A neutralizing antibody that blocks delivery of the enzymatic cargo of Clostridium difficile toxin TcdB into host cells.
ABSTRACT: Clostridium difficile infection is the leading cause of hospital-acquired diarrhea and is mediated by the actions of two toxins, TcdA and TcdB. The toxins perturb host cell function through a multistep process of receptor binding, endocytosis, low pH-induced pore formation, and the translocation and delivery of an N-terminal glucosyltransferase domain that inactivates host GTPases. Infection studies with isogenic strains having defined toxin deletions have established TcdB as an important target for therapeutic development. Monoclonal antibodies that neutralize TcdB function have been shown to protect against C. difficile infection in animal models and reduce recurrence in humans. Here, we report the mechanism of TcdB neutralization by PA41, a humanized monoclonal antibody capable of neutralizing TcdB from a diverse array of C. difficile strains. Through a combination of structural, biochemical, and cell functional studies, involving X-ray crystallography and EM, we show that PA41 recognizes a single, highly conserved epitope on the TcdB glucosyltransferase domain and blocks productive translocation and delivery of the enzymatic cargo into the host cell. Our study reveals a unique mechanism of C. difficile toxin neutralization by a monoclonal antibody, which involves targeting a process that is conserved across the large clostridial glucosylating toxins. The PA41 antibody described here provides a valuable tool for dissecting the mechanism of toxin pore formation and translocation across the endosomal membrane.
Project description:Clostridioides difficile is the causative bacterium in 15-20% of all antibiotic associated diarrheas. The symptoms associated with C. difficile infection (CDI) are primarily induced by the two large exotoxins TcdA and TcdB. Both toxins enter target cells by receptor-mediated endocytosis. Although different toxin receptors have been identified, it is no valid therapeutic option to prevent receptor endocytosis. Therapeutics, such as neutralizing antibodies, directly targeting both toxins are in development. Interestingly, only the anti-TcdB antibody bezlotoxumab but not the anti-TcdA antibody actoxumab prevented recurrence of CDI in clinical trials. In this work, 31 human antibody fragments against TcdB were selected by antibody phage display from the human naive antibody gene libraries HAL9/10. These antibody fragments were further characterized by in vitro neutralization assays. The epitopes of the neutralizing and non-neutralizing antibody fragments were analyzed by domain mapping, TcdB fragment phage display, and peptide arrays, to identify neutralizing and non-neutralizing epitopes. A new neutralizing epitope within the glucosyltransferase domain of TcdB was identified, providing new insights into the relevance of different toxin regions in respect of neutralization and toxicity.
Project description:The Clostridium difficile toxins A and B are primarily responsible for symptoms of C. difficile associated disease and are prime targets for vaccine development. We describe a plasmid-based system for the production of genetically modified toxins in a non-sporulating strain of C. difficile that lacks the toxin genes tcdA and tcdB. TcdA and TcdB mutations targeting established glucosyltransferase cytotoxicity determinants were introduced into recombinant plasmids and episomally expressed toxin mutants purified from C. difficile transformants. TcdA and TcdB mutants lacking glucosyltransferase and autoproteolytic processing activities were ~10?000-fold less toxic to cultured human IMR-90 cells than corresponding recombinant or native toxins. However, both mutants retained residual cytotoxicity that could be prevented by preincubating the antigens with specific antibodies or by formalin treatment. Such non-toxic formalin-treated mutant antigens were immunogenic and protective in a hamster model of infection. The remaining toxicity of untreated TcdA and TcdB mutant antigens was associated with cellular swelling, a phenotype consistent with pore-induced membrane leakage. TcdB substitution mutations previously shown to block vesicular pore formation and toxin translocation substantially reduced residual toxicity. We discuss the implications of these results for the development of a C. difficile toxoid vaccine.
Project description:Clostridium difficile infections (CDIs) are the leading cause of hospital-acquired infectious diarrhea and primarily involve two exotoxins, TcdA and TcdB. Actoxumab and bezlotoxumab are human monoclonal antibodies that neutralize the cytotoxic/cytopathic effects of TcdA and TcdB, respectively. In a phase II clinical study, the actoxumab-bezlotoxumab combination reduced the rate of CDI recurrence in patients who were also treated with standard-of-care antibiotics. However, it is not known whether the antibody combination will be effective against a broad range of C. difficile strains. As a first step toward addressing this, we tested the ability of actoxumab and bezlotoxumab to neutralize the activities of toxins from a number of clinically relevant and geographically diverse strains of C. difficile. Neutralization potencies, as measured in a cell growth/survival assay with purified toxins from various C. difficile strains, correlated well with antibody/toxin binding affinities. Actoxumab and bezlotoxumab neutralized toxins from culture supernatants of all clinical isolates tested, including multiple isolates of the BI/NAP1/027 and BK/NAP7/078 strains, at antibody concentrations well below plasma levels observed in humans. We compared the bezlotoxumab epitopes in the TcdB receptor binding domain across known TcdB sequences and found that key substitutions within the bezlotoxumab epitopes correlated with the relative differences in potencies of bezlotoxumab against TcdB of some strains, including ribotypes 027 and 078. Combined with in vitro neutralization data, epitope modeling will enhance our ability to predict the coverage of new and emerging strains by actoxumab-bezlotoxumab in the clinic.
Project description:Clostridium difficile (C. difficile) infection (CDI) is the main cause of nosocomial antibiotic-associated colitis and increased incidence of community-associated diarrhea in industrialized countries. At present, the primary treatment of CDI is antibiotic administration, which is effective but often associated with recurrence, especially in the elderly. Pathogenic strains produce enterotoxin, toxin A (TcdA), and cytotoxin, toxin B (TcdB), which are necessary for C. difficile induced diarrhea and gut pathological changes. Administration of anti-toxin antibodies provides an alternative approach to treat CDI, and has shown promising results in preclinical and clinical studies. In the current study, several humanized anti-TcdA and anti-TcdB monoclonal antibodies were generated and their protective potency was characterized in a hamster infection model. The humanized anti-TcdA (CANmAbA4) and anti-TcdB (CANmAbB4 and CANmAbB1) antibodies showed broad spectrum in vitro neutralization of toxins from clinical strains and neutralization in a mouse toxin challenge model. Moreover, co-administration of humanized antibodies (CANmAbA4 and CANmAbB4 cocktail) provided a high level of protection in a dose dependent manner (85% versus 57% survival at day 22 for 50 mg/kg and 20 mg/kg doses, respectively) in a hamster gastrointestinal infection (GI) model. This study describes the protective effects conferred by novel neutralizing anti-toxin monoclonal antibodies against C. difficile toxins and their potential as therapeutic agents in treating CDI.
Project description:Various bacterial protein toxins, including Clostridium difficile toxins A (TcdA) and B (TcdB), attack intracellular target proteins of host cells by glucosylation. After receptor binding and endocytosis, the toxins are translocated into the cytosol, where they modify target proteins (e.g., Rho proteins). Here we report that the activity of translocated glucosylating toxins depends on the chaperonin TRiC/CCT. The chaperonin subunits CCT4/5 directly interact with the toxins and enhance the refolding and restoration of the glucosyltransferase activities of toxins after heat treatment. Knockdown of CCT5 by siRNA and HSF1A, an inhibitor of TRiC/CCT, blocks the cytotoxic effects of TcdA and TcdB. In contrast, HSP90, which is involved in the translocation and uptake of ADP ribosylating toxins, is not involved in uptake of the glucosylating toxins. We show that the actions of numerous glycosylating toxins from various toxin types and different species depend on TRiC/CCT. Our data indicate that the TRiC/CCT chaperonin system is specifically involved in toxin uptake and essential for the action of various glucosylating protein toxins acting intracellularly on target proteins.
Project description:Clostridium difficile toxins A and B are members of an important class of virulence factors known as large clostridial toxins (LCTs). Toxin action involves four major steps: receptor-mediated endocytosis, translocation of a catalytic glucosyltransferase domain across the membrane, release of the enzymatic moiety by autoproteolytic processing, and a glucosyltransferase-dependent inactivation of Rho family proteins. We have imaged toxin A (TcdA) and toxin B (TcdB) holotoxins by negative stain electron microscopy to show that these molecules are similar in structure. We then determined a 3D structure for TcdA and mapped the organization of its functional domains. The molecule has a "pincher-like" head corresponding to the delivery domain and two tails, long and short, corresponding to the receptor-binding and glucosyltransferase domains, respectively. A second structure, obtained at the acidic pH of an endosome, reveals a significant structural change in the delivery and glucosyltransferase domains, and thus provides a framework for understanding the molecular mechanism of LCT cellular intoxication.
Project description:As the major cause of antibiotic-associated diarrhea, Clostridium difficile is a serious problem in health care facilities worldwide. C. difficile produces two large toxins, TcdA and TcdB, which are the primary virulence factors in disease. The respective functions of these toxins have been difficult to discern, in part because the cytotoxicity profiles for these toxins differ with concentration and cell type. The goal of this study was to develop a cell culture model that would allow a side-by-side mechanistic comparison of the toxins. Conditionally immortalized, young adult mouse colonic (YAMC) epithelial cells demonstrate an exquisite sensitivity to both toxins with phenotypes that agree with observations in tissue explants. TcdA intoxication results in an apoptotic cell death that is dependent on the glucosyltransferase activity of the toxin. In contrast, TcdB has a bimodal mechanism; it induces apoptosis in a glucosyltransferase-dependent manner at lower concentrations and glucosyltransferase-independent necrotic death at higher concentrations. The direct comparison of the responses to TcdA and TcdB in cells and colonic explants provides the opportunity to unify a large body of observations made by many independent investigators.
Project description:Clostridioides (Clostridium) difficile is the most commonly recognized cause of infectious diarrhea in healthcare settings. Currently there is no vaccine to prevent initial or recurrent C. difficile infection (CDI). Two large clostridial toxins, TcdA and TcdB, are the primary virulence factors for CDI. Immunological approaches to prevent CDI include antibody-mediated neutralization of the cytotoxicity of these toxins. An understanding of the sequence diversity of the two toxins expressed by disease causing isolates is critical for the interpretation of the immune response to the toxins. In this study, we determined the whole genome sequence (WGS) of 478 C. difficile isolates collected in 12 countries between 2004 and 2018 to probe toxin variant diversity. A total of 44 unique TcdA variants and 37 unique TcdB variants were identified. The amino acid sequence conservation among the TcdA variants (?98%) is considerably greater than among the TcdB variants (as low as 86.1%), suggesting that different selection pressures may have contributed to the evolution of the two toxins. Phylogenomic analysis of the WGS data demonstrate that isolates grouped together based on ribotype or MLST code for multiple different toxin variants. These findings illustrate the importance of determining not only the ribotype but also the toxin sequence when evaluating strain coverage using vaccine strategies that target these virulence factors. We recommend that toxin variant type and sequence type (ST), be used together with ribotype data to provide a more comprehensive strain classification scheme for C. difficile surveillance during vaccine development objectives.
Project description:Toxin A (TcdA) and toxin B (TcdB) of Clostridium difficile cause gross pathological changes (e.g., inflammation, secretion, and diarrhea) in the infected host, yet the molecular and cellular pathways leading to observed host responses are poorly understood. To address this gap, we evaluated the effects of single doses of TcdA and/or TcdB injected into the ceca of mice, and several endpoints were analyzed, including tissue pathology, neutrophil infiltration, epithelial-layer gene expression, chemokine levels, and blood cell counts, 2, 6, and 16 h after injection. In addition to confirming TcdA's gross pathological effects, we found that both TcdA and TcdB resulted in neutrophil infiltration. Bioinformatics analyses identified altered expression of genes associated with the metabolism of lipids, fatty acids, and detoxification; small GTPase activity; and immune function and inflammation. Further analysis revealed transient expression of several chemokines (e.g., Cxcl1 and Cxcl2). Antibody neutralization of CXCL1 and CXCL2 did not affect TcdA-induced local pathology or neutrophil infiltration, but it did decrease the peripheral blood neutrophil count. Additionally, low serum levels of CXCL1 and CXCL2 corresponded with greater survival. Although TcdA induced more pronounced transcriptional changes than TcdB and the upregulated chemokine expression was unique to TcdA, the overall transcriptional responses to TcdA and TcdB were strongly correlated, supporting differences primarily in timing and potency rather than differences in the type of intracellular host response. In addition, the transcriptional data revealed novel toxin effects (e.g., altered expression of GTPase-associated and metabolic genes) underlying observed physiological responses to C. difficile toxins.
Project description:Clostridium difficile is the leading cause of hospital-acquired diarrhea and pseudomembranous colitis worldwide. The organism produces two homologous toxins, TcdA and TcdB, which enter and disrupt host cell function by glucosylating and thereby inactivating key signalling molecules within the host. As a toxin-mediated disease, there has been a significant interest in identifying small molecule inhibitors of the toxins' glucosyltransferase activities. This study was initiated as part of an effort to identify the mode of inhibition for a small molecule inhibitor of glucosyltransferase activity called apigenin. In the course of trying to get co-crystals with this inhibitor, we determined five different structures of the TcdA and TcdB glucosyltransferase domains and made use of a non-hydrolyzable UDP-glucose substrate. While we were able to visualize apigenin bound in one of our structures, the site was a crystal packing interface and not likely to explain the mode of inhibition. Nevertheless, the structure allowed us to capture an apo-state (one without the sugar nucleotide substrate) of the TcdB glycosyltransferase domain that had not been previously observed. Comparison of this structure with structures obtained in the presence of a non-hydrolyzable UDP-glucose analogue have allowed us to document multiple conformations of a C-terminal loop important for catalysis. We present our analysis of these five new structures with the hope that it will advance inhibitor design efforts for this important class of biological toxins.