Effects of botulinum neurotoxin-associated proteins on cellular organelle dynamics in NRK-52E cells

Clostridium botulinum produces seven distinct serotypes (A-G) of botulinum neurotoxin (BoNT), a potent biological toxin that affects the nervous system. In its natural state, BoNT forms a progenitor toxin complex with neurotoxin-associated proteins (NAPs), substantially enhancing its stability and oral toxicity. Recent studies have shown that NAPs in certain serotypes induce vacuolation and death in various epithelial cell types. In this study, we examined the effects of the serotype D NAPs complex (NAPs/D) on intracellular organelles in NRK-52E cells, a rat kidney cell line. NRK-52E cells were selected because they exhibit a unique response, showing vacuolation without undergoing cell death upon exposure to NAPs/D. Our findings indicated that NAPs/D-induced vacuole formation was associated with the reduced size of the Golgi apparatus. Additionally, vacuoles were associated with endocytic vesicles, implying that NAPs/D modulate endocytic pathways and affect intracellular transport. Furthermore, NAPs/D exposure activated early autophagic signaling, as evidenced by increased LC3 expression, although vacuole formation appeared to occur independently of the complete autophagy process. These findings provide valuable insights into the cellular mechanisms underlying BoNT toxicity and highlight the complex interplay between NAPs/D-induced vacuolation, endocytosis, and autophagy. This study highlights potential therapeutic approaches to mitigate the effects of botulism by targeting these pathways.