Epsilon toxin is essential for the virulence of Clostridium perfringens type D infection in sheep, goats, and mice.

Clostridium perfringens type D causes disease in sheep, goats, and other ruminants. Type D isolates produce, at minimum, alpha and epsilon (ETX) toxins, but some express up to five different toxins, raising questions about which toxins are necessary for the virulence of these bacteria. We evaluated the contribution of ETX to C. perfringens type D pathogenicity in an intraduodenal challenge model in sheep, goats, and mice using a virulent C. perfringens type D wild-type strain (WT), an isogenic ETX null mutant (etx mutant), and a strain where the etx mutation has been reversed (etx complemented). All sheep and goats, and most mice, challenged with the WT isolate developed acute clinical disease followed by death in most cases. Sheep developed various gross and/or histological changes that included edema of brain, lungs, and heart as well as hydropericardium. Goats developed various effects, including necrotizing colitis, pulmonary edema, and hydropericardium. No significant gross or histological abnormalities were observed in any mice infected with the WT strain. All sheep, goats, and mice challenged with the isogenic etx mutant remained clinically healthy for ≥24 h, and no gross or histological abnormalities were observed in those animals. Complementation of etx knockout restored virulence; most goats, sheep, and mice receiving this complemented mutant developed clinical and pathological changes similar to those observed in WT-infected animals. These results indicate that ETX is necessary for type D isolates to induce disease, supporting a key role for this toxin in type D disease pathogenesis.

The enteric toxins of Clostridium perfringens.

The Gram-positive pathogen Clostridium perfringens is a major cause of human and veterinary enteric disease largely because this bacterium can produce several toxins when present inside the gastrointestinal tract. The enteric toxins of C. perfringens share two common features: (1) they are all single polypeptides of modest (approximately 25-35 kDa) size, although lacking in sequence homology, and (2) they generally act by forming pores or channels in plasma membranes of host cells. These enteric toxins include C. perfringens enterotoxin (CPE), which is responsible for the symptoms of a common human food poisoning and acts by forming pores after interacting with intestinal tight junction proteins. Two other C. perfringens enteric toxins, epsilon-toxin (a bioterrorism select agent) and beta-toxin, cause veterinary enterotoxemias when absorbed from the intestines; beta- and epsilon-toxins then apparently act by forming oligomeric pores in intestinal or extra-intestinal target tissues. The action of a newly discovered C. perfringens enteric toxin, beta2 toxin, has not yet been defined but precedent suggests it might also be a pore-former. Experience with other clostridial toxins certainly warrants continued research on these C. perfringens enteric toxins to develop their potential as therapeutic agents and tools for cellular biology.

Osmotic stabilizers differentially inhibit permeability alterations induced in Vero cells by Clostridium perfringens enterotoxin.

Using a sensitive Vero (African green monkey kidney) cell model system, studies were performed to further investigate whether Clostridium perfringens enterotoxin acts via disruption of the colloid-osmotic equilibrium of sensitive cells. Enterotoxin was shown to cause a rapid loss of intracellular 86Rb+ (Mr approx. 100) with time- and dose-dependent kinetics. The enterotoxin-induced release of intracellular 86Rb+ preceded the loss of two larger labels, 51Cr label (Mr approx. 3500) and 3H-labeled nucleotides (Mr less than 1000). The osmotic stabilizers, sucrose and poly(ethylene glycol), differentially inhibited enterotoxin-induced larger label loss versus 86Rb+ loss. Further, enterotoxin was shown to cause a rapid influx of 24Na+ that was not significantly inhibited by osmotic stabilizers. Additional studies demonstrated that lysosomotropic agents were not protective against characteristic enterotoxin-induced membrane permeability alterations or morphological damage. Taken collectively, these results are consistent with an action for enterotoxin which involves a disruption of the osmotic equilibrium.

Characterization of membrane permeability alterations induced in Vero cells by Clostridium perfringens enterotoxin.

Alterations in plasma membrane permeability induced by Clostridium perfringens enterotoxin were studied using Vero (African green monkey kidney) cells which were radioactively labeled with four markers of different molecular size. The markers were alpha-amino[14C]isobutyric acid (Mr 103), 3H-labeled nucleotide (Mr approx. 300), 51Cr label (Mr approx. 3000) and [3H]RNA (Mr>25000). Over a 2h period, enterotoxin caused significant release of aminoisobutyric acid, nucleotides and 51Cr label but not RNA. The effects of enterotoxin on label release were dose- and time-dependent. The rate of release of markers was dependent upon their size. Permeability alterations could be detected within 15 min with a high dose of enterotoxin. Gel chromatography of released material was used to determine that markers of Mr 3000 but not 25000 leaked from permeabilized cells. It was concluded that enterotoxin is producing functional 'holes' of limited size in the membrane. Permeability changes due to enterotoxin treatment differed between confluent and nonconfluent (growing) cells. We propose that the primary action of the enterotoxin is to interact with the plasma membrane and produce functional 'holes' of defined size. The resultant alterations in membrane permeability cause the loss of essential cellular substances which inhibits processes such as macromolecular synthesis and eventually leads to cell deterioration and death.

Protective effects of osmotic stabilizers on morphological and permeability alterations induced in Vero cells by Clostridium perfringens enterotoxin.

Culture medium made hypertonic by the addition of osmotic stabilizers such as sucrose, poly(ethylene glycol), dextran and bovine serum albumin protected against changes in morphology and plasma membrane permeability induced by Clostridium perfringes enterotoxin. The protection did not appear to be due to binding inhibition. Results of these studies support an osmotic disruption mechanism for the action of the enterotoxin. A comprehensive model of the enterotoxin's action based on an osmotic disruption mechanism is proposed.

Development and preliminary evaluation of a slide latex agglutination assay for detection of Clostridium perfringens type A enterotoxin.

A slide latex agglutination (SLA) assay was developed for rapid screening for Clostridium perfringens type A enterotoxin (CPE). SLA specifically detected CPE added to buffer or normal feces (sensitivity limit of 1 microgram CPE/g feces). Using clinical fecal samples from C. perfringens food poisoning cases, a strong correlation was shown between SLA results and results from other CPE assays and between SLA results and illness status.

An overview of Clostridium perfringens enterotoxin.

Clostridium perfringens enterotoxin (CPE) is considered to be the virulence factor responsible for causing the symptoms of C. perfringens type A food poisoning and may also be involved in other human and veterinary illnesses. CPE has a unique four-step membrane action that apparently involves: (1) CPE binding to a 50,000 mol. wt mammalian protein receptor, forming a small complex of 90,000 mol. wt; (2) the development of a post-binding physical change to this small complex; this physical change could represent either the insertion of CPE into the membrane or a conformational change to small complex; (3) an interaction between this physically changed small complex and a 70,000 mol. wt mammalian protein, forming a large, 160,000 mol. wt complex in membranes; and (4) a breakdown in normal plasma membrane permeability properties for small (< 200,000 mol. wt) molecules. Structure-function analyses have identified a receptor binding region at the C-terminus of CPE and indicate that residues in the N-terminal half of CPE are required for the second step in CPE action to occur. Finally, cpe genetic studies are in their infancy but already indicate that cpe can be either chromosomal or plasmid-borne and that only a tiny minority of the global C. perfringens population is cpe positive. CPE expression appears to be transcriptionally regulated during sporulation, at least in part, by regulatory factors that are common to all C. perfringens isolates.

The complex interactions between Clostridium perfringens enterotoxin and epithelial tight junctions.

Clostridium perfringens enterotoxin (CPE) is responsible for the diarrheal symptoms of C. perfringens type A food poisoning and antibiotic-associated diarrhea. The CPE protein consists of a single 35 kDa polypeptide with a C-terminal receptor-binding region and an N-terminal toxicity domain. Under appropriate conditions, CPE can interact with structural components of the epithelial tight junctions, including certain claudins and occludin. Those interactions can affect tight junction structure and function, thereby altering paracellular permeability and (possibly) contributing to CPE-induced diarrhea. However, the tight junction effects of CPE require cellular damage as a prerequisite. CPE induces cellular damage via its cytotoxic activity, which results from plasma membrane permeability alterations caused by formation of a approximately 155 kDa CPE-containing complex that may correspond to a pore. Thus, CPE appears to be a bifunctional toxin that first induces plasma membrane permeability alterations; using the resultant cell damage, CPE then gains access to tight junction proteins and affects tight junction structure and function.

Clostridium perfringens enterotoxin acts by producing small molecule permeability alterations in plasma membranes.

Clostridium perfringens enterotoxin (CPE) appears to utilize a unique mechanism of action to directly affect the plasma membrane permeability of mammalian cells. CPE action involves a multi-step action which culminates in cytotoxicity. Initially CPE binds to a protein receptor on mammalian plasma membranes. The membrane-bound CPE then becomes progressively more resistant to release by proteases (a phenomenon consistent with the insertion of CPE into membranes). This 'inserted' CPE then participates in the formation of a large complex in plasma membranes which contains one CPE: one 70 kDa membrane protein: one 50 kDa membrane protein. Upon formation of large complex, plasma membranes become freely permeable to small molecules such as ions and amino acids. This CPE-induced disruption of the cellular colloid-osmotic equilibrium then causes secondary cellular effects and cell death.

An ultrastructural comparison of spores from various strains of Clostridium perfringens and correlations with heat resistance parameters.

It has been shown that Clostridium perfringens isolates associated with food poisoning carry a chromosomal cpe gene, whereas nonfood-borne human gastrointestinal disease isolates carry a plasmid cpe gene. In addition, the chromosomal cpe gene isolates exhibit greater heat resistance as compared with the plasmid cpe strains. Therefore, the current study conducted ultrastructural measurements of spores from several plasmid and chromosomal cpe-positive C. perfringens isolates. In support of the dehydration mechanism of spore heat resistance, the C. perfringens spore core average size was found to show a negative correlation with D-values for spores obtained at 100 degrees C. Dipicolinic acid (DPA) concentrations assayed for the spores did not correlate well with C. perfringens spore core averages nor with D(10)-values at 100 degrees C. Spore core thickness might be a distinguishing phenotypic characteristic used to identify heat resistance and survival potential of C. perfringens in improperly cooked foods.

The effect of Clostridium perfringens type C strain CN3685 and its isogenic beta toxin null mutant in goats.

Clostridium perfringens type C is an important cause of enteritis and/or enterocolitis in several animal species, including pigs, sheep, goats, horses and humans. The disease is a classic enterotoxemia and the enteric lesions and associated systemic effects are thought to be caused primarily by beta toxin (CPB), one of two typing toxins produced by C. perfringens type C. This has been demonstrated recently by fulfilling molecular Koch's postulates in rabbits and mice. We present here an experimental study to fulfill these postulates in goats, a natural host of C. perfringens type C disease. Nine healthy male or female Anglo Nubian goat kids were inoculated with the virulent C. perfringens type C wild-type strain CN3685, an isogenic CPB null mutant or a strain where the cpb null mutation had been reversed. Three goats inoculated with the wild-type strain presented abdominal pain, hemorrhagic diarrhea, necrotizing enterocolitis, pulmonary edema, hydropericardium and death within 24h of inoculation. Two goats inoculated with the CPB null mutant and two goats inoculated with sterile culture media (negative controls) remained clinically healthy during 24h after inoculation and no gross or histological abnormalities were observed in the tissues of any of them. Reversal of the null mutation to partially restore CPB production also increased virulence; 2 goats inoculated with this reversed mutant presented clinical and pathological changes similar to those observed in goats inoculated with the wild-type strain, except that spontaneous death was not observed. These results indicate that CPB is required for C. perfringens type C to induce disease in goats, supporting a key role for this toxin in natural C. perfringens type C disease pathogenesis.

Recent progress in understanding the pathogenesis of Clostridium perfringens type C infections.

Clostridium perfringens type C causes necrotizing enteritis in humans and several other animal species. Type C isolates must produce at least beta toxin (CPB) and alpha toxin (CPA) and most strains produce several other toxins including perfringolysin O (PFO) and TpeL. However, current evidence indicates that CPB is the main virulence factor for type C infections. Most of this evidence is based upon the loss of virulence shown by isogenic type C CPB knock out mutants on cells, and also in rabbit intestinal loops and in mouse models. This virulence is regained when these mutants are complemented with the wild-type cpb gene. Many type C isolates respond to close contact with enterocyte-like Caco-2 cells by producing all toxins, except TpeL, much more rapidly than occurs during in vitro growth. This in vivo effect involves rapid transcriptional upregulation of the cpb, cpb2, pfoA and plc toxin genes. Rapid Caco-2 cell-induced upregulation of CPB and PFO production involves the VirS/VirR two-component system, since upregulated in vivo transcription of the pfoA and cpb genes was blocked by inactivating the virR gene and was reversible by complementation to restore VirR expression.

Clostridium Perfringens Toxins Involved in Mammalian Veterinary Diseases.

Clostridium perfringens is a gram-positive anaerobic rod that is classified into 5 toxinotypes (A, B, C, D, and E) according to the production of 4 major toxins, namely alpha (CPA), beta (CPB), epsilon (ETX) and iota (ITX). However, this microorganism can produce up to 16 toxins in various combinations, including lethal toxins such as perfringolysin O (PFO), enterotoxin (CPE), and beta2 toxin (CPB2). Most diseases caused by this microorganism are mediated by one or more of these toxins. The role of CPA in intestinal disease of mammals is controversial and poorly documented, but there is no doubt that this toxin is essential in the production of gas gangrene of humans and several animal species. CPB produced by C. perfringens types B and C is responsible for necrotizing enteritis and enterotoxemia mainly in neonatal individuals of several animal species. ETX produced by C. perfringens type D is responsible for clinical signs and lesions of enterotoxemia, a predominantly neurological disease of sheep and goats. The role of ITX in disease of animals is poorly understood, although it is usually assumed that the pathogenesis of intestinal diseases produced by C. perfringens type E is mediated by this toxin. CPB2, a necrotizing and lethal toxin that can be produced by all types of C. perfringens, has been blamed for disease in many animal species, but little information is currently available to sustain or rule out this claim. CPE is an important virulence factor for C. perfringens type A gastrointestinal disease in humans and dogs; however, the data implicating CPE in other animal diseases remains ambiguous. PFO does not seem to play a direct role as the main virulence factor for animal diseases, but it may have a synergistic role with CPA-mediated gangrene and ETX-mediated enterotoxemia. The recent improvement of animal models for C. perfringens infection and the use of toxin gene knock-out mutants have demonstrated the specific pathogenic role of several toxins of C. perfringens in animal disease. These research tools are helping us to establish the role of each C. perfringens toxin in animal disease, to investigate the in vivo mechanism of action of these toxins, and to develop more effective vaccines against diseases produced by these microorganisms.

Clostridium perfringens enterotoxin damages the human intestine in vitro.

In vitro, Clostridium perfringens enterotoxin (CPE) binds to human ileal epithelium and induces morphological damage concurrently with reduced short-circuit current, transepithelial resistance, and net water absorption. CPE also binds to the human colon in vitro but causes only slight morphological and transport changes that are not statistically significant.

Characterization of calcium involvement in the Clostridium perfringens type A enterotoxin-induced release of 3H-nucleotides from Vero cells.

This report characterizes the involvement of Ca2+ in the release of nucleotides from Vero cells caused by Clostridium perfringens enterotoxin (CPE). A positive linear correlation was observed between increased CPE-induced nucleotide-release and increased extracellular calcium over the range 0.01 to 10 mM calcium. Above 5 mM Ca2+, CPE-specific lysis (i.e. disintegration of cells as monitored by light microscopy) was observed. Addition of 1.7 mM Ca2+ to Vero cells previously CPE-treated in Ca2+-free buffer rapidly increased nucleotide-release, even when cells had been previously incubated for 1 h at 37 degrees C in Ca2+-free buffer. Withdrawal of Ca2+, even after the onset of nucleotide-release, halted further CPE-induced nucleotide-release. These results indicate that Ca2+ must be continuously present for significant CPE-induced nucleotide-release. However, withdrawal of Ca2+ did not reverse membrane bleb formation by CPE. This differentiates bleb formation and nucleotide-release (both Ca2+-dependent CPE effects) and suggests that nucleotide-release does not result simply from bleb formation. Lastly, it was shown that other ions besides physiologic Ca2+ (1.7 mM) are required for CPE-induced nucleotide-release. Interestingly, a role for other ions (but not physiologic Ca2+) is also shown for 86Rb-release by CPE (an early Ca2+-independent CPE effect). This indicates that extracellular ions other than physiologic Ca2+ can be required for both Ca2+-independent and Ca2+-dependent CPE effects.

A recombinant C-terminal toxin fragment provides evidence that membrane insertion is important for Clostridium perfringens enterotoxin cytotoxicity.

Clostridium perfringens enterotoxin (CPE) is believed to be involved in several important gastrointestinal illnesses. Recent studies have identified a number of distinct molecular events which occur after CPE treatment of eukaryotic cells or isolated membranes. Additional studies are underway to determine the temporal order and intrinsic importance of each CPE event for cytotoxicity. We now demonstrate that a truncated CPE fragment binds to membranes, but is unable to insert into membranes or cause any other subsequent post-insertion event. This is the first experimental evidence supporting the importance of membrane insertion for CPE cytotoxicity. Binding of the CPE fragment is also shown to be irreversible, strongly suggesting that the irreversible binding of wild-type CPE is not due solely to insertion of CPE into membranes.

The effects of Clostridium perfringens enterotoxin on intracellular levels or transport of uridine, thymidine and leucine do not fully explain enterotoxin-induced inhibition of macromolecular synthesis in Vero cells.

Clostridium perfringens type A enterotoxin (CPE) has been shown previously to inhibit the incorporation of radiolabeled precursors into acid-insoluble material but the mechanism of inhibition is unknown. It has also been shown that extracellular calcium is required for some CPE effects. In this report, it is shown that CPE completely and virtually simultaneously inhibits incorporation of precursors into RNA, DNA and protein in either the presence or absence of extracellular divalent cations and that changes in intracellular precursor levels did not consistently correlate with this CPE-induced inhibition of incorporation. These results strongly suggest that CPE can inhibit macromolecular synthesis, not just inhibit precursor transport. It is inferred from this that CPE can affect DNA and RNA synthesis, and possibly protein synthesis, by altering other cellular processes besides, or in addition to, precursor transport and these effects then lead to a shutdown of macromolecular synthesis.