Complete subunit structure of serotype C and D botulinum progenitor toxin complex induces vacuolation in the specific...

Clostridium botulinum produces seven botulinum neurotoxin (BoNT) serotypes. In nature, BoNT exists as a part of the progenitor toxin complex (PTC) through associations with neurotoxin associated proteins (NAPs), including nontoxic nonhemagglutinin and hemagglutinin (HA) complex, consists of HA-70, HA-17 and HA-33. Because PTC displays higher oral toxicity than pure BoNTs, NAPs play a critical role in food poisoning. In a previous study, we demonstrated that the NAP complex in mature large-sized PTC (L-PTC) from serotypes C and D concomitantly induced cell death and cytoplasmic vacuolation in the rat intestinal epithelial cell line IEC-6. Here, we found that the serotype D NAP complex induces only cytoplasmic vacuolation in the normal rat kidney cell line NRK-52E without reducing cell viability. NAP complexes from serotype A and B L-PTCs did not affect cell viability or cytoplasmic vacuolation in IEC-6 and NRK-52E cells. Furthermore, we assessed the effect of immature L-PTCs with fewer HA-33/HA-17 trimers (two HA-33 and one HA-17) than mature L-PTCs on cell viability and cytoplasmic vacuolation in IEC-6 and NRK-52E cells. As a result, mature L-PTCs with the maximum number of HA-33/HA-17 trimers displayed the greatest potency. Consequently, the reduction in cell viability and vacuolation induction are related to the number of HA-33/HA-17 trimers in PTC. The discovery of an epithelial cell model where botulinum PTC specifically induces vacuolization may help clarify the unknown cytotoxicity of PTC, which plays an important role in the trans-epithelial transport of the toxin.