Clostridium perfringens enterotoxin

Clostridium perfringens enterotoxin

28

Archana Shrestha and Bruce A. McClane Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Introduction Enterotoxin-producing C. perfringens The Gram-positive, anaerobic, spore former Clostridium perfringens causes an impressive collection of enteric and histotoxic diseases in humans and domestic animals. The pathogenic versatility of this bacterium is largely attributable to its prodigious toxin-producing abilities, with at least 16 different C. perfringens toxins now reported in the literature. However, individual C. perfringens isolates produce only subsets of this toxin arsenal, forming the basis for a commonly used toxinotype classification scheme [1] that assigns C. perfringens isolates to one of five types (A–E), based upon their ability to produce alpha, beta, epsilon and iota toxins (Table 28.1). About 1%–5% of C. perfringens isolates, mainly (but not exclusively) belonging to type A [1], express another biomedically important, 35-kDa single polypeptide toxin named C. perfringens enterotoxin (CPE). Interesting features of CPE action, genetics, and expression represent the major focus of this chapter.

Biomedical importance of enterotoxin-producing C. perfringens Clostridium perfringens enterotoxin (CPE)–positive type A isolates are most notable for causing C. perfringens type A food poisoning, which ranks among the most common of all foodborne human GI diseases [1]. For example, the Centers for Disease Control and Prevention estimate that 1 million cases of this single food poisoning occur annually in the United States [2–4]. C. perfringens type A food poisoning results from ingestion of foods (usually meat or poultry products) contaminated with high levels of vegetative cells of a CPEpositive type A isolate [1]. Those ingested bacteria later sporulate in the intestines, allowing in vivo CPE production (discussed further later in this chapter). Symptoms

The Comprehensive Sourcebook of Bacterial Protein Toxins. DOI: http://dx.doi.org/10.1016/B978-0-12-800188-2.00028-8 © 2015 Elsevier Ltd. All rights reserved.

816

Table 28.1 

Bacterial Protein Toxins Active on the Surface of Target Cells

C. perfringens Toxinotypes Toxins Produced

Type

Alpha

Beta

Epsilon

Iota

A B C D E

+ + + + +

− + + − −

− + − + −

− − − − +

of C. perfringens type A food poisoning typically include diarrhea and abdominal cramps [1], which subside within 24 h and are followed by a full recovery; however, this can be a fatal illness in the elderly or debilitated. During the past decade, two unusually lethal outbreaks of this food poisoning occurred in American psychiatric institutions and resulted in multiple fatalities, mainly in nonelderly and physically healthy people [5,6]. Psychoactive medications taken by those individuals caused a severe constipation that is thought to have prevented the onset of diarrhea, causing prolonged contact between CPE and the intestines; this promoted absorption of the toxin, and so it damaged internal organs such as the liver. Recent studies with a mouse model support this hypothesis and suggest that death from CPE enterotoxemia results from a hyperpotassemia that causes cardiac failure [7]. CPE is well established as the major virulence factor responsible for the symptoms of C. perfringens type A food poisoning. For example, this toxin is detectable in the feces of virtually all people suffering from C. perfringens type A food poisoning and human volunteer feeding studies showed that ingestion of purified CPE is sufficient to reproduce the diarrhea and cramping symptoms of the natural food poisoning [1]. Most importantly, Molecular Koch’s postulate analyses showed that CPE expression is required for the enteric virulence of food poisoning isolates in rabbit ileal loops [8]. In the mid-1980s, enterotoxin-producing type A isolates also became linked to nonfoodborne human GI diseases [9], including about 5–10% of all antibioticassociated diarrhea (AAD) and sporadic diarrhea (SD) cases. CPE appears to play an important role in the pathogenesis of most or all AAD/SD cases since specific inactivation of the cpe gene in SD isolate F4969 eliminated that isolate’s ability to cause enteric pathology in rabbit ileal loops [8]. Compared to C. perfringens type A food poisoning, the GI symptoms of CPE-associated AAD/SD tend to be more severe and of longer duration [9]. CPE may also contribute to other human and animal infections. Recent studies suggest that by acting synergistically with beta toxin, CPE may also contribute to some cases of human enterititis necroticans [10]. Furthermore, CPE-positive isolates have also been linked to certain veterinary GI diseases [11]. For example, enterotoxinproducing type A isolates are implicated in canine enteritis [12].

Clostridium perfringens enterotoxin

817

The genetics and expression of CPE CPE genetics The cpe gene

P4? ~196 bp upstream P3 SigE ~142 bp upstream P2 SigE ~73 bp upstream P1 SigK ~58 bp upstream

45 bp insert ?

The enterotoxin gene (cpe) includes an open reading frame (ORF) encoding a 35-kDa protein with a unique, primary, 319-amino-acid sequence [13]. The cpe ORF is remarkably conserved among most cpe-positive type A isolates [14]. Interestingly, some type E isolates carry cpe ORF sequences that are silent, in part due to the presence of nonsense and frame-shift mutations [15] (for more, see next paragraph). However, other type E isolates were recently identified [16] that produce a variant CPE, although that toxin retains 96% identity with the classical CPE made by type A strains. The DNA region immediately upstream of the cpe ORF is also reasonably conserved among type A isolates, except for the presence in some type A strains of a 45-nucleotide insert about 265 bp upstream of the cpe start codon [17]; this insert has no apparent effect on CPE expression levels [18]. As shown in Figure 28.1, primer extension and deletion analyses [19] identified at least three promoters upstream of the cpe ORF in most or all type A strains (see the section entitled “Regulation of CPE Expression,” later in this chapter, for further discussion). However, one of these promoters (P3) is missing from those type E strains expressing the variant CPE [16]. The promoter region in type E strains with silent cpe sequences has been significantly

5’

cpe ORF

(957 bp) Cytotoxic domain (135 to 162 bp)

3’ Inverted repeats

TTTTTTTTT poly T tail

Binding domain (870 to 957 bp) C. perfringens cpe gene

Figure 28.1  Arrangement of the cpe gene in type A strains. Note the presence of at least three promoters (P1, P2, and P3) immediately upstream of the cpe ORF, as confirmed by primer extension analyses, RNase T2 protection assays and deletion mutagenesis [17,19]; a fourth potential promoter (P4) identified by primer extension analysis could not be confirmed by other techniques [17,19]. A 45-nucleotide insert present upstream of the cpe promoters in some type A isolates [17] has no effect on CPE expression [18]. The 957 nucleotide cpe ORF encodes a unique protein of 35 kDa that lacks a signal peptide [13]. Regions of the cpe ORF encoding cytotoxic or receptor-binding activity are also indicated here. Finally, a region containing inverted repeats followed by an oligo-T tract is located immediately downstream of the cpe termination codon; those sequences may be involved in transcriptional termination, message stability, or both [13].

818

Bacterial Protein Toxins Active on the Surface of Target Cells

disrupted by the apparent insertion of a mobile genetic element carrying iota toxin genes [15]. A putative stem loop structure, followed by an oligo dT tract, is located about 36 bp downstream of the cpe ORF in most, if not all, isolates [13]. This stem loop–containing region possesses characteristics of a rho-independent transcriptional terminator and may also contribute to cpe messenger RNA (mRNA) stability.

Association of the cpe gene with mobile genetic elements In type A isolates, the cpe gene can be present on either the chromosome or a large plasmid [20,21], but no single type A isolate has yet been found to carry both a chromosomal and plasmid-borne cpe gene. In all other types, the cpe gene (or silent cpe sequences in some type E strains) is always present on plasmids [22]. The cpe plasmid in type A strain F4969 was shown to transfer conjugatively [23], likely due to the presence of a tcp (transfer clostridial plasmids) locus that is known to mediate conjugative transfer of another C. perfringens plasmid named pCW3 [24,25]. Most, if not all, other plasmids carrying the cpe gene or silent cpe sequences also carry the tcp locus [22,26–28], indicating that they should also be conjugative. There are two major families of cpe plasmids in type A and E strains; i.e., the pCP5603-like and pCPF4969-like plasmids [29]. These plasmid families share about 50% identity due to the presence of a conserved region containing the tcp locus and other sequences. The variable region of these plasmids carries a mix of metabolic genes, bacteriocin genes, and potential virulence genes; e.g., pCP5603-like plasmids also carry a beta2 toxin gene. Interestingly, some plasmids encoding epsilon toxin or beta toxin are also related to pCP5603 or pCPF4969, suggesting a common evolution [22]. As shown in Figure 28.2, cpe gene or silent cpe sequences are closely associated with insertion sequences in all strains. When located on the chromosome, the cpe gene may be present on a 6.3-kb transposon named Tn5565 [22]. This putative transposon, which carries IS1469 sequences directly upstream of the cpe gene, is flanked on both ends by IS1470 sequences. Some evidence [32] suggests that Tn5565 can excise from the chromosome and then form a circular intermediate, which could facilitate cpe gene movement. However, actual movement/insertion of Tn5565 from one DNA location to another has not yet been demonstrated. Additionally, Figure 28.2 indicates that various insertion sequences are also present upstream of the plasmid-borne cpe gene in type A, C, D, and E isolates [22]. However, the specific insertion sequences associated with those cpe loci, and their arrangement, is variable among different types. As for the chromosomal cpe gene, PCR experiments showed these insertion sequences can excise the cpe gene from plasmids to form circular intermediates [22]. Thus an emerging view is that the cpe gene is highly mobile due to its association with both insertion sequences and conjugative plasmids.

Relationships between type a strain cpe genotypes and human GI disease Approximately 70% of C. perfringens type A food poisoning cases involve a type A strain carrying a chromosomal cpe gene [1]. In addition to causing the remaining about

Clostridium perfringens enterotoxin

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Figure 28.2  Organization of the plasmid cpe locus in various C. perfringens types versus the chromosomal cpe locus of food poisoning isolate NCTC8239 (C). This image is based upon results in several studies [16,28,30,31]. Note that the cpe ORF sequence is identical to the cpe ORF of NCTC8239 in most type C and D strains [31]. Reproduced with permission from [31].

30% of food poisoning cases, type A isolates with a plasmid-borne cpe gene cause virtually all cases of CPE–associated AAD and SD [20,21,28,30,33]. Explanations have emerged for these strong associations between cpe genotypes (i.e., whether a type A isolate carries a plasmid-borne or chromosomal cpe gene) and specific CPEassociated human GI diseases. First, spores of type A isolates carrying a chromosomal cpe gene are much more resistant to food environment stresses (including heat/cooking, freezing/refrigeration, and food preservatives) than type A isolates carrying a plasmid cpe gene [34,35]; this spore resistance phenotype likely favors survival of the chromosomal cpe isolates in the incompletely cooked or inadequately held foods that cause food poisoning outbreaks. This spore resistance is mediated in large part by chromosomal cpe type A strains producing a unique variant of small acid soluble protein-4 that binds exceptionally tightly to spore DNA, thus offering protection from heat and other stresses [36]. A second explanation is that the most common food vehicles for C. perfringens type A food poisoning (i.e., meats, poultry, and seafood), are most commonly contaminated with type A isolates carrying a chromosomal cpe gene rather than a plasmid cpe gene [37]. The strong linkage between type A isolates carrying a cpe plasmid and CPEassociated AAD/SD may also be important for those nonfoodborne human GI diseases. For example, while C. perfringens type A food poisoning results from ingestion of >106–107 C. perfringens vegetative cells/ml, some evidence suggests that CPEassociated AAD/SD often results from ingestion of a low C. perfringens dose [9].

820

Bacterial Protein Toxins Active on the Surface of Target Cells

Under low infecting doses, establishment of CPE-associated AAD/SD might be assisted by the presence of the cpe gene on a conjugative plasmid, as already shown for SD isolate F4969 [23], since this would allow the ingested cpe-positive C. perfringens cells to transfer their cpe plasmid to the cpe-negative C. perfringens commonly found in the normal human GI flora, This putative in vivo cpe plasmid transfer would convert the recipient normal flora C. perfringens isolates to enteric virulence, thus amplifying the original infective dose and thereby increasing the probability of colonization and disease. In addition, this process would result in the presence of the cpe gene in normal flora C. perfringens isolates that are presumably under selective pressure for persistence in the human GI tract. Therefore, conjugative acquisition of the cpe plasmid by colonization-proficient normal GI flora C. perfringens might help to explain clinical observations [9] indicating that the GI symptoms of CPEassociated AAD/SD typically persist longer than the GI symptoms of C. perfringens type A food poisoning.

CPE expression Features of CPE expression The pioneering work of Duncan’s group back in the 1970s, reviewed by Melville et al. [38], first identified a strong linkage between CPE expression and sporulation. Those early studies showed that a stage 0 sporulation mutant of C. perfringens food poisoning isolate NCTC8798 lost the ability to produce CPE. Western blot studies [13] later confirmed a relationship between sporulation and CPE production by demonstrating more than 1500-fold higher CPE production by sporulating versus vegetative cells of food poisoning isolate NCTC 8239. Other Western blot studies [14,21] then demonstrated that CPE expression from both the chromosomal and plasmid-borne cpe genes is strongly sporulation-associated. Several other studies using genetic approaches added further support to a relationship between CPE expression and sporulation by showing that isogenic CcpA and Agr mutants prepared in SM101, a transformable derivative of a CPE-positive food poisoning strain, exhibit reduced sporulation and CPE production [17,39,40]. The interactions between these regulators and sporulation or CPE production require further study. Another pathogenesis-relevant feature of CPE expression is the extremely large amounts of this toxin produced by many type A isolates. CPE often accounts for more than 15% of the total protein present inside sporulating cells of those C. perfringens isolates [13].

Regulation of CPE expression The sporulation-associated nature of CPE expression involves strong regulation at the transcriptional level. Northern blots and RNA slot blots detected cpe transcripts in sporulating, but not vegetative, cultures of C. perfringens food poisoning isolates [19,38,41]. As introduced earlier, the cpe gene is transcribed from three strong promoters (P1, P2 and P3) that are located within 200 bp upstream of the cpe start codon.

Clostridium perfringens enterotoxin

821

It has become clear why CPE expression is strongly sporulation-dependent. Four alternative sigma factors (SigE, SigF, SigG and SigK) control C. perfringens sporulation (Figure 28.1), which is important because the P1 promoter is a SigK-dependent promoter, while P2 and P3 are SigE-dependent promoters [19,42]. Other studies [43] showed that SigF controls the expression of both SigE and SigK, explaining why SigF is also required for cpe expression. In addition, to these alternative sigma factors, cpe expression and sporulation also requires Spo0A, which (when phosphorylated) is critical for initiating alternative sigma factor expression. The strong production of CPE during sporulation involves at least two factors. First, the presence of multiple promoters upstream of the cpe start codon contributes to high levels of transcripts. Second, cpe mRNA shows exceptional stability in sporulating cells. An older study detected a half-life of about 45 min for cpe mRNA [44].

Figure 28.3  Sporulation-associated sigma factor regulation of CPE production. SigF controls the expression of SigE and SigK, which are needed to drive CPE expression. However, all four sigma factors are needed for sporulation [42,43]. Modified from [31].

822

Bacterial Protein Toxins Active on the Surface of Target Cells

Release of CPE from sporulating C. perfringens cells The cpe gene does not encode a signal peptide that could be used for CPE secretion. Instead, the enterotoxin accumulates in the mother cell cytoplasm until the completion of sporulation. At that time, the toxin is finally released when the mother cell lyses to free its mature endospore.

The intestinal action of CPE CPE is classified as an enterotoxin because it induces fluid and electrolyte losses from the GI tract of many mammalian species [45]. It was once thought that only the small intestine is affected by CPE [45], with the ileum being the most sensitive small intestinal region [45]. However, recent studies demonstrated that, at least in the rabbit, the colon is also damaged by the enterotoxin [46]. CPE can act very quickly on both the small intestines and colon, with high toxin doses causing visible damage within 30 min or 1 h, respectively, of treatment [47]. Three observations indicate that this initial histopathologic damage, which starts at the villus tips in the small intestine, triggers CPE-induced small intestinal fluid/electrolyte transport alterations. First, the onset of fluid/electrolyte transport alterations occurs concurrently with the development of damage in the CPE-treated rabbit ileum [47]. Second, only those CPE doses capable of inducing intestinal damage can induce fluid/ electrolyte transport alterations [48]. Finally, binding-capable CPE variants that cannot induce damage cause no fluid accumulation in the lumen of small intestinal loop [49]. CPE exerts biphasic alterations in ileal fluid/electrolyte transport [47]. Initially, the CPE-treated ileum shows only an inhibition of fluid/electrolyte absorption; as described previously, this initial inhibition of absorption probably occurs when histopathologic damage begins in the CPE-treated ileum. However, as treatment time increases, frank ileal fluid/electrolyte secretion develops in the CPE-treated ileum. This intestinal secretion likely reflects continued development of histopathologic damage that eventually desquamates the epithelium of the CPE-treated ileum and causes severe villus shortening [8].

The cellular action of CPE Step one: CPE binds to cells, forming a small, SDS-sensitive complex Considerable evidence indicates that receptors play a critical role in initiating CPE action under pathophysiologic conditions. For example, cultured mouse fibroblasts, which are unable to specifically bind CPE due to the lack of receptors, fail to respond to the moderate CPE concentrations typically present in the intestines during GI disease [50–52]. However, those fibroblasts do specifically bind, and become highly sensitive to, CPE when they are transfected to express certain claudin receptors.

Clostridium perfringens enterotoxin

823

Claudins are a 24-odd-member family of small (about 22-kDa) proteins that play key roles in mediating both the barrier and gate functions of epithelial tight junctions (TJs). It is now clear that several claudins can serve as functional CPE receptors. Expression cloning approaches using mouse fibroblast transfectants indicate that claudins −3, −4, −6, −7, −8, and −14 are functional CPE receptors, while claudins −1, −2, −5 and −10 cannot bind CPE [51,53–55]. Claudins consist of four transmembrane domains, two extracellular loops and a C-terminal cytoplasmic tail [56], which can mediate signalling but is not important for CPE action [57]. It is now clear that CPE binds to the second extracellular loop of receptor claudins [54,58]. The presence of an Asp near the middle of the second extracellular loop is a key factor determining the ability of a claudin receptor to bind the enterotoxin [54]. Residues adjacent to this Asp then determine the affinity of toxin binding, which varies considerably among the CPE claudin receptors [55]. Recent structural biology studies have elucidated the binding interaction between CPE and the second extracellular loop of claudin receptors (Figure 28.4). Those studies revealed that the second extracellular loop forms a helix-turn-helix that docks with a binding pocket in the C-terminal binding domain of the toxin (Figure 28.4; also see further discussion of this later in this chapter). Immediately upon binding to claudin receptors, CPE localizes in a small (about 90-kDa), sodium dodecyl sulfate (SDS)–sensitive complex [60]. Formation of this small CPE complex (Figure 28.5) appears to be important for the enterotoxin’s action since this complex forms in every CPE-sensitive cell line tested to date. The composition and stoichiometry of small CPE complex is not yet completely clear, but immunoprecipitation analyses demonstrated the presence of both receptor and nonreceptor claudins associated with this CPE species [57]. When present in the small complex, CPE quickly becomes trapped on the surface of the host cell membrane [61]. For example, CPE localized in small complex shows relatively limited dissociation from mammalian cells. However, CPEs in small complexes do not appear to be internalized or to insert in lipid bilayers as this toxin remains highly susceptible to external protease treatments.

Figure 28.4  Claudin interaction with CPE. The second extracellular loop of receptor claudins (shown in white in the e-version) interacts with a pocket in the C-terminal region of CPE. Reproduced with permission from [59].

824

Bacterial Protein Toxins Active on the Surface of Target Cells RT CPE

−

4C +

−

+

Figure 28.5  Formation of CPE-containing complexes at room temperature (RT) or 4°C. For this experiment, 125I-CPE was incubated with brush border membranes in the presence (+) or absence (−) of excess native toxin. After extraction with Triton X-100, lysates were subjected to nondenaturing electrophoresis and gels were then autoradiographed. CPE represents free 125 I-CPE dissolved in Triton X-100 that had never been exposed to membranes. The single arrow depicts the location of the 90-kDa small complex, while the double arrow indicates migration of higher Mr complexes. Reproduced with permission from [60].

Step two: CPE becomes localized in large, SDS-resistant complexes At 4°C, CPE binds and forms small complexes, but it does not induce any cytotoxic effects [60,62]. However, if host cells containing CPE bound at 4°C are subsequently warmed to 37°C, cytotoxicity quickly develops. Concurrent with this development of cytotoxicity, much of the CPE bound to those cells shifts (Figure 28.5) from the small complex to higher Mr material [60,62]. Collectively, these observations indicate that (i) although important, formation of the small CPE complex is insufficient to induce cytotoxicity; (ii) small complex is a precursor for formation of the higher Mr, CPEcontaining material; and (iii) some or all of the higher Mr, CPE-containing material is essential for cytotoxicity. The importance of the higher Mr, CPE-containing material for CPE-induced cytotoxicity received further support from deletion mutagenesis, random mutagenesis, and site-directed mutagenesis studies [63–65]. Collectively, those studies identified several CPE fragments or point mutants that can bind CPE and form small complexes yet fail to form the higher Mr, CPE-containing material; in all cases, those CPE fragments/point mutants were noncytotoxic. In addition, some CPE fragments could bind normally yet formed increased amounts of the higher Mr, CPE-containing material; those fragments also consistently exhibited 2–3-fold greater cytotoxic activity than native CPE.

Clostridium perfringens enterotoxin

825 Time of CPE treatment

1 10 20 30 40 60 90 120 CPE Cells

kDa

212

122 CPE antibody

Figure 28.6  The kinetics of CPE large complex formation in suspensions of CaCo-2 cells. CPE was added to a suspension of CaCo-2 cells for the indicated time at 37°C. After washing to remove unbound toxin, cells were lysed with SDS. The resultant lysates were analyzed by SDS-PAGE (no sample boiling) using 4% acrylamide gels. The gels were then Western blotted with either CPE antibodies (left) or occludin antibodies (right). The double, open, and closed arrows represent the migration of the 200-kDa, 155-kDa, or 135-kDa complexes, respectively. Reproduced with permission from [52].

Western blot studies [52] examined suspensions of human enterocyte-like CaCo-2 cells treated with CPE at 37°C and resolved (Figure 28.6) the higher Mr, CPEcontaining material into two major SDS-resistant CPE complexes that run on SDS-PAGE with apparent sizes of about 155 kDa and about 200 kDa. Later coimmunoprecipitation studies [57] showed that these complexes, now named CH-1 and CH-2, each contain six copies of CPE and both receptor and nonreceptor claudins. In addition, CH-2 contains another TJ protein named occludin. Later, CH-1 and CH-2 were also sized more accurately as 425–500 kDa and 550–600 kDa, respectively [57]. In CaCo-2 cell monolayers grown in Transwell cultures (to produce a polarized epithelium) and then treated with CPE on their apical surface, formation of the CH-2 complex requires about 2 h [66]. In contrast, 20 min of CPE treatment on the apical surface is enough time for these CaCo-2 cell Transwell cultures to form CH-1 and exhibit the substantial 86Rb-release indicative of CPE-induced cytotoxicity (see the next section). Thus, it appears that, under experimental conditions resembling in vivo GI disease, CH-1 formation is sufficient to initiate CPE-induced cytotoxicity in polarized monolayers of human enterocyte-like CaCo-2 cells. Recent studies identified the formation of a CH-1-like complex in CPE-treated rabbit small intestine [7]. However, no CH-2 complex formed even though the presence of occludin was confirmed in this tissue. This finding highlights the importance of confirming in vitro results in animal models. It remains conceivable that a CH-2 complex could form in human intestines during disease.

826

Bacterial Protein Toxins Active on the Surface of Target Cells

Step three: CPE causes plasma membrane permeability changes Back in 1979, CPE was first shown to disrupt normal plasma membrane permeability in sensitive mammalian cells [67]. Within 15 min of CPE treatment, sensitive mammalian cells become dramatically more permeable to small molecules (<200 Daltons), including ions and amino acids [67–72]. Over 1–2 h of CPE treatment, cells gradually become permeable to larger molecules up to about 3000 Daltons [70,72]. Considerable evidence indicates that CPE-induced permeability alterations are caused by pore formation. Electrophysiology studies demonstrated that CPE forms cation-selective pores in both artificial membranes and CaCo-2 cells [73,74]. Furthermore, when CPE is present in large complexes, it is substantially protected from external proteases [61], which is fully consistent with substantial portions of the CPE protein inserting into lipid bilayers to form pores after the toxin becomes localized in the large CPE-containing complexes responsible for CPE-induced cytotoxicity.

Step Four: CPE-induced cell death Characterization of CPE-induced cell death CPE is highly cytotoxic for many mammalian epithelial cells [67,75]. When treated with this toxin, cells rapidly lose viability and experience a simultaneous shutdown of DNA, RNA, and protein synthesis. Coincident with those effects, the CPE-treated cells develops substantial morphologic damage, including cell rounding, bleb formation, and detachment. Cell death pathways activated by CPE-treatment of CaCo-2 cell monolayers are toxin dose-dependent [76]. Briefly, a 1-μg/ml CPE dose causes CaCo-2 cells to develop classical caspase 3/7-mediated apoptosis within 60 min, with effects including morphologic damage, DNA fragmentation in a ladderlike cleavage pattern, and caspase-3/7 activation. Those effects can be blocked by caspase-3/7 inhibitors, but not by caspase-1 or oncosis inhibitors. The 1-μg/ml CPE dose also induces rapid mitochondrial membrane depolarization and cytochrome C release in CaCo-2 cells, but those mitochondrial-related effects cannot be inhibited by any caspase inhibitors or glycine. In contrast, CaCo-2 cells treated with a 10 μg/ml CPE dose for 30 min develop characteristics of a form of necrosis known as oncosis, with effects including morphologic damage and a smeared pattern of DNA cleavage that can be transiently blocked by glycine, but not by any caspase inhibitors (even though modest caspase-1 activation occurs in these cells). This high CPE dose has no effect on mitochondrial membrane depolarization or cytochrome C release. Since CPE concentrations present in feces from victims of CPE-associated GI disease range from several nanograms per milliliter to >100 μg/ml [1], both apoptosis and oncosis probably contribute to the histopathologic damage that initiates CPE action in vivo.

The role of calcium in CPE-induced cell death Matsuda et  al. first noted the importance of extracellular Ca2+ for obtaining CPEinduced morphologic damage [75]. That Ca2+ requirement was later explained by a

Clostridium perfringens enterotoxin

827

study [77] demonstrating that CPE treatment of CaCo-2 cells in the absence of extracellular Ca2+ still results in membrane permeability alterations; i.e., the extracellular Ca2+ requirement for CPE-induced cell death occurs after the enterotoxin has bound, formed the CH-1 complex, and induced plasma membrane permeability alterations. Furthermore, evidence was presented demonstrating that extracellular Ca2+ is specifically necessary for the normal development of all events linked to either CPE-induced apoptosis or oncosis (see subsection Step Four above). The extracellular Ca2+ requirement for CPE-induced cell death was then shown to involve an influx of Ca2+, a potent second messenger capable of mediating cell death [78]. This CPE-induced Ca2+ influx quickly raises cell calcium levels in CaCo-2 cells. Both the onset and magnitude of those cellular calcium increases are CPE dose-dependent; i.e., a faster and stronger increase in cell calcium levels occur using a 10- versus 1-μg/ml CPE dose. Increases in cellular Ca2+ levels appear to play a pivotal role in normal CPEinduced cell death. For example, caspase inhibitors and glycine (which are capable of blocking CPE-induced apoptosis or oncosis, respectively) fail to block CPE-induced increases in CaCo-2 cell Ca2+ levels [76,78], supporting the idea that increased calcium levels are not merely a consequence of CPE-induced cell death. Furthermore, Chakrabarti et al. showed that the cell death pathway activated by a given CPE dose can be experimentally shifted from apoptosis to oncosis (or vice versa) simply by manipulating the calcium content of treatment buffers to obtain greater (or lesser) increases in cell calcium levels [76,78]; i.e., cell calcium levels directly select which death pathway becomes activated in a CPE-treated cell. How do CPE-induced changes in cellular Ca2+ levels trigger cell death? Inhibitor studies [78] indicated that two calcium-binding cytosolic proteins (i.e., calmodulin and calpain) play essential roles in CPE-induced apoptosis and oncosis. Furthermore, stronger and faster calpain activation occurs in CaCo-2 cells treated with 10- versus 1-μg/ml CPE doses. The pathway by which calcium-induced activation of calpain and calmodulin leads to death of the CPE-treated cell is currently under study.

Consequences of CPE-induced cell death: initiation of GI disease CPE-induced apoptosis and oncosis produce morphologic damage, which likely initiates the histopathologic damage responsible for starting fluid and electrolyte transport alterations in the CPE-treated intestines. Supporting this model, a CPE fragment that can bind but is noncytotoxic does not cause any damage or fluid accumulation in rabbit small intestinal loops [49].

Consequences of CPE-induced cell death: TJ effects The morphologic damage resulting from CPE-induced apoptosis and oncosis exposes the basolateral membrane of both intoxicated and adjacent (but still healthy) enterocytes. Exposing enterocyte basolateral membranes likely has pathophysiologic relevance since it provides free (unbound) CPE access to (previously hidden) CPE receptors present on basolateral membranes. This effect could be important since

828

Bacterial Protein Toxins Active on the Surface of Target Cells

Transwell CaCo-2 cell studies detected considerably more CPE receptors on the basolateral versus apical surface of those enterocyte-like human polarized cells [66]. CPE binding to receptors on the basolateral surface of enterocyte-like cells (and, probably, enterocytes) is likely to increase CH-1 complex formation, further promoting cell death. Second, it should allow bound CPE to interact with (previously inaccessible) claudins and possibly occludin. In Caco-2 cells, those interactions with CPE prompt the removal of these TJ proteins in to the cytotoplasm. This removal of claudins, and possibly occludin, from TJs probably helps to explain CPE’s ability to damage TJs [79], which (in turn) may explain the increased paracellular permeability observed [80] in polarized monolayers treated with CPE fragments (and, presumably, native CPE). Those CPE-induced increases in paracellular permeability could contribute to the fluid/electrolyte secretion that occurs later in CPE action.

Consequences of CPE-induced cell death: inflammation? Inflammation contributes to the symptoms of many infectious GI diseases (e.g., C. difficile–associated infections). Since oncosis is proinflammatory, the discovery that high CPE doses kill cells via oncosis suggests that intestinal inflammation could also contribute to the diarrheal and cramping symptoms of CPE-associated GI disease. As mentioned earlier, this possibility is supported by studies determining that fecal CPE concentrations from C. perfringens food poisoning victims often exceed the 10-μg/ml CPE dose found to induce oncosis in CaCo-2 cells [76]. Studies are currently underway to directly evaluate the potential proinflammatory properties of CPE.

Summary: a current model for CPE action The results described in the previous sections indicate that the intestinal effects of CPEs are the result of a multistep process. The current model proposes CPE action starts (Figure 28.7A) with the binding of CPE to claudin receptors (and associated nonreceptors) to form the 90-kDa small complex. Through a still-unidentified process, the small CPE complex then interacts with other proteins to form the CH-1 complex. That complex forms pores that render the plasma membrane permeable to small molecules, including Ca2+. Low CPE doses cause a moderate Ca2+ influx that, via modest activation of calmodulin and calpain, induces mitochondrial membranes to release cytochrome C, which leads to caspase 3/7 activation and cell death from classical apoptosis. At higher CPE doses, a massive Ca2+ influx occurs that causes, via strong activation of calmodulin and calpain, cell death via oncosis. As shown in Figure 28.7B, both oncosis and apoptosis result in morphologic damage that exposes the basolateal membranes of the intoxicated cell and adjacent neighbors. This allows CPE to bind to newly exposed basolateral claudin receptors; the basolaterally bound toxin then forms more CH-1 complex and also interacts with occludin to form the about 200-kDa complex, at least in vitro. Formation of these complexes triggers internalization of occludin and claudins, which damage TJs. The CPE-damaged TJs can no longer properly regulate paracellular permeability; along

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Figure 28.7  Model for CPE action: Early steps. (A) Steps leading to CPE-induced cell death: CPE binds to receptors (possibly claudins) to form a small complex. The small complex then interacts with additional proteins to form a 155-kDa large complex whose formation leads to calcium influx, probably through a CPE-containing pore. At low CPE doses, modest calcium influx leads to activation of a classical caspase-3 dependent apopotosis mediated by mitochondria. High CPE doses cause a massive calcium influx that kills cells via oncosis. Both death pathways are mediated by activation of calpain and calmodulin (not shown) and lead to morphologic damage of the CPE-treated cell. (B) Secondary consequences of CPEinduced morphologic damage. Morphologic damage induced by CPE-induced cytotoxicity exposes the basolateral surface on the intoxicated cell and adjacent cells. The basolateral surface contains many CPE receptors; exposure of this surface allows more CPE binding to form more 155-kDa complex formation and also permits initial formation of the 200-kDa complex containing occludin. Formation of the 200-kDa complex probably facilitates further TJ damage.

with gross desquamation of the intestinal epithelium and severe villus shortening, altered paraceullar permeability may contribute to CPE-induced diarrhea. When high CPE doses cause enterocytes to die from oncosis, inflammation may also contribute to diarrhea and other GI symptoms (not shown). Finally, new evidence indicates that both CPE and beta toxin could be present in the intestines during some cases of type C enteritis necroticans in humans [10]. This may be important since those two enterically active toxins were shown to act together synergistically to cause intestinal damage and fluid accumulation in rabbit small intestinal loops. This same synergism may also occur during some cases of human enteritis necroticans.

CPE structure/function relationships The CPE structure/function relationship has been extensively examined using a combination of structural biology and mutagenesis approaches. Structurally, this 35-kDa

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Figure 28.8  CPE structure/function relationships. The N-terminal domain (blue-green in the e-version) of CPE mediates oligomerization and membrane insertion, while the C-terminal domain (yellow-red in the e-version) mediates binding to claudin receptors. Modified with permission from [81].

single polypeptide toxin (Figure 28.8) consists of two major domains: (i) a C-terminal domain that mediates binding activity and (ii) an N-terminal domain, structurally resembling portions of aerolysin, that conveys oligomerization and membrane insertion activity [81,82].

The C-terminal CPE receptor-binding region Early studies, where CPE was chemically cleaved at its single cysteine (residue 186), suggested that the C-terminal half of CPE might contain receptor-binding activity [83]. That assignment was rigorously confirmed by recombinant DNA approaches, which demonstrated that a recombinant CPE (rCPE) fragment consisting of enterotoxin amino acid residues 171–319 is an extremely efficient receptor-binding competitor for native CPE [84]. Subcloning approaches and synthetic peptide studies further localized this CPE receptor-binding activity to the C-terminal 30 amino acids of the native enterotoxin [85]. Later work [86] identified specific C-terminal CPE residues important for receptor binding, including Tyr (306), Tyr (310), Tyr (312), and Val (259), and the other including residues Leu (254), and Leu (315) of CPE. Recent structural biology analyses (Figure 28.4) indicated that the interaction between CPE and claudin receptors is strongly hydrophobic in nature and involves claudin extracellular loop two residues packing into two CPE binding pockets, one defined by the three Tyr residues plus Ile (258) and Val (259), and the other including residues Leu (254) and Leu (315) of CPE.

The CPE N-terminal cytotoxicity Region Despite their ability to bind to receptors, C-terminal CPE fragments fail to induce any cytotoxicity [84,85]. Those observations indicated that sequences present in the N-terminal half of the enterotoxin are required for biologic activity.

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However, those N-terminal cytotoxicity sequences do not include the extreme N-terminal amino acids of the native CPE protein. Early studies [87,88] reported that removing the 25 or 36 N-terminal amino acids from CPE using the intestinal proteases trypsin or chymotrypsin, respectively, produces a twofold to threefold more active toxin. This proteolytic activation, which may occur in the GI tract during disease, is not due to an enhancement of receptor binding [89], which is consistent with localization of receptor binding activity to the CPE C-terminus (as discussed earlier in this chapter). Deletion mutagenesis studies [63] later determined that rCPE fragments lacking up to the 44 N-terminal amino acids of the native enterotoxin still exhibit this activated toxicity phenotype and also determined that this activation effect is due to enhanced formation of the large CPE complexes. However, that study also found that deleting the first 52 amino acids produces an rCPE fragment that can bind to receptors but is completely nontoxic. This observation strongly suggested that the CPE region between residues D45 and G53 is important for toxicity. Random mutagenesis studies [64] supported this conclusion by identifying a G49D rCPE mutant that is binding-proficient but nontoxic. Furthermore, those random mutagenesis studies also determined that the G49D rCPE mutant is specifically blocked for formation of large SDS-resistant complexes (including the about 155-kDa complex responsible for cytotoxicity), implying that the D45-G53 region of native CPE participates in formation of those complexes. Alanine-scanning mutagenesis approaches later fine-mapped the D45-G53 region of CPE to identify the amino acid residues involved in cytotoxicity [65]. Those studies identified two CPE residues (i.e., D48A and I51A) as being specifically important for formation of the CH-1 and CH-2 large CPE complexes. Follow-up saturation mutagenesis studies [65] then determined that both side chain length and charge are required for the D48 CPE residue to participate in large complex formation and cytotoxicity. Similar saturation mutagenesis analyses also found that residue size and hydrophobicity are necessary for the I51 CPE residue to participate in large complex formation and cytotoxicity [65]. Amino acids 81–106 in the N-terminal half of the native CPE protein form a predicted β-hairpin with amphipathic characteristics [90]. Cysteine-scanning mutagenesis of this region confirmed that it is important for cytotoxicity [90]. Further characterization of those Cys mutants determined they bind and form CH-1 similarly as wild-type CPE. However, the inactive mutants were blocked for membrane insertion or formation of a functional ion channel. Therefore, this region, named TM1, is functionally important for pore formation [65,90].

Development of a CPE vaccine? Epitope-mapping studies, where C-terminal rCPE fragments were reacted with CPEspecific monoclonal antibodies, localized the linear epitope recognized by MAb3C9 to the extreme C-terminus of the enterotoxin [89]. Since MAb3C9 is a neutralizing antibody that blocks the binding of the enterotoxin to its receptor [91], this finding

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provides additional evidence for the involvement of the C-terminal CPE region in receptor binding. Determining that a neutralizing linear epitope is present in noncytotoxic C-terminal CPE fragments opened the possibility of using those toxin fragments as CPE vaccine candidates. The vaccine potential of C-terminal CPE fragments received support by conjugating a 30-mer synthetic peptide (corresponding to the extreme C-terminal CPE sequence) to a thryoglobulin carrier [92]. Mice immunized intraperitoneally with that conjugate developed high titers of CPE-neutralizing serum IgG antibodies, while mice immunized with thyroglobulin alone failed to develop any protective antibody response against CPE. While these initial results are promising, additional work is required to fully evaluate the vaccine potential of C-terminal CPE fragments in protecting against CPE-mediated intestinal disease. For example, can these fragments induce the IgA response that is probably required to prevent CPE-mediated GI disease? Such studies will be pursued only when a clear need is demonstrated for a CPE medical or veterinary vaccine.

Potential applications of CPE: cancer and more Botulinum toxins, the most potent clostridial toxins, can be safely harnessed for a variety of therapeutic uses. Over the past decade, studies have explored a similar potential use of CPE as a therapeutic agent against certain tumors. The impetus for this work stemmed from studies indicating that many pancreatic, breast, and prostate cancers overexpress claudin-3 and/or claudin-4 [93–95]. The fact that those two claudins are high-affinity CPE receptors [50,51,80] suggested that the enterotoxin might be useful as a new modality for treating some or all of those cancers. Results from several lab studies have supported that hypothesis [93–98]. For example, pancreatic, prostate, and breast carcinoma cancer cells expressing high levels of claudin CPE receptors were found to be highly sensitive to the enterotoxin in vitro. In contrast, cancer cells failing to express claudins are CPE-insensitive. Perhaps even more exciting are results using animal tumor models [94,95]. For example, intratumoral injections of CPE into xenografts of panc-1 cells (human pancreatic cancer cells; Figure 28.9) or T47D cells (human breast cancer cells, not shown in the figure) cause significant tumor shrinkage, accompanied by necrosis. Equally important, mice carrying the tumor xenografts treated with CPE showed no ill effects from their therapy [95]. While work continues toward transitioning these preliminary findings to the clinic, obvious potential challenges remain to be addressed. For example, given the widespread distribution of claudin reeptors on epithelial and endothelia cells, will CPE toxicity be a problem? Two alternative approach to overcome this potential concern involve (i) the use of a C-terminal CPE binding fragment that enhances paracellular permeability and increases delivery of chemotherapeutic agents to tumor cells [99] and (ii) the use of C-terminal CPE fragments fused with activity domains of other proteins, such as other toxins [100]. Other potential limitations of using CPE for

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Figure 28.9  CPE effects on pancreatic cancer cells expressing claudin-4. Left: Effect of 1- or 7-day treatment of CPE or 0.9% NaCl on tumor volume in Panc-1 nude tumor xenograft models. Right: Histologic effects of treating claudin-4-expressing Panc-1 xenografts or claudin-4-negative HT-1080 xenografts with CPE or 0.9% NaCl. Necrotic areas appear as lighter-shaded areas after H&E staining. Reproduced with permission from [95].

cancer therapy concern the potential development of tumor resistance to CPE treatment or immune response to the CPE protein limiting effectiveness. Nonetheless, there is significant momentum supporting the potential of CPE (or binding-capable CPE derivatives) in clinical application as an antitumor agent. Finally, CPE is also being explored for its potential to enhance muscoal vaccine delivery based on the fact that M cells bind high levels of CPE [101].

Concluding remarks This chapter has emphasized the relatively unique nature of CPE, from its sporulation-associated expression to its ability to interact with TJ proteins to produce pores. The coming years are likely to reveal important new insights into this intriguing protein (including CPE cell death pathways induced in vivo), including the question of whether blocking those cell death pathways in vivo can ameliorate GI disease and interfere with the composition and formation of CPE pores. The potential application

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of CPE or cpe promoters for therapeutics and vaccines, respectively, remains promising; perhaps someday CPE will be a friend as well as a foe.

Acknowledgments Preparation of this chapter was supported by Public Health Service grant AI 19844-32 from the National Institute of Allergy and Infectious Diseases. The author thanks James G. Smedley III, Sameera Sayeed, Jihong Li, and Ganes Chakrabarti for preparing the figures.

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Clostridium perfringens enterotoxin

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Bacterial Protein Toxins Active on the Surface of Target Cells

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