Botulinum Neurotoxin X: A New Tool to Study Intracellular Transport and Membrane Trafficking

The recent discovery of a new serotype, BoNT/X has created an exciting opportunity to characterise the function of SNAREs which were not previously amenable to the action of these toxins. BoNT/X has been shown to cleave multiple RSNAREs in vitro (VAMP1, 2, 3, 4, 5, and Ykt6) and VAMPs 2 and 4 in vivo. However, almost nothing is known about the impact of cleaving these SNAREs on intracellular trafficking. The aim of my thesis was to validate and extend BoNT/X’s substrate specificity by looking at endogenous SNAREs and determine how the loss of these proteins impacts intracellular trafficking using a range of quantitative endocytic and biosynthetic transport assays. I have shown that BoNT/X is active when expressed in cells and has a similar specificity as the native toxin (cleaves endogenous VAMPs 3, 4, and Ykt6). BoNT/X intoxication leads to dramatic alterations in the localisation, morphology, and staining intensity of numerous markers of the endocytic and biosynthetic pathways suggesting that the toxin is impacting many different trafficking pathways. Using a flow cytometry-based endocytic assay I have shown that there is a significant reduction in TF-R endocytosis most likely caused by the loss of the receptor from the cell surface. I have also shown that the surface levels of many proteins are also significantly reduced (CD63, CD/CI-MPR, EGF-R, and TGN46) suggesting a global defect in cell surface trafficking. I have also examined the impact of BoNT/X intoxication on constitutive secretion using a novel flow cytometry-based assay I have developed during my Ph.D. I have found that BoNT/X intoxication blocks the delivery of soluble and membrane-anchored proteins to the cell surface by causing them to accumulate in the early biosynthetic pathway. Taken together, my data suggests that BoNT/X is a potent inhibitor of intracellular trafficking and behaves very differently from any of the well-characterized botulinum serotypes. In the future, it will be important to determine if the observed phenotypes can be rescued using cleavageresistant SNAREs.