C. perfringens alpha toxin

C. perfringens alpha toxin (CPA) is a 42.5 kDa single polypeptide with two domains. The ~250-amino-acid N-terminal domain is responsible for catalytic activity (described below), although it is not immunoprotective. The ~120-amino-acid C-terminal domain mediates host cell-binding activity in the presence of calcium and is immunoprotective. Recent studies reported that alpha toxin can bind to ganglioside GM1 on host cells.

CPA was the first bacterial toxin shown to have enzymatic properties. This toxin is a zinc metallophospholipase exhibiting both phospholipase C (lecithinase) and sphingomyelinase activities. CPA is lethal, with an LD₅₀ in mice of 3 μg/kg (Table 5.2). At high concentrations, the toxin can substantially damage plasma membranes to directly lyse host cells. However, at sublytic concentrations, CPA induces more limited cleavage of phosphotidylcholine and sphingomylelin, creating diacylglycerol and ceramide (Figure 5.1). Those molecules then activate several host signal transduction pathways which, via potent second messengers, induce hemolysis and a myriad of other effects, some of which are described below. An interesting recent study suggests that CPA may also be endocytosed, where it might act intracellularly.

Based on results from molecular Koch’s postulate analyses, CPA is considered to be the most important toxin when C. perfringens causes gas gangrene (clostridial myonecrosis). During histotoxic disease, CPA impairs leukocyte migration and increases the adhesion of leukocytes to form intravascular aggregates that impair blood flow and thereby promote necrosis. Since this necrotic tissue environment is anaerobic, it favors C. perfringens growth. Similar analyses using alpha toxin null mutants of NetB-positive type A or type C natural intestinal disease strains suggest CPA is much less important when C. perfringens causes disease originating in the intestines.

CPA is encoded by the cpa (also referred to as plc) gene, which is chromosomally located. This toxin is produced by almost all C. perfringens strains, regardless of their type. Production of CPA is regulated by the VirS/VirR two-component regulatory system (TCRS). The cpa (plc) gene does not have VirR boxes upstream of its promoter, so this VirS/VirR regulation is indirect, involving a regulatory RNA named VR-RNA. Recently, expression of CPA was also shown to involve regulation by the Agr-like quorum-sensing (QS) system.

C. perfringens beta toxin

C. perfringens beta toxin (CPB) is expressed as a 336-amino-acid prototoxin, containing a 27-amino-acid leader sequence that is subsequently removed during secretion from the cell to generate a mature ~35 kDa polypeptide. The structure of CPB has not been resolved, but based upon its partial sequence homology with other toxins, CPB is considered a member of the β-pore-forming toxin (β-PFT) family. Like all β-PFTs, CPB is predicted to contain three functional domains: binding, oligomerization, and membrane insertion/pore formation (see below).

C. perfringens epsilon toxin

C. perfringens type B and D strains secrete a relatively inactive ~33 kDa single polypeptide prototoxin that is proteolytically activated, creating the mature, ~29 kDa epsilon toxin (ETX). This activation can be caused by trypsin, chymotrypsin, or, possibly, C. perfringens lambda toxin in the intestine, and removes both N- and C-terminal amino acids from the prototoxin, but it is removal of the C-terminal amino acids that is critical for ETX activation. Once activated, ETX is the third most potent of all clostridial toxins, with an LD₅₀ of ~100 ng/kg that ranks it behind only the botulinum and tetanus toxins.

ETX belongs structurally to the aerolysin family of pore-forming toxins. The mature ETX protein consists of three domains. Domain I is thought to be the region interacting with receptors on host cells, while Domain II contains an amphipathic loop that inserts into membranes during pore formation.

The first step in ETX action involves the binding of this toxin to a receptor. The identity of this receptor(s) is not yet clear, although hepatitis A virus cellular receptor 1 (HAVCR-1) has some properties expected of an ETX receptor. Once bound, ETX uses lipid rafts and caveolins to oligomerize into a heptameric pre-pore on the host cell membrane surface. It then extends a β-hairpin loop into the lipid bilayer to form an active pore. This pore formation results in a rapid decrease in cytoplasmic K⁺ levels, with a corresponding influx of Na⁺ and Cl⁻ into these cells (Figure 5.1).

The rapid loss of K⁺ is believed to cause cellular necrosis involving a rapid decrease in nucleus size without any DNA fragmentation. This process is not yet fully understood but involves ATP depletion. However, methyl beta cyclodextrin reportedly prevents pore formation but not cell necrosis, suggesting additional processes beyond pore formation may contribute to ETX-induced cytotoxicity.

During infection, ETX prototoxin is produced by type B or D strains growing in the intestines. After its activation by intestinal proteases, as discussed earlier, or possibly C. perfringens lambda toxin, active ETX can affect the intestines (in goats) or it can be absorbed into the circulation (goats, sheep, and mice). The ability of ETX to traverse the intestinal epithelium even in the absence of intestinal damage suggests it may exploit paracellular permeability routes to reach the circulation. Once present in the circulation, ETX binds to internal organs, including the brain and kidneys. In the brain, ETX binds to endothelial cells and then alters the blood–brain barrier, which leads to the hallmark lesion of ETX brain effects; that is, perivascular edema that can develop rapidly. In animals with sub-acute and chronic disease, the brain lesions become necrotic and hemorrhagic and are known as focal symmetrical encephalomalacia. Another important brain effect of ETX involves increasing release of the stimulatory neurotransmitter glutamate. This effect is considered the major cause of neurologic symptoms observed during type B or D infections.

Insertion sequences are located adjacent to the etx gene in both type B and D strains and those sequences can excise the etx gene from plasmids to form small circular DNAs that could be transposition intermediates. It is possible that those putative transposons may mobilize the etx gene to move between plasmids in C. perfringens. This etx mobilization could help to explain the diversity of etx plasmids detected amongst type D strains. However, only a single etx plasmid is present in type B strains, which could reflect plasmid incompatibility issues – in other words, perhaps only certain combinations of etx plasmids and cpb plasmids can be stably maintained together in type B strains.

ETX is produced during log-phase growth. The regulation of ETX production is not well understood, despite some recent progress in this area. Contact with host Caco-2 cells was shown to upregulate ETX production and the Agr-like QS system is necessary for this host-cell-induced increase in ETX production by type D strain CN3718. However, ETX production is independent of the Agr-like QS system in two type B strains, indicating strain-to-strain (and perhaps type-to-type) differences in ETX regulatory control. Interestingly, the VirS/VirR TCRS does not appear to control ETX production by CN3718, suggesting there is not always regulatory cross-talk between the Agr-like QS and the VirS/VirR systems. Very recently, the CodY protein has been identified as a global regulator of virulence-associated properties of CN3718, including ETX production and bacterial adhesion to host cells.

Iota toxin

Iota toxin (ITX) has several distinguishing properties amongst C. perfringens toxins. First, ITX is one of only two toxins made by this bacterium (TpeL is the other) that clearly acts intracellularly. Second, ITX is the only C. perfringens toxin comprised of more than one polypeptide. ITX belongs to the clostridial binary toxin family because it consists of two polypeptides named IA and IB. Other members of this family include CDT of Clostridium difficile, the iota-like toxin of Clostridium spiroforme, and C2 toxin of C. botulinum.

Both the IA and IB components of ITX are produced as propeptides that are proteolytically activated by intestinal enzymes or, perhaps, C. perfringens lambda toxin. Once activated, the ~74 kDa IB binds as a monomer to receptors on host cells. Recent reports indicate that IB interacts with the lipolysis-stimulated lipoprotein (LSR), which is also a receptor for C. difficile and C. spiroforme binary toxins (although LSR is not a C2 toxin receptor). The presence of multifunctional mammalian surface protein CD44 also reportedly increases ITX binding, so CD44 could be a second receptor or a co-receptor for ITX. Once bound to host cells, IB monomers use lipid rafts to oligomerize into a heptamer. This heptamer then binds the ~48 kDa IA, followed by internalization of the entire ITX into cells by endocytosis. IA escapes from the early endosome and enters the cytoplasm, where it ADP ribosylates actin at residue Arg-177, causing a collapse of the cytoskeleton and cell death (Figure 5.1).

ITX is produced by the iap and ipb genes, which are co-transcribed in an operon and present on very large plasmids. Those plasmid-borne ITX genes are located near IS1151 sequences and either functional or silent cpe gene sequences. The ITX plasmids carry a tcp region, suggesting that they are conjugative. In addition, IS1151 sequences are associated with the iap/ibp genes, leading to the suggestion that the ITX genes have inserted onto cpe plasmids, converting progenitor isolates from type A to type E.

The involvement of ITX in disease remains unclear, although type E strains have been associated with enterotoxemias in calves, lambs, and, perhaps, rabbits.

C. perfringens enterotoxin

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