The Microbiology and Botulism Research Unit (MBRU) puts a major focus on the development of therapies for treating botulism, a disease caused by a family of highly potent neurotoxins produced by Clostridia botulinum (for more information, see the CDC botulism website). Botulism is usually contracted through the consumption of contaminated food, particularly canned foods. Because of their toxicity and ease of production, the botulinum family of neurotoxins is classified as a Category A bioterrorism threat. The toxins cause flaccid paralysis by entering peripheral neurons and cleaving specific synaptosomal proteins involved in exocytosis, thus blocking neurotransmitter release. Seven different botulinum neurotoxin serotypes (A-G) are known in nature, many serotypes having a number of isotypic variants. These toxins are the most potent known to man and consist of a 100 kDa heavy chain (HC) and a 50 kDa light chain (LC). The HC contains the translocation and receptor binding domains while the LC contains the catalytic protease domain.

Since the Botulism Therapeutics Program began in 2004, we have established an approved BL2 select agent laboratory and core technologies such as an in vitro cell bioassay laboratory and an in vivo mouse bioassay laboratory. Four major research projects have been approved and are underway.

Overview of Program:

Botulism Therapeutics Projects:

Tufts in vivo toxin testing core (Project leader, Dr. Jean Mukherjee)
This project, entitled "Establishment of a Botulism Therapeutics Testing Facility", involves the construction and commissioning of a testing facility at Tufts that is capable of testing the efficacy of therapeutic agents to treat botulism post-exposure or to reverse existing intoxication.

Tufts in vitro bioassay core (Project leader, Dr. Jong-Beak Park)
This project entitled "Development of In Vitro Assays to Evaluate the Anti-BoNT Effects of Potential Therapeutic Agents" involves the development and performance of a core facility that performs botulism bioassays in neuronal cell culture systems. (See project entitled "Recombinant Nontoxic Botulinum Neurotoxins and Chains", B. Singh, project leader)

High affinity BoNT clonal antibodies (Project leaders, Drs. Jean Mukherjee and Charles Shoemaker)
We have one project entitled "High Affinity BoNT Clonal Antibodies". This project is generating a large number of monoclonal antibodies (mAbs). In collaboration with the Knight Lab at Loyola University and with Bioventix in the UK, selected mAbs will be tested for their BoNT affinity and their efficacy as therapeutic antitoxins. Recombinant single-chain Abs (scFvs) will also be produced and characterized. This project is generating a number of useful reagents for our program and for the larger BoNT research community.

New small molecule anti-toxin drugs (Project leader, Dr. Charles Shoemaker)
Two separate botulism drug discovery projects are underway.

1. One project, entitled "Discovery of Inhibitors of Botulinum Proteases" is in collaboration with the Laboratory for Drug Discovery in Neurodegeneration (LDDN) at Harvard (Dr. Ross Stein). Assay development and high throughput screening (HTS) is being done at LDDN, X-ray crystallography in the Laboratory of Dr. Allen at Boston University, and medicinal chemistry at Absolute Science (Dr. Alan Jacobson). This project is using a combination of HTS and rational drug design to develop BoNT drugs.

2. A second project, entitled "Development of Small Molecule Inhibitors of Botulinum Neurotoxin Protease Using Computational Methods in Conjunction with Combinatorial Library Design", is in collaboration with the Laboratory of Dr. Kim Janda at Scripps Institute. This project is primarily employing computer-aided rational drug design to develop BoNT drugs.

Agents that target toxins for proteasome degradation (Project leader, Dr. Charles Shoemaker)
Two closely linked botulism "designer E3 ligase" projects are underway. One project is entitled "Curing botulinum intoxication by targeted toxin proteolysis" and the other is "Creating BoNT-specific ubiquitin ligases for botulism therapy". These projects are a collaborative effort between Tufts and Veritas Laboratories (Drs. George Oyler and Randy Kincaid). We are developing designer E3 ligases that bind to BoNT protease and direct its ubiquitination and its subsequent accelerated proteasomal destruction. We are also developing protein and peptide systems that will facilitate the uptake of biomolecules to intoxicated neurons and their delivery to the cytosol. Two-hybrid and GST pulldown studies are being done to identify neuronal proteins that interact with the BoNT protease.

Proteomics of BoNT intoxication (Project leader, Dr. Charles Shoemaker)
This project, entitled "Proteomic Analysis of Neuronal Cell Responses to BoNT/A, B and E" is a collaboration with Dr. Ira Herman at Tufts. We are using Difference Gel Electrophoresis (DIGE) to identify proteomic changes that take place during BoNT intoxication of neurons. We are also looking for proteomic differences between neurons intoxicated with BoNT/A, B and E, particularly those that might explain the differences in duration.