1.C.5 The Channel-forming ε-toxin (ε-toxin) Family
The ε-toxin family consists of ε-toxin produced by Clostridium perfringens types B and D, which are responsible for a rapidly fatal enterotoxemia in sheep and other animals, as well as the mosquitocidal toxins, Mtx2 and Mtx3, produced by Bacillus sphaericus. These proteins are synthesized as relatively inactive prototoxins which are converted to active mature toxins by proteolytic removal of basic N-terminal peptides. ε-toxin increases intestinal and kidney cell permeability, forming a membrane complex of about 155 kDa which promotes efflux of intracellular K+ from target animal cells. The asymmetric pore complex (Nestorovich et al., 2010) is permeable to propidium ions and forms preferentially in the apical rather than the basolateral membrane (Petit et al., 2003). The mechanism of action of the mosquitocidal toxin is not known, but as these proteins are 20 to 27% identical to the ε-toxin of C. perfringens, they presumably function by a similar mechanism.
A 120-residue region of the ε-toxin of Clostridium perfringens (TC #1.C.5.1.1) shows significant sequence similarity to the pore-forming region of the pesticidal crystal protein Cry15Aa (insecticidal δ-endotoxin CryXVA(a)), and the first 77 residues of the epsilon toxin show sequence similarity with the first 82 residues of the beta-2 toxin of C. perfringens (TC #1.C.69). Cry15Aa is not demonstrably homologous to members of the channel-forming δ-endotoxin insecticidal crystal protein (ICP) family (TC #1.C.2). ε-toxin consists of a beta-barrel of 14 amphipatic beta strands (Popoff, 2011). The evidence presented by Knapp et al. (2009) suggests that the Aerolysin and RTX superfamilies may be distantly related.
ε-toxin (ETX) acts by heptamer formation and rapid permeabilization of target cell membranes for monovalent ions with later entry of calcium. Knapp et al. (2009) compared the primary structure of ETX with that of the channel-forming stretches of a variety of binding components of A-B-types of toxins such as Anthrax protective antigen (PA), C2II of C2-toxin and Ib of Iota-toxin and found homology to amino acids 151-180 of ETX. Site-directed mutagenesis of amino acids within the putative channel-forming domain resulted in changes of cytotoxicity and effects on channel characteristics in lipid bilayer experiments including changes in selectivity and partial channel block by methanethiosulfonate (MTS) reagents and antibodies against His(6)-tags from the trans-side of the lipid bilayer membranes.
The generalized transport reaction probably catalyzed by ε-toxin family members is:
Small solutes (in) small solutes (out)
ε-toxin (epsilon toxin; ETx) type B precursor, EtxB, of 328 aas. Forms heptameric pores (Miyata et al. 2002). However, it has been reported to act on oligodendrocytes causing demyelination without forming pores (Wioland et al. 2015). The toxin acts on the brain, affecting vascular permeability, but also damaging neurons, astrocytes and oligodendrocytes (Freedman et al. 2016). The pore-forming regions are initially folded up on the surfaces of the soluble precursors. To create the transmembrane pores, these regions must extend and refold into membrane-inserted beta-barrels (Tilley and Saibil 2006).
EtxB of Clostridium perfringens
Uncharacterized protein of 319 aas, Sip1A
Sip1A of Lysinibacillus sphaericus
Parasporal crystal protein C53
C53 of Bacillus thurengiensis (Q45728)