TOXIN ANTITOXIN Flashcards
(64 cards)
1
Q
basic concepts of the TA system
A
- an unstable antitoxin is produced in large amounts and a stable toxin is produced in small amounts
- the antitoxin neutralises the toxin
- the target has specific host targets that it degrades or modifies
- the antitoxin is degraded by host proteases
- if the TA system is lost then the toxin is free to degrade target -so only cells with the TA system will survive
2
Q
explain the type 1 TA system
A
the toxin mRNA is bound by the antitoxin antisense mRNA to form a duplex that is degraded by RNase
3
Q
explain the type 2 TA system
A
the toxin and antitoxin are both proteins that form a complex that inhibits the action of the toxin and blocks transcription from the TA locus
4
Q
explain the type 3 TA system
A
the non coding RNA antitoxin directly binds the toxin
5
Q
proposed model for TA functioning in bacteria
A
the reversible stasis model ie; reversible bacteriostasis
-when the cell is exposed to environmental stressors; the toxin induces stasis in the cell instead of killing it so that when the conditions improve the cell can re-enter the population and grow
-this model is supported by the idea that bacteria that live in relatively constant environments lack the TA system whereas bacteria living in constantly changing environments eg; obligate intracellular parasites do have these TA modules
6
Q
what are a toxins 2 modes of action
A
- destructive- where the toxin degrades the target leading to cell death
2.constructive-where the toxin modifies or sequesters the target
7
Q
what are the 2 roles of TA systems on plasmids
A
- phage stabilisation(maintenance) of low copy plasmids
-to ensure these plasmids are not lost/cured from the environment - phage stabilisation(maintenance) of multidrug resistance - the TA system confers multidrug resistance onto the plasmid
8
Q
what are the roles of the TA system on bacterial genomes
A
- reduction of bacterial growth under environmental stress- reversible bacteriostasis
- biofilm production
- bacterial pathogenicity
- phage inhibition
-these all have strong evidence - bacterial persistence
- programmed cell death
-these have weak evidence
9
Q
where has the ccd locus been found
A
- in the F plasmid
- in the genomes of E. coli( homologs of ccd)
10
Q
what TA system type is the ccdAB system
A
type 2
11
Q
what has the role of the ccdAB TA system been found to be and what role was it previously linked to
A
-previously linked to playing a role in plasmid stabilization because F plasmids have a very low cell copy number of 1-2 per bacterial cell
-recently linked to bacterial persistence
12
Q
explain how the ccd system ensures plasmid stabilization
A
-after cell division one cell will inherit the F plasmid and the other daughter cell wont
-the cell that doesnt inherit the plasmid lacks the TA system and so no fresh toxin and antitoxin is made-thus toxin prevails and can kill the cell
13
Q
why is it important that ccdA and ccB are translationally coupled
A
because it means ccdA is always made first and then only can ccdB be made
-and so ccdA is always produced in excess to ccdB
14
Q
explain how the toxin ccdB targets DNA gyrase host
A
- the toxin will target the GYRA core of gyrase
- the gyrase will nick the ds circular DNA but before the nick can be resealed into a supercoil the toxin will bind and inhibit the resealing
- this forms linear DNA
- so there is a reduction in DNA synthesis and activation of SOS response system - can be fatal
15
Q
explain the process of rejuvenation
A
the antitoxin will remove ccdB from the DNA:gyrase complex so that DNA synthesis can resume and the cell is not killed
16
Q
what process is mechanistically linked to rejuvenation
A
autoregulation of the ccd operon
17
Q
explain autoregulation of the ccd operon
A
- so first the ratio of ccdA:ccdB > 1
-this means that the toxin antitoxin will form a complex of ratio 2:2 that can bind to the ccd promoter region and repress transcription of the ccd operon
-when this happens no more fresh toxin or antitoxin is produced
-so antitoxin gets degraded by lon protease - so now the ratio is ccdA:ccdB< 1
-now there is more toxin and so the toxin binds to antitoxin in ratio 4:2; removing the complex from the promoter to allow for derepression
-so now fresh antitoxin is produced that can bind to the toxin; preventing it from targeting host gyrase
-now ratio is reset
18
Q
what is the high affinity binding site of ccdB used for
A
-used in rejuvenation
-where the ccdA toxin binds to the ccdB toxins high affinity binding sites to remove the toxin from the DNA:gyrase complex
-first only a segment of ccdA can bind to the ccdB but then once ccdB is cleaved; then the rest of ccdA can bind to the high affinity region
19
Q
what is the low affinity binding site of ccdB used for
A
-essential in autoregulation
20
Q
explain what persisters are
A
these are bacterial cells that are able to survive antibiotic treatment and they dont influence the MIC
21
Q
what are the 2 types of persistence cells
A
- stochastic/spontaneous persistence
-where the cells become persistent before any change in environmental conditions and can adapt to the new conditions - induced/triggered persistence
-the cells are induced by the environmental stress to become persisters
22
Q
bacterial tolerance vs bacterial persistence
A
-tolerance is where the cells will eventually all be killed by the antibiotic but they tolerate the antibiotic for longer and so it simply takes longer to kill them
-persistent cells survive from the killed population after treatment with antibiotic
23
Q
why are persisters important
A
because it means a small subpopulation will sacrifice fast proliferation for the overall survival of the population in adverse conditions
-thus; entire population not lost
24
Q
what type of antibiotic treatment generates more persisters
A
periodic antibiotic treatment
-it allows for the overall generation of more persisters overtime
25
what was the experiment they used to show the role of the ccd operon in persistence
they took native ccd from F plasmid and native ccd from the E.coli 0157 strain and placed into BW strains
=BWccdO157 and BWccdF
-also generated a control
=BWccdinactive
-they gave all 3 strains antibiotic treatment
-the results showed that the strains with functional ccd inserted survived antibiotic treatment through the formation of persisters
- concluded that the F plasmid can be used as a transmissible persistence factor
26
why is it difficult to study TA systems
because the overexpression of toxin will kill the cell
27
what was the methodology created to regulate the levels of ccdB toxin in a dose dependent fashion
-this methodology uses the generation of an inactive active site ccdB mutant protein
-this protein has a high affinity for ccdA but not for host DNA gyrase
28
explain the steps in the conditional regulated expression of ccdB toxin
1. introduce an inactive active site ccdB mutant toxin in a plasmid into an E.coli that contains a TA system
2. when the mutant toxin is induced it will displace the wild type ccdB from ccdA therefore; freeing it and it will derepress the ccd operon and so more wild type toxin is produced
3. the increased levels of wild type cddB in the cell can now bind the target and kill the cell
29
what induces the inactive active site ccdB mutant protein
induced by arabinose
-because the ccdB mutant is under control of an arabinose inducible promoter
-so to control the expression of this mutant you can change the concentration of arabinose in the cell
30
what is the point of using the mutant ccdB toxin
-it allows us to regulate the expression of wild type ccdB in the cell so that it is only overexpressed when we need it to be
-so when the mutant expression is not induced then we wont get overexpression of toxin and so we see normal rejuvenation and autoregulation occuring - persisters can form
-but when the mutant expression is induced then toxin is overexpressed which either leads to cell death or cell growth inhibition-allows persisters to form
31
what is the effect of lon and recA on ccdB mediated persister generation
1. when ccdB poisons the DNA:gyrase complex this leads to induction of the SOS response which is mediated by recA
-so if we delete recA then the SOS response wont occur and the cell will become particularly sensitive to antibiotics that target DNA
2. deletion of either lon or recA results in a decrease in persister formation
32
experiment done to determine the effect of lon and recA on ccdB mediated persister formation
-they took strains containing wild type ccd system and low levels of the mutant toxin
-this means growth inhibition would occur
- persisters can form in the face of antibiotic treatment
1. deleted recA- saw reduction in persister survival rate by 100 fold compared to the wild type
2.deleted lon-saw reduction in persister survival rate by 10 fold to the wild type but also saw reduction in surival rate decline by 50-200 fold compared to uninduced control
3. in a double knockout mutant the rate reduced to similar levels as the single knockouts
33
explain the significance that the double knockouts produced similar levels to the single knockouts
indicative that the downstream pathways activated by lon and recA are partially independent
34
how does the environmental stress trigger the TA systems to produce persisters
via alarmone (p)ppGpp
- alarmone is produced in response to environmental stress like nutrient starvation
-synthesised by 2 synthetases relA and spoT
-relA is synthesised in response to amino acid starvation and heat shock whereas spoT is produced in response to carbon/nitrogen starvation
-note that exponential growth can also trigger alarmone and lead to the production of stochastic persisters
35
explain the step by step mechanism of how alarmone leads to persistence
1. alarmone triggered by environmental stress
2. alarmone inhbits PPX
3.this allows for the accumulation of polyphosphate
4. polyposhphate increases the rate of lon protease activity
5. lon protease degrades more antitoxin
6. toxin is free target host cellular processes and thereby inhibit growth
7. this leads to persister formation
36
where is the hok/sok type 1 system found
on R1 plasmids
37
define hok, sok and mok
hok-host killing
mok-modulation of host killing
sok-supression of host killing
38
what is the brief concept behind the hok/sok system
short lived antidote but long lived poison
39
explain the gene organisation of hok/sok
1. the mok and hok are under control by the same Pmok promoter -this means that hok will only be produced once mok is produced
2. there is an overlap between the mok and hok protein produced
3. the sok antisense mRNA is controlled by psok promoter
40
regulation of the hok/sok killing system
1. if the hok/sok R1 plasmid is not inherited by the daughter cell after cell division then that cell will die
-because the sok protein will degrade rapidly so hok is free to then bind to the host cell membrane where it can cause cell death or cause the cell to stop growing= ghost cells
41
how does the plasmid containing cell survive hok killing
the sok antisense mRNA will bind to the hok mRNA forming a complex that is degraded by RNase and so hok cannot target the membrane
42
how does the daughter cell with no plasmid "inherit" hok
well because it previously had the hok/sok system now when it is lost; the unstable sok is degraded in 30seconds whereas the hok mRNA is more stable and lasts up to 20 minutes
-so hok mRNA prevails and will form hok protein that can target the cell membrane
43
explain the steps of how the hok/sok system operates
1. first the mok/hok mRNA is transcribed from a weak promoter
-translationally coupled
2. the hok mRNA is stable and initially inactive when it is transcribed and lasts around 20 minutes
3.it is inactive because it forms a closed loop and so is not accessible to ribosomes for translation or accessible to sok antisense mRNA
4. so hok can accumulate without being inactivated by sok and without killing the cell
5. then the 3 prime end of the hok mRNA is processed by RNase 2 which activates the transcript
6. now the transcript can either be bound by ribosomes for translation or bound by sok antisense mRNA for degradation
7. now unstable sokantisense mRNA is produced from the psok promoter and lasts only 30 seconds -just it must be produced in excess to hok mRNA
8. it has such a short halflife because it is rapidly decayed by RNase E.
9. sok indirectly inhibits hok by inhibiting mok translation at the mok TIR region
10. binding of antisense sok mRNA to mok TIR is irreversible
44
in summary; what is the killing of plasmid free cells based on
based on the differential decay between hok mRNA(20 minutes) and sok mRNA(30 seconds)
45
how does the hok/sok system lead to bacterial persistence
1. if the hok protein is produced in large amounts then it will bind to the cell membrane; resulting in complete collapse of the membrane and cell death
2.but if the hok protein is expressed in moderate amounts then it will bind to the cell membrane causing depolarization/reduction in membrane potential and so the cell growth is inhibited =persistence
46
what causes hok/sok mRNA to be transcribed from the R1 plasmid in the first place
this is due to alarmone and obg
47
what is obg
obg gene translates into a universally conserved GTPase
-this gene is essential for viability in most bacteria
-activation of obg is done via alarmone
-so overexpression of obg is shown to significantly increase persister formation
48
explain the steps of how obg activation leads to persister formation
1. environmental stressors like nutrient deficiency results in increased alarmone levels
2. this alarmone activates obg
3. active obg increases transcription of hok mRNA
4. so increased levels of hok mRNA can overrride the sok antisense inhibition
5. hok protein levels increase
6. so hok can decrease the membrane potential and cause persister formation
49
what regulates hokB mRNA expression
A proposed feedback mechanism that relies on the acitivity of RNase E
50
regulation of hokB expression in normal conditions
in normal cell conditions the growth continues and so alarmone remains low
-this means the membrane potential increases; causing RNase E to anchor into the membrane and continue degrading sok mRNA
-because the production of sok is steady; it can continue to block hok mRNA
-so no hok protein can disturb the membrane
51
regulation of hokB expression in stress conditions
under stress the cell growth stops and alarmone levels are high
-this alarmone activates obg
-active obg can increase hokB mRNA production so it overrides the sok inhibition
-so hok protein is translated and anchors into the membrane; decreasing the membrane potential
-so this decline in membrane potential causes detachment of RNase E from the membrane
-so sok is no degraded by RNase E and can bind to hokB to prevent more translation and thus; the hokB protein levels wont rise by too much and cause further damage; only persister formation
52
how does hokB causes a decline in membrane potential once it binds
1. when hokB anchors to the membrane it uses dsbA to catalyze disulfide bridges between 2 hokB monomers
2. this allows for pore formation in the membrane so the cell stops growing = persistence
3. then upon waking; dsbC is used to degrade the disulfide bridge between the monomers and so the pore is destabilized and membrane is repolarized by the ETC
4. eventual regrowth of the persister cells
53
why is it important to understand the concept of persister formation and awakening due to TA systems
1. allows us to identify targets that could inhibit persister formation and thus; allow us to decrease persister formation
2. allow us to develop therapeutics that prevent the awakening process to prevent chronic infection
3. the targets of the toxins could be used to develop new antibiotics
4. important to understand the role of TA systems in antimicrobial resistance
54
what is the function of the toxIN system
this type 3 TA system has been found to function in phage inhibition
-protects against infection by phages 2,4,5 and 6
55
explain what abortive infection is in bacteria
this is when an infected bacterial cell by a phage will kill itself to prevent spreading of the phage particles to neighbouring cells
56
where is the toxIN system found
in E.coli genomes
57
explain the genome organization of the toxIN system
it is a bicistronic operon and so toxI and toxN genes are found under control by the same promoter with a rho independent terminatory located between these 2 genes
-because of the infrequency of transcription through the rho terminator; more toxI is always produced compared to toxN
-toxI produces an sRNA antitoxin
-toxN produces an RNase toxin
- the half life of toxI is much shorter than that of toxN
-toxN toxin degrades any mRNA target that has sequence GAAAU in it.
58
explain what happens after sRNA and RNase are produced
1. initially the RNase willd cleave the sRNA antitoxin at its GAAAU sites to produce sRNA repetitive monomers
2. these monomers form pseudoknots
3. the pseudoknot is a loop in which the loop forms interactions with bases outside of the loop
4. 3 sRNA monomer pseudoknots will come together to bind 3 toxNs
=heterohexamer structure where 1 sRNA monomer binds to the head and tail of 2 seperate toxins
- this inhibits the function of the toxN
59
how is toxN expression regulated
1. due to the bicistronic operon structure; toxI always produced in excess
2. because of the infrequent read through of rho- always less toxN
3. 1 toxI molecule can inhibit 3 toxNs
60
so how does this toxIN system inhibit phage infection
1. well when the phage infects the cell it inhibits all cellular transcriptional processes; including transcription of the toxIN mRNA
2. and so toxI is degraded much quicker; resulting in more toxN
3.toxN will target mRNA GAAAU sequences found on the viral mRNA
4. this stops phage maturation from occuring
61
what happens in cells with no toxIN system
1. upon T4 phage infection the cellular transcription processes will be shut off
2. because there is no toxIN system; the phage will degrade all the host genomic DNA and use host cellular processes to replicate and produce more phage mRNA for capsid formation
3. thus more phage particles are produced and released
62
when does cell death occur following phage infection
cell death only occurs if the phage causes too much damage to the cell before toxN is activated
-abortive mechanism prevents neighbouring cells from being infected
63
what is the advantage of host transcription shutoff by phages
this allows the cell to detect infection without risk of autoimmunity
64
potential ways that the T4 phage can escape the toxIN
1. the t4 phage can produce extended short viral sequences that are very similiar to that of the toxI monomer repeats and so they bind to the toxN; freeing the phage
