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United States Department of Agriculture

Agricultural Research Service

Research Project: Technologies for Detecting and Determining the Bioavailability of Bacterial Toxins

Location: Foodborne Toxin Detection and Prevention

Title: Purification and characterization of neurotoxin complex from a dual toxin gene containing Clostridium botulinum strain PS-5

Authors
item Singh, Ajay
item Sachdeva, Amita
item Degrasse, Jeffery
item Croley, Timothy
item Stanker, Larry
item Hodge, David
item Sharma, Shashi

Submitted to: The Protein Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 5, 2013
Publication Date: April 27, 2013
Citation: Singh, A.K., Sachdeva, A., Degrasse, J.A., Croley, T.R., Stanker, L.H., Hodge, D., Sharma, S.K. 2013. Purification and characterization of neurotoxin complex from a dual toxin gene containing Clostridium botulinum strain PS-5. The Protein Journal. 32(4):288-296. DOI: 10.1007/S10930-013-9486-1.

Interpretive Summary: Botulism is a serious, often fatal neuroparalytic disease in humans and animals caused by a protein toxin (botulinum toxin, BoNT) produced by the bacterium Clostridium botulinum. BoNT is considered the most toxic biological toxin known and is considered a class A bioterrorism agent. BoNT is secreted as part of a protein complex consisting of a toxin molecule surrounded by non-toxin associated proteins, the toxic complex (TC). The exact role of the TC in disease is not known, but it is thought that they protect the toxin as it passes through the harsh environment of the stomach. Seven immunologically distinct types (serotypes A-G) of toxin are known. We describe a strain of Cloistridium botulinum, designated PS-5, that carries the genes for two toxin serotypes, A and B and methods to purify the TC. It is possible that organisms that produce more than one type of toxin may cause a more severe illness because of synergism between the toxins. Thus, it is critically important to purify and characterize TC’s produced by dual toxin producing strains. These studies increase our understanding to dual toxin producing strains and improve our ability to detect this lethal foodborne agent resulting in a safer food supply.

Technical Abstract: Botulinum neurotoxins (BoNTs) are produced as a toxin complex (TC) which consists of neurotoxin (NT) and neurotoxin associated proteins (NAPs). The characterization of NT in its native state is an essential step for developing diagnostics and therapeutic countermeasures against botulism. The presence of NT genes was validated by PCR amplification of toxin specific fragments from genomic DNA (gDNA) of Clostridium botulinum strain PS-5 which indicated the presence of both serotype A and B genes on PS-5 genome. Further, TC was purified and characterized by Western blotting, Digoxin-Enzyme Linked Immunosorbent Assay (DIG-ELISA), endopeptidase activity assay, and Liquid Chromatography-Mass Spectrometry (LC-MS). The data showed the presence of serotype A specific neurotoxin. Based on the analysis of neurotoxin genes and characterization of TC, PS-5 strain appears as a serotype A(B) strain of C. botulinum which produces only serotype A specific TC in the cell culture medium

Last Modified: 12/20/2014
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