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Clostridium botulinum (Botulism)

Botulism is a Category A bioweapon. It has a highly stable spore capable of surviving autoclaving, produces a potent toxin (175ng kills a 150lb man), and there are no effective treatments after toxin production. It has good medical applications, though, such as Botox. In 1989, FDA approved the use of BoNT-A (BOTOX, Allergan, Inc.) for strabismus (crossed eyes), dystonias (twitches and ticks), and hemifacial spasms (sustained contraction). The botulism virulence factor is a toxin, which is unusual. Botulism infections can be prevented by safe food handling and preparation.

Clinical Manifestations

There are 10-30 botulism cases reporter per year in the United States. After an incubation time of 12-26 hours (characterized by general muscular weakness and paralysis), flaccid descending paralysis occurs. Flaccid descending paralysis begins in ocular and throat muscles and later extends to all skeletal muscles. Some GI tract symptoms include cramps and constipation. There are four different forms of botulism infections:
  • Food-borne: most common, ingestion of toxin
  • Infant: ingestion of spores which germinate in G.I.
  • Botulinal toxic infection: similar to infant form, but >1 year old
  • Wound botulism: spores contaminating wound injuries (rare).

Morphology

Caused by Clostridium botulinum, C. butyricum, or C. barati, the infectious agent for botulism is a large gram-positive obligate aerobe motile bacillus 3-8 micrometers long. It forms subterminal spores.

Identifying Virulence Genes

The virulence factor for botulism is the botulism neurotoxin (BoNT). There are 7 BoNT types (BoNT-A through BoNT-G). Botulism and tetanus are examples of the simplest model of bacterial pathogenicity, where bacteria produce a toxin which results in disease. The BoNT type A gene is chromosomally located and found to be associated with several other genes, including 3 hemagglutinins, a non-toxic non hemagglutinin (NTNH) and a putative transcriptional regulator (BotR). They are hypothesized to protect the toxin from acidic and proteolytic degradation through the GI tract following consumption of contaminated foods. All of these genes are collectively referred to as the BoNT type A gene cluster. There is not a similar gene cluster (eg. NTNH and/or HA) found associated with the gene encoding TeNT.

Bradshaw et al. (1998) used a Clostridium/E. coli shuttle plasmid to make two recombinant constructs involving the BoNT gene cluster. One construct contained the genes encoding for ORFX-NTNH-BoNT (5" encoding region of BoNT) and the other construct contained the gene encoding only for BoNT (5" encoding region of BoNT). Both of these recombinant plasmids were transformed into wild type C. botulinum and a mutant of C. botulinum whose BoNT gene cluster was knocked out by Tn mutagenesis. Bradshaw cloned only the 5" coding region of the BoNT gene on each of the plasmid constructs so that in a wild type C. botulinum background, expression of plasmid encoded toxin could be differentiated from expression of full length toxin expressed from the chromosomal gene. Transformations using the recombinant plasmids revealed that the monocistronic message that encoded BoNT was not a post-transcriptional modification of the bi-cistronic message that encoded both NTNH and BoNT and ORFX/p21 was the transcription regulator of the BoNT type A gene cluster.

Biochemical Mechanism of Botulism and Tetanus Toxicity: Spastic vs Flaccid Paralysis

Tetanus Neurotoxin (TeNT) and Botulism Neurotoxin (BoNT) cause a spastic paralysis in one case and a flaccid paralysis in the other, despite cleaving the same target molecules which results in the inability to release neurotransmitters. This phenomenon arises because TeNT and BoNT bind different membrane receptors. BoNT enters the motor neuron by receptor mediated endocytosis after specific binding to the 58 kDa synaptotagmin protein by the Hc domain of BoNT. The BoNT-containing vesicle stays at the neuromuscular junction and the catalytic domain (L) is released when the disulfide bond is reduced following acidification of the vesicle. The catalytic domain then cleaves one of the docking protein machinery (either VAMP, syntaxin or SNAP-25, depending on which BoNT toxin it is). Cleavage of these proteins prevents release of neurotransmitters into the neuromuscular junction → no stimulation of the muscle → flaccid paralysis.

Both BoNT and TeNT bind to the same primary receptor (GT1b ganglioside) via the Hn region of their heavy chains. However, both bind to different secondary receptors via the Hc region of their heavy chains. Both these events result in receptor-mediated endocytosis, but the endosomes containing BoNT remain at the NMJ, whereas the endosomes containing TeNT undergo retrograde axonal transport to the central nervous system and localize to inhibitory neurons. Both toxins effect neurotransmitter release by cleaving proteins associated with the docking machinery. BoNT does this at NMJ, resulting in flaccid paralysis, while TeNT does this at the inhibitory neurons resulting in uncontrolled neural excitation causing spastic paralysis.

Diagnosis and treatment

Botulism infections are diagnosed via:

  • Clinical symptoms
  • Bacterial culture
  • PCR
  • Monoclonal anti-botulism antibody
  • Direct toxin detection

Before toxin production, botulism infections are treated by a heavy dose of antibiotics such as penicillin, tetracycline, or clindamycin. When toxins are in circulation, a heavy-dose I.V. administration of anti-toxin from pooled hyperimmune human donors or recombinant anti-toxin antibody. When toxins are bound to nerve receptors, intensive supportive care (including mechanical breathing assistance) is the only treatment.

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