Clostridium difficile infection (CDI) causes antibiotic-associated diarrhea and pseudomembranous colitis. Hypervirulent strains of the pathogen, which are responsible for increased morbidity and mortality of CDI, produce the binary actin-ADP ribosylating toxin Clostridium difficile transferase (CDT) in addition to the Rho-glucosylating toxins A and B. CDT depolymerizes the actin cytoskeleton, increases adherence and colonization of Clostridia by induction of microtubule-based cell protrusions and, eventually, causes death of target cells. Using a haploid genetic screen, we identified the lipolysis-stimulated lipoprotein receptor as the membrane receptor for CDT uptake by target cells. Moreover, we show that Clostridium perfringens iota toxin, which is a related binary actin-ADP ribosylating toxin, enters target cells via the lipolysis-stimulated lipoprotein receptor. Identification of the toxin receptors is essential for understanding of the toxin uptake and provides a most valuable basis for antitoxin strategies.

Clostridium difficile toxin A (TcdA) and toxin B (TcdB), C. sordellii lethal toxin (TcsL) and C. novyi α-toxin (TcnA) are important pathogenicity factors, which represent the family of the clostridial glucosylating toxins (CGTs). Toxin A and B are associated with antibiotic-associated diarrhea and pseudomembraneous colitis. Lethal toxin is involved in toxic shock syndrome after abortion and α-toxin in gas gangrene development. CGTs enter cells via receptor-mediated endocytosis and require an acidified endosome for translocation of the catalytic domain into the cytosol. Here we studied the endocytic processes that mediate cell internalization of the CGTs. Intoxication of cells was monitored by analyzing cell morphology, status of Rac glucosylation in cell lysates and transepithelial resistance of cell monolayers. We found that the intoxication of cultured cells by CGTs was strongly delayed when cells were preincubated with dynasore, a cell-permeable inhibitor of dynamin, or chlorpromazine, an inhibitor of the clathrin-dependent endocytic pathway. Additional evidence about the role of clathrin in the uptake of the prototypical CGT family member toxin B was achieved by expression of a dominant-negative inhibitor of the clathrin-mediated endocytosis (Eps15 DN) or by siRNA against the clathrin heavy chain. Accordingly, cells that expressed dominant-negative caveolin-1 were not protected from toxin B-induced cell rounding. In addition, lipid rafts impairment by exogenous depletion of sphingomyelin did not decelerate intoxication of HeLa cells by CGTs. Taken together, our data indicate that the endocytic uptake of the CGTs involves a dynamin-dependent process that is mainly governed by clathrin.

Summary: Clostridium difficiletoxins A and B bind to eukaryotic target cells, are endocytosed and then deliver their N‐terminal glucosyltransferase domain after processing into the cytosol. Whereas glucosyltransferase, autoprocessing and cell‐binding domains are well defined, structural features involved in toxin delivery are unknown. Here, we studied structural determinants that define membrane insertion, pore formation and translocation of toxin B. Deletion analyses revealed that a large region, covering amino acids 1501–1753 of toxin B, is dispensable for cytotoxicity in Vero cells. Accordingly, a chimeric toxin, consisting of amino acids 1–1550 and the receptor‐binding domain of diphtheria toxin, caused cytotoxic effects. A large N‐terminal part of toxin B (amino acids 1–829) was not essential for pore formation (measured by⁸⁶Rb⁺release in mammalian cells). Studies using C‐terminal truncation fragments of toxin B showed that amino acid residues 1–990 were still capable of inducing fluorescence dye release from large lipid vesicles and led to increased electrical conductance in black lipid membranes. Thereby, we define the minimal pore‐forming region of toxin B within amino acid residues 830 and 990. Moreover, we identify within this region a crucial role of the amino acid pair glutamate‐970 and glutamate‐976 in pore formation of toxin B.

Significance: Glycosylating toxins are major virulence factors of pathogenic Clostridia , including Clostridium difficile toxins A and B, which cause antibiotics-associated diarrhea and pseudomembranous colitis. Host cell uptake of these toxins is suggested to be mediated via C-terminal combined repetitive oligopeptides (CROP). Here, we identified LDL receptor-related protein 1 (LRP1) as a host cell receptor of Clostridium perfringens TpeL toxin, a recently identified clostridial glycosylating toxin, which has no CROP domain. TpeL binds to LRP1 with a C-terminal receptor-binding domain, which is conserved in all other clostridial glycosylating toxins and precedes the CROP domain. Thus, our study identifies LRP1 as TpeL receptor and suggests a two-receptor model for clostridial glycosylating toxins, which offers additional perspectives in antitoxin strategies.