Clostridium perfringens is a prolific, toxin-producing anaerobe causing multiple diseases in humans and animals. One of these toxins is epsilon, a 33 kDa protein produced by Clostridium perfringens (types B and D) that induces fatal enteric disease of goats, sheep and cattle. Epsilon toxin (Etx) belongs to the aerolysin-like toxin family. It contains three distinct domains, is proteolytically-activated and forms oligomeric pores on cell surfaces via a lipid raft-associated protein(s). Vaccination controls Etx-induced disease in the field. However, therapeutic measures are currently lacking. This review initially introduces C. perfringens toxins, subsequently focusing upon the Etx and its biochemistry, disease characteristics in various animals that include laboratory models (in vitro and in vivo), and finally control mechanisms (vaccines and therapeutics).

Some hypervirulent strains of Clostridium difficile produce the binary actin-ADP-ribosylating toxin C. difficile transferase (CDT) in addition to Rho-glucosylating toxins A and B. It has been suggested that the presence of CDT increases the severity of C. difficile -associated diseases, including pseudomembranous colitis. CDT contains a binding and translocation component, CDTb, that mediates the transport of the separate enzyme component CDTa into the cytosol of target cells, where CDTa modifies actin. Here we investigated the mechanism of cellular CDT uptake and found that bafilomycin A1 protects cultured epithelial cells from intoxication with CDT, implying that CDTa is translocated from acidified endosomal vesicles into the cytosol. Consistently, CDTa is translocated across the cytoplasmic membranes into the cytosol when cell-bound CDT is exposed to acidic medium. Radicicol and cyclosporine A, inhibitors of the heat shock protein Hsp90 and cyclophilins, respectively, protected cells from intoxication with CDT but not from intoxication with toxins A and B. Moreover, both inhibitors blocked the pH-dependent membrane translocation of CDTa, strongly suggesting that Hsp90 and cyclophilin are crucial for this process. In contrast, the inhibitors did not interfere with the ADP-ribosyltransferase activity, receptor binding, or endocytosis of the toxin. We obtained comparable results with the closely related iota-toxin from Clostridium perfringens . Moreover, CDTa and Ia, the enzyme component of iota-toxin, specifically bound to immobilized Hsp90 and cyclophilin A in vitro . In combination with our recently obtained data on the C2 toxin from C. botulinum , these results imply a common Hsp90/cyclophilin A-dependent translocation mechanism for the family of binary actin-ADP-ribosylating toxins.

Some pathogenic spore-forming bacilli employ a binary protein mechanism for intoxicating the intestinal tracts of insects, animals, and humans. These Gram-positive bacteria and their toxins include Clostridium botulinum (C2 toxin), Clostridium difficile (C. difficile toxin or CDT), Clostridium perfringens (ι-toxin and binary enterotoxin, or BEC), Clostridium spiroforme (C. spiroforme toxin or CST), as well as Bacillus cereus (vegetative insecticidal protein or VIP). These gut-acting proteins form an AB complex composed of ADP-ribosyl transferase (A) and cell-binding (B) components that intoxicate cells via receptor-mediated endocytosis and endosomal trafficking. Once inside the cytosol, the A components inhibit normal cell functions by mono-ADP-ribosylation of globular actin, which induces cytoskeletal disarray and death. Important aspects of each bacterium and binary enterotoxin will be highlighted in this review, with particular focus upon the disease process involving the biochemistry and modes of action for each toxin.

In osteoporosis, bone structure can be improved by the introduction of therapeutic molecules inhibiting bone resorption by osteoclasts. Here, biocompatible hydrogels represent an excellent option for the delivery of pharmacologically active molecules to the bone tissue because of their biodegradability, injectability, and manifold functionalization capacity. The present study reports the preparation of a multifunctional hybrid hydrogel from chemically modified human serum albumin and rationally designed DNA building blocks. The hybrid hydrogel combines advantageous characteristics, including rapid gelation through DNA hybridization under physiological conditions and a self‐healing and injectable nature with the possibility of specific loading and spatiotemporally controlled release of active proteins, making it an advanced biomaterial for the local treatment of bone diseases, for example, osteoporosis. The hydrogels are loaded with a recombinant Rho‐inhibiting C3 toxin, C2IN‐C3lim‐G205C. This toxin selectively targets osteoclasts and inhibits Rho‐signaling and, thereby, actin‐dependent processes in these cells. Application of C2IN‐C3lim‐G205C toxin‐loaded hydrogels effectively reduces osteoclast formation and resorption activity in vitro, as demonstrated by tartrate‐resistant acid phosphatase staining and the pit resorption assay. Simultaneously, osteoblast activity, viability, and proliferation are unaffected, thus making C2IN‐C3lim‐G205C toxin‐loaded hybrid hydrogels an attractive pharmacological system for spatial and selective modulation of osteoclast functions to reduce bone resorption.

Bacillus anthracis lethal toxin consists of the protective antigen (PA) and the metalloprotease lethal factor (LF). During cellular uptake PA forms pores in membranes of endosomes, and unfolded LF translocates through the pores into the cytosol. We have investigated whether host cell chaperones facilitate translocation of LF and the fusion protein LF_NDTA. LF_N mediates uptake of LF_NDTA into the cytosol, where DTA, the catalytic domain of diphtheria toxin, ADP-ribosylates elongation factor-2, allowing for detection of small amounts of translocated LF_NDTA. Cyclosporin A, which inhibits peptidyl-prolyl cis/trans isomerase activity of cyclophilins, and radicicol, which inhibits Hsp90 activity, prevented uptake of LF_NDTA into the cytosol of CHO-K1 cells and protected cells from intoxication by LF_NDTA/PA. Both inhibitors, as well as an antibody against cyclophilin A blocked the release of active LF_NDTA from endosomal vesicles into the cytosol in vitro. In contrast, the inhibitors did not inhibit cellular uptake of LF. In vitro, cyclophilin A and Hsp90 bound to LF_NDTA and DTA but not to LF, implying that DTA determines this interaction. In conclusion, cyclophilin A and Hsp90 facilitate translocation of LF_NDTA, but not of LF, across endosomal membranes, and thus they function selectively in promoting translocation of certain proteins, but not of others.

There are many pathogenic Clostridium species with diverse virulence factors that include protein toxins. Some of these bacteria, such as C. botulinum, C. difficile, C. perfringens and C. spiroforme, cause enteric problems in animals as well as humans. These often fatal diseases can partly be attributed to binary protein toxins that follow a classic AB paradigm. Within a targeted cell, all clostridial binary toxins destroy filamentous actin via mono-ADP-ribosylation of globular actin by the A component. However, much less is known about B component binding to cell-surface receptors. These toxins share sequence homology amongst themselves and with those produced by another Gram-positive, spore-forming bacterium also commonly associated with soil and disease: Bacillus anthracis. This review focuses upon the iota and C2 families of clostridial binary toxins and includes: 1) basics of the bacterial source; 2) toxin biochemistry; 3) sophisticated cellular uptake machinery; and 4) host-cell responses following toxin-mediated disruption of the cytoskeleton. In summary, these protein toxins aid diverse enteric species within the genus Clostridium.