| Abstract: | Large glycosylating toxins are major virulence factors of various species of pathogenic Clostridia. Prototypes are Clostridium difficile toxins A and B, which cause antibiotics-associated diarrhea and pseudomembranous colitis. The current model of the toxins’ action suggests that receptor binding is mediated by a C-terminal domain of combined repetitive oligopeptides (CROP). This model is challenged by the glycosylating Clostridium perfringens large cytotoxin (TpeL toxin) that is devoid of the CROP domain but still intoxicates cells. Using a haploid genetic screen, we identified LDL receptor-related protein 1 (LRP1) as a host cell receptor for the TpeL toxin. LRP1-deficient cells are not able to take up TpeL and are not intoxicated. Expression of cluster IV of LRP1 is sufficient to rescue toxin uptake in these cells. By plasmon resonance spectroscopy, a KD value of 23 nM was determined for binding of TpeL to LRP1 cluster IV. The C terminus of TpeL (residues 1335–1779) represents the receptor-binding domain (RBD) of the toxin. RBD-like regions are conserved in all other clostridial glycosylating toxins preceding their CROP domain. CROP-deficient C. difficile toxin B is toxic to cells, depending on the RBD-like region (residues 1349–1811) but does not interact with LRP1. Our data indicate the presence of a second, CROP-independent receptor-binding domain in clostridial glycosylating toxins and suggest a two-receptor model for the cellular uptake of clostridial glycosylating toxins.Clostridial glycosylating toxins are major pathogenicity factors that are responsible for numerous severe diseases in humans and animals. Prototypes of these toxins are Clostridium difficile toxins A and B, the causative agents of antibiotics-associated diarrhea and pseudomembranous colitis (1, 2). During recent years, morbidity and mortality of C. difficile-induced infections (CDI) largely increased (3, 4). In fact, CDI advanced to one of the most important nosocomial infections in developed countries. Other members of the family of clostridial glycosylating toxins are Clostridium sordellii lethal and hemorrhagic toxin, and the α-toxin from Clostridium novyi, which cause gas gangrene syndromes (5). All these toxins have a very similar primary structure comprising an amino acid sequence identity of 40–90% (1, 5). Recently, an ABCD model has been proposed for these toxins with an N-terminally located glycosyltransferase domain (domain A), a subsequent cysteine protease domain for autoproteolytic cleavage (domain C), a putative pore-forming and delivery domain (domain D), and a C-terminal binding domain (domain B) (6). After binding to cell surface receptors, the toxins are endocytosed in a clathrin-dependent and dynamin-dependent manner (7). At a low pH of endosomes, the toxins insert into endosomal membranes and form pores, which probably allow translocation of the glycosyltransferase (GT) and cysteine protease domains into the cytosol where inositol hexakisphosphate activates the protease for autoproteolytic cleavage and release of the GT into the cytosol (8–10). In the cytosol, Rho and/or Ras proteins are glucosylated or modified by GlcNAcylation, resulting in inhibition of these switch proteins and, eventually, in inflammation and cell death (11–13). Although autoproteolytic processing and toxin-induced glycosylation of Rho/Ras proteins are well characterized, the interaction of the toxins with cell surface receptors is still enigmatic. Cell surface-binding is suggested to be mediated by C-terminal polypeptide repeats (B domain) termed combined repetitive oligopeptides (CROP) that might recognize cell surface carbohydrate structures (14–17). Recombinant fragments of this C-terminal toxin part blocks toxin binding and cytotoxicity (18). Moreover, monoclonal antibodies raised against this toxin portion prevent cell intoxication (19). Putative receptors have been described for C. difficile toxin A, including carbohydrates, glycophospholipids, and proteins (16, 17, 20, 21). The hypothesis that the C-terminal part of clostridial glycosylating toxins is solely responsible for receptor interaction has been challenged. For example, after removal of the CROP domain of toxin A or toxin B, the toxins were still cytotoxic (10, 22). Thus, we and others hypothesized that a receptor binding site different from the CROP domain might be involved in toxin binding.Recently, TpeL, the most recent member of the family of clostridial glycosylating toxins that is produced by Clostridium perfringens type A, B, and C strains, was described (23). TpeL exhibits 30–40% amino acid sequence identity with other clostridial glycosylating toxins and shares with them the glycosyltransferase domain, the cysteine protease domain, and the delivery domain. However, it does not possess a CROP domain. Nevertheless, TpeL intoxicates cells and kills mice (23). Toxic effects of TpeL are probably due to GlcNAcylation of Ras proteins at threonine-35 (24). In addition, TpeL-induced modification of Rac has been reported (25).Thus, TpeL is a model toxin to unravel the interaction of clostridial glycosylating toxins with target cells. Here, we identify the low-density lipoprotein receptor-related protein 1 (LRP1) as a target molecule for binding and cell entry of TpeL. We report that the C-terminal part of TpeL binds to LRP1 and represents the receptor-binding domain. Furthermore, we show that the respective part within C. difficile toxin B, which resembles to the receptor-binding domain of TpeL, binds to cells. Our study strongly supports a two-receptor model of clostridial glycosylating toxins and offers an additional perspective in the understanding of the pathogenicity of this group of clinically important toxins. |
