%0 Journal Article
%T Retargeting Clostridium difficile Toxin B to Neuronal Cells as a Potential Vehicle for Cytosolic Delivery of Therapeutic Biomolecules to Treat Botulism
%A Greice Krautz-Peterson
%A Yongrong Zhang
%A Kevin Chen
%A George A. Oyler
%A Hanping Feng
%A Charles B. Shoemaker
%J Journal of Toxicology
%D 2012
%I Hindawi Publishing Corporation
%R 10.1155/2012/760142
%X Botulinum neurotoxins (BoNTs) deliver a protease to neurons which can cause a flaccid paralysis called botulism. Development of botulism antidotes will require neuronal delivery of agents that inhibit or destroy the BoNT protease. Here, we investigated the potential of engineering Clostridium difficile toxin B (TcdB) as a neuronal delivery vehicle by testing two recombinant TcdB chimeras. For AGT-TcdB chimera, an alkyltransferase (AGT) was appended to the N-terminal glucosyltransferase (GT) of TcdB. Recombinant AGT-TcdB had alkyltransferase activity, and the chimera was nearly as toxic to Vero cells as wild-type TcdB, suggesting efficient cytosolic delivery of the AGT/GT fusion. For AGT-TcdB-BoNT/A-Hc, the receptor-binding domain (RBD) of TcdB was replaced by the equivalent RBD from BoNT/A (BoNT/A-Hc). AGT-TcdB-BoNT/A-Hc was >25-fold more toxic to neuronal cells and >25-fold less toxic to Vero cells than AGT-TcdB. Thus, TcdB can be engineered for cytosolic delivery of biomolecules and improved targeting of neuronal cells. 1. Introduction Clostridial toxins in nature are remarkably efficient cell cytosol delivery vehicles with highly evolved cell-specific delivery features that may be ideal for therapeutic applications. Specifically these toxins (1) gain entry to animals; (2) survive in blood; (3) bind to target cells expressing a specific receptor; (4) penetrate the target cells; (5) deliver an enzymatically active cargo to the cytosol. C. difficile toxins A and B (TcdA and TcdB) contain a receptor-binding domain (RBD) that binds to receptors that are broadly expressed on cells and then enters by endocytosis. Once in the endosome, the toxins employ a translocation domain (TD) to deliver a glucosyltransferase (GT) to the cytosol which inactivates Rho GTPases and leads to cell death [1]. The toxins also contain a cysteine protease (CPD), located between GT and TD, that cleaves the GT enzymatic ¡°cargo¡± from the ¡°delivery vehicle¡± at the endosomal membrane and liberates it into the cytosol [2¨C4]. C. difficile bacteria generally reside in the gut where the released toxins intoxicate intestinal epithelial cells and cause the disruption of tight junctions of epithelium and its barrier function. It is likely that in severe cases of the infection, the toxins penetrate into the submucosa and disseminate systemically [5]. We recently identified C. difficile toxins in the blood of the experimentally infected animals [6, 7], suggesting that the toxins may be reasonably stable in serum. Recent developments have enabled the application of TcdA and TcdB as therapeutic
%U http://www.hindawi.com/journals/jt/2012/760142/