During bacterial infection, many protein toxins released by the bacteria have cytotoxic activity. This toxicity is often accomplished by disrupting protein synthesis or cytoskeletal structure or by compromising the integrity of the plasma membranes. In some cases, a toxin forms pores to deliver an enzymatic component into the cytoplasm (Tilley & Saibil, 2006
Pore-forming toxins (PFTs) are classified according to their architecture; for example, they can form an α-helical channel or a β-barrel. PFTs are a group of cytotoxic proteins with divergent protein structures (Anderluh & Lakey, 2008
) which have the same activity, i.e.
excessive permeability of a cell membrane, that ultimately leads to cell death. The loss in membrane fidelity caused by PFTs leads to the uncontrolled efflux of essential cytoplasmic components, such as amino acids, nucleotides, cofactors etc
., combined with an equally uncontrolled influx of Ca2+
ions and water, which can lead to the activation of cell-death pathways and unsustainable cell swelling, which can cause cell lysis (Gonzalez et al.
(CP) is a Gram-positive anaerobic rod-shaped spore-forming bacillus which is ubiquitous in the environment and is a natural inhabitant of the gastrointestinal tract of humans and animals (Songer & Uzal, 2005
). The bacterium CP is an aetiological agent that causes a wide variety of diseases in humans and domestic animals and has become a paradigm species because of its environmental ubiquity, fast growth rate and oxygen tolerance and its ability to produce a range of extracellular protein toxins, e.g.
-toxin, ι-toxin, enterotoxin (CPE) and several others. Some of these toxins are lethal and cause disease in humans and animals (Songer & Uzal, 2005
; Brynestad & Granum, 2002
). The ability of different strains of CP to cause disease has been ascribed mainly to differential production of these exotoxins (McClane et al.
). However, individual isolates produce a specific subset of these toxins and based on the production of α-, β-,
- and ι-toxins the bacterium CP can be classified into five different serotypes (A–E).
The enterotoxin (CPE) is the virulence determinant of type A food poisoning and other gastrointestinal (GI) diseases (McClane & Chakrabarti, 2004
). CPE is produced by type A strains and these strains also cause hospital-acquired and community-acquired antibiotic associated diarrhoea (AAD) and sporadic diarrhoea (SD), which are more severe than normal type A food-borne diseases. CPE has also been linked to some veterinary GI diseases (Meer et al.
CPE (UniProt P01558) is a single polypeptide chain consisting of 319 amino acids and with a molecular mass of ~35 kDa. This protein is produced in large amounts by C. perfringens
during sporulation (~15% of dry cell mass). In common with other PFTs, it is synthesized as a less active pre-protein and is probably activated upon release into the host gut by endogenous proteases such as trypsin and chymotrypsin (Granum, 1986
). One difference between CPE and other PFTs is that CPE is not actively secreted; rather, it is released upon cell lysis when the spore is released. After release and proteolytic activation in the mammalian gut, CPE associates with its receptor(s), which include members of the claudin family of transmembrane proteins that are found in tight junctions (McClane, 2001
Whilst some molecular events leading to CPE pore formation are presently unclear, SDS-resistant CPE-containing complexes have been isolated with masses of 425–500 and 550–660 kDa and are thought to consist of a CPE hexamer and differing amounts of various CPE-receptor claudins, nonreceptor claudins and (for the 550–660 kDa complex) occludin (Robertson et al.
). Claudins and occludin are found in tight junctions between cells in gut epithelia and are involved in maintaining the integrity of the tight junction (Hossain & Hirata, 2008
). In addition to this, they also act as functional receptors for CPE, allowing toxin concentrations to be maximized at the cell membrane. This in turn facilitates CPE pore formation.
CPE also contains a sequence pattern containing alternating hydrophobic and hydrophilic amino-acid residues (residues 81–106) that is characteristic of amphipathic β-strands seen in β-pore-forming toxins, and pre-pore complexes have also been identified biochemically (Smedley et al.
). Because of these observations, CPE is presumed to be a novel variant of the β-pore-forming toxins. The structure of the claudin-binding domain (residues 197–319) has recently been solved (Van Itallie et al.
); however, since the N-terminal CPE sequences are required for oligomerization and pore formation (Kokai-Kun & McClane, 1996
; Smedley et al.
), it is important to solve the structure of the native CPE protein in order to fully understand this toxin’s action.
Over the last few decades, CPE has been the subject of intense research; however, how the CPE protein mediates its action during disease is not yet fully understood at the molecular level. Here, we report the crystallization and preliminary X-ray diffraction data characterization of CPE.