Role of Neurotoxin Associated Proteins in the Low pH Induced Structural Changes in the Botulinum Neurotoxin Complex

Abstract

BoNT (Botulinum Neurotoxin) produced by the bacterium Clostridium botulinum as a complex with NAPs causes botulism. It has been known that the NAPs protect the toxin from both extremes of pHs and proteases of the GI tract. In an attempt to emulate the physiological conditions encountered by the toxin, we examined BoNT/A, BoNT/A complex, and NAPs under different pH conditions and monitored their structural characteristics by far-UV CD and thermal denaturation analysis. BoNT/A complex showed the maximum CD signal with a molar ellipticity of −1.8 × 105 deg.cm2/dmol at 222 nm at both acidic and neutral pHs. Thermal denaturation analysis revealed NAPs to be the most stable amongst the three protein samples examined. Interestingly and quite uniquely, at pH 2.5, there was an increase in CD signal for BoNT complex as a function of temperature, which correlated with the NAPs profile, indicating a shielding effect of NAPs on BoNT complex at low pH. Calculation of the weighted mean of the ellipticities at the Tm for thermal unfolding of toxin and NAPs at neutral and acidic pHs showed variation with that of BoNT complex, suggesting structural reorganization in BoNT complex upon the association of NAPs and BoNT. In conclusion, this study reveals the structural behavior of BoNT complex and NAPs with pH changes substantially, which could be quite relevant for BoNT survival under extreme pH conditions in vivo.

Dissociation mechanics and stability of type A botulinum neurotoxin complex by means of biophysical evaluation

“…The present work supports the earlier studies that BoNT/A within its complex at acidic pH is structurally more stable against heat stress than in a free state 24,25,42 . Most of the marketed lyophilized formulations exist in a complex form with a molecular weight ranging from 500 kDa in Dysport® to 900 kDa in Botox® 16 .…”

“…Neurotoxins can readily dissociate from the complex within a minute, especially when exposed to alkaline pH, suggesting the existence of a free form (i.e., dissociated) of botulinum neurotoxin as reconstituted 22,23 . At acidic pH, the protective activity of NAPs on the neurotoxin indicates they maintain their associated state with the neurotoxin 7,24,25 .…”

Clostridium botulinum type C, D, C/D, and D/C: An update

“…After ingestion, the toxin is absorbed by the duodenum and jejunum and then passes into the bloodstream. BoNT resists gastric acidity and digestive enzymes because it forms a complex with a group of neurotoxin associated proteins or NAPs ( Chellappan et al, 2014 ). BoNT/A, B and E are associated with foodborne botulism.…”