Introduction
Clostridium perfringens is a Gram-positive, endospores anaerobe that is pathogenic to humans and mammals (1). It produces up to 18 distinct enterotoxins that cause diverse histotoxic conditions, including necrotic enteritis and enterotoxemia (2). Freedman, Shrestha, and McClane (1) found that the toxin arsenal of C. perfringens isolates has substantial variability, ranging from alpha to iota cytotoxin. C. perfringens enterotoxin (CPE) is a 35-kDa polypeptide toxin that is the main pathogenic agent for most type-A strains (6). CPE-positive variants are the leading cause of food-borne gastrointestinal (GI) diseases in humans, especially in the developed world (3). This paper examines the mechanism of CPE pathogenicity in mammals through a review of peer-reviewed scientific literature.
CPE and Food-borne Diseases
C. perfringens type-A strains account for most food-borne GI illnesses that manifest as diarrhoea and abdominal discomfort (1). They account for the high prevalence of food poisoning that presents as enteritis or enterotoxemia diagnosis in humans. Among the toxins implicated in these diseases is CPE. It is the product of the sporulation of C. perfringens vegetative cells. The virulence of the CPE-positive variants involves either a plasmid-encoded or a chromosomal cpe gene, as is the case with strains implicated in type-A food poisoning (4).
The role of CPE in enteric disease causation in mammals comes from genetic studies and positive faecal tests. In vitro attenuation of this locus has been shown to suppress CPE output, rendering isolates non-pathogenic (1). The enterotoxin is also detectable in the faecal matter of people diagnosed with type-A food-borne diseases. Although the toxin is highly variable, the basic sequence (319 amino acids) is common to all CPE-producing isolates. Structurally, the cytotoxicity of CPE lies in its two functional domains: receptor-binding C-terminal tyrosine residues and N-terminal hairpin loops implicated in oligomerisation and penetration (6).
CPE Release and Regulation
The initiation of CPE production occurs when nutrient stress-induced sporulation triggers CPE gene expression in C. perfringens. During this reproductive stage, the pathogen produces dormant and heat-resistant spores that cause food poisoning (1). Type-A endospores in contaminated food develop into many vegetative cells. Yasugi et al. (5) state that spores that are not destroyed by the stomach acid reach the intestines where they divide into more non-infective structures. CPE production starts when enteric vegetative cells begin to sporulate. Sporulation is followed by cell lysis that produces CPE, which causes type-A enteric diseases (5).
Two-component regulatory systems (TCRS) are implicated in C. perfringens sporulation. According to Chen, Ma, Uzal, and McClane (4), TCRS involve signal transduction, whereby external stimuli, e.g., nutrients, trigger autophosphorylation of a membrane sensor to activate the response regulator (Spo0A) that induces the expression of virulence genes. The inorganic phosphate released in the phosphorylation step is the primary signal involved in this process. Phosphorylated Spo0A attaches to upstream promoter regions to trigger the expression of a sigma factor, SigF, which “activates SigG, SigK, and SigE” that cause sporulation and active enterotoxin release (1).
Another TCRS model studied in C. perfringens type-A strains is the VirS/VirR system regulated by the virS/virR operon (4). Autophosphorylation of its N-terminal VirS transmembrane sensor activates the VirR regulator that attaches to C-terminal binding sites to regulate the expression of toxin genes (4). Quorum sensing (QS) is also implicated in type-A enterotoxin production. Agr-like QS has been shown to control CPE release by CPE-positive strains in broth cultures by regulating the plasmid-borne agriB gene (4).
Cellular Action of CPE
CPE action in food poisoning begins with the binding of the enterotoxin to claudins – fibril-forming host proteins that regulate tight junctions (TJs) of the intestinal epithelial cells (6). CPE-induced cytotoxicity is triggered when the toxin binds to these TJ-localized receptors. The claudin family comprises 27 variable polypeptides, each composed of 4 membrane-bound motifs, a C-terminal tail, and 2 β-hairpin loops (1). Upon binding to claudins, CPE forms oligomeric complexes through a series of reactions that culminate in its release into host cells. According to Shrestha, Uzal, and McClane (6), about six conjugates containing the toxin and claudins (receptors) oligomerise on the epithelial cell surface to create an initial pore. The pre-pore is the primary port of entry of CPE into the cells.
The β-hairpin loops of this structure then undergo conformational changes to form a β-barrel that creates a transmembrane channel called CH-1 (2). This CPE pore is semi-permeable, allowing small-sized cations to enter the host cells. As such, its formation causes a massive calcium influx that stimulates the release of calmodulin and calpain (6). These proteins cause oncosis or apoptotic events (caspase-mediated cell death). The apoptosis manifests as necrotic enteritis that is characterised by diarrhoea and GI cramps.
The morphology of moribund CPE-exposed cells also changes, exposing claudins in nearby cells to CPE. As a result, more CPE binds to the receptors to form many CH-1 pores, which causes a further calcium influx that triggers substantial apoptotic cell death (6). CPE-induced apoptosis harms the intestinal endothelial, resulting in epithelia bleeding and villi numbing. As described by Shrestha and McClane (2), the damage to the intestines may also result from excessive fluid and salt loss from enteric loops through diarrhoea. Thus, CPE poisoning manifests as epithelial desquamation of the small intestines and the colon and diarrhoeal symptoms in patients.
CPE-Claudins Interactions in Food-borne Diseases
CPE-mediated GI illnesses involve receptor-toxin complex structures that precede pore formation. Twenty-four proteins of the claudin family are critical to this process. Most CPE-binding members belong to claudin-3, 4, 6, 7, 8, 9, and 14 receptors (6). However, the relative affinity to CPE differs between them with claudin-3 and 4 bindings most strongly to the enterotoxin. Their function relates to reinforcing the TJs to regulate cellular permeability to ions by creating ion-selective pores (6). Claudin expression differs between epithelial cells, and thus, multiple receptors may be expressed at the same time.
The C-terminal part of this receptor family plays a role in toxin binding and subsequent cytotoxicity. Yelland et al.’s (3) study of the X-ray crystallographic information of CPE found that the enterotoxin binds to a binding groove on the C-terminal half of the claudin-2. Leucine and valine residues enter this site to initiate conformational interactions between the toxin and the receptor. Other amino acids involved in the receptor-binding process include aspartate and proline (3).
As stated above, the CPE binding properties differ between claudins depending on the presence of a motif responsible for enterotoxin affinity. Further, receptor expression levels also affect CPE-mediated GI disease. If claudin-3 and 4 are highly expressed in mammalian intestines, CPE-induced cytotoxicity levels will be high since these receptors bind strongly to CPE to form CH-1 (3). However, claudin-8 and claudin-14 exhibit weaker binding to this enterotoxin, especially at lower toxin doses. Therefore, in vivo C. perfringens type-A food poisoning primarily involves two proteins, namely, claudin-3 and 4, which mediate GI disease at significant CPE doses (6). CPE action in intestinal epithelial cells mainly occurs through these receptors, which are expressed on the surface of tight junctions. Their expression levels are high, and thus, they are the primary binding sites for CPE.
Structural biology studies have shown that the two hairpin loops of claudins – denoted as ECL-1 and ECL-2 – are involved in CPE-receptor interactions (6). ECL-1 is considered responsible for membrane permeability that triggers ionic fluxes and subsequent apoptotic events. The receptors have a C-terminal and N-terminal tail that play a role in receptor binding. They show significant sequence variability, which is a challenge to vaccine development. The C-terminal of the CPE toxin is the motif that binds to the claudins.
Studies attribute receptor affinity to the enterotoxin to diverse hydrophobic forces (6). Notably, the C-terminal of CPE and the N-terminal of claudin-2 binding motif interact by forming hydrogen bonds within a specific groove in the receptor (6). Therefore, hydrophobic contact determines if a receptor has an affinity for CPE and its binding strength. The interaction is critical to CPE-mediated food-borne diseases. It precedes pore formation that fosters calcium influx, which causes intestinal cell apoptosis that characterises histotoxic conditions associated with C. perfringens food poisoning.
Conclusion
C. perfringens is a virulent bacillus associated with GI disease linked to food poisoning. Ingested vegetative cells divide in vivo to attain a critical mass that then produces endospores. In CPE-mediated cytotoxicity, sporulation induces the phosphorylation of a specific regulator (Spo0A) that promotes sigma gene expression, resulting in the production of CPE. Upon its release, the enterotoxin binds to claudin receptors (usually claudin-3 and 4) expressed in mammalian intestinal TJs. This binding results in the formation of a transmembrane pore (CH-1) that fosters calcium influx into endothelial cells to cause calmodulin/calpain-mediated cell death that manifests as symptoms of CPE food-borne disease, i.e., diarrhoea and GI cramps.
References
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