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Anaerobe 30 (2014) 102e107

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Clostridium perfringens epsilon toxin: The third most potent bacterialtoxin known

Guilherme Guerra Alves a, *, Ricardo Andrez Machado de �Avila b,Carlos Delfin Ch�avez-Ol�ortegui c, Francisco Carlos Faria Lobato a, *

a Veterinary School, Universidade Federal de Minas Gerais (UFMG), Antonio Carlos Avenue, 6627, Belo Horizonte, MG CEP 31.270-901, Brazilb Universidade do Extremo Sul Catarinense (UNESC), Universit�aria Avenue, 1105, Criciúma, SC CEP 88.806-000, Brazilc Instituto de Ciencias Biol�ogicas, Universidade Federal de Minas Gerais (UFMG), Antonio Carlos Avenue, 6627, Belo Horizonte, MG CEP 31.270-901, Brazil

a r t i c l e i n f o

Article history:Received 12 June 2014Received in revised form18 August 2014Accepted 19 August 2014Available online 16 September 2014

Keywords:Clostridium perfringensEnterotoxemiaEpsilon toxinPore-forming

* Corresponding authors. Tel.: þ55 31 3409 2103; fE-mail addresses: guilhermeguerra.vet@gmail.c

francisco@yahoo.com.br, flobato@vet.ufmg.br (F.C.F. L

http://dx.doi.org/10.1016/j.anaerobe.2014.08.0161075-9964/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

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. ETXbelongs to the aerolysin-like pore-forming toxin family. Although ETX has striking similarities to othertoxins in this family, ETX is often more potent, with an LD50 of 100 ng/kg in mice. Due to this highpotency, ETX is considered as a potential bioterrorism agent and has been classified as a category Bbiological agent by the Centers for Disease Control and Prevention (CDC) of the United States. Theprotoxin 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 otherorgans, including the kidneys, lungs and brain. The importance of this toxin for veterinary medicine andits possible use as a biological weapon have drawn the attention of researchers and have led to a largenumber of studies investigating ETX. The aim of the present work is to review the existing knowledge onETX from C. perfringens type B and D.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Clostridium perfringens, which was previously named Bacilluscapsulatus aerogenes then Clostridium welchii, is a Gram-positivenon-motile anaerobic bacillus that is capable of transforming intoa more resistant form called a spore [1]. Under certain conditions,this bacterium can become pathogenic, causing gas gangrene,gastrointestinal disorders and enterotoxemias in domestic rumi-nants [2]. Although C. perfringens can produce up to 16 differenttoxins, it is classified in 5 toxinotypes based on the production of 4major toxins, as shown in Table 1 [3].

Epsilon toxin (ETX) is produced by C. perfringens type B and Dstrains and is responsible for the development of several importantdiseases in domestic animals [3]. For instance, C. perfringens type Bis the etiologic agent of dysentery in newborn lambs but can alsocause other diseases [4]. ETX is the primary virulence factor ofC. perfringens type D and is responsible for the clinical signs and

ax: þ55 31 3409 2086.om (G.G. Alves), lobato.obato).

lesions associated with enterotoxemias in domestic ruminants [5].The aim of the present article is to review the existing literature onETX from C. perfringens type B and D strains and to provide themostup-to-date information on the structural and pathogenic charac-teristics of this toxin.

2. Development

2.1. Toxin

ETX is the primary cause of enterotoxemia in domestic rumi-nants and is a member of the aerolysin-like b-pore-forming toxinfamily, which also includes the alpha toxin (a) of Clostridium sep-ticum. 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 potentbacterial toxin known after the botulinum and tetanus toxins [7,8].There have been a few reported cases of human illness related toC. perfringens type D and/or ETX [9e11]. ETX is currently consideredas a potential agent of bioterrorism or biological warfare [11,12] andis classified as a category B biological agent by the Centers forDisease Control and Prevention (CDC) of the United States [13].

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

Major toxins

Toxinotype Alpha (CPA) Beta (CPB) Epsilon (ETX) Iota (ITX)

A þ � � �B þ þ þ �C þ þ � �D þ � þ �E þ � � þ

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

G.G. Alves et al. / Anaerobe 30 (2014) 102e107 103

2.2. Genetics

The etx gene, which encodes the information for the synthesis ofETX, is harbored on plasmids. In fact, the etx gene can be found onfive or more different plasmids, whose sizes range from 48 kb to110 kb. At least two of these plasmids are conjugative; i.e., theyreplicate autonomously and mediate the conjugation and transferof genetic material. A single bacterial isolate can harbor multipleplasmids, each of which may carry up to three different genesencoding additional toxins or other accessory virulence factors.That these plasmids also frequently harbor the tcp locus, whichmediates the transfer of the tetracycline resistance plasmid(pCW3), is consistent with the fact that the conjugative transfer ofplasmids encoding the etx gene can occur between twoC. perfringens isolates [14e16].

The location of the etx gene on extrachromosomal elements andthe conjugative nature and genetic makeup of these plasmidsenable intra-species horizontal transfer of toxin genes and theconsequent acquisition or loss of virulence factors, whichcontribute to changes in toxigenic types observed in someC. 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 ofb-sheets. Domain I contains a large a-helix followed by a loop andan a-helix. Between domains I and II, there is another a-helix fol-lowed by a loop. Domain II is a b-sandwich consisting of twoantiparallel b-sheets and a b-hairpin. Domain III is also a b-sand-wich with two b-sheets, and the second of these two sheets formsthe carboxyl-terminus of the toxin [6].

ETX has remarkable structural similarities with other membersof the aerolysin-like pore-forming toxin family, such as the aero-lysin produced by Aeromonas hydrophila, parasporin-2 produced byBacillus thuringiensis and pore-forming lectin (LSL) produced byLaetiporus sulphureus. Although the primary structures of theseproteins have less than 20% sequence similarity, the proteins havehigh structural similarity in terms of their shapes and b-sheet ar-rangements. All of the examples listed above are pore-formingtoxins, with ETX and aerolysin forming heptameric pores and LSLforming hexameric pores. Furthermore, ETX, aerolysin andparasporin-2 are secreted as protoxins and activated by the pro-teolytic cleavage of their amino- and carboxyl-terminal sequences[12].

Despite the striking structural similarities of these toxins, ETX isfar more potent than the others, with a lethal activity in miceapproximately 100 times greater than that of aerolysin or theC. septicum a toxin, another member of this particular toxin family[11]. The large differences in toxicity among these toxins likelyreflect their distinct amino acid compositions but are primarily dueto the interaction of these proteins with distinct receptor-bindingsites [6].

The functions of the different regions of some pore-formingtoxin family members, particularly aerolysin, are well character-ized. Based on the similarities between the domains of ETX andother family members, as well as the findings of direct studies, thefunctional characteristics of certain regions of ETX have beendetermined.

Table 2Characteristics of the activation of Clostridium perfringens epsilon toxin by differentproteases.

Protease N-Terminalpeptide

C-Terminalpeptide

Potency(LD50 in mice)

Reference

Trypsin 13 residues 22 residues 320 ng/kg [23]Chymotrypsin e 29 residues e [12]l-Protease 10 residues 29 residues 110 ng/kg [23]Trypsin þ chymotrypsin 13 residues 29 residues 50e65 ng/kg [21e23]

G.G. Alves et al. / Anaerobe 30 (2014) 102e107104

Domain I of ETX appears to be critical for its initial interactionwith the cell. This domain shares limited but some similarity withdomain II of aerolysin, which is important for interacting withsugar moieties of glycosylphosphatidylinositol (GPI) anchors. Acluster of spatially proximal aromatic amino acids (Tyr42, Tyr43,Tyr49, Tyr209 and Phe212) in the Domain I of ETX, alongwith the onlytryptophan (important for receptor binding), may be directlyinvolved in the binding of the protein to its cellular receptors [6].Domain II of aerolysin and the receptor-interacting domains ofC. septicum alpha toxin and C. perfringens enterotoxin, members ofthe aerolysin-like pore-forming toxin family, also include surfacearomatic residues. Mutagenesis of these aromatic amino acids tonon-aromatic greatly compromise binding to its respective re-ceptors [6,11,18].

Alignment of the sequences of Domain II of ETX and similardomains in the a toxin of C. septicum, aerolysin and two mosqui-tocidal toxins (MT-X 2 and MT-X 3) reveals the amphipathic areasin these regions. Areas with these characteristics are essential forprotein insertion into cell membranes and are found in other toxinsin the aerolysin-like pore-forming toxin family. Based on studiesincluding other proteins in this family, Domain II of ETX is likely tobe the membrane insertion region and has been implicated inprotein oligomerization and pore formation and stabilization [6,12].Amino acid mutations within Domain II of ETX result in changes incytotoxicity and affect the characteristics of the channels formed bythe toxin. Residues Ser124 and Thr143 appear to be particularlyimportant for these characteristics and the activity of the channels.An analysis of the sequence and hydrophobicity of this regionindicated that the His119eAla149 segment forms an amphipathic b-hairpin that is a constituent component of the transmembranechannel of ETX heptamers [19]. Moreover, this same segment alsohas one or more neutralizing epitope regions [20].

Although Domain III of ETX, which contains the carboxyl-terminal portion of the toxin, has also been implicated in mem-brane insertion, it is primarily associated with the heptamerizationof the protein. In the epsilon and aerolysin protoxins, the carboxyl-terminal 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 subsequentlyblocks oligomerization [6,12]. The epsilon protoxin is able to bind toits cellular receptors, but without protoxin activation, these re-ceptors are unable to form heptameric complexes in the cellmembrane. As described above, the carboxyl-terminal peptide inDomain III must be removed to permit the formation of the hep-tameric complexes. Thus, this terminal peptide functions similarlyto an intramolecular chaperone by aiding protein folding and pre-venting active ETX molecules from aggregating in solution [21].

2.4. Activation

ETX is secreted as a 32.981 kDa protoxin, which is then con-verted into an active toxin that is nearly 1000 times more toxic thanthe progenitor. The activation of the protoxin causes a small changein 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 pro-teases trypsin, chymotrypsin and lambda toxin (l), a thermolysin-like zinc metalloprotease synthesized by C. perfringens [3,22].

For activation to occur, both the amino-terminal and carboxyl-terminal peptides need to be removed from the protoxin. Proteo-lytic cleavage results in the removal of 10e13 amino-terminal and22e29 carboxy-terminal residues, depending on the protease thatcatalyzes the cleavage. Therefore the primary sequence of theresulting cleaved protein and its toxic potency may vary alsodepending on the proteases [22,23]. Maximum potency occurswhen the toxin is activated by the combined action of trypsin and

chymotrypsin, resulting in the loss of 13 amino-terminal and 29carboxy-terminal residues [21,23]. Table 2 presents how proteasescatalyze the cleavage of ETX, and its resulting toxic potency.

A l-toxin negative C. perfringens strain was recently discoveredand was found to process the epsilon protoxin intracellularly. Usingan as yet unidentified protease, this strain cleaves the amino- andcarboxyl-terminal portions of the protoxin and releases a toxicproduct upon bacterial lysis, which begins to occur when the cul-ture reaches the stationary growth phase [8]. An activation methoddistinct from classical activation of clostridial toxins was discoveredusing the cell line mpkCCDc14, in which the epsilon protoxin isprocessed and completely activated by proteases present on theapical surface of the plasmamembrane of these cells [24]. Althougha 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 likelydue to the presence of specific receptors on sensitive cells.

ETX has long been known to bind to receptors present on theouter surface of the vascular endothelial cell membranes ofparticular organs, including the brain, kidneys and liver [30]. It wasalso established early on that the interaction of the toxinwith thesereceptors depends on a lipid environment [31]. Nevertheless, theexact identity of the cellular receptor of ETX remains unknown.

ETX binds to receptors restricted to certain regions with anaverage area of 0.35e0.45 mm2. These receptors are dependent oncholesterol and sphingomyelin and occur in specific cell membraneregions known as lipid rafts or detergent-resistant membranes(DRMs) [32]. The number of cellular receptors appears to be higheron the apical membrane than on the basolateral membrane of cells[24,33]. The restricted nature of these receptors leads to a largeconcentration of ETX monomers, which facilitates their oligomer-ization 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 studiessubstantiating this hypothesis, and the domains of ETX and aero-lysin that interact with cellular receptors are very different [19].

Recently, Ivie et al. [29] demonstrated that ETX binds to thehepatitis A virus cellular receptor 1 (HAVCR1) in vitro andconfirmed that these receptors contribute to the cytotoxicity of theprotein. According to the authors, HAVCR1may be the ETX receptoror may act as a co-receptor for the toxin. However, although somecell lines expressing HAVCR1 are sensitive to the toxin, there is stillno definitive evidence that the toxin directly binds to these re-ceptors [36]. Subsequent studies characterizing the interactions ofETX, HAVCR1 and the sensitive cell line MDCK (MadineDarbycanine kidney) indicated that the tyrosine residues Y42, Y43, Y49 andY209 are essential for these interactions [18].

Bokori-Brown et al. [36] further confirmed the importance ofthese tyrosine residues, which are present in Domain I, for ETX

Table 3Cell lines sensitive to the effects of Clostridium perfringens epsilon toxin.

Cell line Description Reference

GPPM Guinea-pig Peritoneal Macrophages [25]MDCK MadineDarby Canine Kidney [26]G402 Caucasian Renal Leiomyoblastoma [27]mpkCCDc14 Mouse Renal Cortical Collecting Duct [24]HRTEC Human Renal Tubular Epithelial Cells [28]ACHN Human Kidney Adenocarcinoma [29]

G.G. Alves et al. / Anaerobe 30 (2014) 102e107 105

binding to MDCK cells. However, they were not found to be ascritical for binding to ACHN (human kidney adenocarcinoma) cells,suggesting that the toxin recognizes different cellular receptors andinteracts with them via distinct binding sites. One of these sitesmay 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 thetype D strains, must first colonize the intestine of affected animalsand produce the toxin. Thereafter, the toxin must act locally and beabsorbed into the bloodstream [37].

Sialidases produced by the bacteriummay play a key role duringthese stages by promoting the adhesion of the microorganism toenteric cells, thereby allowing C. perfringens type D to colonize theintestine. Sialidases also increase the sensitivity of cells to ETXthrough increased binding and increased oligomerization of thetoxin. These changes likely involve one or both of the followingmechanisms: the sialidases expose additional receptors for ETX onthe cell surfaces, and/or they modify non-receptor components ofthe cell surface and transform them into de facto receptors for thetoxin. Trypsin also plays a key role during these stages: in additionto activating ETX, trypsin activates and increases the activity ofsialidases [37].

C. perfringens type D grows and produces toxins in both thesmall and large intestines. All intestinal segments absorb the ETXproduced, but it is absorbed to a greater extent in the colon. Inaddition, factors such as low pH and high intestinal concentrationsof glucose and sodium chloride, which are likely present duringenterotoxemia, contribute to greater absorption of the toxin inruminant intestines [38].

Although intestinal lesions caused by ETX result in changes inorgan function and allow absorption of the toxin, these lesions arenot frequently observed in cases of enterotoxemia caused byC. perfringens type D. Thus, the toxin may alter the permeability ofthe intestine through mechanisms independent of morphologiclesions [39].

Studies have shown that ETX induces fluid accumulation andincreased intestinal permeability by opening the tight junctions ofthe mucosa, dilating intercellular spaces and causing degenerativechanges in the lamina propria; all of these changes affect fluidhomeostasis, possibly through a paracellular pathway [39,40].Consequently, water, electrolytes and macromolecules are releasedinto the intestinal lumen, which explains the occurrence of diar-rhea or aqueous content in the intestines of some animals withenterotoxemia. Although ETX binds to enterocytes, the direct ef-fects of this binding are not well defined; it is also unknownwhether the toxin interacts with other components of the mucosaand/or submucosa to physiologically modulate the enterocytes[39].

Two additional mechanisms may contribute to the intestinalabsorption of ETX. First, ETX causes direct damage to the endo-thelial cells lining the intestine and increases the permeability ofthe vascular and intestinal walls [39]. Second, ETX is capable of

inhibiting gastrointestinal transit in mice, which may furthercontribute to the multiplication of C. perfringens type D and in-crease toxin production and absorption. Two hypotheses have beenproposed to explain howgastrointestinal transit is inhibited: it mayresult from local action of the toxin in the intestine and/or action inthe central nervous system resulting in neuronal damage andimpaired intestinal motor function [41].

2.7. Toxic action

The current general model of ETX action is as follows. (1) Theepsilon protoxin is activated primarily by the proteases trypsin,chymotrypsin and l toxin [3]. (2) The activated toxin binds to un-identified receptors, possibly HAVCR1, located in the cell mem-brane regions known as lipid rafts [29,42]. (3) Oligomerization alsooccurs within these regions [42], and large protein complexes witha molecular mass of approximately 155 kDa or 220 kDa are formed[24,33]. These complexes consist of ETX monomers that have oli-gomerized in a pre-pore state and have not inserted into the cellmembrane [43]. (4) Toxin heptamers are inserted into the sameregions of the membrane where they were assembled, formingactive 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 alsointernalized and cause intracellular damage [24,45,46].

In addition to the intestine, where ETX is absorbed, the othertarget organs of the toxin include the kidneys, lungs and brain. Thetoxin has also been found in the spinal cord, eyes, spleen, liver andnasal turbinates [30,47]. The role of the toxin at these sites will helpshed light on the clinical signs and lesions observed as well as onthe course of enterotoxemia in ruminants.

In the intestine, the action of ETX can result in fluid accumula-tion, mucosal hemorrhaging and hemorrhagic content. The histo-pathological changes in the small and large intestines includemucosal congestion and erosion, with hemorrhaging affecting themucosa, lamina propria and submucosa, and moderate poly-morphonucleocyte infiltration. Ultrastructurally, the toxin causesepithelial cell detachment and cell necrosis, lamina propria hem-orrhaging and polymorphonucleocyte infiltration. Opening of thetight junctions and dilation of intercellular spaces also occur,particularly in the large intestine [39,40]. Along with ETX, beta-2toxin (b2) appears to contribute to the pathogenesis of the le-sions observed in the intestine. This toxin may therefore beresponsible for the necrosis and ulcer formation observed in somecases of enterotoxemia, most notably in goats [48].

Upon entering the bloodstream, ETX is distributed to all of itstarget organs but primarily accumulates in the kidneys. Except forthe medulla and proximal tubules, the toxin can be detectedthroughout the kidney and accumulates primarily in the glomeruli,capillaries and collecting ducts. Within the renal system, ETX canalso be detected in bladder epithelial cells [47,49]. Histologically,dilatation of Bowman's space is observed along with degenerativechanges in the distal tubules and collecting ducts, including re-ductions in the height of the epithelial cells, dilation of the lumenand cell exfoliation. Despite the large accumulation of ETX in thekidneys, however, renal lesions fromwhich the name “pulpy kidneydisease” comes are likely to be postmortem changes and theresulting clinical signs are fairly unimportant in terms of thedevelopment of enterotoxemia. In fact, bilateral nephrectomy ofmice injected with ETX reduces their survival times. It is possiblethat the kidneys help reduce the amount of toxin in circulation,thereby mitigating the harmful effects of the toxin when it accu-mulates in other organs [3,47].

In the cardiorespiratory system and pleura, ETX acts on theendothelium of blood vessels, leading to the formation of pores and

G.G. Alves et al. / Anaerobe 30 (2014) 102e107106

increased vascular permeability. Consequently, fluid extravasation,hydropericardium, hydrothorax and pulmonary edema areobserved [11,50].

When large amounts of ETX reach the brain, the lesions areprimarily microscopic and are accompanied by severe and wide-spread vasogenic edema.When smaller doses of the toxin reach theorgan or when the intoxicated animal exhibits partial immunity tothe toxin, the lesions are frequently macroscopic, and bilaterallysymmetrical focal malacia is observed. The tissue softening mayspread to the basal ganglia, internal capsule, thalamus, substantianigra, hippocampus or cerebellar peduncles, a condition that hasbeen termed focal symmetrical encephalomalacia (FSE). Althoughless common, lysis and liquefaction of certain regions of whitematter have also been known to occur [51]. A microscopic changecharacteristic and pathognomonic of ETX action in the nervoustissue is proteinaceous perivascular edema, which is characterizedby the accumulation of fluid and proteins around small- andmedium-sized arteries and veins. This change is likely caused bytoxin-induced damage to the endothelial cells in these vessels [50].

The luminal surface of brain endothelial cells represents thelikely binding site of ETX, and the vascular endothelium is wherethe main changes occur. The endothelial cells damaged by the toxininitially exhibit edema, loss of cytoplasmic organelles, vesicle for-mation, a reduction in cytoplasm and nuclear pyknosis. Due to theformation of vascular lesions, there is a loss of the bloodebrainbarrier (BBB), increased vascular permeability, generalized cerebraledema and increased intracranial pressure. Intramyelinic edemaand periaxonal edema also occur in the white matter of the cere-bellum, accompanied by swelling of axonal terminals and dendritesin adjacent gray matter and swelling of astrocytes of the cerebellargranular layer. In addition to the endothelium, the toxin also ap-pears to act on the synaptosomal membranes [51,52].

Vascular events in the brain appear to be involved in thedevelopment of regions with FSE. For instance, endothelial damagemay cause microthrombosis and vessel occlusion via platelet ag-gregates; together with the adjacent edema, these changes maylead to collapse, capillary obstruction and blood stasis. In turn,blood perfusion failure and focal ischemia or hypoxia may occur. Itis also possible that after disrupting the BBB, ETX exerts its cyto-toxic effects by acting directly on neurons and other neural ele-ments [51,52].

Evidence strongly supporting the hypothesis of direct ETX ac-tion was recently reported by Lonchamp et al. [53]. Specifically, theauthors showed that ETX binds to various brain structures, pri-marily the cerebellum but also the hippocampus, thalamus, stria-tum, substantia nigra, olfactory bulb, colliculi and cerebral whitematter. In the cerebellum, the toxin binds to oligodendrocytes andgranule cells then induces the release of glutamate, an excitatoryneurotransmitter. It also induces the release of dopamine by hip-pocampal neuronal cells that are exposed to the toxin [54].

The induction of glutamate release by granule cells is likely anindirect effect of the damage to neuronal morphology and physi-ology. ETX reduces the resistance of neuronal membranes, inducesthe opening of pores and membrane depolarization and increasesintracellular Ca2þ levels, all of which culminate in the firing of theneuronal machinery and the stimulation of neurotransmitterrelease. Ultimately, the effects of glutamate release in the centralnervous systemmay contribute to the neurological signs frequentlyobserved in animals suffering from enterotoxemia [53].

2.8. ETX and human health

Little is known about the importance of C. perfringens ETX forhuman health. There are only two case reports of illness in humansrelated to the epsilon toxin, both from 1955 [55,56]. Furthermore,

considering that some human cell lines (G402, HRTEC and ACHN)are sensitive to ETX, it is almost undeniable that the contact ofpeople with this toxin, either in natural conditions, such as dis-eases, or in an induced manner may pose risks to human health. Infact, ETX is currently considered as a potential bioterrorism orbiological warfare agent, and is classified as a category B biologicalagent by the Centers for Disease Control and Prevention (CDC) ofUnited States [11,12].

As a bioterrorism agent, ETX has potential for mass dissemina-tion, primarily via aerosol. Inhalation of the agent may result indamage to pulmonary vascular endothelial cells, with consequentprimary lung lesion characterized by increased vascular perme-ability and pulmonary edema. Then the absorbed toxin can reachother organs, causing renal, cardiac and central nervous systemlesions. Considering an estimated LD50 of 1 mg/kg in humans, bythe respiratory route, sufficient doses of ETX can theoreticallyrapidly weaken large civilian and military populations, or evencause death of a considerable number of people [57].

Although sera containing antibodies against ETX can neutralizeit, there is no approved treatment in humans against the effects ofthis toxin. Moreover, the rapid onset of symptoms and the highlethality can make treatment unfeasible. Thus, the potential use ofETX as a biological weapon along with a lack of an effective andviable treatment emphasize the need to develop safe and effectivevaccines for humans [12,58]. However, there are no reports ofvaccine trials in humans or other primates [59]. Most of immuno-gens licensed for veterinary use are produced from crude orpartially purified supernatant from cultures of C. perfringens.Therefore, it is unlikely that these vaccines, consisting primarily oftoxoids, would be approved for human use [58].

Even if there was a vaccine for humans in a possible biologicalwarfare or bioterrorist attack, it is likely that the effectiveness ofimmunization would be compromised if a large amount of ETXwere inhaled. The pulmonary and local concentration of circulatingimmunoglobulins can be insufficient to fight the action and effectsof the toxin. In this case, the development of auxiliary and pro-phylactic treatment is of utmost importance. Antibodies of humanor animal origin, or Fab fragments via aerosol or administeredintravenously could be used as prophylactic and curative treatmentagainst ETX [60]. Another option is based on the development oftherapies with drugs or toxin subunits to compete with the nativeprotein for binding to cell receptors.

3. Conclusion

As the cause of enterotoxemias in domestic ruminants, ETXrepresents an essential component in commercial clostridial vac-cines and is thus particularly important in the field of veterinarymedicine. ETX has also attracted the attention of researchersworldwide due to its potential use as a biological weapon and itsinclusion 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 ETXand greatly increased our understanding of the toxin.

Conflict of interest

The authors declare that they have no conflicts of interest.

Acknowledgments

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

G.G. Alves et al. / Anaerobe 30 (2014) 102e107 107

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