Botulinum neurotoxin Light Chain/A1 uses fast synaptic vesicle cycling to cleave plasma membrane bound SNAP-25

Botulinum neurotoxins (BoNT) are the most potent protein toxins for humans, yet how BoNT-Light Chain/A1 (LC/A1) journeys to cleave intracellular SNAP-25 is understudied. Here we use a cell-based assay to measure cytosolic EGFP-LC/A1 intracellular trafficking and SNAP-25 cleavage in Neuro-2A cells. Intracellular LC/A1 associated on microtubules and co-localized with Rab GTPases involved in fast synaptic vesicles and endosome recycling. Multiple Dominant Negative (DN) Rabs GTPases involved in fast synaptic vesicles or endosome recycling inhibited LC/A1 trafficking to the intracellular plasma membrane and SNAP-25 cleavage. A cytosolic LC/A1 variant that bound the plasma membrane from the cytosol was insensitive to DNRab GTPases involved in fast synaptic vesicle recycling. LC/A1 traffics on fast synaptic vesicles to the intracellular plasma membrane to cleave SNAP-25. Our data suggest, like Heavy Chain host cell entry and LC catalysis, LC intracellular trafficking to target host substrates can contribute to bacterial toxin potency.