Clostridium difficile toxins: mediators of inflammation

Abstract

Clostridium difficile is a significant problem in hospital settings as the most common cause of nosocomial diarrhea worldwide. C. difficile infections (CDIs) are characterized by an acute intestinal inflammatory response with neutrophil infiltration. These symptoms are primarily caused by the glucosylating toxins, TcdA and TcdB. In the past decade, the frequency and severity of CDIs have increased markedly due to the emergence of so-called hypervirulent strains that overproduce cytotoxic glucosylating toxins relative to historical strains. In addition, these strains produce a third toxin, binary toxin or C. difficile transferase (CDT), that may contribute to hypervirulence. Both the glucosylating toxins and CDT covalently modify target cell proteins to cause disassembly of the actin cytoskeleton and induce severe inflammation. This review summarizes our current knowledge of the mechanisms by which glucosylating toxins and CDT disrupt target cell function, alter host physiology and stimulate immune responses.

Fig. 1

Pathogenesis of Clostridium difficile. When C. difficile spores are ingested, they are stimulated to germinate by bile salts (e.g. taurocholate) in the small intestine. C. difficile can productively colonize the colon of individuals whose normal intestinal flora has been disrupted (e.g. by antibiotic treatment). Colonization likely depends upon adherence of the bacterium to the epithelium, although little is known about the factors that mediate adherence. CDT-producing strains may increase their adherence to intestinal epithelial cells by inducing microtubule protrusions that trap C. difficile. Glucosylating toxin-producing strains stimulate inflammation of the colonic lining by inducing cytoskeletal changes that compromise the epithelial barrier and inflammatory cytokine production. Disruption of tight junctions allows the toxins to cross the epithelium, where they can further induce inflammatory cytokine production in lymphocytes and mast cells. This leads to escalation of the inflammatory response due to neutrophil and lymphocyte influx, which can lead to pseudomembrane formation. Whether glucosylating toxins enter the bloodstream remains unclear, although in a zebrafish model of infection, the toxins can become systemic [78]. During colonization of the host, C. difficile produces spores that are shed by the patient and facilitate transmission of C. difficile to susceptible hosts.

Fig. 2

aABCD structure of glucosylating toxins TcdA and TcdB. Functional and structural domain boundaries are marked, with active site residues being indicated. A = ‘Activity’ domain of Glc (blue); C = ‘cutting’ domain/autoprocessing CPD (pink); D = ‘delivery’ domain/translocation domain (orange); B = ‘binding’ domain/receptor binding domain (grey). For the D domain, the minimal pore-forming region consists of residues 830–990, while residues 1–1,850 are sufficient to mediate intoxication of target cells. The B domain contains multiple repeat sequences (CROPs), which range in size from 21–50 residues and are repeated throughout the C-terminus of the protein. The CROPs are more divergent and less frequent in TcdB than in TcdA. The dashed arrow indicates a putative minor receptor binding domain (residues 1,500–1,850) identified by deletion analyses [38, 40]. b Intoxication of target cells by TcdA and TcdB. Binding of the B domain to unknown receptors on target cells results in clathrin-dependent receptor-mediated endocytosis. During acidification of the endosome, conformational changes occur within the toxin that result in pore formation by the D domain and translocation of the A domain into the cytosol. Exposure of the C domain to InsP₆ (yellow) activates its protease function, resulting in toxin autoprocessing and release of the A domain into the cytosol. The A domain glucosylates Rho GTPases (Rho, Rac, Cdc42 family) on a conserved Thr residue, which prevents Rho GTPases from interacting with their cognate effectors and sequesters them at the membrane.

Fig. 3

aCDT is comprised of 2 polypeptides, CDTa (ADP-ribosyltransferase, green) and CDTb (receptor binding domain, grey). Signal peptide sequences of both polypeptides are shown. CDTb contains a propeptide whose proteolytic removal allows the activated binding domain (CDTb’ in b) to interact with as yet unidentified receptors on target cells. b Intoxication of target cells by CDT. Following proteolytic activation on the target cell surface, the binding domain oligomerizes and binds to cell receptors. Binding results in receptor-mediated endocytosis. During acidification of the endosome, the binding domain undergoes conformational rearrangements that result in pore formation and translocation of the ADP-ribosyltransferase domain into the target cell cytosol in an Hsp90-dependent manner. The ADP-ribosyltransferase domain ADP-ribosylates monomeric G-actin on Arg177, which blocks actin polymerization and ultimately leads to the dissolution of the actin cytoskeleton. CDTa also induces formation of microtubule projections, which may enhance the adherence of C. difficile to the intestinal epithelium.