REVIEW ARTICLE
Molecular basis of toxicity of Clostridium perfringens
epsilon toxin
Monika Bokori-Brown
1
, Christos G. Savva
2
,Se
´
rgio P. Fernandes da Costa
1
, Claire E. Naylor
2
,
Ajit K. Basak
2
and Richard W. Titball
1
1 Biosciences, College of Life and Environmental Sciences, University of Exeter, UK
2 Department of Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck College, London, UK
Introduction
The Clostridium genus encompasses more than 80 spe-
cies that form a diverse group of rod-shaped, Gram-
positive bacteria with the ability to form spores [1].
These organisms are principally obligate anaerobes,
although some species are able to survive in the pres-
ence of trace amounts of oxygen [2,3]. Clostridia are
omnipresent bacteria that can be found in the environ-
ment, particularly in soil and water, as well as in
decomposing animal and plant matter. In addition,
some clostridial species can be found in the gastroin-

also associated with enteritis and enterotoxaemia in goats, calves and foals.
It is considered to be a potential biowarfare or bioterrorism agent by the
US Government Centers for Disease Control and Prevention. The rela-
tively inactive 32.9 kDa prototoxin is converted to active mature toxin by
proteolytic cleavage, either by digestive proteases of the host, such as tryp-
sin and chymotrypsin, or by C. perfringens k-protease. In vivo, the toxin
appears to target the brain and kidneys, but relatively few cell lines are sus-
ceptible to the toxin, and most work has been carried out using Madin–
Darby canine kidney (MDCK) cells. The binding of e-toxin to MDCK cells
and rat synaptosomal membranes is associated with the formation of a sta-
ble, high molecular weight complex. The crystal structure of e-toxin reveals
similarity to aerolysin from Aeromonas hydrophila, parasporin-2 from
Bacillus thuringiensis and a lectin from Laetiporus sulphureus. Like these
toxins, e-toxin appears to form heptameric pores in target cell membranes.
The exquisite specificity of the toxin for specific cell types suggests that it
binds to a receptor found only on these cells.
Abbreviations
DRM, detergent resistant membrane; GPI, glycosylphosphatidylinositol; LD
50
, 50% lethal dose; LSL, pore-forming lectin;
MTS, methanethiosulfate; MDCK, Madin–Darby canine kidney; PS, parasporin-2.
FEBS Journal 278 (2011) 4589–4601 ª 2011 The Authors Journal compilation ª 2011 FEBS 4589
cause virulence, their individual significance and roles
in disease can be difficult to interpret.
e-toxin is produced by C. perfringens toxinotypes B
and D. C. perfringens type B, which also produces
b-toxin, is the aetiological agent of dysentery in new-
born lambs, but is also associated with enteritis and
enterotoxaemia in goats, calves and foals (Table 2)
[5,10]. C. perfringens type D affects mainly sheep and

lights the need to understand the molecular basis of
toxicity in order to develop an effective vaccine.
Molecular biology of e-toxin
The e-toxin gene, etx, is located on plasmids in both
toxinotypes B and D [15]. In toxinotype B isolates, the
etx gene is carried on a  65 kb plasmid that may also
carry the cpb2 gene for b2-toxin [16,17], while the cpb
gene encodes b-toxin resides on a separate plasmid. In
toxinotype D isolates, the etx gene is present on plas-
mids ranging from 48 to 110 kb [18]. Interestingly, the
larger plasmids have been found to carry up to three
different toxin-encoding genes (etx, cpe and cpb2) [18].
A common theme in both toxinotypes is the associa-
tion of the etx gene with insertion sequences. The
transposable element IS1151 has been found upstream
of the etx gene in plasmids from both toxinotypes,
although in opposite orientations [16]. This association
has led to speculation about possible virulence gene
mobilisation and exchange between plasmids. Support
for this hypothesis was provided by the identification
of circular transposition intermediates containing
IS406-etx-IS1151 [18]. These findings have implications
for the evolution of C. perfringens and help to explain
why some plasmids carry multiple toxin genes. Addi-
tional evidence for genetic exchange among toxino-
types is provided by the finding that the tcp locus,
required for conjugation [19], is present in some etx
plasmids from both toxinotype B and D isolates
[17,18]. Hughes et al. demonstrated conjugative trans-
fer of an etx plasmid from a toxinotype D to a type A

B Enterotoxaemia in sheep
Chronic enteritis in lambs (pine)
Enteritis in calves, goats and foals
Dysentery in lambs
D Enterotoxaemia in sheep (pulpy kidney disease,
overeating disease), calves and goats
Molecular basis of toxicity of C. perfringens e toxin M. Bokori-Brown et al.
4590 FEBS Journal 278 (2011) 4589–4601 ª 2011 The Authors Journal compilation ª 2011 FEBS
produced ten times more e-toxin than the strain from
which the etxB gene was isolated (NCTC 8533) [22].
The relatively inactive secreted prototoxin of 296
amino acids (32.9 kDa) is converted to the fully active
mature toxin by proteolytic cleavage in the gut lumen,
either by digestive proteases of the host, such as tryp-
sin and chymotrypsin [23], or by C. perfringens k-pro-
tease [14,24]. Proteolytic activation of the toxin can
also be achieved in the laboratory by controlled
enzyme digestion [25].
Depending on the protease, proteolytic cleavage
results in the removal of 10–13 amino-terminal and 22–
29 carboxy-terminal amino acids (Fig. 1) [14,23]. Maxi-
mal activation of the toxin occurs with a combination
of trypsin and chymotrypsin, resulting in the loss of
13 N-terminal residues and 29 C-terminal residues, pro-
ducing a mature toxin that is > 1000-fold more toxic
than the prototoxin [26], with an LD
50
of 50–65 ngÆkg
)1
in mice [14,27]. This makes e-toxin the most potent

sequence identity to a number of putative bacterial
proteins of unknown function, identified by genome
sequencing projects, including a number of proteins
from Bacillus thuringiensis (UniProt ID: C3GC23 or
C3FC62).
Effects of e-toxin on cultured cells
Over the past few decades, a number of cell lines have
been tested in order to identify a suitable in vitro
model for the study of e-toxin. The Madin–Darby
canine kidney (MDCK) cell line of epithelial origin,
derived from the distal collecting tubule, was initially
identified to be toxin-sensitive by microscopic examina-
tion of intoxicated cells [30]. Cytotoxicity assays on a
further 11 kidney cell lines of animal origin failed to
identify additional cell lines sensitive to the toxin [31].
Cytotoxicity assays on 17 human cell lines (originating
from kidney, brain, skin, bone, respiratory and intesti-
nal tracts) identified the Caucasian renal leiomyoblas-
toma (G-402) cell line to be toxin-sensitive, albeit to a
lesser extent than the MDCK cell line [32].
In MDCK cells the dose of e-toxin needed to kill
50% of cells is reported to be 15 ngÆmL
)1
[31]. Intoxi-
cated cells undergo morphological changes including
swelling and formation of membrane blebs [33].
The rapid death of cells exposed to the toxin [34]
results in the formation of a large membrane complex
on the target cell surface [33], leading to pore forma-
tion, an efflux of K

plasm without structure [39]. Mixed glial primary cell
cultures, isolated from mice brains, are also toxin-sen-
sitive [40]. Primary cultures of mice cerebellar cortex
identified granule cells targeted and affected by e-toxin
[41], leading to membrane severing, Ca
2+
influx and
glutamate efflux [41]. Primary cultures of human renal
tubular epithelial cells also showed toxin-induced
swelling of cells and formation of membrane blebs
[42].
Effects of e-toxin on animals and
tissues
Enterotoxaemia in naturally infected animals is usually
characterised by enterocolitis in goats and systemic
lesions in sheep. It is postulated that proteolytic activa-
tion of the toxin in the gastrointestinal tract compro-
mises the intestinal barrier of intoxicated animals,
allowing the dissemination of toxin via the bloodstream
to the main target organs of the kidneys and brain. The
mechanism of e-toxin absorption from the gastrointesti-
nal tract is not well defined. Histological analysis of
ligated intestinal loops of sheep and goats exposed to
e-toxin revealed necrosis of the colonic epithelium in
both species, suggesting that alteration of large intesti-
nal permeability might play a role in toxin absorption
[43]. In mice and rats, transmission electron microscopy
studies revealed that the toxin alters the small intestinal
permeability predominantly by opening the mucosa
tight junction, indicating that the small intestine might

hosts and in experimental animal models [51]. Several
studies provide evidence that neurological damage in
intoxicated animals is induced by increased vascular
permeability in brain blood vessels, leading to vasogenic
oedema, a common feature of animals suffering from
C. perfringens enterotoxaemia. There is also evidence
that the toxin acts directly on neuronal tissues of
intoxicated animals. For example, in mice and rat
brains, intoxication causes both selective and extensive
neurotoxicity, depending on the dose of toxin adminis-
tered [52,53]. Extensive neuronal damage was observed
in the rat brain after intravenous toxin administration
at a minimal lethal dose, while sub-lethal dose caused
neuronal damage predominantly in the hippocampus,
including the mossy fibre layers, that was not due to
alteration of cerebral blood flow [53]. Subacute or
chronic intoxication of rats also produced degeneration
and necrosis of neuronal cells [54].
In intravenously injected mice, pre-injection of pro-
totoxin inhibited preferential accumulation and lethal
activity of radiolabelled toxin in the brain, indicating
that the toxin specifically binds to, and acts on, the
brain [55]. High affinity binding of radiolabelled toxin
to rat brain homogenates and synaptosomal membrane
fractions also suggested the presence of specific binding
sites in brain tissue [56]. Pre-treatment of synaptoso-
mal membrane fractions with pronase, heat and neur-
aminidase decreased toxin binding, indicating that the
interaction of toxin with cell membranes in the brain is
facilitated by a sialoglycoprotein [56]. Pre-treatment of

synaptosomal membrane fractions, where binding of
radiolabelled toxin to rat synaptosomes was associated
with the formation of a stable, high molecular weight
complex, leading to pore formation [27,56,59]. Dorca-
Arevalo’s recent study [50] also disputes the direct
action of GFP-tagged epsilon toxin on nerve terminals,
based on the failure of the toxin to trigger glutamate
release from toxin-treated mouse brain synaptosomal
fractions. In this study, synaptosomal preparations
were found to be contaminated by myelin structures,
identified as the main toxin binding sites in these prep-
arations [50].
Crystal structure of e-toxin
The three-dimensional structure of e-toxin has been
determined [60] by multiwavelength anomalous disper-
sion ( PDB ID: 1UYJ). The
crystal structure revealed that e-toxin is a very elon-
gated molecule (100 A
˚
· 20 A
˚
· 20 A
˚
) and is com-
posed of mainly b-sheets (Fig. 2). The toxin structure
can be divided into three domains. Domain I contains
an a-helix and a three-stranded anti-parallel sheet,
upon which the large helix lies. The second domain is
a b-sandwich, containing a five-stranded sheet and a
b-hairpin (both of which are anti-parallel). The third

receptor binding. In aerolysin, the N-terminal domain
has been postulated to be responsible for the initial
interaction with cells [66]. Aerolysin binds to glycosyl-
phosphatidylinositol (GPI)-anchored proteins that are
found in detergent resistant membranes (DRMs) via
domain II. The crystal structure of an oligomerising,
but not pore-forming, mannose-6-phosphate bound
aerolysin is now available (PDB ID: 3C0O). Domains
Iofe-toxin and PS (Fig. 2) are similar, and have some
limited similarity to aerolysin. It has been suggested
that this domain performs a similar function in e-toxin
[60] and PS [62]. However, none of the residues
involved in sugar-binding in aerolysin are present in e-
toxin or PS. Therefore, it seems likely that these pro-
teins have a different cell-surface receptor. In complete
contrast, domain I of LSL has a b-trefoil lectin fold
(Fig. 2), in which lactose and N-acetyl-d-lactosamine
have been observed crystallographically. It is probable
that the major reported differences in the target cell
specificities of aerolysin and e-toxin, and the different
function of LSL, is the result of the different structures
and properties of these domains.
The second and third domains of e-toxin exhibit
obvious structural similarity to the third and fourth
domains of aerolysin, and to the second and third
domains of LSL and PS. As described previously,
domain II is composed of a five-stranded sheet with an
amphipathic b-hairpin (residues 124–146) lying against
it, while domain III is a b-sandwich composed of
four- and three-stranded b-sheets. This amphipathic

final amino acid in the hairpin. Numbering corresponds to PDB file, except for C. septicum a-toxin, where numbering corresponds to UniProt
ID: Q53482. ETX, C. perfringens e-toxin (1UYJ); AERO, A. hydrophilus aerolysin (1PRE); LSL, L. sulphureus lectin (1W3A); PS2, B. thuringien-
sis parasporin-2 (2ZTB); NONTOX, B. thuringiensis 26 kDa non-toxic protein (2D42); ATOX, C. septicum a-toxin. Boxing and letter colouring
indicate regions of higher sequence conservation.
Molecular basis of toxicity of C. perfringens e toxin M. Bokori-Brown et al.
4594 FEBS Journal 278 (2011) 4589–4601 ª 2011 The Authors Journal compilation ª 2011 FEBS
the bilayer. This hydrophobic sequence is proposed to
drive membrane insertion and possibly act as a rivet,
stabilising the pore. However, the effect was not seen in
e-toxin, where turn residues could be accessed from the
trans-side by antibodies [67]. In Clostridium septicum a-
toxin, a protein with significant sequence homology to
aerolysin, the region equivalent to this hairpin was
tested for membrane insertion using sequential cysteine
mutation, modified with a fluorescent probe sensitive to
changes from an aqueous to a lipid environment [70].
This technique showed that, alternately, these residues
point into a lipid and then an aqueous environment
when bound to a membrane, indicating insertion of
the two-stranded sheets in a similar manner to Staphy-
lococcus aureus a-toxin.
The final domain of e-toxin has been associated with
heptamerisation [27]. In the precursor forms of both
e-toxin and aerolysin the C-terminal peptides appear
to block oligomerisation. The electron microscope
structure of the water-soluble, non-pore-forming hept-
amer formed by an aerolysin mutant, Y221G, shows
that the interface between a pair of monomers in the
heptamer is made up of one face from one monomer
and the opposite face from the other [71], as is the case

cates not only the presence of pores within the mem-
brane after the addition of e-toxin, but also that these
pores are long-lived, with no association–dissociation
equilibrium. These results showed that pores could be
formed in the absence of a membrane receptor.
Although various lipids have been used in these experi-
ments, the toxin has not been shown to have any lipid
preference [35]. However, lipids with low melting
points seem to favour membrane insertion under the
same experimental conditions [74]. This group reported
a 100-fold lower sensitivity of the toxin to carboxy-
fluorescein loaded liposomes compared with MDCK
cells. This is not surprising, considering the absence of
a receptor in liposomes. The same study also demon-
strated the existence of heptameric assemblies formed
in liposomes. However, the heptamers were not stable,
as evidenced by the presence of intermediate species on
an SDS ⁄ PAGE gel.
e-toxin appears to target the DRMs in membranes.
This is also the case for aerolysin [75] and PS [65].
Both monomeric and heptameric e-toxin accumulates
in DRMs, and depletion of cholesterol, a major
constituent of DRMs, has an inhibitory effect on both
e-toxin [59] and PS. e-prototoxin, unable to form
heptamers, also binds mainly to DRMs, indicating that
heptamerisation is not a prerequisite for interactions
with susceptible cells. Therefore, the putative receptor
for both e-prototoxin and e-toxin is thought to be
present mainly in the DRMs. All steps, from binding
to membrane insertion, are thought to occur in

0.4 nm on the side of toxin insertion and 1.0 nm on
the opposing side. High-throughput screen methods
identified some e-toxin inhibitors that appear to work
by blocking the pore [79], as they do not work by
inhibiting cell-binding or oligomerisation and are effec-
tive in cells pre-treated with toxin.
In summary, the likely mechanism of pore formation
by e-toxin is predicted to be as follows. The prototoxin
is secreted by the bacterium and activated, possibly
locally, by C. perfringens k-protease or by host prote-
ases such as trypsin and ⁄ or chymotrypsin. Receptor
binding may occur prior to or after activation. Once
activated, heptamerisation occurs on the membrane,
which may lead to formation of a pre-pore complex.
This has been observed in cholesterol-dependent cytol-
ysins [80,81] and in S. aureus a-toxin [82]. In fact,
under certain conditions, heptamerisation of both aer-
olysin [71] and e-toxin [68] is possible without pore
formation. The final step of pore formation might
involve unfolding of the amphipathic hairpin and its
insertion into the membrane to form the walls of the
pore composed of 14 b-strands.
Prevention of disease
A number of commercially available vaccines exist for
the prevention of C. perfringens enterotoxaemia, and
these have been used extensively over the past decades
to prevent disease in domesticated livestock. The vac-
cines are typically prepared by treating C. perfringens
type D culture filtrate with formaldehyde to toxoid
components. Because relatively crude culture filtrates

reported to be immunogenic in rabbits, sheep, goats
and cattle, and to give rise to > 5 IU of antitoxin
after two doses [85,88]. This recombinant toxoid was
reported to be a superior immunogen to the commer-
cially available vaccines available in Brazil [85].
An alternative approach to the development of a
toxoid vaccine would involve generating a gene encod-
ing a non-toxic variant, which can then be expressed
in E. coli or another easily cultured host. e-toxin con-
sists of three domains (Fig. 2) that are dependent on
two strands traversing the entire molecule [60]. There-
fore, expression of the individual domains of e-toxin,
which are likely to be non-toxic, is not straightfor-
ward. Site-directed mutants of the toxin have been
produced, which show markedly reduced toxicity
towards MDCK cells, and these could be exploited as
vaccines [68,89]. The evaluation of these mutants in
mice has not been reported by Pelish and McClain
[68]. However, the H106P variant protein (H119P, fol-
lowing the numbering system for prototoxin without
signal peptide) reported by Oyston et al. [89] has been
shown to be non-toxic to mice. Mice immunised
with H106P developed an antibody response against
e-toxin. More importantly, these immunised mice were
protected against a subsequent challenge with 1000
minimum lethal doses of wild-type e-toxin [89]. These
findings suggest that H106P could form the basis of a
vaccine.
The reasons why the H106P protein is not toxic are
not known. However, it may be relevant that chemical

known whether other neutralising monoclonal anti-
bodies recognise this loop. Any of these antibodies
could have utility for the prevention or treatment of
disease.
An intriguing alternative to the use of antibodies is
the use of dominant-negative inhibitors of toxicity.
This approach involves generating variant forms of
e-toxin which are inactive but are still able to oligome-
rise. In the work reported by Pelish and McClain [68],
variants were generated in which the putative mem-
brane-insertion loop was locked into the folded confor-
mation by the introduction of cysteines, which were
then able to form disulfide bridges. Mixtures of the
variant and wild-type toxin, in a ratio of at least 1:8,
were non-toxic towards MDCK cells. Although these
mixtures were able to form oligomers and bind to cells,
they were unable to form heat-resistant and sodium
dodecyl sulfate resistant oligomers [68]. It is conceiv-
able that these variant forms of the toxin could be
used to limit toxicity, but they may need to be given at
the same time as exposure to the wild-type toxin,
which would limit their therapeutic value.
Conclusion
All of the evidence indicates that C. perfringens e-toxin
intoxicates cells by forming pores in cell membranes,
and in this respect the toxin is similar to many other
bacterial pore-forming toxins. The toxin monomer
appears to be structurally related to a range of bacte-
rial and eukaryotic pore-forming toxins, although the
low degree of sequence homology suggests that conver-

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