Aptamer Screening of HA33 for the Diagnose of Botulism Neurotoxin

Aptamer Screening of HA33 for the Diagnose of Botulism Neurotoxin

Norma Arroyo – Fall 2013

Abstract

Botulism is an illness that leads to flaccid paralysis of the muscles and even causes death from respiratory failure. Botulism is caused by a neurotoxin produced by the Bacterium Clostridium botulinum.  HA33 is a nontoxic neurotoxin-associated protein (NAPs). HA33 helps protect the Clostridium Botulinum neurotoxin (BoNTs) from acid denaturation in the stomach and from attacks from proteolytic enzymes found in the gastro intestinal tract. HA33 have been discovered to potentially serve as internalization or an activator for BoNTs [1].

Botulism’s symptoms can be very similar to other illnesses and disease of muscle paralysis, therefore many tests may be performed to eliminate other possible illnesses. Botulism is diagnosed through a mouse inoculation test. This and other test may take up to days or weeks to finalize a concrete diagnosis of botulism[2]. An alternative diagnostic method for diagnosing botulism could be through utilizing aptamers.

Aptamers have a high specifically affinity to their target, HA33 in this case. Modifying an aptamer to carry a fluorescein will allow HA33 to be label and track in the small intestine. Similar to the CFP/YEP-based sensors used to detect the activity of BoNTs in cells as shown in Figure 1. [3]. Screening for the botulism neurotoxin can possibly help to estimate the amount of Heptavalent Botulism Antitoxin necessary for the treatment. This can also prevent the waste of Heptavalent Botulism Antitoxin. 

Figure 1. CFP/YFP-based sensors for detecting BoNT protease activity in vitro. [3]

Specific Aim 1:  Identify an aptamer against HA33 that can be modified to carry a fluorescein and be allowed to be tracked within the small intestine. Identifying the binding locations of these BoNTs can not only help for a sceening to diagnose botulism but possibly to deliver a pay load to the specific region.
Link to proposal:  https://docs.google.com/file/d/0BwaR2WWmhjiOVnJTejJUc2pmMzQ/edit?usp=sharing
Link to first progress report:  https://docs.google.com/file/d/0BwaR2WWmhjiON1Z5NWQ3TThfdVE/edit?usp=sharing
Link to second progress report:
 https://drive.google.com/file/d/0BwaR2WWmhjiOUlZScVRWTjZuSlk/edit?usp=sharing
Link to final progress report:
https://drive.google.com/file/d/0BwaR2WWmhjiOZDA0ZFp1b3A2akk/edit?usp=sharing

Target Order Information:

Vendor: Ellington Lab

Vendor Website: https://www.ellingtonlab.org

Central Lab Telephone: 512-471-6445

Cost per Round: $0.00 (Available in Lab)
Cost of Target: $0.00

References

1.      Arndt, J.W., Gu, J., Jaroszewski, L., Schwarzenbacher, R., Hanson, H.A., Lebeda, F.J., and Stevens, R.C. (2005). The Structure of the Neurotoxin-associated Protein HA33/A from Clostridium botulinum Suggests a Reoccurring β-Trefoil Fold in the Progenitor Toxin Complex. Jornal of Molecular Biology. 365(4): 1083-1093.

2.       Centers for Disease Control and Prevention [CDC], National Center for Emerging and Zoonotic Infectious Diseases [NCEZID], Division of Foodborne, Waterborne, and Environmental Diseases [DFWED] (2011) Botulism.

3.      Dong, M., Tepp, W.H., Johnson, E.A., and Chapman, E.R. (2004). Using fluorescent sensors to detect botulinum neurotoxin activity in vitro and in living cells. Proc Natl Acad Sci USA. 10(41): 14701-14706

4.       Davis, R.G., (2004). The ABCs of bioterrorism for veterinarians focusing on Category A Agents. J Am Vet Med Assoc 2004; 224:1084-1095

5.      Davis, Charles P., Stöppler, Melissa C. (2012) Botulism. WebMD

6.      Hutson R.A., Zhou Y., Collins M.D., Johnson E.A., Hatheway C.L., and Sugiyama H. J., (1996). Genetic characterization of Clostridium botulinum type A containing silent type B neurotoxin gene sequences. Biol. Chem. 271:10786-10792

7.       Pestronk, Alan ( 2012). Botulism. Neuromuscular Disease Center. Washington University, St. Louis, MO USA

8.       Hoon S., Zhou B.., Janda KD., Brenner S., Scolnick J., (2011). Aptamer selection by high-throughput sequencing and informatics analysis. BioTechniques. 51(6):413-6