Clostridium perfringens Iota-Toxin: Mapping of Receptor Binding and Ia Docking Domains on Ib

Abstract

Clostridium perfringens iota-toxin is a binary toxin consisting of iota a (Ia), an ADP-ribosyltransferase that modifies actin, and iota b (Ib), which binds to a cell surface protein and translocates Ia into a target cell. Fusion proteins of recombinant Ib and truncated variants were tested for binding to Vero cells and docking with Ia via fluorescence-activated cytometry and cytotoxicity experiments. C-terminal residues (656 to 665) of Ib were critical for cell surface binding, and truncated Ib variants containing >200 amino acids of the C terminus were effective Ib competitors and prevented iota cytotoxicity. The N-terminal domain (residues 1 to 106) of Ib was important for Ia docking, yet this region was not an effective competitor of iota cytotoxicity. Further studies showed that Ib lacking just the N-terminal 27 residues did not facilitate Ia entry into a target cell and subsequent cytotoxicity. Five monoclonal antibodies against Ib were also tested with each truncated Ib variant for epitope and structural mapping by surface plasmon resonance and an enzyme-linked immunosorbent assay. Each antibody bound to a linear epitope within the N terminus (residues 28 to 66) or the C terminus (residues 632 to 655). Antibodies that target the C terminus neutralized in vitro cytotoxicity and delayed the lethal effects of iota-toxin in mice.Clostridium perfringens type E produces iota-toxin, a binary toxin that consists of two nonlinked proteins implicated in sporadic diarrheic outbreaks among animals (6, 28, 32). The 47-kDa enzymatic component, designated iota a (Ia), docks with and is translocated by the 80-kDa iota b (Ib) into a target cell. Iota-toxin belongs to an A-B toxin family possessing nonlinked proteins like Clostridium botulinum C2 toxin (12), Clostridium spiroforme iota-like toxin (23, 29), anthrax toxin from Bacillus anthracis (14), and the vegetative insecticidal protein (VIP) produced by Bacillus cereus (11).The current understanding of iota-toxin is mainly centered around the Ia molecule (16,18,34,35), while the structurefunction relationship of Ib in iota toxicity is poorly understood. Like many clostridial toxins, Ia and Ib are activated by proteases (10). The protoxin form of Ib contains a 20-kDa aminoterminal peptide released after proteolysis. Upon binding to a protein receptor (30), activated Ib docks with Ia and facilitates entry of Ia into a cell via receptor-mediated endocytosis (23). Once inside a cell, an Ia molecule ADP-ribosylates monomeric actin and consequently disrupts formation of actin filaments necessary for the cytoskeleton, with resultant cell rounding and death (18,35). Either Ia or Ib functionally complements the serologically related iota-like toxin proteins (Sa and Sb) produced by C. spiroforme (29). There is no biological synergy of either iota or iota-like components with other A-B proteins from anthrax (20) or C2 (22, 24, 25) toxins. However, there is a 34 and 41% sequence homology between Ib and the cellbinding proteins of anthrax toxin (protective antigen; PA) and C2 toxin (...

Binary Bacterial Toxins: Biochemistry, Biology, and Applications of CommonClostridiumandBacillusProteins

“…Other studies of domain 4 reveal that truncations of only 5 to 12 amino acids from the far C terminus of PA prevent binding to cells (401,449), suggesting an important role in direct binding and/or conformational integrity of PA (230). Similar investigations with C2II and Ib also unveil a pattern of exquisite sensitivity regarding deletions within the C terminus and subsequent effects on biological activity (48,252), thus demonstrating a conserved structural trait among "B" components from this binary-toxin family.…”

“…For instance, domains 1 (amino acids 1 to 259) and 4 (amino acids 597 to 735) of PA83 are respectively responsible for (i) docking with EF and/or LF and (ii) binding to the cell surface via protein receptors recently identified as variants 1 and 2 of tumor endothelium marker 8, as well as human capillary morphogenesis protein 2 (60,64,80,86,92,243,245,326,361,379,401,449). There is relatively little amino acid homology of PA domains 1 and 4 to their equivalents from other Clostridium and Bacillus binary toxins, which is not surprising since these regions respectively afford a unique docking site for distinct "A" components and receptor-binding specificity (33,48,252,325,418). Domain 1 contains the proteolytic cleavage site (R 167 ) that subsequently triggers the release of a 20-kDa precursor peptide and formation of PA63 heptamers (211).…”

“…Like PA and C2II, the importance of various domains for Ib activity has been ascertained via deletion mutagenesis and antibody studies (252,420). Similar to PA and C2II, truncation of 10 amino acids from the C terminus (domain 4) abrogates Ib binding to Vero cells, and Ib peptides containing Ն200 Cterminal residues represent competitive inhibitors of cytotoxicity in vitro (252).…”

Bacterial protein toxins and lipids: pore formation or toxin entry into cells

“…The C-terminal domain (domain 4) is involved in the cell-surface-receptor recognition. The C-terminus of Ib and C2-II, B components of iota and C2 toxins respectively, corresponding to domain 4 of PA, have also been identified as receptor-binding domains (Blöcker et al, 2001;Marvaud et al, 2001). Domain 3, which is the smallest, is likely to play a role in oligomerization, and domain 2 contains long β-strands and, in particular, an amphipathic flexible loop (amino acids 302-325) that forms a β-hairpin, playing a central role in pore formation (Petosa et al, 1997;Cunningham et al, 2002;Mogridge et al, 2002a).…”

“…The flexible loop of PA domain 2 partially detaches from the monomer and associates with the six other loops of the neighbouring monomers to form a β-barrel that inserts into the membrane, similar to α-haemolysin and leukocidin from S. aureus. Interestingly, leukocidins are also binary toxins involved in pore formation, but remain on the cell surface, whereas PA enables the translocation of partially unfolded enzymatic components into the cytosol through a pH gradient (Blöcker et al, 2001;Marvaud et al, 2001;Cunningham et al, 2002;Mogridge et al, 2002aMogridge et al, , 2002bAbrami et al, 2003).…”