Anaerobe 30 (2014) 102e107

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Clostridium perfringens epsilon toxin: The third most potent bacterial toxin known b
Guilherme Guerra Alves a, *, Ricardo Andrez Machado de Avila ,
vez-Olo
rtegui c, Francisco Carlos Faria Lobato a, * Carlos Delfin Cha ^nio Carlos Avenue, 6627, Belo Horizonte, MG CEP 31.270-901, Brazil Veterinary School, Universidade Federal de Minas Gerais (UFMG), Anto
ria Avenue, 1105, Criciúma, SC CEP 88.806-000, Brazil Universidade do Extremo Sul Catarinense (UNESC), Universita c
gicas, Universidade Federal de Minas Gerais (UFMG), Anto ^nio Carlos Avenue, 6627, Belo Horizonte, MG CEP 31.270-901, Brazil Instituto de Ci^ encias Biolo a

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 June 2014 Received in revised form 18 August 2014 Accepted 19 August 2014 Available online 16 September 2014

Epsilon toxin (ETX) is produced by Clostridium perfringens type B and D strains and causes enterotoxemia, a highly lethal disease with major impacts on the farming of domestic ruminants, particularly sheep. ETX belongs to the aerolysin-like pore-forming toxin family. Although ETX has striking similarities to other toxins in this family, ETX is often more potent, with an LD50 of 100 ng/kg in mice. Due to this high potency, ETX is considered as a potential bioterrorism agent and has been classified as a category B biological agent by the Centers for Disease Control and Prevention (CDC) of the United States. The protoxin is converted to an active toxin through proteolytic cleavage performed by specific proteases. ETX is absorbed and acts locally in the intestines then subsequently binds to and causes lesions in other organs, including the kidneys, lungs and brain. The importance of this toxin for veterinary medicine and its possible use as a biological weapon have drawn the attention of researchers and have led to a large number of studies investigating ETX. The aim of the present work is to review the existing knowledge on ETX from C. perfringens type B and D.

Keywords: Clostridium perfringens Enterotoxemia Epsilon toxin Pore-forming

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction Clostridium perfringens, which was previously named Bacillus capsulatus aerogenes then Clostridium welchii, is a Gram-positive non-motile anaerobic bacillus that is capable of transforming into a more resistant form called a spore [1]. Under certain conditions, this bacterium can become pathogenic, causing gas gangrene, gastrointestinal disorders and enterotoxemias in domestic ruminants [2]. Although C. perfringens can produce up to 16 different toxins, it is classified in 5 toxinotypes based on the production of 4 major toxins, as shown in Table 1 [3]. Epsilon toxin (ETX) is produced by C. perfringens type B and D strains and is responsible for the development of several important diseases in domestic animals [3]. For instance, C. perfringens type B is the etiologic agent of dysentery in newborn lambs but can also cause other diseases [4]. ETX is the primary virulence factor of C. perfringens type D and is responsible for the clinical signs and

* Corresponding authors. Tel.: þ55 31 3409 2103; fax: þ55 31 3409 2086. E-mail addresses: [email protected] (G.G. Alves), lobato. [email protected], fl[email protected] (F.C.F. Lobato). http://dx.doi.org/10.1016/j.anaerobe.2014.08.016 1075-9964/© 2014 Elsevier Ltd. All rights reserved.

lesions associated with enterotoxemias in domestic ruminants [5]. The aim of the present article is to review the existing literature on ETX from C. perfringens type B and D strains and to provide the most up-to-date information on the structural and pathogenic characteristics of this toxin. 2. Development 2.1. Toxin ETX is the primary cause of enterotoxemia in domestic ruminants and is a member of the aerolysin-like b-pore-forming toxin family, which also includes the alpha toxin (a) of Clostridium septicum. Although these toxins have striking structural similarities, they lack homology in their amino acid sequences [6]. With an LD50 of 100 ng/kg in mice, ETX is the third most potent bacterial toxin known after the botulinum and tetanus toxins [7,8]. There have been a few reported cases of human illness related to C. perfringens type D and/or ETX [9e11]. ETX is currently considered as a potential agent of bioterrorism or biological warfare [11,12] and is classified as a category B biological agent by the Centers for Disease Control and Prevention (CDC) of the United States [13].

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Table 1 Toxinotypes of Clostridium perfringens and their respective major toxins produced. Major toxins Toxinotype

Alpha (CPA)

Beta (CPB)

Epsilon (ETX)

Iota (ITX)

A B C D E

þ þ þ þ þ

 þ þ  

 þ  þ 

    þ

2.2. Genetics The etx gene, which encodes the information for the synthesis of ETX, is harbored on plasmids. In fact, the etx gene can be found on five or more different plasmids, whose sizes range from 48 kb to 110 kb. At least two of these plasmids are conjugative; i.e., they replicate autonomously and mediate the conjugation and transfer of genetic material. A single bacterial isolate can harbor multiple plasmids, each of which may carry up to three different genes encoding additional toxins or other accessory virulence factors. That these plasmids also frequently harbor the tcp locus, which mediates the transfer of the tetracycline resistance plasmid (pCW3), is consistent with the fact that the conjugative transfer of plasmids encoding the etx gene can occur between two C. perfringens isolates [14e16]. The location of the etx gene on extrachromosomal elements and the conjugative nature and genetic makeup of these plasmids enable intra-species horizontal transfer of toxin genes and the consequent acquisition or loss of virulence factors, which contribute to changes in toxigenic types observed in some C. perfringens strains [17]. 2.3. Structure The structure of ETX was definitively resolved by Cole et al. [6] using X-ray crystallography (Fig. 1). The protein is elongated [(100  20  20) Å] and has three domains consisting primarily of b-sheets. Domain I contains a large a-helix followed by a loop and an a-helix. Between domains I and II, there is another a-helix followed by a loop. Domain II is a b-sandwich consisting of two antiparallel b-sheets and a b-hairpin. Domain III is also a b-sandwich with two b-sheets, and the second of these two sheets forms the carboxyl-terminus of the toxin [6]. ETX has remarkable structural similarities with other members of the aerolysin-like pore-forming toxin family, such as the aerolysin produced by Aeromonas hydrophila, parasporin-2 produced by Bacillus thuringiensis and pore-forming lectin (LSL) produced by Laetiporus sulphureus. Although the primary structures of these proteins have less than 20% sequence similarity, the proteins have high structural similarity in terms of their shapes and b-sheet arrangements. All of the examples listed above are pore-forming toxins, with ETX and aerolysin forming heptameric pores and LSL forming hexameric pores. Furthermore, ETX, aerolysin and parasporin-2 are secreted as protoxins and activated by the proteolytic cleavage of their amino- and carboxyl-terminal sequences [12]. Despite the striking structural similarities of these toxins, ETX is far more potent than the others, with a lethal activity in mice approximately 100 times greater than that of aerolysin or the C. septicum a toxin, another member of this particular toxin family [11]. The large differences in toxicity among these toxins likely reflect their distinct amino acid compositions but are primarily due to the interaction of these proteins with distinct receptor-binding sites [6].

Fig. 1. Structure of Clostridium perfringens epsilon toxin with domains labeled. The Nterminal (N) and C-terminal (C) regions are also labeled.

The functions of the different regions of some pore-forming toxin family members, particularly aerolysin, are well characterized. Based on the similarities between the domains of ETX and other family members, as well as the findings of direct studies, the functional characteristics of certain regions of ETX have been determined.

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Domain I of ETX appears to be critical for its initial interaction with the cell. This domain shares limited but some similarity with domain II of aerolysin, which is important for interacting with sugar moieties of glycosylphosphatidylinositol (GPI) anchors. A cluster of spatially proximal aromatic amino acids (Tyr42, Tyr43, Tyr49, Tyr209 and Phe212) in the Domain I of ETX, along with the only tryptophan (important for receptor binding), may be directly involved in the binding of the protein to its cellular receptors [6]. Domain II of aerolysin and the receptor-interacting domains of C. septicum alpha toxin and C. perfringens enterotoxin, members of the aerolysin-like pore-forming toxin family, also include surface aromatic residues. Mutagenesis of these aromatic amino acids to non-aromatic greatly compromise binding to its respective receptors [6,11,18]. Alignment of the sequences of Domain II of ETX and similar domains in the a toxin of C. septicum, aerolysin and two mosquitocidal toxins (MT-X 2 and MT-X 3) reveals the amphipathic areas in these regions. Areas with these characteristics are essential for protein insertion into cell membranes and are found in other toxins in the aerolysin-like pore-forming toxin family. Based on studies including other proteins in this family, Domain II of ETX is likely to be the membrane insertion region and has been implicated in protein oligomerization and pore formation and stabilization [6,12]. Amino acid mutations within Domain II of ETX result in changes in cytotoxicity and affect the characteristics of the channels formed by the toxin. Residues Ser124 and Thr143 appear to be particularly important for these characteristics and the activity of the channels. An analysis of the sequence and hydrophobicity of this region indicated that the His119eAla149 segment forms an amphipathic bhairpin that is a constituent component of the transmembrane channel of ETX heptamers [19]. Moreover, this same segment also has one or more neutralizing epitope regions [20]. Although Domain III of ETX, which contains the carboxylterminal portion of the toxin, has also been implicated in membrane insertion, it is primarily associated with the heptamerization of the protein. In the epsilon and aerolysin protoxins, the carboxylterminal peptides appear to block the oligomerization of the toxins. When these peptides fail to be removed during protoxin activation, the disrupted interaction between the monomers subsequently blocks oligomerization [6,12]. The epsilon protoxin is able to bind to its cellular receptors, but without protoxin activation, these receptors are unable to form heptameric complexes in the cell membrane. As described above, the carboxyl-terminal peptide in Domain III must be removed to permit the formation of the heptameric complexes. Thus, this terminal peptide functions similarly to an intramolecular chaperone by aiding protein folding and preventing active ETX molecules from aggregating in solution [21]. 2.4. Activation ETX is secreted as a 32.981 kDa protoxin, which is then converted into an active toxin that is nearly 1000 times more toxic than the progenitor. The activation of the protoxin causes a small change in its molecular mass and a large change in its isoelectric point (pI), from 8.02 to 5.36 or 5.74. This activation is catalyzed by the proteases trypsin, chymotrypsin and lambda toxin (l), a thermolysinlike zinc metalloprotease synthesized by C. perfringens [3,22]. For activation to occur, both the amino-terminal and carboxylterminal peptides need to be removed from the protoxin. Proteolytic cleavage results in the removal of 10e13 amino-terminal and 22e29 carboxy-terminal residues, depending on the protease that catalyzes the cleavage. Therefore the primary sequence of the resulting cleaved protein and its toxic potency may vary also depending on the proteases [22,23]. Maximum potency occurs when the toxin is activated by the combined action of trypsin and

chymotrypsin, resulting in the loss of 13 amino-terminal and 29 carboxy-terminal residues [21,23]. Table 2 presents how proteases catalyze the cleavage of ETX, and its resulting toxic potency. A l-toxin negative C. perfringens strain was recently discovered and was found to process the epsilon protoxin intracellularly. Using an as yet unidentified protease, this strain cleaves the amino- and carboxyl-terminal portions of the protoxin and releases a toxic product upon bacterial lysis, which begins to occur when the culture reaches the stationary growth phase [8]. An activation method distinct from classical activation of clostridial toxins was discovered using the cell line mpkCCDc14, in which the epsilon protoxin is processed and completely activated by proteases present on the apical surface of the plasma membrane of these cells [24]. Although a cell line being able to activate bacterial toxins is not uncommon, ETX is the only clostridial toxin proven to present this feature so far. 2.5. Cellular receptors Only a few cell lines are sensitive to the harmful effects of ETX (Table 3). This restricted range of susceptible cell lines is most likely due to the presence of specific receptors on sensitive cells. ETX has long been known to bind to receptors present on the outer surface of the vascular endothelial cell membranes of particular organs, including the brain, kidneys and liver [30]. It was also established early on that the interaction of the toxin with these receptors depends on a lipid environment [31]. Nevertheless, the exact identity of the cellular receptor of ETX remains unknown. ETX binds to receptors restricted to certain regions with an average area of 0.35e0.45 mm2. These receptors are dependent on cholesterol and sphingomyelin and occur in specific cell membrane regions known as lipid rafts or detergent-resistant membranes (DRMs) [32]. The number of cellular receptors appears to be higher on the apical membrane than on the basolateral membrane of cells [24,33]. The restricted nature of these receptors leads to a large concentration of ETX monomers, which facilitates their oligomerization and partially explains the high toxicity of the protein [32]. As with aerolysin and C. septicum alpha toxin receptors [34,35], ETX receptors are thought to be glycosylphosphatidylinositol (GPI)anchored proteins present in DRMs. However, there are no studies substantiating this hypothesis, and the domains of ETX and aerolysin that interact with cellular receptors are very different [19]. Recently, Ivie et al. [29] demonstrated that ETX binds to the hepatitis A virus cellular receptor 1 (HAVCR1) in vitro and confirmed that these receptors contribute to the cytotoxicity of the protein. According to the authors, HAVCR1 may be the ETX receptor or may act as a co-receptor for the toxin. However, although some cell lines expressing HAVCR1 are sensitive to the toxin, there is still no definitive evidence that the toxin directly binds to these receptors [36]. Subsequent studies characterizing the interactions of ETX, HAVCR1 and the sensitive cell line MDCK (MadineDarby canine kidney) indicated that the tyrosine residues Y42, Y43, Y49 and Y209 are essential for these interactions [18]. Bokori-Brown et al. [36] further confirmed the importance of these tyrosine residues, which are present in Domain I, for ETX

Table 2 Characteristics of the activation of Clostridium perfringens epsilon toxin by different proteases. Protease

N-Terminal C-Terminal peptide peptide

Potency Reference (LD50 in mice)

Trypsin Chymotrypsin l-Protease Trypsin þ chymotrypsin

13 residues 22 residues 320 ng/kg e 29 residues e 10 residues 29 residues 110 ng/kg 13 residues 29 residues 50e65 ng/kg

[23] [12] [23] [21e23]

G.G. Alves et al. / Anaerobe 30 (2014) 102e107 Table 3 Cell lines sensitive to the effects of Clostridium perfringens epsilon toxin. Cell line

Description

Reference

GPPM MDCK G402 mpkCCDc14 HRTEC ACHN

Guinea-pig Peritoneal Macrophages MadineDarby Canine Kidney Caucasian Renal Leiomyoblastoma Mouse Renal Cortical Collecting Duct Human Renal Tubular Epithelial Cells Human Kidney Adenocarcinoma

[25] [26] [27] [24] [28] [29]

binding to MDCK cells. However, they were not found to be as critical for binding to ACHN (human kidney adenocarcinoma) cells, suggesting that the toxin recognizes different cellular receptors and interacts with them via distinct binding sites. One of these sites may occur between two of the b-sheets present in Domain III [36]. 2.6. Intestinal absorption For ETX to exert systemic effects, C. perfringens, particularly the type D strains, must first colonize the intestine of affected animals and produce the toxin. Thereafter, the toxin must act locally and be absorbed into the bloodstream [37]. Sialidases produced by the bacterium may play a key role during these stages by promoting the adhesion of the microorganism to enteric cells, thereby allowing C. perfringens type D to colonize the intestine. Sialidases also increase the sensitivity of cells to ETX through increased binding and increased oligomerization of the toxin. These changes likely involve one or both of the following mechanisms: the sialidases expose additional receptors for ETX on the cell surfaces, and/or they modify non-receptor components of the cell surface and transform them into de facto receptors for the toxin. Trypsin also plays a key role during these stages: in addition to activating ETX, trypsin activates and increases the activity of sialidases [37]. C. perfringens type D grows and produces toxins in both the small and large intestines. All intestinal segments absorb the ETX produced, but it is absorbed to a greater extent in the colon. In addition, factors such as low pH and high intestinal concentrations of glucose and sodium chloride, which are likely present during enterotoxemia, contribute to greater absorption of the toxin in ruminant intestines [38]. Although intestinal lesions caused by ETX result in changes in organ function and allow absorption of the toxin, these lesions are not frequently observed in cases of enterotoxemia caused by C. perfringens type D. Thus, the toxin may alter the permeability of the intestine through mechanisms independent of morphologic lesions [39]. Studies have shown that ETX induces fluid accumulation and increased intestinal permeability by opening the tight junctions of the mucosa, dilating intercellular spaces and causing degenerative changes in the lamina propria; all of these changes affect fluid homeostasis, possibly through a paracellular pathway [39,40]. Consequently, water, electrolytes and macromolecules are released into the intestinal lumen, which explains the occurrence of diarrhea or aqueous content in the intestines of some animals with enterotoxemia. Although ETX binds to enterocytes, the direct effects of this binding are not well defined; it is also unknown whether the toxin interacts with other components of the mucosa and/or submucosa to physiologically modulate the enterocytes [39]. Two additional mechanisms may contribute to the intestinal absorption of ETX. First, ETX causes direct damage to the endothelial cells lining the intestine and increases the permeability of the vascular and intestinal walls [39]. Second, ETX is capable of

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inhibiting gastrointestinal transit in mice, which may further contribute to the multiplication of C. perfringens type D and increase toxin production and absorption. Two hypotheses have been proposed to explain how gastrointestinal transit is inhibited: it may result from local action of the toxin in the intestine and/or action in the central nervous system resulting in neuronal damage and impaired intestinal motor function [41]. 2.7. Toxic action The current general model of ETX action is as follows. (1) The epsilon protoxin is activated primarily by the proteases trypsin, chymotrypsin and l toxin [3]. (2) The activated toxin binds to unidentified receptors, possibly HAVCR1, located in the cell membrane regions known as lipid rafts [29,42]. (3) Oligomerization also occurs within these regions [42], and large protein complexes with a molecular mass of approximately 155 kDa or 220 kDa are formed [24,33]. These complexes consist of ETX monomers that have oligomerized in a pre-pore state and have not inserted into the cell membrane [43]. (4) Toxin heptamers are inserted into the same regions of the membrane where they were assembled, forming active pores that allow the passage of ions and molecules up to ~0.5 kDa, which in turn causes cellular changes and damage [42,44]. There is some evidence that these heptamers are also internalized and cause intracellular damage [24,45,46]. In addition to the intestine, where ETX is absorbed, the other target organs of the toxin include the kidneys, lungs and brain. The toxin has also been found in the spinal cord, eyes, spleen, liver and nasal turbinates [30,47]. The role of the toxin at these sites will help shed light on the clinical signs and lesions observed as well as on the course of enterotoxemia in ruminants. In the intestine, the action of ETX can result in fluid accumulation, mucosal hemorrhaging and hemorrhagic content. The histopathological changes in the small and large intestines include mucosal congestion and erosion, with hemorrhaging affecting the mucosa, lamina propria and submucosa, and moderate polymorphonucleocyte infiltration. Ultrastructurally, the toxin causes epithelial cell detachment and cell necrosis, lamina propria hemorrhaging and polymorphonucleocyte infiltration. Opening of the tight junctions and dilation of intercellular spaces also occur, particularly in the large intestine [39,40]. Along with ETX, beta-2 toxin (b2) appears to contribute to the pathogenesis of the lesions observed in the intestine. This toxin may therefore be responsible for the necrosis and ulcer formation observed in some cases of enterotoxemia, most notably in goats [48]. Upon entering the bloodstream, ETX is distributed to all of its target organs but primarily accumulates in the kidneys. Except for the medulla and proximal tubules, the toxin can be detected throughout the kidney and accumulates primarily in the glomeruli, capillaries and collecting ducts. Within the renal system, ETX can also be detected in bladder epithelial cells [47,49]. Histologically, dilatation of Bowman's space is observed along with degenerative changes in the distal tubules and collecting ducts, including reductions in the height of the epithelial cells, dilation of the lumen and cell exfoliation. Despite the large accumulation of ETX in the kidneys, however, renal lesions from which the name “pulpy kidney disease” comes are likely to be postmortem changes and the resulting clinical signs are fairly unimportant in terms of the development of enterotoxemia. In fact, bilateral nephrectomy of mice injected with ETX reduces their survival times. It is possible that the kidneys help reduce the amount of toxin in circulation, thereby mitigating the harmful effects of the toxin when it accumulates in other organs [3,47]. In the cardiorespiratory system and pleura, ETX acts on the endothelium of blood vessels, leading to the formation of pores and

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increased vascular permeability. Consequently, fluid extravasation, hydropericardium, hydrothorax and pulmonary edema are observed [11,50]. When large amounts of ETX reach the brain, the lesions are primarily microscopic and are accompanied by severe and widespread vasogenic edema. When smaller doses of the toxin reach the organ or when the intoxicated animal exhibits partial immunity to the toxin, the lesions are frequently macroscopic, and bilaterally symmetrical focal malacia is observed. The tissue softening may spread to the basal ganglia, internal capsule, thalamus, substantia nigra, hippocampus or cerebellar peduncles, a condition that has been termed focal symmetrical encephalomalacia (FSE). Although less common, lysis and liquefaction of certain regions of white matter have also been known to occur [51]. A microscopic change characteristic and pathognomonic of ETX action in the nervous tissue is proteinaceous perivascular edema, which is characterized by the accumulation of fluid and proteins around small- and medium-sized arteries and veins. This change is likely caused by toxin-induced damage to the endothelial cells in these vessels [50]. The luminal surface of brain endothelial cells represents the likely binding site of ETX, and the vascular endothelium is where the main changes occur. The endothelial cells damaged by the toxin initially exhibit edema, loss of cytoplasmic organelles, vesicle formation, a reduction in cytoplasm and nuclear pyknosis. Due to the formation of vascular lesions, there is a loss of the bloodebrain barrier (BBB), increased vascular permeability, generalized cerebral edema and increased intracranial pressure. Intramyelinic edema and periaxonal edema also occur in the white matter of the cerebellum, accompanied by swelling of axonal terminals and dendrites in adjacent gray matter and swelling of astrocytes of the cerebellar granular layer. In addition to the endothelium, the toxin also appears to act on the synaptosomal membranes [51,52]. Vascular events in the brain appear to be involved in the development of regions with FSE. For instance, endothelial damage may cause microthrombosis and vessel occlusion via platelet aggregates; together with the adjacent edema, these changes may lead to collapse, capillary obstruction and blood stasis. In turn, blood perfusion failure and focal ischemia or hypoxia may occur. It is also possible that after disrupting the BBB, ETX exerts its cytotoxic effects by acting directly on neurons and other neural elements [51,52]. Evidence strongly supporting the hypothesis of direct ETX action was recently reported by Lonchamp et al. [53]. Specifically, the authors showed that ETX binds to various brain structures, primarily the cerebellum but also the hippocampus, thalamus, striatum, substantia nigra, olfactory bulb, colliculi and cerebral white matter. In the cerebellum, the toxin binds to oligodendrocytes and granule cells then induces the release of glutamate, an excitatory neurotransmitter. It also induces the release of dopamine by hippocampal neuronal cells that are exposed to the toxin [54]. The induction of glutamate release by granule cells is likely an indirect effect of the damage to neuronal morphology and physiology. ETX reduces the resistance of neuronal membranes, induces the opening of pores and membrane depolarization and increases intracellular Ca2þ levels, all of which culminate in the firing of the neuronal machinery and the stimulation of neurotransmitter release. Ultimately, the effects of glutamate release in the central nervous system may contribute to the neurological signs frequently observed in animals suffering from enterotoxemia [53].

considering that some human cell lines (G402, HRTEC and ACHN) are sensitive to ETX, it is almost undeniable that the contact of people with this toxin, either in natural conditions, such as diseases, or in an induced manner may pose risks to human health. In fact, ETX is currently considered as a potential bioterrorism or biological warfare agent, and is classified as a category B biological agent by the Centers for Disease Control and Prevention (CDC) of United States [11,12]. As a bioterrorism agent, ETX has potential for mass dissemination, primarily via aerosol. Inhalation of the agent may result in damage to pulmonary vascular endothelial cells, with consequent primary lung lesion characterized by increased vascular permeability and pulmonary edema. Then the absorbed toxin can reach other organs, causing renal, cardiac and central nervous system lesions. Considering an estimated LD50 of 1 mg/kg in humans, by the respiratory route, sufficient doses of ETX can theoretically rapidly weaken large civilian and military populations, or even cause death of a considerable number of people [57]. Although sera containing antibodies against ETX can neutralize it, there is no approved treatment in humans against the effects of this toxin. Moreover, the rapid onset of symptoms and the high lethality can make treatment unfeasible. Thus, the potential use of ETX as a biological weapon along with a lack of an effective and viable treatment emphasize the need to develop safe and effective vaccines for humans [12,58]. However, there are no reports of vaccine trials in humans or other primates [59]. Most of immunogens licensed for veterinary use are produced from crude or partially purified supernatant from cultures of C. perfringens. Therefore, it is unlikely that these vaccines, consisting primarily of toxoids, would be approved for human use [58]. Even if there was a vaccine for humans in a possible biological warfare or bioterrorist attack, it is likely that the effectiveness of immunization would be compromised if a large amount of ETX were inhaled. The pulmonary and local concentration of circulating immunoglobulins can be insufficient to fight the action and effects of the toxin. In this case, the development of auxiliary and prophylactic treatment is of utmost importance. Antibodies of human or animal origin, or Fab fragments via aerosol or administered intravenously could be used as prophylactic and curative treatment against ETX [60]. Another option is based on the development of therapies with drugs or toxin subunits to compete with the native protein for binding to cell receptors. 3. Conclusion As the cause of enterotoxemias in domestic ruminants, ETX represents an essential component in commercial clostridial vaccines and is thus particularly important in the field of veterinary medicine. ETX has also attracted the attention of researchers worldwide due to its potential use as a biological weapon and its inclusion on the CDC list of possible bioterrorism agents. As a result, a considerable number of studies have investigated the structural, physiological, pathogenic and immunological characteristics of ETX and greatly increased our understanding of the toxin. Conflict of interest The authors declare that they have no conflicts of interest.

2.8. ETX and human health Acknowledgments Little is known about the importance of C. perfringens ETX for human health. There are only two case reports of illness in humans related to the epsilon toxin, both from 1955 [55,56]. Furthermore,

This work was supported by Fapemig (APQ-01620-12), CAPES, CNPq and PRPq-UFMG.

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References [1] Hatheway CL. Toxigenic clostridia. Clin Microbiol Rev 1990;3(1):66e98. [2] Lobato FCF, Salvarani FM, Assis RA. Clostridiosis of small ruminants. Rev Port ^ncias Vet 2007;102:23e34. Cie [3] Uzal FA, Vidal JE, Mcclane BA, Gurjar AA. Clostridium perfringens toxins involved in mammalian veterinary diseases. Open Toxinol J 2010;3:24e42. [4] Songer JG. Clostridial enteric diseases of domestic animals. Clin Microbiol Rev 1996;9(2):216e34. [5] Garcia JP, Adams V, Beingesser J, Hughes ML, Poon R, Lyras D, et al. Epsilon toxin is essential for the virulence of Clostridium perfringens type D infection in sheeps, goats, and mice. Infect Immun 2013;81(7):2405e14. [6] Cole AR, Gibert M, Popoff M, Moss DS, Titball RW, Basak AK. Clostridium perfringens ε-toxin shows structural similarity to the pore-forming toxin aerolysin. Nat Struct Mol Biol 2004;11(8):797e8. [7] Gil DM. Bacterial toxins: a table of lethal amounts. Microbiol Rev 1982;46(1): 86e94. [8] Harkness JM, Li J, Mcclane BA. Identification of a lambda toxin-negative Clostridium perfringens strain that processes and activates epsilon prototoxin intracellularly. Anaerobe 2012;18:546e52. [9] Morinaga G, Nakamura T, Yoshizawa J, Nishida S. Isolation of Clostridium perfringens type D from a case of gas gangrene. J Bacteriol 1965;90(3):826. [10] Miller C, Florman S, Kim-Schluger L, Lento P, Garza J, Wu J, et al. Fulminant and fatal gas gangrene of the stomach in a healthy live liver donor. Liver Transpl 2004;10(10):1315e9. [11] Popoff MR. Epsilon toxin: a fascinating pore-forming toxin. FEBS J 2011;278(23):4602e15. [12] Bokori-Brown M, Savva CG, Costa SPF, Naylor CE, Basak AK, Titball RW. Molecular basis of toxicity of Clostridium perfringens epsilon toxin. FEBS J 2011: 1e13. [13] Centers for Disease Control. Biological and chemical terrorism: strategic plan for preparedness and response. Recommendations of the CDC Strategic Planning Workgroup. MMWR Recomm Rep 2000;9:1e14. [14] Hughes ML, Poon R, Adams V, Sayeed S, Saputo J, Uzal FA, et al. Epsilon-toxin plasmids of Clostridium perfringens type D are conjugative. J Bacteriol 2007;189(21):7531e8. [15] Sayeed S, Li J, Mcclane BA. Virulence plasmid diversity in Clostridium perfringens type D isolates. Infect Immun 2007;75(5):2391e8. [16] Miyamoto K, Li J, Sayeed S, Akimoto S, Mcclane BA. Sequencing and diversity analyses reveal extensive similarities between some epsilon-toxin-encoding plasmids and the pCPF5603 Clostridium perfringens enterotoxin plasmid. J Bacteriol 2008;190(21):7178e88. [17] Petit L, Gibert M, Popoff MR. Clostridium perfringens: toxinotype and genotype. Trends Microbiol 1999;7(3):104e10. [18] Ivie SE, Mcclain MS. Identification of amino acids important for binding of Clostridium perfringens epsilon toxin to host cells and to HAVCR1. Biochemistry 2012;51(38):7588e95. [19] Knapp O, Maier E, Benz R, Geny B, Popoff MR. Identification of the channelforming domain of Clostridium perfringens epsilon-toxin (ETX). Biochim Biophys Acta 2009;1788:2584e93. [20] Mcclain MS, Cover TL. Functional analysis of neutralizing antibodies against Clostridium perfringens epsilon-toxin. Infect Immun 2007;75(4):1785e93. [21] Miyata S, Matsushita O, Minami J, Katayama S, Shimamoto S, Okabe A. Cleavage of a C-terminal peptide is essential for heptamerization of Clostridium perfringens ε-toxin in the synaptosomal membrane. J Biol Chem 2001;276(17):13778e83. [22] Worthington RW, Mülders MSG. Physical changes in the epsilon prototoxin molecule of Clostridium perfringens during enzymatic activation. Infect Immun 1977;18(2):549e51. [23] Minami J, Katayama S, Matsushita O, Matsushita C, Okabe A. Lambda-toxin of Clostridium perfringens activates the precursor of epsilon-toxin by releasing its N- and C-terminal peptides. Microbiol Immunol 1997;41(7):527e35. [24] Chassin C, Bens M, Barry J, Courjaret R, Bossu JL, Cluzeaud F, et al. Poreforming epsilon toxin causes membrane permeabilization and rapid ATP depletion-mediated cell death in renal collecting duct cells. Am J Physiol Renal Physiol 2007;293(3):F927e37. [25] Buxton D. In-vitro effects of Clostridium welchii type-D epsilon toxin on guinea-pig, mouse, rabbit and sheep cells. J Med Microbiol 1978a;11: 299e302. [26] Payne DW, Willianson ED, Havard H, Modi N, Brown J. Evaluation of a new cytotoxicity assay for Clostridium perfringens type D epsilon toxin. FEMS Microbiol Lett 1994;116(2):161e7. [27] Shortt SJ, Titball RW, Lindsay CD. An assessment of the in vitro toxicology of Clostridium perfringens type D ε-toxin in human and animal cells. Hum Exp Toxicol 2000;19:108e16. [28] Fernandez-Miyakawa ME, Zabal O, Silberstein C. Clostridium perfringens epsilon toxin is cytotoxic for human renal tubular epithelial cells. Hum Exp Toxicol 2010;30(4):275e82. [29] Ivie SE, Fennessey CM, Sheng J, Rubin DH, Mcclain MS. Gene-trap mutagenesis identifies mammalian genes contributing to intoxication by Clostridium perfringens ε-toxin. PLoS One 2011;6(3):e17787. [30] Buxton D. The use of an immunoperoxidase technique to investigate by light and electron microscopy the sites of binding of Clostridium welchii type-D epsilon toxin in mice. J Med Microbiol 1978b;11:289e92.

107

[31] Nagahama M, Sakurai J. High-affinity binding of Clostridium perfringens epsilon-toxin to rat brain. Infect Immun 1992;60(3):1237e40. [32] Türkcan S, Masson JB, Casanova D, Mialon G, Gacoin T, Boilot JP, et al. Observing the confinement potential of bacterial pore-forming toxin receptors inside rafts with nonblinking Eu(3þ)-doped oxide nanoparticles. Biophys J 2012;102(10):2299e308. [33] Petit L, Gibert M, Gillet D, Laurent-Winter C, Boquet P, Popoff MR. Clostridium perfringens epsilon-toxin acts on MDCK cells by forming a large membrane complex. J Bacteriol 1997;179(20):6480e7. [34] Nelson KL, Raja SM, Buckley JT. The glycosylphosphatidylinositol-anchored surface glycoprotein Thy-1 is a receptor for the channel-forming toxin aerolysin. J Biol Chem 1997;272(18):12170e4. [35] Gordon VM, Nelson KL, Buckley JT, Stevens VL, Tweten RK, Elwood PC, et al. Clostridium septicum alpha toxin uses glycosylphosphatidylinositol-anchored protein receptors. J Biol Chem 1999;274(38):27274e80. [36] Bokori-Brown M, Kokkinidou MC, Savva CG, Costa SPF, Naylor CE, Cole AR, et al. Clostridium perfringens epsilon toxin H149A mutant as a platform for receptor binding studies. Protein Sci 2013;22:650e9. [37] Li J, Sayeed S, Robertson S, Chen J, Mcclane BA. Sialidases affect the host cell adherence and epsilon toxin-induced cytotoxicity of Clostridium perfringens type D strain CN3718. PLoS Pathog 2011;7(12):e1002429. [38] Losada-Eaton DM, Uzal FA, Fernandez-Miyakawa ME. Clostridium perfringens epsilon toxin is absorbed from different intestinal segments of mice. Toxicon 2008;51:1207e13. [39] Goldstein J, Morris WE, Loidl CF, Tironi-Farinatti C, Mcclane BA, Uzal FA, et al. Clostridium perfringens epsilon toxin increases the small intestinal permeability in mice and rats. PLoS One 2009;4(9):e7065. [40] Morris WE, Dunleavy MV, Diodati J, Berra G, Fernandez-Miyakawa ME. Effects of Clostridium perfringens alpha and epsilon toxins in the bovine gut. Anaerobe 2012;18(1):143e7. [41] Losada-Eaton DM, Fernandez-Miyakawa ME. Clostridium perfringens epsilon toxin inhibits the gastrointestinal transit in mice. Res Vet Sci 2010;89: 404e8. [42] Miyata S, Minami J, Tamai E, Matsushita O, Shimamoto S, Okabe A. Clostridium perfringens ε-toxin forms a heptameric pore within the detergent-insoluble microdomains of Madin-Darby canine kidney cells and rat synaptosomes. J Biol Chem 2002;277(42):39463e8. [43] Robertson SL, Li J, Uzal FA, Mcclane BA. Evidence for a prepore stage in the action of Clostridium perfringens epsilon toxin. PLoS One 2011;6(7):e22053. [44] Nestorovich EM, Karginov VA, Bezrukov SM. Polymer partitioning and ion selectivity suggest asymmetrical shape for the membrane pore formed by epsilon toxin. Biophys J 2010;99:782e9. [45] Nagahama M, Itohayashi Y, Hara H, Higashihara M, Fukatani Y, Takagishi T, et al. Cellular vacuolation induced by Clostridium perfringens epsilon-toxin. FEBS J 2011;278:3395e407. [46] Fennessey CM, Ivie SE, Mcclain MS. Coenzyme depletion by members of the aerolysin family of pore-forming toxins leads to diminished ATP levels and cell death. Mol Biosyst 2012;8:2097e105. [47] Tamai E, Ishida T, Miyata S, Matsushita O, Suda H, Kobayashi S, et al. Accumulation of Clostridium perfringens epsilon-toxin in the mouse kidney and its possible biological significance. Infect Immun 2003;71(9):5371e5. [48] Uzal FA, Fisher DJ, Saputo J, Sayeed S, Mcclane BA, Songer G, et al. Ulcerative enterocolitis in two goats associated with enterotoxin- and beta2 toxin-positive Clostridium perfringens type D. J Vet Diagn Invest 2008;20: 668e72.
valo J, Martín-Satue
M, Blasi J. Characterization of the high affinity [49] Dorca-Are binding of epsilon toxin from Clostridium perfringens to the renal system. Vet Microbiol 2012;157:179e89. [50] Uzal FA, Songer JG. Diagnosis of Clostridium perfringens intestinal infections in sheep and goats. J Vet Diagn Invest 2008;20:253e65. [51] Finnie JW. Neurological disorders produced by Clostridium perfringens type D epsilon toxin. Anaerobe 2004;10:145e50. [52] Wioland L, Dupont J, Bossu J, Popoff MR, Poulain B. Attack of the nervous system by Clostridium perfringens epsilon toxin: from disease to mode of action on neural cells. Toxicon 2013;75:122e35. [53] Lonchamp E, Dupont J, Wioland L, Courjaret R, Mbebi-Liegeois C, Jover E, et al. Clostridium perfringens epsilon toxin targets granule cells in the mouse cerebellum and stimulates glutamate release. PLoS One 2010;5(9):e13046. [54] Miyamoto K, Sumitani K, Nakamura T, Yamagami S, Miyata S, Itano T, et al. Clostridium perfringens epsilon toxin causes excessive release of glutamate in the mouse hippocampus. FEMS Microbiol Lett 2000;189(1):109e13. [55] Gleeson-White MH, Bullen JJ. Clostridium welchii epsilon toxin in the intestinal contents of man. Lancet 1955;268:384e5. [56] Kohn J, Warrack GH. Recovery of Clostridium welchii type D from man. Lancet 1955;189:385. [57] Greenfield RA, Brown BR, Hutchins JB, Iandolo JJ, Jackson R, Slater LN, et al. Microbiological, biological, and chemical weapons of warfare and terrorism. Am J Med Sci 2002;323(6):326e40. [58] Titball RW. Clostridium perfringens vaccines. Vaccine 2009;27(4):D44e7. [59] Mantis NJ. Vaccines against the category B toxins: Staphylococcal enterotoxin B, epsilon toxin and ricin. Adv Drug Deliv Rev 2005;57:1424e39. [60] Paddle BM. Therapy and prophylaxis of inhaled biological toxins. J Appl Toxicol 2003;23:139e70.