41:1167-73
57 | P a g e
Sobko AA, Kotova EA, Antonenko YN et al. Effect of lipids with different spontaneous
curvature on the channel activity of colicin E1: evidence in favor of a toroidal pore. FEBS Lett.
2004;576:205-10
Sobko AA, Kotova EA, Antonenko YN et al. Lipid dependence of the channel properties of a
colicin E1-lipid toroidal pore. J Biol Chem. 2006;281:14408-16
Song L, Hobaugh MR, Shustak C et al. Structure of staphylococcal α-hemolysin, a heptameric
transmembrane pore. Science. 1996;274:1859-66
Songer JG. Bacterial phospholipases and their role in virulence. Trends Microbiol. 1997;5:15661
Sousa MV, Richardson M, Fontes W et al. Homology between the seed cytolysin enterolobin
and bacterial aerolysins. J Protein Chem. 1994;13:659-67
Spangler BD. Structure and function of cholera toxin and the related Escherichia coli heatlabile enterotoxin. Microbiol Rev. 1992;56:622-49
Starr T, Sun Y, Wilkins N et al. Rab33 and Rab6 are functionally overlapping regulators of
Golgi-homeostasis and trafficking. Traffic. 2010;11:626-36
Stevens DL. The toxic shock syndromes. Infect Dis Clin North Am. 1996;10:727–46.
Sugawara N, Tomita T, Sato T et al. Assembly of Staphylococcus aureus leukocidin into a
pore-forming ring-shaped oligomer on human polymorphonuclear leukocytes and rabbit
erythrocytes. Biosci Biotech Biochem. 1999;63:884-891
Sugawara-Tomita N, Tomita T, Kamio Y. Stochastic assembly of two-component
staphylococcal gamma-hemolysin into heteroheptameric transmembrane pores with alternate
subunit arrangements in ratios of 3:4 and 4:3. J Bacteriol. 2002;184:4747-56
Sutkowski N, Conrad B, Thorley-Lawson DA et al. Epstein-Barr virus transactivates the human
endogenous retrovirus HERV-K18 that encodes a superantigen. Immunity. 2001;15:579–89.
Suttorp N, Hessz T, Seeger W et al. Bacterial exotoxins and endothelial permeability for
water and albumin in vitro. AM J Physiol. 1988;255;C368-76
Suttorp N, Seeger W, Dewein E et al. 1985. Staphylococcal a-toxin-induced-PGI2 in
endothelial cells: role of calcium. Am J Physiol. 1985;248:C127–34
Swaminathan S, Eswaramoorthy S. Structural analysis of the catalytic and binding sites of
Clostridium botulinum neurotoxin B. Nat Struct Biol. 2000;7:693–9
Tabas I, Ron D. Integrating the mechanisms of apoptosis induced by endoplasmic reticulum
stress. Nat Cell Biol. 2011;13:184-90
Takao T, Shimonishi Y, Kobayashi M et al. Amino acid sequence of heat-stable enterotoxin
produced by Vibrio cholerae non-O1. FEBS Lett. 1985b;193:250–4.
Takao T, Tominaga S, Yoshimura Y et al. Isolation, primary structure and synthesis of heatstable enterotoxin produced by Yersinia enterocolitica. Eur J Biochem. 1985a;152:199–206.
Takenouchi H, Kiyokawa N, Taguchi T et al. Shiga toxin binding to globotriaosyl ceramide
induces intracellular signals that mediate cytoskeleton remodelling in human renal carcinomaderived cells. J Cell Sci. 2004;117:3911-22
Talbot JC, Thiaudiere E, Vincent M et al. Dynamics and orientation of amphipathic peptides
in solution and bound to membranes: a steady-state and time-resolved fluorescence study of
staphylococcal deltatoxin and its synthetic analogues. Eur Biophys J. 2001;30:147-61
Tam P, Mahfoud R, Nutikka A et al. Differential intracellular transport and binding of
verotoxin 1 and verotoxin 2 to globotriaosylceramide-containing lipid assemblies. J Cell Physiol.
2008;216:750-63
Tamai K, Semenov M, Kato Y et al. LDL-receptor-related proteins in Wnt signal transduction.
Nature. 2000;407:530-5
Tanoue T, Nishida E. Molecular recognitions in the MAP kinase cascades. Cell Signal.
2003;15:455-62
Thelestam M, Mollby R. Determination of toxin-induced leakage of different size nucleotides
through the plasma membrane of human diploid fibroblasts. Infect Immun. 1975;11:640–8.
58 | P a g e
Thompson MR, Steinberg MS, Gemski P et al. Inhibition of in vitro protein synthesis by
Shigella dysenteriae 1 toxin. Biochem Biophys Res Commun. 1976;71:783-8
Torrens JK. Clostridium botulinum was named because of association with ‘‘sausage
poisoning’’. BMJ. 1998;316:151
Tournier JN, Quesnel-Hellman A, Mathieu J et al. Anthrax edema toxin cooperates with lethal
toxin to impair cytokine secretion during infection of dendritic cells. J Immunol. 2005;174:4934-41
Tousson A, Fuller CM, Benos DJ. Apical recruitment of CFTR in T-84 cells is dependent on
cAMP and microtubules but not Ca2+ or microfilaments. J Cell Sci. 1996;109:1325-34
toxin-binding glycolipid from rabbit jejunum and HeLa cells and its identification as
globotriaosylceramide. J Exp Med. 1986;163:1391-404.
Trujillo C, Taylor-Parker J, Harrison R et al. Essential lysine residues within transmembrane
helix 1 of diphtheria toxin facilitate COPI binding and catalytic domain entry. Mol Microbiol.
2010;76:1010-9
Tsai B, Rapoport T. Unfolded cholera toxin is transferred to the ER membrane and release
from protein disulphide isomerase upon oxidation by Ero1. J Cell Biol. 2002;159:207-15
Tsai B, Rodighiero C, Lencer WI et al. Protein disulfide isomerase acts as a redox-dependent
chaperon to unfold cholera toxin. Cell. 2001;104:937-948
Tsukamoto K, Kozai Y, Ihara H, et al. Identification of the receptor binding sites in the
carboxyl-terminal half of the heavy chain of botulinum neurotoxin types C and D. Microb. Pathog.
2008;44:484–93
Uchiyama T, Yan XJ, Imanishi K et al. Bacterial super antigens: mechanism of T cell activation
by the superantigens and their rol in the pathogenesis of infections diseases. Microbiol Immunol.
1994;38:245-56
Ulrich RG, Bavari S, Olson MA. Bacterial superantigens in human disease: structure, function
and diversity. Trends Microbiol. 1995;3:463-8
Utskarpen A, Slagsvold HH, Dyve AB et al. SNX1 and SNX2 mediate retrograde transport of
shiga toxin. Biophys Res Commun. 2007;358:566-70
Vaandrager AB, Schulz S, de Jonge HR et al. Guanylyl cyclase C is an N-linked glycoprotein
receptor that accounts for multiple heat-stable enterotoxin-binding proteins in the intestine. J Biol
Chem. 1993;268:2174-9.
Vaandrager AB, Smolenski A, Tilly BC et al. Membrane targeting of cGMP-dependent protein
kinase is required for cystic fibrosis transmembrane conductance regulator Cl- channel activation.
PNAS USA. 1998;95:1466-71
Vaandrager AB, Tilly BC, Smolenski A et al. cGMP stimulation of cystic fibrosis
transmembrane conductance regulator CL- channels co-expressed with cGMP0dependent protein
kinase type II but not type Ibeta. J Biol Chem. 1997;272:4195-200
Vaandrager AB. Structure and function of the heat-stable enterotoxin receptor/guanylyl
cyclase C. Mol Cell Biochem. 2002;230:73-83
Valeva A, Palmer M, Bhakdi S. Staphylococcal α-toxin: Formation of the heptameric pore is
partially cooperative and proceeds through multiple intermediate stages. Biochemistry.
1997;36:13298-304
Van der Goot FG, González-Mañas, Lakey JM et al. A molten-globule membrane insertion
intermediate of the pore-forming domain of colicin A. Nature. 1991;354:408-10
Van der Goot FG, Lakey JK, Pattus F et al. Spectroscopic study of the activation and
oligomerization of the channel-forming toxin aerolysin: Identification of the site of proteolytic
activation. Biochemistry. 1992;31:8566-70
Van der Wal F, Luirink J, Oudega B. Bacteriocin release proteins: mode of action, structure
and biotechnological application. FEMS Microbiol Rev. 1995;17:381-99
Van Leeuwenhoek, A. The collected letters of Antoni van Leeuwenhoek. 1941;2:91-95
Vanden Broeck D, Horvath C, De Wolf MJS. Vibrio cholerae: Cholera toxin. Int J Biochem Cell
Biol. 2007;39:1771-5
59 | P a g e
Vander Spek JC, Howland K, Friedman T et al. Maintenance of the hydrophobic face of the
diphtheria toxin amphipathic transmembrane helix 1 is essential for the effecient delivery of the
catalytic domain to the cytosol of target cells. Protein Eng. 1994;7:985-9
Vetter IR, Parker MW, Tucker AD et al. Crystal structure of a colicin N fragment suggests a
model for toxicity. Structure. 1998;6:863-74
Von Bartheld CS. Axonal transport and neuronal transcytosis of trophic factors, tracers, and
pathogens. J Neurobiol. 2004;58:295-314
Wälchli S, Skånland SS, Gregers TF et al. The mitogen-activated protein kinase p38 links
Shigatoxin-dependent signaling and trafficking. Mol Biol Cell. 2008;19:95-104
Weaver C, Redborg AH, Konisky J. Plasmid-determined immunity of Escherichia coli K-12 to
colicin Ia is mediated by a plasmid-encoded membrane protein. J Bacteriol. 1981;148:817-28
Wedel BJ, Garbers DL. The guanylyl cyclase family at Y2K. Annu Rev Physiol. 2001;63:215233.
Weiglmeier PR, Rösch P, Berkner H. Cure and curse: E. coli heat-stable enterotoxin and its
receptor guanylyl cyclase C. Toxins. 2010;2:2213-29
Werner SB, Passaro DJ, McGee J et al. Wound botulism in California, 1951–1998: a recent
epidemic in heroin injectors. Clin Infect Dis. 2000;31:1018–24.
White J, Johannes L, Mallard F et al. Rab6 coordinates a novel Golgi to ER retrograde
transport pathway in live cells. J Cell Biol. 1999;147:743-60
Wiegmann K, Schütze S, Machleidt T et al. Functional dichotomy of neutra land acidic
spingomyelinases in tumor necrosis factor signaling. Cell. 1994;78:1005-15
Wiener M, Freymann D, Ghosh P et al. Crystal structure of colicin Ia. Nature. 1997:385:461-4
Wigelsworth DJ, Krantz BA, Christensen KA et al. Binding stoichiometry and kinetics of the
interaction of a human anthrax toxin receptor, CMG2, with protective antigen. J Biol Chem.
2004;279:23349-56
Williamson LC, Halpern JL, Montecucco C et al. Clostridial neurotoxins and substrate
proteolysis in intact neurons. J Biol Chem. 1996;271:7694–9
Williamson LC, Neale EA. Bafilomycin A1 inhibits the action of tetanus toxin in spinal cord
neurons in cell culture. J Neurochem. 1994;63:2342–5.
Willing BP, Finlay BB. The role of the immune system in regulating the microbiota. Gut
microbes. 2010;1:213-23
Wilmsen HU, Leonard KR, Tichelaar W et al. The aerolysin membrane channel is formed by
heptamerization of the monomer. EMBO J. 1992;11:2457-63
Windschiegl B, Orth A, Romer W et al. Lipid reorganization induced by Shiga toxin clustering
on planar membranes. PLoS ONE. 2010;4:e6238
Wolf AA ,Jobling MG, Wimer-Mackin S et al. Ganglioside structure dictates signal
transduction by cholera toxin in polarized epithelia and association with caveolae-like membrane
domains. J Cell Biol. 1998;141:917-27
Wolf AA, Fujinaga Y, Lencer WI. Uncoupling of the cholera toxin-G(M1) ganglioside receptor
complex from endocytosis, retrograde Golgi trafficking, and downstream signal transduction by
depletion of membrane cholesterol. J Biol Chem. 2002;277:16249:56
Woltmann A, Hamann L, Ulmer AJ et al. Molecular mechanisms of sepsis. Langenbecks Arch
Surg. 1998;383:2–10.
Wong SKF, Garbers DL. Receptor guanylyl cyclases. J Clin Invest. 1992;90:299-305.
Yoshino K, Miyachi M, Takao T et al. Purification and sequence determination of heat-stable
enterotoxin elaborated by a cholera toxin producing strain of Vibrio cholerae O1. FEBS Lett.
1993;181:138-42.
Yoshino K, Xiaozhe, Miyachi M et al. Amino acid sequence of a novel heat-stable enterotoxin
produced by a yst gene-negative strain of Yersinia enterocolitica. Lett Pept Sci. 1994;1:95–105.
Zakharov SD, Cramer WA. Colicin crystal structures: pathways and mechanisms for colicin
insertion into membranes. Biochim Biophys Acta. 2002;1565:333-46
60 | P a g e
Appendix
Table 1: Membrane damaging toxins
Bacteria
Actinobacillus suis
Actinobacillus
actinomycetemcomitans
Actinobacillus
pleuropneumoniae
Aeromonas hydrophila
Aeromonas sobria
Bacillus alvei
Bacillus cereus
Bacillus subtilis
Bordetella pertussis
Clostridium botulinum
Clostridium histolyticum
Clostridium perfringens
Clostridium septicum
Clostridium sordelli
Clostridium tetani
Corynebacterium bovis
Corynebacterium ulcerans
Escherichia coli
Gardnerella vaginalis
Legionella pneumophila
Listeria Ivanovii
Listeria monocytogenes
Listeria seeligeri
Morganella morganii
Pasteurella haemolytica A1
Proteus mirabilis
Proteus vulgaris
Pseudomonas aeruginosa
Serratia marcescens
Staphylococcus aureus
Toxin
Hemolysin (AshA)
Leukotoxin (AaltA)
Group
Pore forming toxin
Pore forming toxin
Hemolysins (Apx IA, IIA, IIIA)
Pore forming toxin
Aerolysin (AerA)
Aerolysin (AerA)
Alveolysin (ALV)
Cerolysin O (CLO)
Phospholipase C
Phospholipase A
hemolysin (CyaA)
Botulinolysin (BLY)
Histolyticolysin O
Perfringolysin O (PFO)
Alpha toxin
Beta toxin
Enterotoxin
Alpha toxin
Septicolysin O
Sordellilysin
Tetanolysin (TLY)
Phospholipase D
Phospholipase D
Hemolysin Hlya
Phospholipase A
Hemolysin (Gvh)
legiolysin
Ivanolysin O
Listeriolysin O (LLO)
Phospholipase C
Seelerigolysin
Hemolysin (MmxA)
Leukotoxin (LktA)
Hemolysin (HmpA)
Hemolysin (PvxA)
Hemolysin (HmpA)
Cytotoxin (leukocidin)
Phospholipase C (PLC-H)
Hemolysin (ShIA)
Alpha toxin
Beta toxin (sphingomyelinase)
Gamma toxin
Delta toxin
Pore forming toxin
Pore forming toxin
Pore forming toxin
Pore forming toxin
Enzymatic cytolysin
Enzymatic cytolysin
Pore forming toxin
Pore forming toxin
Pore forming toxin
Pore forming toxin
Enzymatic cytolysin
Pore forming toxin
Pore forming toxin
Pore forming toxin
Pore forming toxin
Pore forming toxin
Pore forming toxin
Enzymatic cytolysin
Enzymatic cytolysin
Pore forming toxin
Enzymatic cytolysin
Pore forming toxin
Pore forming toxin
Pore forming toxin
Pore forming toxin
Enzymatic cytolysin
Pore forming toxin
Pore forming toxin
Pore forming toxin
Pore forming toxin
Pore forming toxin
Pore forming toxin
Pore forming toxin
Enzymatic cytolysin
Pore forming toxin
Pore forming toxin
Enzymatic cytolysin
Pore forming toxin
Detergent-like toxin
61 | P a g e
Streptococcus agalactiae
Streptococcus mutans
Streptococcus pneumoniae
Streptococcus pyogenes
Vibrio cholerae (non-O1)
Vibrio cholerae (el Tor)
Vibrio damsela
Vibrio mimicus
Vibrio parahaemolyticius
Vibrio vulnificus
Leukocidin S&F (incl Panton
Valentine leukocidin)
Streptolysin S-similar toxin
Streptolysin S-similar toxin
Pneumolysin (PLY)
Streptolysin O (SLO)
Streptolysin S
Heat-stable hemolysin (TDH)
Heat-labile cytolysin
Damselysin
Heat-stable hemolysin (TDH)
Heat-labile hemolysin
Heat-stable hemolysin (TDH)
Phospholipase A2
Heat-labile cytolysin
Pore forming toxin
Detergent-like toxin?
Detergent-like toxin
Pore forming toxin
Pore forming toxin
Detergent-like toxin
Pore forming toxin
Pore forming toxin
Enzymatic cytolysin
Pore forming toxin
Pore forming toxin
Pore forming toxin
Enzymatic cytolysin
Pore forming toxin
Table 2: Receptor targeting toxins
Bacteria
Citrobacter freundii
Escherichia coli
Mycoplasma arthritidis
Staphylococcus aureus
Streptococcus pyogenes
Yersinia enterocolitica
Toxin
Heat-stable toxin (STa)
Heat-stable toxin (STa)
Heat-stable toxin (STb)
M. arthr. Mitogen (MAM)
Enterotoxins (SE) A, B, C1-3, D, E
(G, H, I)
Epidermolytic toxins A, B
Toxic shock syndrome tx (TSST)
Erythrogenic toxins A, C
Heat-stable toxin (STa)
Group
E. coli enterotoxin
E. coli enterotoxin
E. coli enterotoxin
Superantigen
Superantigen
Superantigen
Superantigen
Superantigen
E. coli enterotoxin
Table 3: Internalised toxins
Bacteria
Aeromonas hydrophila
Bacillus anthracis
Bacillus cereus
Bordetella pertussis
Campylobacter jejuni
Clostridium botulinum
Clostridium difficile
Toxin
Cholera-like enterotoxin
Edema factor and PA
Lethal factor and PA
Exoenzyme
Adenylate cyclase (AC)
Pertussis toxin (PT)
Cytolethal distending toxin (AC)
Neurotoxin A
Neurotoxin B-G
C2 toxin
C3 toxin
Toxin A
Target
G proteins (ADP-ribosylation)
Adenylate cyclase
MEK
G proteins (ADP-ribosylation)
Adenylate cyclase
G proteins (ADP-ribosylation)
Cell cycle
SNAP (t-SNARE)
SNAP/syntaxin (t-SNARE) or
synaptobrevin (v-SNARE)
Actin (ADP-ribosylation)
G proteins (ADP-ribosylation)
Rho (glucosylation)
62 | P a g e
Clostridium limosum
Clostridium perfringens
Clostridium sordellii
Clostridium spiroforme
Clostridium tetani
Corynebacterium diphteriae
Corynebacterium
pseudotuberculosis
Escherichia coli
Helicobacter pylori
Pseudomonas aeruginosa
Burkholderia (Pseudomonas)
pseudomallei
Salmonella spec
Shigella dysenteriae
Staphylococcus aureus
Vibrio cholerae
Vibrio mimicus
Toxin B
exoenzyme
Iota toxin
Hemorrhagic toxin
Lethal toxin
Iota-like toxin
Tetanus toxin (TeTx)
Diphtheria toxin
Diphtheria toxin
Rho (glucosylation)
G proteins (ADP-ribosylation)
Actin (ADP-ribosylation)
Rho (glucosylation)
Small GTP binding proteins “
Actin (ADP-ribosylation)
Synaptobrevin (v-SNARE)
EF-2 (ADP-ribosylation)
EF-2 (ADP-ribosylation)
Shiga-like toxins (SLT I,II)
Heat-labile toxins (LT I,II)
Vacuolating cytotoxin (VacA)
Exotoxin A (ETA)
Exoenzyme S
Exotoxin
rRNA (N-glycosidasis)
G proteins (ADP-ribosylation)
Vacuolisation
Ef-2 (ADP-ribosylation)
G proteins (ADP-ribosylation)
EF-2
Cholera-like enterotoxin
Shiga toxin
EDIN
Cholera toxin (CT)
Cholera-like enterotoxin
G proteins (ADP-ribosylation)
rRNA (N-glycosidase)
G proteins (ADP-ribosylation)
G proteins (ADP-ribosylation)
G proteins (ADP-ribosylation)
63 | P a g e