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Retargeting Clostridium difficile Toxin B to Neuronal Cells as a Potential Vehicle for Cytosolic Delivery of Therapeutic Biomolecules to Treat Botulism

DOI: 10.1155/2012/760142

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Abstract:

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 [2–4]. 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

References

[1]  T. Jank and K. Aktories, “Structure and mode of action of clostridial glucosylating toxins: the ABCD model,” Trends in Microbiology, vol. 16, no. 5, pp. 222–229, 2008.
[2]  M. Egerer, T. Giesemann, T. Jank, K. J. Fullner Satchell, and K. Aktories, “Auto-catalytic cleavage of Clostridium difficile toxins A and B depends on cysteine protease activity,” Journal of Biological Chemistry, vol. 282, no. 35, pp. 25314–25321, 2007.
[3]  T. Giesemann, M. Egerer, T. Jank, and K. Aktories, “Processing of Clostridium difficile toxins,” Journal of Medical Microbiology, vol. 57, no. 6, pp. 690–696, 2008.
[4]  M. Egerer, T. Giesemann, C. Herrmann, and K. Aktorles, “Autocatalytic processing of Clostridium difficile toxin B: binding of inositol hexakisphosphate,” Journal of Biological Chemistry, vol. 284, no. 6, pp. 3389–3395, 2009.
[5]  D. E. Voth and J. D. Ballard, “Clostridium difficile toxins: mechanism of action and role in disease,” Clinical Microbiology Reviews, vol. 18, no. 2, pp. 247–263, 2005.
[6]  X. He, X. Sun, J. Wang et al., “Antibody-enhanced, Fc γ receptor-mediated endocytosis of Clostridium difficile toxin A,” Infection and Immunity, vol. 77, no. 6, pp. 2294–2303, 2009.
[7]  J. Steele, H. Feng, N. Parry, and S. Tzipori, “Piglet models of acute or chronic Clostridium difficile illness,” Journal of Infectious Diseases, vol. 201, no. 3, pp. 428–434, 2010.
[8]  G. Yang, B. Zhou, J. Wang et al., “Expression of recombinant Clostridium difficile toxin A and B in Bacillus megaterium,” BMC Microbiology, vol. 8, article 192, 2008.
[9]  L. L. Simpson, “Identification of the major steps in botulinum toxin action,” Annual Review of Pharmacology and Toxicology, vol. 44, pp. 167–193, 2004.
[10]  B. Davletov, M. Bajohrs, and T. Binz, “Beyond BOTOX: advantages and limitations of individual botulinum neurotoxins,” Trends in Neurosciences, vol. 28, no. 8, pp. 446–452, 2005.
[11]  C. Verderio, O. Rossetto, C. Grumelli, C. Frassoni, C. Montecucco, and M. Matteoli, “Entering neurons: botulinum toxins and synaptic vesicle recycling,” EMBO Reports, vol. 7, no. 10, pp. 995–999, 2006.
[12]  J. J. Schmidt, R. G. Stafford, and K. A. Bostian, “Type A botulinum neurotoxin proteolytic activity: development of competitive inhibitors and implications for substrate specificity at the S1' binding subsite,” FEBS Letters, vol. 435, no. 1, pp. 61–64, 1998.
[13]  J. M. Tremblay, C. L. Kuo, C. Abeijon et al., “Camelid single domain antibodies (VHHs) as neuronal cell intrabody binding agents and inhibitors of Clostridium botulinum neurotoxin (BoNT) proteases,” Toxicon, vol. 56, no. 6, pp. 990–998, 2010.
[14]  J. Dong, A. A. Thompson, Y. Fan et al., “A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic α-exosite binding region,” Journal of Molecular Biology, vol. 397, no. 4, pp. 1106–1118, 2010.
[15]  Y. C. Tsai, R. Maditz, C. L. Kuo et al., “Targeting botulinum neurotoxin persistence by the ubiquitin-proteasome system,” Proceedings of the National Academy of Sciences of the United States of America, vol. 107, no. 38, pp. 16554–16559, 2010.
[16]  C. L. Kuo, G. A. Oyler, and C. B. Shoemaker, “Accelerated neuronal cell recovery from botulinum neurotoxin intoxication by targeted ubiquitination,” PLoS ONE, vol. 6, no. 5, 2011.
[17]  A. Keppler, S. Gendreizig, T. Gronemeyer, H. Pick, H. Vogel, and K. Johnsson, “A general method for the covalent labeling of fusion proteins with small molecules in vivo,” Nature Biotechnology, vol. 21, no. 1, pp. 86–89, 2003.
[18]  H. F. LaPenotiere, M. A. Clayton, and J. L. Middlebrook, “Expression of a large, nontoxic fragment of botulinum neurotoxin serotype A and its use as an immunogen,” Toxicon, vol. 33, no. 10, pp. 1383–1386, 1995.
[19]  M. C. Shoshan, T. Bergman, M. Thelestam, and I. Florin, “Dithiothreitol generates an activated 250,000 mol. wt form of Clostridium difficile toxin B,” Toxicon, vol. 31, no. 7, pp. 845–852, 1993.
[20]  G. Lalli, J. Herreros, S. L. Osborne, C. Montecucco, O. Rossetto, and G. Schiavo, “Functional characterisation of tetanus and botulinum neurotoxins binding domains,” Journal of Cell Science, vol. 112, no. 16, pp. 2715–2724, 1999.
[21]  J. B. Park and L. L. Simpson, “Inhalational poisoning by botulinum toxin and inhalation vaccination with its heavy-chain component,” Infection and Immunity, vol. 71, no. 3, pp. 1147–1154, 2003.
[22]  G. Pfeifer, J. Schirmer, J. Leemhuis et al., “Cellular uptake of Clostridium difficile toxin B. Translocation of the N-terminal catalytic domain into the cytosol of eukaryotic cells,” Journal of Biological Chemistry, vol. 278, no. 45, pp. 44535–44541, 2003.
[23]  S. Genisyuerek, P. Papatheodorou, G. Guttenberg, R. Schubert, R. Benz, and K. Aktories, “Structural determinants for membrane insertion, pore formation and translocation of Clostridium difficile toxin B,” Molecular Microbiology, vol. 79, no. 6, pp. 1643–1654, 2011.
[24]  A. Sundriyal, A. K. Roberts, R. Ling, J. McGlashan, C. C. Shone, and K. R. Acharya, “Expression, purification and cell cytotoxicity of actin-modifying binary toxin from Clostridium difficile,” Protein Expression and Purification, vol. 74, no. 1, pp. 42–48, 2010.
[25]  T. Dingle, S. Wee, G. L. Mulvey et al., “Functional properties of the carboxy-terminal host cell-binding domains of the two toxins, TcdA and TcdB, expressed by Clostridium difficile,” Glycobiology, vol. 18, no. 9, pp. 698–706, 2008.
[26]  L. A. Barroso, J. S. Moncrief, D. M. Lyerly, and T. D. Wilkins, “Mutagenesis of the Clostridium difficile toxin B gene and effect on cytotoxic activity,” Microbial Pathogenesis, vol. 16, no. 4, pp. 297–303, 1994.
[27]  S. M. Kern and A. L. Feig, “Adaptation of Clostridium difficile toxin A for use as a protein translocation system,” Biochemical and Biophysical Research Communications, vol. 405, no. 4, pp. 570–574, 2011.
[28]  P. Zhang, R. Ray, B. R. Singh, D. Li, M. Adler, and P. Ray, “An efficient drug delivery vehicle for botulism countermeasure,” BMC Pharmacology, vol. 9, article 12, 2009.
[29]  M. Ho, L.-H. Chang, M. Pires-Alves et al., “Recombinant botulinum neurotoxin A heavy chain-based delivery vehicles for neuronal cell targeting,” Protein Engineering, Design and Selection, vol. 24, no. 3, pp. 247–253, 2011.
[30]  A. B. Maksymowych and L. L. Simpson, “Structural features of the botulinum neurotoxin molecule that govern binding and transcytosis across polarized human intestinal epithelial cells,” Journal of Pharmacology and Experimental Therapeutics, vol. 310, no. 2, pp. 633–641, 2004.

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