A-B toxins Flashcards
simple A-B toxin
Encoded on a single genes
- Single polypeptide cleaved into A and b portions
Then these two portions get held together by a disulfide bond
it’s only once the toxin gets inside a cell and that disulfide Bond breaks that the toxin
becomes active and carries out its function.
compound A-B toxin
multiple b subunits
single A subunit
A portion
The A portion is the enzymatic part. This is the bit that carries out the toxic function.
- The A portion targets a protein inside the host cell
B portion
B subunits are the bits that are responsible for binding to host cell receptors. So they give the cell specificity of the toxin.
common mechanism for binding and entry of A-B toxins into host cells
the B portion binds to a receptor on the host cell and that causes endocytosis and the whole toxin ends up inside an endosome.
- once it’s in the endosome you get a pH drop that causes a conformational change of the toxin and the A portion is allowed to translocate Across the endosomal membrane and then to the cytoplasm of the host cell
- when it goes into the host cell the disulfide bond gets broken and this A portion now becomes active.
- It’s now free in the cytoplasm active and carries out its effect
why is there huge diversity in the actions of AB toxins
there’s a huge diversity in the actions of AB toxins and that’s due to
- The receptors that B portion binds to so that gives cell specificity
- and it’s to do with the target proteins of the a portion. They can all affect different proteins in the cell.
most common action of A-B toxins
A portions remove the ADP ribosyl group from nicotinamide adenine dinucleotide and causes ADP- ribosylation of a target host protein
and when that gets attached to the Target protein it either inactivates that protein
or Alters its function somehow.
example disease - diphtheria
-caused by Corynebacterium diphtheriae - gram-negative, non-spore-forming, facultative anaerobic rod
- humans are only host
-it’s able to colonise the throat and it gets transmitted usually by inhalation of aerosols. So it’s through person-to-person transmission.
- vaccine is a toxoided (chemically inactivated) version of diphtheria toxin
symptoms of diphtheria
first symptoms (2-4 days post colonisation): malaise, low-grade fever, tonsilitis, sore throat, loss of appetite
typically a grey- white membrane develops on the tonsils, soft palate and pharangeal wall: adheres to underlying tissue
diphtheria toxin expression and regulation
The toxin is encoded on a bacteriophage - only strains of Karine bacterium diphtheria that have been infected by this bacteriophage can produce a toxin.
- the gene that encodes diphtheria toxin is called tox and its on a bacteriophage.
- For the tox Gene to be expressed by the bacteria, the bacteria have to be in low iron conditions.
- So in the absence of iron you get expression of the tox Gene.
- But in the presence of iron, there’s a regulator that binds to the iron and when it binds iron, it binds to the promoter of the toxin Gene and represses transcription.
-So to get diphtheria toxin expression the bacteria need to possess the bacteriophage and they need to be in a low iron environment.
proteolytic cleavage of the secreted form of diphtheria toxin
it’s encoded on the single Gene and you end up with this 60kg Dalton polypeptide
- this gets cleaved into the A and the B portions
- the A portion is a catalytic domain and the B portion is The Binding domain.
- the B portion actually has two parts
○ It has a translocation region and it has a receptor binding region.
structure of diphtheria toxin - 3 functional regions
A region - catalytic domain - contains enzyme activity
T region - translocation domain - the movement of toxin into the host cell cytoplasm
R region - receptor domain (binds to target cell)
binding, endocytic uptake and translocation of diphtheria toxin in host cells:
1. how does the diphtheria toxin get cleaved
Toxin has been secreted from the bacteria ,
- first it needs to be cleaved and it can be cleaved by a protease on the surface of the host cell but if not, there’s now evidence that it can also get cleaved by a furin protease in the endosome
- So it gets cleaved and the A portion stays held to the B Portion by the disulfide bond
- and the B portion is split into the translocation domain and the receptor binding region
binding, endocytic uptake and translocation of diphtheria toxin in host cells:
2. B portion
B recognizes the receptor on our host cell and the receptor it recognize is the HP EGF receptor
○ . This is a hormone that gets released by epithelial cells and when the hormone gets released the receptor hangs around a bit in the membrane and it’s just that diphtheria toxin hijacks that receptor
- So it recognizes the HP EGF receptor binds to it and that causes endocytosis.
- So the toxin is now within the endosome.
binding, endocytic uptake and translocation of diphtheria toxin in host cells:
3. once it is in the endosome
there is a protease that can cleave the toxin if it hasn’t already happened.
- And then in the endosome you get a pH drop and that causes a conformational change in the toxin.
- That allows the translocation domain to now becomes exposed and because it’s hydrophobic it inserts into the endosomal membrane.
- As it does that it translocates the A chain into the cytoplasm of the host cell
- disulfide Bond breaks and the A portion becomes active. So now it’s got its enzymatic activity and it catalyzes the ADP ribosylation of elongation factor 2 protein.
role of the A chain of diphtheria toxin
it adds an ADP ribosyl group from NAD onto elongation Factor 2
- elongation Factor 2 is involved in protein synthesis.
- when it gets adp-ribosylated. It becomes non-functional so protein synthesis stops.
- the A chain of diphtheria toxin is an enzyme so it can keep catalyzing the same reaction
○ . You just need one molecule in there and it will keep doing the same reaction.
○ So it will do that till all the elongation Factor 2 is used up
which completely halts protein synthesis and leads to cell death.
why is the A chain of diphtheria toxin completely specific to elongation Factor 2
the reason is because elongation
Factor 2 has an unusual histidine residue within it.
- after it’s been synthesized it gets post-translationally modified and it gets this side chain added to a histidine.
○ Called the dipthimide residue of elongation Factor 2.
the a chain of diphtheria toxin recognizes that unusual histidine and attaches the adp-ribosyl -group to it and that inactivates it and Halt’s protein synthesis.
therapeutic use of toxin-derived immunotoxins - diphtheria toxin derived immunotoxins
use targeting molecules (receptor-specific ligands) conjugated to a modified toxin - targets a toxin to cancer cells
- cancer cells frequently have specific growth factor receptors and antigens overexpressed on surface which allows selective targeting of immunotoxins
- targeting molecules are typically antibodies, antibody fragments, cytokines or growth factors that recognise a specific cell surface receptor that is either absent on the surface of normal cells or highly up-regulated on cancer cells
therapeutic use of toxin-derived immunotoxins - strategies based on the controlled tumour-specific expression of diphtheria toxin
targeted killing of cancer cells by delivering botulinum A chain to cells and limiting its expression to within cancer cells through transcriptional regulation
botulism
intoxication caused by Clostridium botulinum
- relatively large, anaerobic, gram positive, rods
forms sub terminal endospores, strictly fermentative metabolism
botulism spores
spores often contaminate food
- disease occurs when spores are able to germinate and bacteria grow in food
can only grow in environments that are anaerobic so no oxygen, but the spores can
Persist in environments with oxygen in
if you’ve got food contaminated with the spores the spores might be able to start to germinate in the intestine, but because of the microbiota
in the intestine, they actually not able to persist and so you don’t get the disease
- the microbiota is enough to compete out those bacteria.
what are most cases of botulinum associated with
most cases associated with consumption of unheated canned food
what happens in the canning process is the food gets heated up, but it doesn’t destroy the spores then the can or jar gets sealed and it becomes anaerobic.- If it’s stored at room temperature, they bacteria can start to replicate in that environment and they produce a toxin
- and then it if you ingest that toxin that’s when you become ill and the disease symptoms are directly proportional to the amount of toxin that you ingest.
botulin toxin
it’s a really potent neurotoxin.
initial symptoms - nausea and vomiting, headache, double vision, slurred speech and other neurological symptoms
generalised flaccid paralysis occurs due to blocking neurotransmitter release preventing muscle stimulation
ends up being fatal - starts to interfere with breathing and heart function
food borne botulism
C.botulinum in food -> toxin produced -> toxin ingested -> toxin in bloodstream -> attacks neurons
infant botulism
infant botulism is if a baby ingests a spores they don’t have a well-developed microbiota.
- And so in that case the bacteria are able to replicate in the anaerobic intestinal tract, release a toxin in the intestinal tract and then that toxin gets absorbed into the bloodstream
-> attacks neurons
infant botulism - has been associated with sudden infant death syndrome.
wound botulism
very rare
spores contaminate wounds
- if it’s an anaerobic wound, so you’ve not got blood flow to that wound, The spores can germinate, bacteria grow, release a toxin and you get the same effect.
what does botulism toxin get secreted as
botulism toxins gets secreted from the bacteria just like diphtheria toxin. But in this case, it gets secreted as a multi protein complex
- and that multi protein complex is called progenitor toxin.
multi protein complex of botulism
that multi protein complex is called progenitor toxin.
and the other proteins in this complex actually protect the toxin so when it gets ingested the toxin is protected as it goes through the gastrointestinal tract,
so it protects it from the low PH in the stomach
what happens if you ingest the derivative toxin of botulism
If you ingest the derivative toxin (when this multi-protein complex is disassembled) and you are just left with the toxin, it’s much less active and it’s thought that that’s because it’s not protected
However If you inject it, It’s actually the derivative toxin that is more potent because that’s going straight into the bloodstream
disassembly of the progenitor complex botulism toxin
the progenitor toxin disassembles and you’re left with the derivative toxin
- And then that gets cleaved by a protease and you end up with A and b chain
- for botulinum, The a chain is called the light chain and The B chain is called the heavy chain.
- So the heavy chain has got the receptor binding domain and the translocation domain
- the light chain is where the enzymatic Activity is
receptor binding domain of botulism toxin
receptor binding domain is actually split into two parts.
- It’s this C terminal part that recognizes
the first receptor
different types of botulinum toxin
you get lots of different Sera types of botulinum toxin and these have been grouped into botulinum toxins A to G.
structure of botulinum toxin type A
- binding domain - beta sheets - good for receptor recognition
- translocation domain - alpha helices
- catalytic domain - alpha helices good for inserting in membranes
action of botulinum toxins
- nicked toxin
- binds neurons at the neuromuscular Junction and targets peripheral nerve endings
- to get into the cell. It uses two receptors:
○ uses a ganglioside that gives it an initial loose Association with the nerve cell
○ and then it uses a second protein receptor that makes a much tighter interaction and drags the toxin into the endosome - just like diphtheria toxin, it gets internalized by receptor-mediated endocytosis and the toxin ends up inside the endosome,
what happens when botulism toxin is inside the endosome
- botulinum toxin is is a zinc metalloprotease.
- So it Cleaves proteins and the proteins that it Cleaves are snare proteins and the different sera types Cleve different snare proteins
- whatever snare proteins they cleave they basically block this acetyl choline containing vesicle from fusing with the cell membrane so you don’t get neurotransmission.
- So you block the acetyl choline release. so you don’t get muscle contraction and you end up with flaccid paralysis.
normal neurotranmission - no botulism
normally for neurotransmission - you’ve got synaptic vesicles which contain the neurotransmitter acetylcholine
- the ach gets released at the neuromuscular Junction and binds to muscle cell receptors and that stimulates contraction.
- For that vesicle to fuse with that membrane, you need snare proteins
○ and the three key snare proteins are synaptobrevin, syntaxin and snap 25
- So all three of these proteins have to be intact for that acetyl choline release to get muscle stimulation
snare proteins
synaptobrevin which is associated with the endosomal membrane
○ syntaxin, which is on the cell surface membrane
○ and snap 25 that brings the two together.
B, D, F & G group of botulinum toxins
cleave a protein called synaptobrevin also called VAMP (vesicle associated membrane protein)
A & E group of botulinum toxin
cleave a protein called snap 25
C group of botulinum toxin
cleaves SNAP -25 and syntaxin 1
action of botulinum toxin type A on motor neurone
- botulinum toxin binds to neurons and is taken up by endocytosis
- the A subunit exits the endosome
- the A subunit cleaves SNAP 25
- vesicle fusion and release
- cell is not stimulated
tetanus
intoxication caused by Clostridium tetani
- relatively large, anaerobic, gram-positive, rods
- forms terminal endospores, strictly fermentative metabolism
- the vaccine is a toxoided version of the tetanus toxin
tetanus toxin
potent neurotoxin
- highly fatal (40-80% mortality)
-early symptoms (4 days to several weeks): painful spasms and rigidity of the voluntary muscles
- progressive rigidity and violent spasms
- death usually from exhaustion, respiratory failure and occlusion of carotid in the neck
tetanus toxin (tetanspasmin)
the sequence of tetanus toxin is very similar to botulinum toxin. The structures are really really similar and it cleaves synaptobrevin at the same point as botulinim toxin
it targets inhibitory neurons, so it Abolishes the release of inhibitory neurotransmitters glycine and GABA
action of tetanus toxin and botulinum toxin
both tetanus toxin and botulinum toxin bind to the gangliosides on the surface.
- They both use the same receptors to get into the cell
- key differences when botulinum toxin goes in you get a pH drop in the endosome and A chain comes out into the cytoplasm at the peripheral nerve ending
- But for tetanus toxin it Associates with lipid RAS, and you don’t get that pH drop.
- The toxin then undergoes retrograde trafficking and then goes into the inhibitory interneurons by trans cytosis
- . So it pops out of the neuron and then re-enters the inhibitory interneuron.
- because it’s in the inhibitory Neuron The Vesicles in there are carrying the blockers of neurotransmission - So Glycine and Gaba
- it stops those vesicles being able to fuse with the cell membrane and release these Inhibitors
these Inhibitors can’t now enter into the neuron to stop neurotransmission - opposite to botulism
botulinum toxins - current therapeutic uses
native toxin administered into peripheral tissue resulting in reversible blockage of the NMJ:
dystonias and other involuntary movements
inappropriate muscle contractions e,g.
- spasticity
- chronic tension (muscle contraction) headaches
- neuropathic pain
- neurogenic bladder
other cosmetic applications:
- wrinkles, brow furrows, frown lines, platysma lines, facial asymmetry
