Clostridium botulinum is a gram-positive bacterium which causes botulism, a very fatal disease that prevents acetylcholine from binding to receptors therefore not removing the muscle’s ability to contract, in people (Shukla 2005). The cells of this bacterium secrete botulinum neurotoxin, (BoNT), which is considered the deadliest toxin up to date and has been deliberated by biodefense researchers as a possible agent for bioterrorism. BoNT, shown in Figure 1, works by binding to the receptor in which acetylcholine is supposed to bind, blocking the binding reaction from occurring. One very important protein in the process of secreting BoNT is HA33.
BoNT forms a toxin complex (TC) that includes HA33 (Ito et all 2011). HA33 is a protein from Clostridium botulinum that was shown to possibly protect and activate BoNT. HA33 is a neurotoxin- associated protein (NAP) that protects BoNT form acid denaturation in the stomach and gastric tract (Arndt et all 2005). These TCs are actually more toxic than pure BoNT. Without NAPs, like HA33, BoNT are actually susceptible to the acid conditions in the gastric tract. This allows the absorbance of BoNT throughout the body to increase due to the more epithelium tissue being available for the BoNT (Ito et all 2011). This means that HA33 is a problem because it allows BoNT to be even more of a threat than it already is just by allowing it to further penetrate the body. A way that has been discovered to possibly stop this protein from helping BoNT is by the use of aptamers.
Specific Aim 1: Use aptamers that can bind to HA33 and change its shape in order to prevent the neurotoxin from causing problems in the future.
The aptamer would look for the specific sequence in HA33 by going through a binding selection. The binding selection would take place in a series of rounds in order to narrow a pool of RNA to a specific sequence that will bind to the HA33 protein.
Figure 1: A crystal structure of BoNT in Clostridium botuinum. The arrow in the picture is pointing towards the HA33 present in the toxin complex (Arndt et all 2005)
Vendor: Ellington Lab
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01). Ha-33 facilitates transport of the serotype d botulinum toxin across a rat intestinal epithelial cell monolayer. Retrieved from http://onlinelibrary.wiley.com/doi/10.1111/j.1574-695X.2011.00779.x/full
4. Nobuko, H., Ellington, A., & Stanton, M. (2001, June 15).Aptamer beacons for the direct detection of proteins. Retrieved from http://www.sciencedirect.com/science/article/pii/S0003269701951693
5. Yoshimasa , S., Ken, I., Shin-Ichiro, M., Keita, M., Tomonori, S., Koichi, N., & Toshihiro, W. (2012, August 22). Botulinum toxin complex: A delivery vehicle of botulinum neurotoxin traveling digestive tract. Retrieved from http://www.intechopen.com/books/structure-and-function-of-food- engineering/botulinum-toxin-complex-a-delivery-vehicle-of-botulinum-neurotoxin-traveling-digestive-
6. Sugiyama, H. (1980). Clostridium botulinum neurotoxin.Microbiological Reviews, 44(3), 419-448. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC373187/
First Progress Report