• Chapter 20 (Schaechter’s)

SBM 2044 Lecture 7

Tetanus and Botulinum Neurotoxins

• Clostridium tetani Clostridium botulinum

• Similar neurotoxins – different diseases

Portrait byCharles Bellc. 1821

Clostridium botulinum

• Gram-positive, spore forming, rods

Clostridium tetani

• Strict anaerobes

• Normal habitat: soil

Diseases

• Pseudomembranous colitis

• gas gangrene

• Cellulitis

• Tetanus

• Botulism

• Food poisoning

Pathogenesis Encounter, entry and multiplication:• C. botulinum spores found in soil or marine sediments,

contaminated meats, vegetables and fish.• C. tetani in GIT of humans and in soil samples – most

cases of tetanus are associated with a traumatic wound• C. perfringens in soil and the intestinal tract of many

animals. Wound site has a low oxidation reduction potential, compromised blood supply, calcium ions and various peptides and amino acids that allows germination of spores.

• Spores are heat resistant – survive food processing and canning – anaerobic spores germinate and release potent toxins– proteolytic enzymes cause spoilage of food

Damaging effects of Clostridium

• Many clostridial diseases are serious and life threatening

• All are caused by exotoxins secreted by the clostridia, often without colonization or invasion by the organism.

• Heating canned foods insufficiently before sealing lids produces a nice anaerobic environment without killing spores – ideal conditions for germination and growth of C. botulinum. – e.g. boiling not sufficient, and many home pressure cookers also inadequate.

• Even if toxin is present in food, boiling for 15 mins or so before eating is usually sufficient to inactivate toxin

Damage by C. botulinum

• 1 microgram can kill a large family; 400g could kill all the people on Earth

• Botulinum toxin prevents the release of neurotransmitter Ach, muscle unable to contract

• Flaccid paralysis within 12-36hrs after ingestion of toxin

Botulism

• Most cases: Toxin ingested in food bloodstream flaccid paralysis

• Infant botulism: (Floppy baby syndrome) In absence of developed gut flora, C. botulinum can grow in colon (anaerobic environment) + produce toxin – less readily absorbed than ingested toxin, but can cause death.

• Wound botulism: RARE – but can occur if deep wounds (anaerobic) very heavily contaminated (spores) with soil – e.g. war

(common source: home-canned foods)

Tetanus

Tetanus:Toxin produced by C.tetani infecting deep wound bloodstream CNS spastic paralysis

(Lockjaw)

Botulinum neurotoxin (BoNT):

Tetanus neurotoxin (TeNT):

Can also produce other toxins – do not confuse the neurotoxins (e.g. C. botulinum C2 & C3 toxins; C. tetani tetanolysin)

Clostridium neurotoxins

• produced by C. botulinum (C. barati, & C. butyricum)

• 8 different variants (serotypes A to G)

• toxin gene carried on phage or plasmid

• produced by C. tetani

• all strains produce same TetNT - no significant variants• gene located on a plasmid

S

S

N

C

BotNT & TetNT: secreted as single 150Kd polypeptides, with internal disulphide bond

nicked by protease

S

S

N

N

C

C

L-chain (50Kd)

H-chain (100Kd)

Hc HN

activates toxin

• In the lab., purified H-chain cleaved in half by papain, to produce separate Hc and HN fragments

• Reduction separates the Heavy and Light chains

S

S

N

N

C

C

L-chain (50Kd)

H-chain (100Kd)

Hc HN

• Studies on purified L-chain, H-chain, & Hc & HN fragments

• Note similarity in overall ‘architecture’ to DTx

Receptor-bindingdomain

neuron-specific

Membrane-translocation domain (probable)

active domain

deduced from abilityto conductance of planar lipid bilayers @ low pH

Mechanisms of action

• By 1948: BotNT blocks neurotransmitter release at neuro-muscular junctions (NMJ)

• In 1960s: TetNT blocks neurotransmitter release by inhibitory interneurons in spinal cord.

Both toxins very similar structures + very similar mechanisms

Very different diseases

BUT

Different sites of action

Neurons

SynapsesConnections

Protagonistmuscle contracting

Antagonist musclerelaxing

Inhibitory (e.g. GABA)

Motor neuronsSensory neuronsInterneurons

Stimulatory (e.g. acetylcholine)

Neurons Neurotransmitters

M (on)

M (off)

I

I

M

S

CNS

muscle relaxed

musclerelaxed

M (on)

M (off)

I

I

M

S

CNS

Botulinum Toxin: Acts at neuro-muscular junctions.

BoNT

BoNT

• binds receptor on end-plates of motor neurons RME

• L-chain translocated from acidified vesicle to cytoplasm

• blocks neurotransmitter release FLACCID PARALYSIS

JAMA 2001; 285;1059-1070

Mechanisms of action of BotTN

M

M

I

I

M

S

CNS

Tetanus Toxin: Acts in the CNS

vesicle inside axon

• binds receptor at terminals of peripheral nerves RME

• Unlike BotNT, active domain NOT translocated at this site (reasons still unclear - distinct receptors/vesicles ??)• Instead, retrograde axonal transport CNS

M

M

I

I

S Vesicle +Tet. toxin Exocytosis

• vesicle reaches post-synaptic dendrites• exocytosis releases toxin into synaptic cleft• binds receptors on pre-synaptic axon terminals of interneurons • internalised again by RME• in the interneuron, TeNT active domain is translocated from endocytic vesicle to cytoplasm blocks release of inhibitory neurotransmitters

TetX X

Motor neuron dendrite

Interneuronaxon terminal

RME + translocation to

cytoplasm

M

M

I

I

M

S

CNS

Tetanus Toxin: Acts in the CNS

• Blocking inhibitory neuro-transmitters disrupts normal control (incl. sensory feedback) of signals to opposing muscles, resulting in continuous stimulation of muscles

SPASTIC PARALYSIS (hence “lockjaw”)

Tet

Tet

Mechanism of action of L-chain

DNA sequencing provided key clue

• Regions of strongest homology between amino acid sequences of the L-chains of TetNT & various BotNTs

included a Zn-binding ‘motif’ similar to that in Zn-dependent proteases called Zn-endopeptidases

Strongly suggested that L-chains act as proteases

Must ‘target’ proteins involved in neurotransmitter release

Axon terminal of motor neuron

Muscle cell

Neurotransmitters insmall synaptic vesicles which have a surfaceprotein called VAMP

Complex incl. SNAP-25, syntaxin on inner surface of axon membrane

‘docks’ with

neurotransmitters releasedby exocytosis at NMJ

Small synaptic vesicle (contain neurotransmitters)

Neuron membrane

Vesicle membrane

In early 1990s, proteins involved in neurotransmitter releaseidentified – later shown to be cleaved by BoNT and TeNT

Other Clostridial diseasesGas gangrene• caused by C. perfringens, produces multiple virulence

factors; highly tissue invasive -toxin damages cell membranes by hydrolyzing

phosphatidylcholine and sphingomyelin, leading to cell death myonecrosis

Other Clostridial diseases

C.difficile

• Cause severe ulcerating disease of the large bowel – pseudomembranous colitis (diarrhoeal disease)

• Antibiotic-associated diarrhoea – milder and less severe than pseudomembranous colitis

Treatment and Prevention• Botulism

– Trivalent antitoxin for types A, B and E. antitoxin from horses

– Botulism can be prevented by proper canning methods

• Tetanus– Tetanus toxoid vaccine

– Antibiotic to kill the organism

– Physiological exercise to limit complications

• Gas gangrene– Prompt treatment of either surgical removal or penicillin

administration to control wound infection

• C. difficile diarrhoea– Antibiotics (cephalopsorins, ampicillin, clindamycin)

Term Paper

1. Discuss how sea cucumber protein could potentially help in infectious diseases.

2. How do animal slaughtering contribute to the spread of zoonosis diseases?

3. Vaccination can be unsafe and ineffective. Give your comments.

4. Tell us about ONE most interesting topic in the microbiology subject. Justify by giving reasons and examples to your chosen topic.

Term Paper

• Choose your question number. (3 students per question)

• Write in word-processed, fully formatted – PLUS legends for tables, figures,

– and REFERENCES

– 1.5 or 2.0 spacing

• Write for ONLY 15 – 30 PAGES. • Remember to write not just a purely scientific

paper, but a philosophical one!• Deadline: FRIDAY, 27 FEBRUARY 2009