A-B toxins

Question 1

simple A-B toxin

Answer 1

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.

Question 2

compound A-B toxin

Answer 2

multiple b subunits
single A subunit

Question 3

A portion

Answer 3

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

Question 4

B portion

Answer 4

B subunits are the bits that are responsible for binding to host cell receptors. So they give the cell specificity of the toxin.

Question 5

common mechanism for binding and entry of A-B toxins into host cells

Answer 5

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

Question 6

why is there huge diversity in the actions of AB toxins

Answer 6

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.

Question 7

most common action of A-B toxins

Answer 7

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.

Question 8

example disease - diphtheria

Answer 8

-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

Question 9

symptoms of diphtheria

Answer 9

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

Question 10

diphtheria toxin expression and regulation

Answer 10

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.

Question 11

proteolytic cleavage of the secreted form of diphtheria toxin

Answer 11

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.

Question 12

structure of diphtheria toxin - 3 functional regions

Answer 12

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)

Question 13

binding, endocytic uptake and translocation of diphtheria toxin in host cells:
1. how does the diphtheria toxin get cleaved

Answer 13

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

Question 14

binding, endocytic uptake and translocation of diphtheria toxin in host cells:
2. B portion

Answer 14

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.

Question 15

binding, endocytic uptake and translocation of diphtheria toxin in host cells:
3. once it is in the endosome

Answer 15

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.

Question 16

role of the A chain of diphtheria toxin

Answer 16

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.

Question 17

why is the A chain of diphtheria toxin completely specific to elongation Factor 2

Answer 17

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.

Question 18

therapeutic use of toxin-derived immunotoxins - diphtheria toxin derived immunotoxins

Answer 18

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

Question 19

therapeutic use of toxin-derived immunotoxins - strategies based on the controlled tumour-specific expression of diphtheria toxin

Answer 19

targeted killing of cancer cells by delivering botulinum A chain to cells and limiting its expression to within cancer cells through transcriptional regulation

Question 20

botulism

Answer 20

intoxication caused by Clostridium botulinum
- relatively large, anaerobic, gram positive, rods

forms sub terminal endospores, strictly fermentative metabolism

Question 21

botulism spores

Answer 21

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.

Question 22

what are most cases of botulinum associated with

Answer 22

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.

Question 23

botulin toxin

Answer 23

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

Question 24

food borne botulism

Answer 24

C.botulinum in food -> toxin produced -> toxin ingested -> toxin in bloodstream -> attacks neurons

Question 25

infant botulism

Answer 25

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.

Question 26

wound botulism

Answer 26

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.

Question 27

what does botulism toxin get secreted as

Answer 27

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.

Question 28

multi protein complex of botulism

Answer 28

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

Question 29

what happens if you ingest the derivative toxin of botulism

Answer 29

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

Question 30

disassembly of the progenitor complex botulism toxin

Answer 30

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

Question 31

receptor binding domain of botulism toxin

Answer 31

receptor binding domain is actually split into two parts.
- It’s this C terminal part that recognizes
the first receptor

Question 32

different types of botulinum toxin

Answer 32

you get lots of different Sera types of botulinum toxin and these have been grouped into botulinum toxins A to G.

Question 33

structure of botulinum toxin type A

Answer 33

• binding domain - beta sheets - good for receptor recognition
• translocation domain - alpha helices
• catalytic domain - alpha helices good for inserting in membranes

Question 34

action of botulinum toxins

Answer 34

• 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,

Question 35

what happens when botulism toxin is inside the endosome

Answer 35

• 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.

Question 36

normal neurotranmission - no botulism

Answer 36

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

Question 37

snare proteins

Answer 37

synaptobrevin which is associated with the endosomal membrane
○ syntaxin, which is on the cell surface membrane
○ and snap 25 that brings the two together.

Question 38

B, D, F & G group of botulinum toxins

Answer 38

cleave a protein called synaptobrevin also called VAMP (vesicle associated membrane protein)

Question 39

A & E group of botulinum toxin

Answer 39

cleave a protein called snap 25

Question 40

C group of botulinum toxin

Answer 40

cleaves SNAP -25 and syntaxin 1

Question 41

action of botulinum toxin type A on motor neurone

Answer 41

1. botulinum toxin binds to neurons and is taken up by endocytosis
2. the A subunit exits the endosome
3. the A subunit cleaves SNAP 25
4. vesicle fusion and release
5. cell is not stimulated

Question 42

tetanus

Answer 42

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

Question 43

tetanus toxin

Answer 43

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

Question 44

tetanus toxin (tetanspasmin)

Answer 44

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

Question 45

action of tetanus toxin and botulinum toxin

Answer 45

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

Question 46

botulinum toxins - current therapeutic uses

Answer 46

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