Chapter 20 (Schaechter’s)

1. Chapter 20 (Schaechter’s)

2. SBM 2044 Lecture 7 Tetanus and Botulinum Neurotoxins • Clostridium tetani • Clostridium botulinum • Similar neurotoxins – different diseases Portrait by Charles Bell c. 1821

3. Clostridium tetani Clostridium botulinum • Gram-positive, spore forming, rods • Strict anaerobes • Normal habitat: soil

4. Diseases • Pseudomembranous colitis • gas gangrene • Cellulitis • Tetanus • Botulism • Food poisoning

5. 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

6. 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

7. 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

8. Most cases: • Toxin ingested in food bloodstream flaccid paralysis Botulism (common source: home-canned foods) • 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

9. Tetanus Tetanus: Toxin produced by C.tetani infecting deep wound bloodstream CNS spastic paralysis (Lockjaw)

10. Clostridium neurotoxins Botulinum neurotoxin (BoNT): • produced by C. botulinum (C. barati, & C. butyricum) • 8 different variants (serotypes A to G) • toxin gene carried on phage or plasmid Tetanus neurotoxin (TeNT): • produced by C. tetani • all strains produce same TetNT - no significant variants • gene located on a plasmid Can also produce other toxins – do not confuse the neurotoxins (e.g. C. botulinum C2 & C3 toxins; C. tetani tetanolysin)

11. BotNT & TetNT: secreted as single 150Kd polypeptides, with internal disulphide bond S S S S L-chain (50Kd) N C H-chain (100Kd) C Hc N HN • In the lab., purified H-chain cleaved in half by papain, to • produce separate Hc and HN fragments N nicked by protease C activates toxin • Reduction separates the • Heavy and Light chains

12. S S • Studies on purified L-chain, H-chain, & Hc & HN fragments • Note similarity in overall ‘architecture’ to DTx active domain L-chain (50Kd) N C H-chain (100Kd) C N deduced from ability to conductance of planar lipid bilayers @ low pH Hc HN Receptor-binding domain neuron-specific Membrane-translocation domain (probable)

13. 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 BUT Different sites of action Very different diseases

14. Neurons Connections Synapses

15. Neurons Neurotransmitters Motor neurons Sensory neurons Stimulatory (e.g. acetylcholine) Inhibitory (e.g. GABA) Interneurons Protagonist muscle contracting CNS M (on) M I S I M (off) Antagonist muscle relaxing

16. Botulinum Toxin: Acts at neuro-muscular junctions. • binds receptor on end-plates of motor neurons RME • blocks neurotransmitter release FLACCID PARALYSIS BoNT BoNT M (on) CNS muscle relaxed M I S muscle relaxed I M (off) • L-chain translocated from acidified vesicle to cytoplasm

17. JAMA 2001; 285;1059-1070

18. Mechanisms of action of BotTN

19. Tetanus Toxin: Acts in the CNS • 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 vesicle inside axon CNS M M I S I M

20. 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 Interneuron axon terminal Motor neuron dendrite X X M Tet I S Vesicle + Tet. toxin RME + translocation to cytoplasm Exocytosis I M

21. Tetanus Toxin: Acts in the CNS Tet Tet • 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”) CNS M M I S I M

22. 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

23. Axon terminal of motor neuron Neurotransmitters in small synaptic vesicles which have a surface protein called VAMP ‘docks’ with Complex incl. SNAP-25, syntaxin on inner surface of axon membrane neurotransmitters released by exocytosis at NMJ Muscle cell

24. In early 1990s, proteins involved in neurotransmitter release identified – later shown to be cleaved by BoNT and TeNT Small synaptic vesicle (contain neurotransmitters) Vesicle membrane Neuron membrane

25. Other Clostridial diseases Gas 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

26. 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

27. 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)

28. Term Paper • Discuss how sea cucumber protein could potentially help in infectious diseases. • How do animal slaughtering contribute to the spread of zoonosis diseases? • Vaccination can be unsafe and ineffective. Give your comments. • Tell us about ONE most interesting topic in the microbiology subject. Justify by giving reasons and examples to your chosen topic.

29. 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