ObjectiveThe Working Group on Civilian Biodefense has developed consensus-based
recommendations for measures to be taken by medical and public health professionals
if botulinum toxin is used as a biological weapon against a civilian population.ParticipantsThe working group included 23 representatives from academic, government,
and private institutions with expertise in public health, emergency management,
and clinical medicine.EvidenceThe primary authors (S.S.A. and R.S.) searched OLDMEDLINE and MEDLINE
(1960–March 1999) and their professional collections for literature
concerning use of botulinum toxin as a bioweapon. The literature was reviewed,
and opinions were sought from the working group and other experts on diagnosis
and management of botulism. Additional MEDLINE searches were conducted through
April 2000 during the review and revisions of the consensus statement.Consensus ProcessThe first draft of the working group's consensus statement was a synthesis
of information obtained in the formal evidence-gathering process. The working
group convened to review the first draft in May 1999. Working group members
reviewed subsequent drafts and suggested additional revisions. The final statement
incorporates all relevant evidence obtained in the literature search in conjunction
with final consensus recommendations supported by all working group members.ConclusionsAn aerosolized or foodborne botulinum toxin weapon would cause acute
symmetric, descending flaccid paralysis with prominent bulbar palsies such
as diplopia, dysarthria, dysphonia, and dysphagia that would typically present
12 to 72 hours after exposure. Effective response to a deliberate release
of botulinum toxin will depend on timely clinical diagnosis, case reporting,
and epidemiological investigation. Persons potentially exposed to botulinum
toxin should be closely observed, and those with signs of botulism require
prompt treatment with antitoxin and supportive care that may include assisted
ventilation for weeks or months. Treatment with antitoxin should not be delayed
for microbiological testing.

https://jamanetwork.com/journals/JAMA/articlepdf/193600/jst00017.pdf

Botulinum Toxin as a Biological Weapon: Medical and Public Health Management

Nerve terminals are specific sites of action of a very large number of toxins produced by many different organisms. The mechanism of action of three groups of presynaptic neurotoxins that interfere directly with the process of neurotransmitter release is reviewed, whereas presynaptic neurotoxins acting on ion channels are not dealt with here. These neurotoxins can be grouped in three large families: 1) the clostridial neurotoxins that act inside nerves and block neurotransmitter release via their metalloproteolytic activity directed specifically on SNARE proteins; 2) the snake presynaptic neurotoxins with phospholipase A(2) activity, whose site of action is still undefined and which induce the release of acethylcholine followed by impairment of synaptic functions; and 3) the excitatory latrotoxin-like neurotoxins that induce a massive release of neurotransmitter at peripheral and central synapses. Their modes of binding, sites of action, and biochemical activities are discussed in relation to the symptoms of the diseases they cause. The use of these toxins in cell biology and neuroscience is considered as well as the therapeutic utilization of the botulinum neurotoxins in human diseases characterized by hyperfunction of cholinergic terminals.

https://www.physiology.org/doi/pdf/10.1152/physrev.2000.80.2.717

Neurotoxins Affecting Neuroexocytosis

Zinc is known to be indispensable to growth and development and transmission of the genetic message. It does this through a remarkable mosaic of zinc binding motifs that orchestrate all aspects of metabolism. There are now nearly 200 three dimensional structures for zinc proteins, representing all six classes of enzymes and covering a wide range of phyla and species. These structures provide standards of reference for the identity and nature of zinc ligands in other proteins for which only the primary structure is known. Three primary types of zinc sites are apparent from examination of these structures: structural, catalytic and cocatalytic. The most common amino acids that supply ligands to these sites are His, Glu, Asp and Cys. In catalytic sites zinc generally forms complexes with water and any three nitrogen, oxygen and sulfur donors with His being the predominant amino acid chosen. Water is always a ligand to such sites. Structural zinc sites have four protein ligands and no bound water molecule. Cys is the preferred ligand in such sites. Cocatalytic sites contain two or three metals in close proximity with two of the metals bridged by a side chain moiety of a single amino acid residue, such as Asp, Glu or His and sometimes a water molecule. Asp and His are the preferred amino acids for these sites. No Cys ligands are found in such sites. The scaffolding of the zinc sites is also important to the function and reactivity of the bound metal. The influence of zinc on quaternary protein structure has led to the identification of a fourth type of zinc binding site, protein interface. In this case zinc sites are formed from ligands supplied from amino acid residues residing in the binding surface of two proteins. The resulting zinc site usually has the coordination properties of a catalytic or structural zinc binding site.

Zinc coordination sphere in biochemical zinc sites

Botulinum toxin is a uniquely potent substance synthesized by the organisms Clostridium botulinum, Clostridium baratii, and Clostridium butyricum. This toxin, which acts preferentially on peripheral cholinergic nerve endings to block acetylcholine release, is both an agent that causes disease (i.e., botulism) as well as an agent that can be used to treat disease (e.g., dystonia). The ability of botulinum toxin to produce its effects is largely dependent on its ability to penetrate cellular and intracellular membranes. Thus, toxin that is ingested or inhaled can bind to epithelial cells and be transported to the general circulation. Toxin that reaches peripheral nerve endings binds to the cell surface then penetrates the plasma membrane by receptor-mediated endocytosis and the endosome membrane by pH-induced translocation. Internalized toxin acts in the cytosol as a metalloendoprotease to cleave polypeptides that are essential for exocytosis. This review seeks to identify and characterize all major steps in toxin action, from initial absorption to eventual paralysis of cholinergic transmission.

Identification of the Major Steps in Botulinum Toxin Action

How botulinum and tetanus neurotoxins block neurotransmitter release.

Botulinum neurotoxin type A (BoNT/A) is the potent disease agent in botulism, a potential biological weapon and an effective therapeutic drug for involuntary muscle disorders. The crystal structure of the entire 1,285 amino acid di-chain neurotoxin was determined at 3.3 A resolution. The structure reveals that the translocation domain contains a central pair of alpha-helices 105 A long and a approximately 50 residue loop or belt that wraps around the catalytic domain. This belt partially occludes a large channel leading to a buried, negative active site--a feature that calls for radically different inhibitor design strategies from those currently used. The fold of the translocation domain suggests a mechanism of pore formation different from other toxins. Lastly, the toxin appears as a hybrid of varied structural motifs and suggests a modular assembly of functional subunits to yield pathogenesis.

D B Lacy

Papers

Crystal structure of botulinum neurotoxin type A and implications for toxicity.