Botulinum neurotoxins (BoNTs) are the most poisonous toxins known and are classified as Category A select agents. BoNTs are the causative agent of human botulism, and are used as efficacious pharmaceuticals for treatment of a myriad of neuronal diseases. BoNTs are conventionally subdivided into 7 serotypes (A - G). In recent years, several subtype BoNTs have been identified for most serotypes based on differences in nucleotide sequences. Five subtypes of BoNT/A (A1 - A5) are recognized. Apart from sequence studies and initial data indicating significant functional differences, little researh has been conducted on the molecular and cell biology of the subtype BoNTs. The goal of this research project is to characterize and discern the molecular and cell biology properties of the BoNT/A1-A5 in vitro and in vivo, and relate subtype specific biological characteristics to structural properties. The proposed studies represent a strong partnership among three laboratories with long term and productive collaborations, and the impetus for this application is based upon recent progress in these laboratories on the subtype BoNTs, including that the subtypes differ in molecular characteristics, kinetics and mechanisms of intoxication, and structural features. This proposal will test the overall hypothesis that the five BoNT/A subtypes possess unique in vivo toxicity profiles.
Aim 1 will examine the hypothesis that each BoNT/A subtype possesses distinct in vitro toxicity characteristics, and that recombinant BoNT/A subtypes can be produced as source materials for genetic modifications to define functional properties.
Aim 2 will test the hypothesis that BoNTs/A1-A5 have distinct cell entry kinetics and in vivo toxicity profiles.
Aim 3 will examine the hypothesis that BoNT/A subtypes have unique cell trafficking mechanisms in neuronal cell models, and that the differences in cellular traffickig and biological properties between BoNT/A subtypes are due to specific structural differences of the toxins. The data resulting from this project have the potential to improve clinical treatments with BoNT based pharmaceuticals, as new and modified pharmaceuticals with targeted characteristics and fewer side effects will become necessary. In addition, the characterization of toxin properties will facilitate future developments of targeted anti-BoNT therapeutics that will b effective against all BoNT/A subtypes.

Public Health Relevance

The proposed work will examine and compare the biological properties of the five botulinum neurotoxin (BoNT) A subtypes and determine which structural features are responsible for specific functions of the toxin. The knowledge gained from this study and the technical systems developed for expression and manipulation of recombinant BoNTs will positively and significantly impact the botulinum toxin research field as well as the medical field by providing the basis for potentially improved and alternative BoNT based therapeutics, which could significantly increase BoNT treatment options and indications.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Ranallo, Ryan
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University of Wisconsin Madison
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United States
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Pellett, Sabine; Bradshaw, Marite; Tepp, William H et al. (2018) The Light Chain Defines the Duration of Action of Botulinum Toxin Serotype A Subtypes. MBio 9:
Moritz, Molly S; Tepp, William H; Bradshaw, Marite et al. (2018) Isolation and Characterization of the Novel Botulinum Neurotoxin A Subtype 6. mSphere 3:
Pellett, Sabine; Tepp, William H; Lin, Guangyun et al. (2018) Substrate cleavage and duration of action of botulinum neurotoxin type FA (""H, HA""). Toxicon 147:38-46
Fredrick, Chase M; Lin, Guangyun; Johnson, Eric A (2017) Regulation of Botulinum Neurotoxin Synthesis and Toxin Complex Formation by Arginine and Glucose in Clostridium botulinum ATCC 3502. Appl Environ Microbiol 83:
Pellett, Sabine; Tepp, William H; Johnson, Eric A et al. (2017) Assessment of ELISA as endpoint in neuronal cell-based assay for BoNT detection using hiPSC derived neurons. J Pharmacol Toxicol Methods 88:1-6
Maslanka, Susan E; L├║quez, Carolina; Dykes, Janet K et al. (2016) A Novel Botulinum Neurotoxin, Previously Reported as Serotype H, Has a Hybrid-Like Structure With Regions of Similarity to the Structures of Serotypes A and F and Is Neutralized With Serotype A Antitoxin. J Infect Dis 213:379-85
Pellett, Sabine; Tepp, William H; Bradshaw, Marite et al. (2016) Purification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified Clostridium botulinum Strain. mSphere 1:
Pellett, Sabine; Tepp, William H; Whitemarsh, Regina C M et al. (2015) In vivo onset and duration of action varies for botulinum neurotoxin A subtypes 1-5. Toxicon 107:37-42
Lin, Guangyun; Tepp, William H; Bradshaw, Marite et al. (2015) Immunoprecipitation of native botulinum neurotoxin complexes from Clostridium botulinum subtype A strains. Appl Environ Microbiol 81:481-91
Pellett, Sabine; Schwartz, Michael P; Tepp, William H et al. (2015) Human Induced Pluripotent Stem Cell Derived Neuronal Cells Cultured on Chemically-Defined Hydrogels for Sensitive In Vitro Detection of Botulinum Neurotoxin. Sci Rep 5:14566

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