Flashcards in L2 - Toxins as Tools Deck (27):
What are the Clostridium bacteria and what do they each cause?
bolulinum - botulism, food poisoning
difficile - fluid accumulation
perfringens - gangrene
tetanus - paralysis from infected wounds
How many strains of toxin does C. botulinum and C. tetani produce?
botulinum - 7, A-G
tetani - 1
What are the features of clostridial neurotoxins?
Domain structure homology
Secreted as 150kD single chain with single Di-S bond
Toxin cleaved into heavy and light chain
What are the functions of the two chains of clostridial toxins?
Heavy - binding (Hc) / translocation (Hn)
Light - enzyme (LC)
What do clostridial toxins bind to?
What are gangliosides and where are they located?
carbonhydrate modified sphingolipids on external leaflet of membrane (lots at synapse)
What is the purpose of gangliosides for the toxin?
Binding to synapse
Binding to correct ganglioside will deliver toxin to correct neuronal type
How do clostridial toxins enter the cell?
Co-docks with synaptotagmin and so is endocytosed with vesicle
How does the clostridial toxin leave the vesicle?
Hn domain reacts to low pH and redox conditions of synaptic vesicle and forms a pore across membrane
Threads light chain through and reduced bond to release
What did Mochida (1990) find?
Injection of light chain mRNA inhibits synaptic transmission
Why do the clostridial toxins induce such different paralytic outcomes?
Site of action of BoTx in motorneurons
TeTx in inhibitory spinal interneurons
What are the symptoms of the tetanus toxin?
What are the symptoms of clostridium toxin?
What happens internally with TeTx?
Retrogradely transported along axon
Trans-synaptically transported into inhibitory interneuron
What happens internally with BoTx?
Internalised by motorneurons
What sequence homology does the light chain share?
What does the sequence homology in the light chain show?
Suggests clostridial toxins might cleave intracellular target
What did Schaivo (1992) show about the clostridium toxins cleavage?
Block NT release by cleavage of synaptobrevin
Why is the Leech Retzius neuron used for testing toxins?
Large with many vesicles
Intact pre and post synaptic neuron pair
Single cell can be used for western blot
How was it shown there are similar structural requirement for toxin binding?
BotA inhibited cleavage of synaptobrevin by BotB,F,D,G or TetX
What proteins are part of the SNARE complex?
Vesicel - synaptobrevin
membrane - syntaxin and SNAP-25
What is a feature of the SNARE'S?
Make a complex resistant to chaotropic agent SDS
So must be thermodynamically stable
What other 2 proteins bind to SNARE?
NSF (ATPase) and SNAP
Which protein binds calcium?
What is the transglutaminase activity hypothesis?
TeTx activates transglutaminase causing covalent linkage of vesicle protein synapsin - blocking movement and release of vesicles
What do synaptobrevin knockouts tell us?
Not essential for fusion
Essntial for rapid fusion and endocytosis