Antibody-Enhanced, Fc Gamma Receptor-Mediated Endocytosis of Clostridium difficile Toxin A

Abstract

Clostridium difficile has emerged as a leading cause of hospital-acquired enteric infections whose annual health care costs are rapidly escalating in the United States (33). The severity of C. difficile-associated infections ranges from mild diarrhea to life-threatening pseudomembranous colitis (2, 3). Several hospital outbreaks of C. difficile-associated diarrhea (CDAD) with high rates of morbidity and mortality in the past few years in North America have been attributed to the widespread use of broad-spectrum antibiotics. The emergence of more virulent C. difficile strains is also contributing to the increased incidence and severity of disease (38, 39).Toxin A (TcdA) and toxin B (TcdB) are the two major virulence factors in pathogenic C. difficile strains. These toxins are enterotoxic, induce intestinal epithelial cell damage, and disrupt epithelium tight junctions, leading to increased mucosal permeability (46,51,55). Moreover, these toxins induce production of immune mediators, leading to subsequent neutrophil infiltration and severe colitis (28,29). TcdA and TcdB are structurally homologous and putatively contain an N-terminal glucosyltransferase domain, a cysteine proteinase domain, a transmembrane domain, and a C-terminal receptor binding domain (21,65,66). Interaction between the C terminus and the host cell receptors is believed to initiate receptormediated endocytosis (11,25,63). Although the intracellular mode of action remains unclear, it has been proposed that the toxins undergo a conformational change at low pH in the endosomal compartment, leading to membrane insertion and channel formation (12,15,17,47). A host cofactor is then required to trigger a second structural change, which is accompanied by immediate autocatalytic cleavage and release of the glucosyltransferase domain into the cytosol (44,49,52). Once the glucosyltransferase domain reaches the cytosol, it inactivates proteins belonging to the Rho/Rac family, leading to alterations in the cytoskeleton and ultimately cell death (23,57).The clinical manifestations of CDAD are highly variable and range from asymptomatic carriage to mild self-limiting diarrhea to the more severe disease pseudomembranous colitis. Systemic complications and death are increasingly common in CDAD patients (58). In life-threatening cases of CDAD, systemic complications that include cardiopulmonary arrest (22), acute respiratory distress syndrome (20), multiple organ failure (9), renal failure (6), and liver damage (53) are observed. The exact reason for these complications is unclear, but the toxin's entry into the circulation and systemic dissemination have been suggested as possible causes (16).Protection against C. difficile appears to be conferred by antitoxin antibodies, which are present in the general population in individuals over 2 years of age; the levels of these antibodies are higher and relapse is less frequent in less severe cases (27,30,35,62,64). Disease progression and recurrence

Masking autoprocessing of Clostridium difficile toxin A by the C-terminus combined repetitive oligo peptides

“…The highly purified recombinant toxins that appeared as a single band on an SDS-PAGE gel were used in this study. Western blot was performed to detect various domains of toxins using mouse poly- and mono-clonal antibodies that were reported previously [17, 18]. These antibodies are: α-TcdA and α-TcdB-I are antibodies respectively against C terminus of TcdA and TcdB (BioDesign Inc.); α-TcdB-II is alpaca serum raised in the lab which is against full length of TcdB; monoclonal mouse antibody A1E6 raised in the lab recognizes the whole C terminus RBD of TcdA.…”

A Chimeric Toxin Vaccine Protects against Primary and Recurrent Clostridium difficile Infection

“…Cytopathic and cytotoxic activities of the toxins were assayed as described previously (20). CT-26 or Vero cells seeded in 96-well plates were treated with wild-type or mutant toxins.…”

Salubrinal protects against toxin B-induced CT26 cell death

“…The cytotoxic effect induced by salubrinal or TcdB was analyzed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] viability assay as described previously [32]. Briefly, 1 × 10 4 CT26…”