Botulism; Botulinum toxin

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BOTULISM^⁴

Clinical features
Clostridium botulinum
Diagnosis
Features of subtypes
Mechanisms of action
Pathology
Prevention
Prognosis
Protein structure
Clinical Syndromes
Foodborne
Iatrogenic: Generalized after local injection
GI colonization
Infantile
Inhalation
Wound

External links
Biological weapon: AMA (pdf)
CDC Manual (pdf)
Movie

Emile Van Ermengem

Causative organisms
- Clostridium botulinum
  - Gram positive bacilli
  - Spore producing
  - Produced under anaerobic conditions: Obligate
  - Toxin production
    - May produce all 7 toxin serotypes: A-G
    - Genetic information
      - Type G on plasmids
      - Types C1 & D on bacteriophages
      - Types A, B, E, and F on the bacterial chromosome
    - Bound to hemagglutinins: ? Inhibits degradation by digestive enzymes
  - External links: Arizona; Illustration
- C. baratii: Produces serotype F
- C. butyricum: Produces serotype E
Botulinum Toxin: Proteins & Transport
- 7 neurotoxic types of botulinum toxin: A , B , C1 , D , E , F , G
- Sequence homology (30% to 40%) to tetanus toxin
- Produced as a single chain protein (protoxin) with molecular weight 150 kDa
- Released from bacteria as part of noncovalent multimeric complex
  - Auxiliary proteins include: Hemagglutinins (HA); Nontoxin, nonhemagglutinin (NTNH)
  - Complex may protect toxin at low pH in GI tract
  - Complex dissociates spontaneously at physiologic pH
  - No direct role of auxiliary proteins in toxin-induced blockade of cholinergic transmission
- Cleavage of Botulinum protoxin: Into 2 chains
  
  Botulinum Toxin
  Type B
  - Heavy (100 kDa) chain
  - Light (L) chain (50 kDa)
    - Light chains have a tetrahedral zinc binding motif: Contains
      - Consensus HELIH amino acid sequence
        
        Common to other zinc dependent metalloproteinases
      - 2 histidines, glutamate & water molecule
      - Structure resembling thermolysin-like endoproteases
    - Contain toxic activity
      - Zinc-dependent endopeptidase
      - Cleave proteins forming synaptic vesicle docking & fusion complex
    - 3-D Geometry: Catalytic sites buried deeply within protein
  - Heavy & Light subunits linked by disulfide bond
  - Double chain product is active form: Inhibits cholinergic transmission
  - Protease producing cleavage
    - Often contained by producing organism
    - May also occur in GI tract
- Toxin passes from GI tract to vasculature: Mechanisms
  - Transport cell: Absorptive enterocyte
  - Binding to the apical surface of epithelial cells
  - Receptor-mediated endocytosis
  - Transcytosis through cells
  - Delivery to the basolateral surface of cells
  - Ability of toxin to cross cells depends on
    - Toxin type
      - Able to cross cells: Botulinum type A & B
      - Not able to cross cells: Botulinum type C & Tetanus
    - Heavy chain of botulinum toxin: C-terminus
- Toxin passes out of vasculature to presynaptic regions
  - ? Mechanism
  - Does not cross blood-brain barrier
- Other absorption routes
  - Botulinum toxin may be absorbed from respiratory system
- General action: Blocks exocytosis
  - Most potent action: Blockade of cholinergic terminals
  - Other blockade of exocytosis: At higher concentrations of toxin
    - Other nerve terminals: Norepinephrine, Serotonin
    - Non-neural cells: If membrane receptors present
- External link: Botulinum proteins

Botulinum toxin: Mechanism of action at presynaptic nerve terminals²

Overall action: Blocks neurotransmitter release at peripheral cholinergic nerve terminals
- Neuromuscular junction
- Autonomic nerve terminals: Sympathetic & Parasympathetic
Endplate electrophysiology
- MEPP frequency: Reduced
- EPP quantal content: Reduced
4 STEPS in development of presynaptic blockade at NMJs

Binding to receptors on unmyelinated presynaptic membrane
- Mediated by: Carboxy (C) terminal of botulinum toxin heavy chain
  - In native state heavy chain is associated with light chain
- Receptors: Double receptor model
  - First receptors
    - Identity of receptors: Gangliosides with more than one neuraminic (sialic) acid, e.g. GT1b
    - Type of binding
      - Lock & Key
      - Little or no change in conformation of bound botulinum neurotoxin
      - Low affinity
    - Role: Bring toxin into proximity with second receptor
  - Second receptor
    - Postulated to be integral membrane protein
    - Candidate molecules: Synaptotagmins I & II
- Susceptibility of cholinergic & non-cholinergic neurons to specific botulinum and tetanus toxins
  - May be related to ability of toxins to bind to receptors on presynaptic terminals
Uptake of toxin into nerve terminals by endocytosis
- Bound toxin is internalized by endocytosis
  - Botulinum toxicity is inhibited by Botulinum anti-toxin only in 1st 30 minutes after toxin exposure
- L-chain is translocated across presynaptic membrane through a channel
- Translocation is mediated by (N) terminal of botulinum toxin heavy chain (H_N)
  - Heavy chain then forms channel releasing light chain into terminal cytoplasm
  - Translocation domain: Kinked α-helices in H_N
  - Low pH in endosome necessary
    - ? Brings amphiphilic/transmembrane segments of H_N together to form transporter channel
    - ? Causes conformation change in N-terminus of heavy chain
Translocation across endosome membrane
- General: Movement of catalytically active domain from lumen of endosome to cytosol
- pH-induced
  - Induces conformational changes in botulinum that expose hydrophobic domains
  - Inhibited by drugs that neutralize endosomal pH (Chloroquine)
- Insertion of toxin into endosomal membrane
  - Mediated by N-terminus of heavy chain
  - Heavy & light chains become separated
  - Heavy chain: Has dual functions
    - Channel & chaperone, for light chain
    - Associated with cleft formation in endosomal membrane
  - Light chain
    - Transported from endosome to cytosol
    - In cytosol: More neutral pH restores light chain structure

Inhibition of transmitter exocytosis from presynaptic terminal

Action of toxin: Zinc dependent proteolysis of protein components of exocytosis (SNARE complex)
- Enzyme class: Endopeptidase
- Toxin actions antagonized by metalloproteinase inhibitors
  - Captopril; Phosphoamidon
- Inhibition produced by: Light chains of botulinum and tetanus toxins
- Protein substrates can only be cleaved before assembly of SNARE complex
- Endopeptidase activity mainly observed with reduction of disulfide bond between 2 chains of botulinum toxin

Molecular targets: Botulinum subtypes and tetanus toxin

Clostridium toxins	Target proteins cleaved
Botulinum A B C D E F G Tetanus	SNAP-25 VAMP (Synaptobrevin) SNAP-25; Syntaxin VAMP (Synaptobrevin) SNAP-25 VAMP (Synaptobrevin) VAMP (Synaptobrevin) VAMP (Synaptobrevin)

General
- Location of substrates: Nerve terminal cytosol
- Specific substrates
  - SNARE presynaptic proteins involved in exocytosis
  - Peptide substrates in each protein target have common motifs
- Mechanism of action: Proteolysis of substrates
Vesicle associated membrane protein (VAMP; Synaptobrevin)
- Present in synaptic transmitter-containing vesicles
- Site cleaved is extravesicle loop of VAMP
  - Botulinum type B: Gln76-Phe77
  - Tetanus: Gln76-Phe77
  - Botulinum type D: Lys59-Leu60
  - Botulinum type F: Gln58-lys59
Synaptosomal-associated protein (SNAP-25 )
- Attached to syntaxin & presynaptic membrane
- Cleaved near C-terminus by
  - Botulinum type A: Gln197-Arg198
  - Botulinum type E: Arg180-Ile181
Syntaxin 1
- Presynaptic membrane protein associated with
  - Ca⁺⁺ channels & SNAP-25
- Cleaved near C-terminus by
  - Botulinum type C1
  - Only when in membrane

Termination of action
- Mechanism unknown
- Type A toxin with longer action than type B

External link: Movie (Shockwave)

Botulinum toxin: Subtypes & Features
- Type A
  - Most common outbreaks in Rocky Mountains & West
  - Elevated Ca⁺⁺ level in synaptosomes overcomes blockade
  - More severe & long-lasting paralysis: 67% with intubation
  - Physiology
    - Miniature endpale potentials (MEPPs)
      - Frequency: Reduced
      - Amplitude: Usually Reduced
    - EPPs: Quantal content reduced
  - Cleavage target: SNAP-25 (t-snare)
  - More blockade of spontaneous transmitter release than type B
  - Repetitive stimulation evokes post-synaptic response
- Type B
  - Most common outbreaks in East, especially Allegheny range
  - Has most structural homology to tetanus toxin
  - Requires assembled intracellular microtubules for action
  - Somewhat less severe paralysis than Type A
  - Physiology
    - Miniature endpale potentials (MEPPs): Normal or Moderate reduction in frequency
    - EPPs: Quantal content reduced; Variable latencies
  - Cleavage target: Synaptobrevin 1 (VAMP) (v-snare)
  - Blocks effect of α-Latrotoxin
  - Repetitive stimulation does not evoke post-synaptic response
- Types C & D
  - Poorly absorbed from intestine
  - Cause disease in animals
    - Avian botulism: Type C; "Limber neck"
    - Equine: Forage (adult) poisoning; Shaker Foal Syndrome
  - Only rarely implicated in human intoxication
  - Botulinum C3: Ribosylates ADP
    - Substrates: RAC1 ; ; RAC2 ; RAC3
- Type E
  - Found in fresh water sediment
    - Most common outbreaks in Baltic, Alaska & Great Lakes states
    - Associated with fish ingestion
- Type F: Produced by C. baratii
Botulism: Clinical features
- Time course
  - Incubation period:
    - Average: 18 to 38 hours
    - Extremes: 2 hours to 1 week
- Weakness
  - Diffuse; Usually symmetric; Proximal > Distal
  - Bulbar: Dysphagia; Dysarthria
  - Extraocular: Ptosis; Extraocular muscle weakness
- Sensory loss: Never prominent
- Tendon reflexes
  - Reduced
  - ? Less involved with type E
- Autonomic
  - Cholinergic
    - Pupils
      - Dilated
      - Blurred vision
      - Many, but not all, patients
    - Bradycardia
    - Hypotension
    - Skin: Hypohydrosis
    - Urinary retention
    - Gastrointestinal
      - Nausea & vomiting associated with contaminated food
      - Constipation: Frequently first sign, especially in infants; Common late
      - Diarrhea may occur early
  - Lab
    - Sympathetic skin response: Absent
    - Plasma norepinephrine: Reduced
- Treatment
  - Supportive care
  - Anti-toxin: Human
    - Usual type used: Trivalent vs A, B, E
    - May be most effective for Type E
    - Infant botulism: Reduces time in hospital, on ventilator & tube feeding
    - US Army anti-toxin: Heptavalent; For bioterrorist attack
Botulism: Diagnosis
- Electrodiagnostic¹
  - Repetitive nerve stimulation
    - Increment: With (40 Hz) or paired stimuli
      - Occurs in 50% to 60% of patients
      - Testing of multiple muscles may be needed to see increment
      - More common with type B botulism
    - Decrement on slow RNS: Occasional patients
  - CMAPs: Small
  - Motor unit potentials: Short
  - Recruitment: Normal, or Mildly reduced
  - SNAPS: Normal
  - EMG: Muscle denervation; Fibrillations & Positive sharp waves
- Muscle biopsy: Scattered angular, small muscle fibers
- Analysis of serum, feces & implicated food
  - Passive transfer of serum or other body fluid to mice
    - Toxicity to mice
    - Selectively prevented by anti-toxin
  - Stool or wound culture: Not useful in Iatrogenic or Inhalation syndromes
Botulism: Clinical syndromes

Foodbourne
GI colonization
Iatrogenic
Inhalation
Wound
- Foodborne botulism
  - Disease mechanism: Related to ingestion of toxin
    - Food
      - Contaminated with Clostridium spores
      - Clostridium grows in Anaerobic conditions: Produces toxin
    - Absorption
      - Stomach: Not absorbed; Toxin complex with accessory proteins is resistant to proteolysis
      - Intestine
        
        Alkaline pH of intestine dissociates toxin from associated proteins
        
        Dissociated toxin is absorbed into circulation by endocytosis & translocation
  - Age: Usually adults
  - Epidemiology: Intoxication most common in California, Colorado, New Mexico, Illinois
  - Associated foods
    - Home canned vegetables, potatoes fish & preserved sea food
    - Recent epidemics: Peyote cactus tea; Mascarpone cream cheese
  - Treatment
    - Supportive care: Respiratory; Wound debridement
    - Early
      - Emetics: Avoid magnesium containing
      - Lavage
      - Enemas: Not when paralytic ileus
    - Antitoxin
      - Most useful in 1st 24 hours
      - Trivalent ABE
      - Obtain from CDC: 404-639-3753 (day); 404-639-2888 (night)
      - Use on clinical diagnosis: Without waiting for lab confirmation
      - Lowers fatality rate & shortens illness (For Type A)
      - Complications (immune): 9%
    - ? 3,4-diaminopyridine
- Wound botulism
  - Disease mechanism: Toxin is produced locally by infecting organisms in wound
    - In wound or abscess
    - After proliferation of bacterial spores
  - Causes
    - Drug abuse most common cause
      - Intramuscular or subcutaneous ("Skin-popping")
      - "Black tar" heroin
      - Subcutaneous abscesses (50%)
      - Most common in California
    - Injuries: Crush; Compound fractures
  - Incubation period 4 to 14 days, longer when wound is debrided
  - Onset: Blurred vision & Bulbar weakness
  - Progression
    - Generalized weakness
    - Dysarthria; Dysphagia
    - Pupillary reactivity: Reduced
  - No GI motility change
  - Most common in California
  - Botulinum types A > B
  - Electrophysiology
    - CMAP amplitude: Reduced (50%)
    - RNS
      - 2 Hz: Decrement in 50%;
      - 30 Hz: Increment in 50%
    - Motor unit potentials: Small
  - Treatment
    - Wound debridement
    - Penicillin
  - Prognosis
    - Ventilator dependence frequency: Type A > B > E
    - Slow improvement in strength over weeks to months
    - 1 year: Most near normal ± fatigue
    - Autonomic function may improve later than weakness
    - Mortality: 5% to 10%
- GI colonization syndromes: Usually Infants (Infant botulism)
  - Disease mechanism: Toxin is produced locally by infecting organisms in GI tract
  - Exposure: Ingested Clostridium botulinum spores
    - Raw honey may be a source of type B organisms
    - Clostridium grows better in GI tract without normal flora
      - Young infants: Before normal flora has developed
      - After treatment with antibiotics: Reduces normal flora
      - Less competition from normal GI organisms
    - Organisms produce toxin in the GI tract
  - Epidemiology
    - Occurs in areas with botulinum spores in soil
    - US: Common in
      - California (50% of cases in US), Utah, Pennsylvania & Hawaii
      - Utah soil features
        
        Alkaline, Low organic content, Sagebrush grasslands
        
        C botulinum type A
      - Eastern US soil features
        
        Slightly acidic, Higher organic content, Cultivated fields
        
        C botulinum type B
  - Onset
    - Age: 1 to 6 months; Median 4 months
    - Adults: ~ 1 to 2 months after ingestion of spores
      - Predisposing factors
        
        Achlorhydria
        
        Chronic antibiotics
        
        Gastrectomy
        
        Intestinal surgery
      - Toxin types: A; B; F (produced by C. baratii)
  - Clinical
    - Onset: Acute
    - Initial signs: Constipation & Weakness
    - Weakness
      - Eye: Ptosis; Ophthalmoplegia; Mydriasis
      - Bulbar: Poor suck & feeding; Reduced gag
      - Weak cry
      - Hypotonia
      - Respiratory distress
    - Parasympathetic changes
      - Hypotension
      - Tachycardia
      - Mydriasis
  - Diagnosis
    - Stool: Culture & Analysis for toxin
    - Repetitive nerve stimulation: Increment
  - Treatment
    
    Increment on rapid repetitive nerve stimulation
    - Botulism immune globulin⁵
      - Shortens hospital stay by~17 days
      - Cost: $45,000 per patient
    - Anti-toxin & antibiotics not effective
    - Supportive care: Respiratory; Nutrition; Fluid
  - Prognosis
    - Improve spontaneously without treatment
    - Time course: Weeks to months
    - Deaths: Rare with adequate support
- Iatrogenic: Generalized signs after local botulinum injection
  - Dose used: Within therapeutic guidelines
  - Weakness: Generalized
  - Autonomic: May be involved
  - Lab: Abnormal single fiber EMG
- Inhalation botulism syndrome³
  - Exposure
    - Aerosolized botulinum toxin
    - Sources: Bioterrorism; Veterinary
  - GI prodrome: None
  - Early signs: Dysphagia; Dilated pupils; Ophthalmoparesis
  - Likely Epidemiology in Bioterrorism
    - Several cases: Multiple simultaneous groups
    - Common temporal & geographical, but not food, exposure
    - Probably not waterbourne
    - Latency: 12 to 80 hours
    - Unusual toxin type for geographic area
  - Treatment
    - Respiratory support
    - Antitoxin: As early as possible
Botulism: Prevention
- Canning or preserving foods with appropriate heat, pressure, & low pH
  - Spores
    - Survive 2 hr at 100 °C
    - Inactivated at 120 °C
    - Boiling food before canning at high elevations may not inactivate the spores
    - Factors favoring spore germination: Low acidity (pH > 5.0); Low O₂; High water content
  - Toxin: Inactivated after 1 minute at 85 °C, or 5 minutes at 80 °C
- Avoid exposure of infants to honey (may contain Clostridium botulinum spores)
- Botulinum intoxication: No immunity from subsequent episodes
- Immunization
  - Pentavalent toxoid
  - Used only for high risk personnel: Laboratory workers; Military
Botulism: Pathology of terminal axons

Terminal axons at Neuromuscular junctions (Rat)
Botulinum toxin induces enlargement of NMJs (middle)
& sprouting of nerve terminals (middle & top)
Also see: Terminal axon sprouting

Botulinum Toxin, Type B

Return to Neuromuscular Home Page
Return to Neurotoxins

References
1. Muscle Nerve 2000;23:1137-1144
2. Nature Structural Biology 2000;7:617-619
3. JAMA 2001;285:1059-1070
4. Annu Rev Pharmacol Toxicol 2004;44:167�193
5. Neurology 2005;64:2029�2032

11/20/2007

BOTULISM4

BOTULISM^⁴