Submitted to: PLoS One
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/27/2013
Publication Date: 1/22/2014
Publication URL: http://handle.nal.usda.gov/10113/59077
Citation: Vazques-Cintron, E.J., Vakulenko, M., Band, P.A., Stanker, L.H., Johnson, E.A., Ichtchenko, K. 2014. Atoxic derivative of botulinum neurotoxin A as a prototype vehicle for targeted delivery to neuronal cytoplasm. PLoS One. 9(1):E85517. DOI: 10.1371/JOURNAL.PONE.0085517. Interpretive Summary: Botulism is a serious, often fatal neuroparalytic disease in humans and animals caused by a protein toxin (botulinum toxin, BoNT) produced by the bacterium Clostridium botulinum. BoNT is considered the most toxic biological toxin known. Because of its high toxicity, the need for a long recovery period requiring extensive treatment, and the ease of producing BoNT, it is considered a class A bioterrorism agent. The high toxicity of BoNT makes it difficult to study the biological mechanisms associated with toxicity. Using an atoxic form of BoNT (adBoNT), described earlier, we describe details of the internalization and intracellular movement of toxin in primary neuron cultures. Furthermore, we evaluated the intracellular stability of internalized ad toxin light chain. These cellular studies further our understanding, at the cellular level, of the steps leading to disease. This information furthers our ability to design intervention approaches, monitor for toxin contamination, and thus improve the safety of the U.S. food supply.
Technical Abstract: We have previously described genetic constructs and expression systems that enable facile production of recombinant derivatives of botulinum neurotoxins (BoNTs) that retain the structural and trafficking properties of wt BoNTs. In this report we describe the properties of one such derivative, BoNT/A ad, which was rendered atoxic by double point mutations to the light chain (LC) of wt BoNT/A, and which is being developed as a molecular vehicle for delivering drugs to the neuronal cytoplasm. The neuronal binding, internalization, and intracellular trafficking of BoNT/A ad in primary hippocampal cultures was evaluated using three complimentary techniques: flow cytometry, immunohistochemistry, and Western blotting. Significant neuronal binding of BoNT ad was only observed when neurons were incubated in depolarizing medium. Flow cytometry demonstrated that BoNT/A ad internalized into neurons but not glia. After 24 hours, the majority of the neuron-bound BoNT/A ad became internalized, as determined by its resistance to pronase E-induced proteolytic degradation of proteins associated with the plasma membrane of intact cells. Significant amounts of atoxic LC accumulated in a Triton X100-extractable fraction of the neurons, and persisted as such for at least 11 days with no evidence of degradation. Immunocytochemical analysis demonstrated that the LC of BoNT/A ad was translocated to the neuronal cytoplasm after uptake and was specifically targeted to SNARE proteins. The atoxic LC consistently co-localized with synaptic markers SNAP-25 and VAMP-2, but was rarely co-localized with markers for early or late endosomes. These data demonstrate that BoNT/A ad mimics the trafficking properties of wt BoNT/A, confirming that our platform for designing and expressing BoNT derivatives provides an accessible system for elucidating the molecular details of BoNT trafficking, and can potentially be used to address multiple medical and biodefense needs.