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.