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MCB3026F > TA Systems & Persistence > Flashcards

TA Systems & Persistence Flashcards

1
Q

What is bacterial antibiotic resistance?

A
  • Inherited trait that allows a resistant bacterial strain to grow in the presence of a [specific antibiotic] > the minimal inhibitory concentration (MIC) compared to a susceptible strain
  • Trait often carried on MGEs
  • MGEs allow for DNA shuffling of antibiotic resistance genes among different bacterial species of clinical importance
  • BAR via MGEs often allows for emergence of multidrug resistant bacteria = superbugs
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2
Q

What is Bacterial Antibiotic Tolerance?

A
  • Homogenously displayed by the whole population and allows the pop to transiently survive antibiotic exposure
  • Most common method of BAT = when entire bacterial pop is slow growing due to environmental limitations or a genetic alteration (auxotroph) = the pop becomes tolerant to a specific class of antibiotic because it requires actively growing/dividing cells, which they aren’t.
  • Tolerant bacteria requires a longer exposure to the antibiotic to observe the same level of death as in susceptible strain
    o Minimal duration of killing (MDK): MDK99 = min duration of antibiotic treatment required to kill 99% of bacterial pop
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3
Q

What is Bacterial Persistence?

A
  • Found only in a fraction (1%) of population and are genetically indistinguishable from sensitive cells within the pop (don’t have a MGE or something that makes it resistant)
    o Persisters are also often multitolerant
  • Transiently survive antibiotic treatment and will not influence the MIC or MDK
  • Can be ‘spontaneous/stochastic persistence’ or ‘triggered/induced persistence’
  • In ‘triggered/induced persistence’ conditions (starvation, oxidative stress, C source transition) DNA damage or antibiotics increase the frequency of persister cells in pop
  • P cells can revert stochastically (randomly) or through environ conditions
  • Upon exit, new pop can be formed when antibiotics are removed from environ
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4
Q

Will Bacterial Persisters influence the MIC or MDK?

A

No

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5
Q

How do persister cells escape antibiotic treatments?

A
  • P cells don’t change genetics to escape antibiotic treatments – survive due to phenotypic bistability
    o Persisters not resistant, considered phenotypic variants that are tolerant to antibiotics (same genetics, different phenotype)
  • Survival due to transition into ‘dormant’ state – & substantial reduction of growth rate & metabolism
    o Most antibiotics kill growing cells -> dormant state protects these ones
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6
Q

Why does a bacterial population have Persister cells?

A

o Considered a bet-hedging strategy in which a subpop of clonal cells sacrifices fast proliferation to ensure survival of the pop in adverse conditions

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7
Q

What does stochastic mean?

A

Having a random probability distribution or pattern that may be analyzed statistically but may not be predicted precisely.

  • i.e. within a pop there will always be persister cells but we can’t predict which they will be
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8
Q

How are persister cells formed?

A
  1. Induced: Occurs due to environmental challenge
  2. Stochastically: Subpop of cells are in a persistent state prior to environmental challenge
    • Responsive Diversification: Stochastic cells adapt to growth under new conditions following environmental cues
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9
Q

Effect of antibiotic on Tolerant vs Persistent cells

A

a) Tolerant are slow growing so the number of cells in the pop is reduced but at much slower rate than wild type. Have different MDK99
b) Reduces at same speed as wild-type till only persistent are left. MDK99 are the same

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10
Q

What is a biofilm?

A
  • Microbial pop growing on a surface where the cells are enclosed by extracellular matrix & exhibit multicellular-like behaviour
  • Involved in 80% of human bacterial chronic inflammatory & infections diseases & are multidrug tolerant & resistant to host immune system
  • Contain increased prevalence of persister cells which appear to be responsible for cause and recalcitrance of chronic infections
  • Are protective habitats for persisters against antibiotics & the immune system
    o Persisters remain viable and repopulate biofilms when the level of antibiotics drops and they can recolonize the host after antibiotic removal
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11
Q

How is a biofilm formed?

A
  1. Bacterial cells settle onto hard surface
  2. Cells proliferate & secrete adhesive extracellular polymer substances (EPS)
  3. Cells detach and spread biofilm to new locations
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12
Q

What is the resistance mechanism of a biofilm?

A
  1. Treatment kills surface cells
  2. Persister cells remain on surface, protected by EPS
    a. EPS protects from immune system
    b. Diffuse antibiotic, reducing its efficacy so some cells survive
  3. Biofilm regrows
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13
Q

Periodic Antibiotic Treatment - Periodic doses of lethal cones. VS Constant sub inhibitory stress

A
  • Constant subinhibitory stress (Enough to select for sensitive cells, but not kill them all) resulted in antibiotic-resistance mutants
  • Periodic doses of lethal antibiotic concs select for high-persistence mutants rather than antibiotic resistance
    o MUCH worse than antibiotic resistant mutants because it allows entire populations to regrow with higher concs of persister cells.
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14
Q

What is the basic concept of toxin-antitoxin systems?

A
  • Requires a low [stable toxin] & a very high [unstable antitoxin]
  • Antitoxin is unstable due to being target of host RNAses or proteases
  • Toxin must attack specific host target to cause cell death
  • Toxin neutralized by antitoxin & cell protected against action of toxin
  • Cognate antitoxin is either a protein or a small RNA mol
  • Antitoxin counteracts the toxin activity by acting as a direct inhibitor or by controlling toxin production
  • Toxins within TA modules cause inhibition of cell growth by interfering with cellular processes (DNA replication, translation, cell division & ATP synthesis)
  • Ensures that only cells with TA system will survive – depends on de novo synthesis of antitoxin for cell survival
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15
Q

What happens when a bacteria loses its TA system?

A
  • When bacteria lose TA system = selectively killed because unstable antitoxin degraded faster than more stable toxin
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16
Q

What is a Type I TA System?

A

mRNA coding toxin | Antisense RNA antitoxin

Translation of toxin prevented by formation of RNA duplex between toxin mRNA & an antisense RNA antitoxin which is degraded by ribonucleases

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17
Q

What is a Type II TA System?

A

Protein toxin | Protein antitoxin: Interact directly

Both toxin & antitoxin are proteins which form a complex, inhibiting toxin activity and repressing transcription of the TA locus

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18
Q

What is a Type III TA System?

A

Protein toxin | Small RNA antitoxin

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19
Q

What is a Type IV TA System?

A

Protein toxin | Protein antitoxin : Don’t interact directly

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20
Q

What is a Type V TA System?

A

mRNA coding toxin | RNAse antitoxin

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21
Q

What is a Type VI TA System?

A

Protein toxin | Protein antitoxin: Interact directly

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22
Q

What is the role of TA systems in Plasmids?

A
  • Many TAs found across numerous prokaryotic genomes in addition to plasmids & bacteriophages
  • Plasmid TA systems showed to be involved in:
    o Plasmid stabilization (maintenance) of low copy plasmids [Plasmid Addiction]:
    • Plasmid not ‘lost’ from pop
    • Allows plasmid to become reservoir of antibiotic resistance genes
    o Plasmid stabilization (maintenance) of multidrug resistance under non-selective pressure:
    • Maintain metabolic burden in bacterial cell in an antibiotic-free environ
    o Bacterial Persistence
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23
Q

What is the role of TA systems in Bacterial Genomes?

A
  • Chromosomal TA systems shown to be involved in:
    1. Bacterial persistence via responding to nutrient stress (stringent response) or DNA damage response to antibiotics
    2. Bacterial pathogenicity
    3. Stabilization of genomic islands (regions on chromosome that often contain virulence genes)
    4. Acting as anti-addiction modules
    5. Anti-phage mechanism
    6. Biofilm formation
    7. Development of fruiting body formation in Myxococcus Xanthus.
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24
Q

What is the role of TA systems in regulating Bacterial Persistence?

A
  1. TA modules were shown to be highly expressed in isolated persister cells of E. coli and Mycobacterium tuberculosis.
  2. The first persister gene – hipA – was later found to be part of the hipBA type II TA system
  3. Ectopic (abnormal, in this case = overexpression) expression of Type II toxins from TA modules increases persistence
  4. Deletion of hipBA TA system = ↓ no. persisters in biofilm, ↑ biofilm sensitivity to antibiotic treatment
  5. Deletion of a Type I TA system -> ↓persister cells + ↓sensitivity to antibiotic treatment
  6. Deletion of single TA modules often insufficient to yield a detectable persistence defect
    o Persister formation & regrowth has functional redundancy due to multiple TA systems
  7. Evidence that stringent response (nutrient starvation) & SOS response (DNA Damage) activate TA systems and thereby ↑bacterial persistence
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25
Q

Why is bacterial persistence as a phenotype very difficult to study?

A

Because there is such a small percentage of the population that displays it.

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26
Q

What is the current hypothesis surrounding the purpose of TA modules?

A

Reversible Stasis Model [They are bacterial metabolic stress managers]

= TA Modules are linked to the metabolic state of the cell & activated/deactivated in response to changes in that metabolic state

  • TA toxins don’t kill cells but induce a reversible stasis to enable some cells to survive episodes of extreme nutritional stress or other environmental challenges
  • When conditions improve, part of pop is capable of recovery & resumes normal cell physiology
  • Consistent with this:
    o TA modules extremely common in genomes of bacteria that are confronted with periodic changes in environ
    o Are absent in organisms that live in constant environ – like obligate intracellular parasites
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27
Q

What is the Structural Organisation of the ccdAB Type II TA system?

A
  • ccd operon [bicistronic = 1 promoter] encodes:
    o ccdA – antitoxin
    o ccdB – toxin, targets DNA Gyrase leading to cell death
  • ccdB is translationally coupled to ccdA
    o Translational coupling = Shine-Dalgarno for ccdB sequestered in hairpin loop, so can only translate after ribosome unwinds hairpin after translation of ccdA
    o Means that ccdA is always produced in excess to ccdB
  • ccdA-ccdB can interact to form a complex:
    o This neutralizes the action of ccdB
    o Can bind to ccd operator-promoter -> Transcriptional repression (default state of operon)
    o ccdA is the DNA binding element of ccdA-ccdB complex
  • ccdA is degraded by the bacterial host Lon protease
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28
Q

What is the cellular action of DNA Gyrase?

A
  • DNA Gyrase has subunits – GyrA subunit targeted by ccdB toxin
    o Gyrases catalyses the ATP-dependant negative super-coiling of ds-closed-circular DNA
    o Introduces transient dsDNA breaks & reseals it
    o GyrA is the catalytic core
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29
Q

What is the action of the CcdB Toxin?

A

Targets GyrA

o If Gyrase is in process of cleaving DNA, it will trap cleaved DNA-Gyrase complex & impede resealing of nicked DNA – promoting cleavage of closed-circular DNA to its linear form

o Binds to free GyrA making Gyrase catalytically inactive

o Results at cellular & morphological level:
• ↓DNA synthesis due to DNA damage
• Activation of SOS response
• Cell filamentation – long cells appearing
• All of above leads to cell death because extent of damage can’t be fixed

30
Q

What is the action of the ccdA Antitoxin?

A

o Releases ccdB from inactive ccdB-DNA-Gyrase complexes = ‘Rejuvenation’
o Prevents ccdB-GyrA complex formation

31
Q

What is the process of autoregulation of the ccd Operon?

A
  • Repressing Operator Complex [CcdA:CcdB >1]: DEFAULT
    o A chain of alternating ccdA2 & ccdB2 dimers is formed where both the high& low-affinity binding sites of ccdB2 are occupied
    o High [CcdA] means they can bind to the low-affinity site forming the chain which binds to the operator site and repression of the ccd operon will start, preventing excessive production of CcdA&B
  • Non-repressing Operator Complex [CcdA:CcdB <1 ]
    o Cellular A:B ratio ↓ due to the proteolytic action of Lon on A -> excess B
    o Because CcdA can only bind to low-affinity site when it is in excess, it will be released from the low-affinity site.
    o Causes this CcdB2:CcdA2:CcdB2 complex to be formed (only bound to high-affinity site)
    o Leads to the breaking up of the repressor chain -> dissociation from the promoter region -> derepression
    o Following transcription, higher levels A than B -> A pool replenished causing empty low-affinity binding sites on CcdB2 to be resaturated and repressive mode restored
32
Q

What is the process of Rejuvenation in the Ccd Operon?

A
  • When ccdB poisons gyrase, only a segment of ccdA can access its binding surface on ccdB
  • Initial interaction of CcdA with this segment induces conformational change in ccdB, releasing it from gyrase
  • After/coincident with ccdB release, a different segment of ccdA locks onto ccdB via high-affinity binding site resulting in CcdB:CcdA complex.
  • This complex is building block of repressor complex & forms chain of alternating ccdA2 & ccdB2 dimers
33
Q

What is the effect of the Ccd Operon on Bacterial Drug Tolerance?

A

It was found that there was increased protection from cell death under antibiotic stress conditions in cells expressing the Ccd Operon through formation of persisters

34
Q

What is the mechanism of conditionally regulated expression of the CcdB Toxin

A
  • The method involves the overproduction of a mutant CcdB toxin that has a high-affinity for the antitoxin but a low-affinity for the cellular target, DNA gyrase.
  • This results in the release of the wild-type toxin from the ccdAB TA complex allowing them to bind to DNA Gyrase
35
Q

What controls the expression of the Inactive Active-site pBAD promoter?

A
  • Inactive Active-site CcdB mutant protein is expressed under control of arabinose inducible pBAD promoter.
    o Therefore level of expression of the CcdB mutant protein can be modulated by varying arabinose conc. in medium & adding glucose to repress further expression
36
Q

What is Alarmone?

A

(p) ppGpp
- Is an important 2nd messenger in bacterial cells – typically produced in response to nutrient starvation & other stress
- Allows cells to reprogram cellular physiology from growth to metabolic homeostasis and survival function
- RelA & SpoT are synthetases that synthesize alarmone in response to different stresses
- Alarmone modulates cellular physiology by transcriptional reprogramming and direct adjustment of target protein activities

37
Q

What is SpoT?

A

Bifunctional enzyme that has hydrolase activity which hydrolyses ppGpp to GDP

38
Q

What are the triggers for Alarmone synthesis?

A
  • Amino acid starvation and/or Heat shock
    o RelA converts GTP to pppGpp via ATP
  • C/N/Fe/P/FA starvation
    o SpoT converts GTP to pppGpp via ATP
  • pppGpp phosphohydrolase then converts pppGpp -> ppGpp
  • ppGpp inhibits PPX which would usually allow adaptation to the stationary phase
39
Q

How does Alarmone interact with the Lon Protease?

A
  • Any nutrient stress triggers Alarmone which inhibits PPX
  • Inhibited PPX = accumulation of polyphosphates (not broken down by PPX)
  • Polyphosphates bind to Lon protease = increased protease activity (degrading ribosomes etc.)
40
Q

What affect does Alarmone have on TA Systems?

A
  • Environmental stress = increased alarmone
  • Alarmone inhibits PPX = accumulation of polyphosphates
  • = Increased rate of Lon Protease Activity = Increased degradation of Antitoxins
  • = Activation of Type II Toxins = Increased Persistence
  • Evidence:
    o RelA & SpoT mutants exhibit defective persister formation during exponential growth, in biofilms & in stationary phase
41
Q

What is the effect of Lon & RecA on CcdB mediated Persister generation?

A
  • Overexpression of mutant CcdB -> increase in level of free WTCcdB
  • Studied:
    o ∆lon & ∆rec strains with F plasmid induced for expression of mutant Ccb toxin for 1hr (Increase [wtCcdB])– then challenged with different antibiotics at ±10X MIC for 4hr
    o Kills all non-persister cells
  • Findings:
    o Decrease in persisters for ∆lon & ∆rec mutants
    o Downstream pathways activated by Lon & RecA to generate persisters appear to be partially independent.
    o Not all antibiotics result in persisters to the same degree
  • Conclusions:
    o Lon also plays important role in CcdB-mediated persister generate, apart from its role in antitoxin degradation
    o Double knockout of recA-Lon mutant showed persisters were further reduced to close to background, but similar levels to individual knockouts suggesting that the downstream pathways activated by Lon & RecA to generate persisters are at least partially independent.
42
Q

What is the model of stress leading to persistence via the ccdTA system?

A
  1. Antibiotic Stress
  2. Increase in Alarmone
  3. Inhibits PPX
  4. Accumulation of polyphosphate
  5. Increased rate of Lon Protease activity
  6. Increased degradation of CcdA antitoxin
  7. CCdB toxin is freed
  8. GyrA:GyrB cleavage complex
  9. DNA Damage
  10. RecA-mediated SOS response
  11. Multidrug persistence
43
Q

tisABTA System General Information

A
  • tisAB is a Type I system
  • Locus encodes tisAB mRNA which expresses the TisB toxin & 2 small RNAs (1stR-1 & 1stR-2) where 1stR-1 is the antisense RNA to tisAB mRNA
  • The expression of TisB is partially controlled by the LexA repressor
  • If expressed, TisB toxin is localized to inner membrane & results in membrane damage & subsequent decrease in DNA replication, RNA transcription & protein synthesis
  • 1stR-1 is antitoxin & inhibits TisB toxicity at RNA level
44
Q

tisABTA system in Non-Stressful conditions

A
  • SOS system is off due to LexA binding to LexA promoters (SOS boxes), including that of the tisAB TA system.
  • IstR-1 antisense RNA is present in excess over its target, tisAB mRNA, which is expressed at low levels because of incomplete repression by LexA.
  • IstR-1 antisense RNA base-pairing with a 23ntide region in the tisAB mRNA results in RNase III-dependent cleavage and inactivates the mRNA for translation of TisB toxin.
  • IstR-1 functions in preventing inadvertent TisB synthesis during normal growth
45
Q

tisABTA system in Stressful conditions

A
  • DNA damage results in induction of SOS response.
  • SOS response -> activated RecA which cleaves the LexA occupying the tisAB promoter
  • Allows tisAB mRNA to be made in excess
  • Leads to depletion of the IstR-1 pool & resultant accumulation of tisAB mRNA.
  • Translation of tisAB allows TisB toxin to accumulate which targets the cell membrane decreasing respiration, slowing down the SOS response and leading to growth arrest of the cell = PERSISTENCE
46
Q

What is the TisAB TA Systems response to DNA damage?

A
  1. Antibiotics (like ciprofloxacin) kill bacteria by damaging their DNA
  2. RecA is activated by the accumulation of ssDNA
  3. Activated RecA interacts with the LexA repressor and LexA is cleaved
  4. SOS genes are induced to repair DNA damage.
  5. Concurrently, the SOS induction results in cleavage of the IstR-1 pool.
  6. Accumulation of tisAB mRNA, further depletion of IstR-1 antisense RNA pool by RNAse III cleavage and expression of TisB toxin.
  7. TisB toxin causes membrane damage and the loss of membrane proton motive force (pmf) and ATP level = decreased pmf & ATP
  8. As a result, drugs are driven out of the cells, leading to persister formation
47
Q

Integrated model of persistence via the ccd and tisABTA systems

A
  1. Stress -> Increased (p)ppGpp levels -> Increased rate of Lon protease activity due to inhibition of PPX
    - Lon protease degrades CcdA allowing CcdB to poison DNA gyrase
    - DNA gyrase can no longer repair DNA replication forks resulting in DNA damage and induction of SOS response.
    - Accumulation of ssDNA from damage activates RecA which cleaves the LexA occupying the tisAB promoter
    - Allows tisAB mRNA to be made in excess leading to depletion of the IstR-1 antisense RNA pool & concomitant accumulation of tisAB mRNA.
    - Translation of tisAB allows TisB toxin to accumulate which targets the cell membrane decreasing respiration, slowing down the SOS response and leading to growth arrest of the cell = PERSISTENCE
48
Q

What type of TA system is the Hok Sok system?

A

Type I

49
Q

Where is the Hok/Sok system found?

A

On the R1 plasmid & E. coli genomes

50
Q

What do ‘Hok’ and ‘Sok’ stand for?

A

Host Killing & Suppression of Killing

51
Q

What are the cis sites on the mok/mok mRNA?

A 
Pmok = Promoter
tac = Transcriptional activator sequence
ucb = Upstream complementary box
fbi = Fold back inhibition element
hok = host killing
sok = suppression of killing
mok = modulation of killing
52
Q

What is mok proteins function in the cell?

A

No function other than to open the hok coding region

  • It is a safety mechanism - you can only translate hok if mok has been translated
53
Q

In what form does hok mRNA accumulate in the cell?

A
  • Hok mRNA accumulates as a stable (20mins) & inactive RNA:
    1. 3’ fbi region binds to 5’ tac region
    2. 5’ ucb region binds to 5’ tac & mok shine-dalgarno regions
    3. This forms closed loop & highly structured RNA
  • Closed loop = inactive mok/hok RNA is NOT accessible to initiating ribosomes (i.e. not translated) NOR binding to antisense Sok RNA.
54
Q

How is hok mRNA transcript activated?

A
  • Slow processing of 3’ end of the hok mRNA (fbi) by host RNAse ll & PNPase activates the transcript and it:
    o Can be translated by ribosome binding OR
    o Can bind to sok antisense RNA at sokT region
55
Q

How can transcription of Hok mRNA be induced?

A

Increased levels of ppGpp + Obg

56
Q

Sok Antisense RNA

A
  • Transcribed in opposite direction to hok/mok mRNA from a strong promoter (Psok)
    o Sok RNA is in molar excess to hok mRNA
  • Has very short half-life (30s) – cleavage by RNAse E initiates rapid decay
  • Target for Sok RNA is mok TIR region (SokT):
    o Indirectly inhibits hok translation by inhibiting mok translation when it binds to sokT
  • Sok RNA can only bind to active hok mRNA (i.e. after 3’ processing)
    o When bound to mok/hok mRNA via sokT it triggers degradation by RNAse III
  • Sok antisense RNA binding to sokT is irreversible
57
Q

How is host killing achieved by the Hok/Sok system?

A
  • If daughter cell lacks R1 -> killed by hydrophobic Hok protein:
    o Hok associates with cell membrane and high or moderate concs.
    o High [Hok] at membrane -> collapses membrane potential -> Cell death {Killing}
58
Q

How is host suppression achieved by the Hok/Sok system?

A

o If daughter cell lacks R1 -> killed by hydrophobic Hok protein:
o Hok associates with cell membrane and high or moderate concs.
o Moderate [Hok] at membrane -> membrane depolarization -> growth halt {Suppression}

59
Q

How is suppression of killing achieved by the Hok/Sok system?

A
  • In R1-containing cells -> high rate of sok antisense RNA transcription + weak transcription & slow processing of hok mRNA = no translation of Hok protein
    o Sok binds to hok -> degraded by RNase III
  • Default state = high production of sok (less stable/shorter halflife – but much more) binds to hok = degradation
60
Q

What are the 5 steps in the operation of the hok/sok killing system?

A
  1. Full length, stable, inactive mok/hok mRNA that cannot be translated or bound by Sok antisense RNA
    a. Due to complementary bas paring between fbi and tac
    b. Due to complementary base paring between ucb and tac/mok Shine-Dalgarno
  2. mRNA is activated via 3’ processing and refolding by RNAse II & PNPase
    a. Very slow process (Makes half life 20mins)
  3. Makes sokT region available & SD of mok mRNA
  4. IF plasmid carrying cell:
    a. Sok Antisense RNA is short-lived due to cleavage by RNAse E
    b. Is complimentary to the sokT region, so can bind and prevents ribosome binding to SD of mok mRNA
  5. IF plasmid-free cell:
    a. Ribosome will bind to mok SD & start translating
    b. SD of hok opens during translation of mok
    c. Leads to NEW ribosome binding to hok SD and translating a protein that will lead to cell death
61
Q

What is Obg?

A
  • Obg is a universally conserved P-loop GTPase – acts at crossroads of maj. cellular processes of protein translation & DNA replication
  • Directly or indirectly plays role in DNA replication initiation, replication fork stabilisation, ribosome maturation
  • Essential for viability in several bacteria -> simple knockout mutant can’t be obtained
62
Q

What is the role of Obg in persistence?

A

Over-expression of Obg increased persistence significantly & silencing of obg expression caused a significantly decreased number of persisters

63
Q

What is the interaction of Alarmone and Obg in persistence?

A
  • Obg-mediated persistence requires (p)ppGpp because Obg no longer increased persistence when overexpressed in a ΔrelA ΔspoT mutant.
    oNeeded the double mutant (not just ΔspoT) because the amount of alarmone plays an important role
64
Q

HokB Transcription and Obg

A
  • Obg-mediated persistence requires (p)ppGpp
  • Obg + (p)ppGpp transcriptionally activates hokB toxin mRNA expression
  • In a cell that has a functional hok/sok TA system, elevated HokB levels are sufficient to overcome Sok RNA and cause membrane depolarization, resulting in persistence but not cell death
65
Q

Obg activation of HokB leading to persistence

A
  1. Stress - decreased energy & nutrients
  2. Increased alarmone
  3. Alarmone associates with Obg
  4. Increases HokB mRNA transcription
  5. Overrides inhibition by Sok RNA
  6. Allows increase in HokB protein
  7. Targets membrane & decreases membrane potential
  8. Causes dormant cells
  9. Persistence
66
Q

How does RNase E control Sok & Hok B Activities?

A
  • Sok RNA is produced in excess to Hok mRNA and prevents its translation
  • Sok RNA degraded by RNAse E
  • RNAse E (an enzyme) is associated and anchored in the inner membrane via the membrane targeting sequence (MTS)
  • RNAse E enzyme activity is correlate with membrane association
    o i.e. RNAse E + membrane = RNAse E enzyme activity
    o i.e. if not associated with membrane it has decreased/no activity and can’t degrade Sok RNA
  • Membrane association plays a regulatory role in determining RNAse E activity
  • Levels of sok RNA is determined by RNAse E degradation and Hok B translation is determined by Sok RNA levels = therefore RNAse E also determines the rate of HokB translation
67
Q

HokB expression under normal growth conditions

A
  1. Cell Growth (V Low alarmone)
  2. Normal membrane potential
  3. RNAse E associates with membrane (default state)
  4. RNase E degrades Sok
  5. Cell is in steady state - Rate of Sok synthesis sufficient to prevent translation of hok mRNA
  6. No HokB Protein
  7. Cell grows
68
Q

Homeostatic control model for Hok & Sok under stress conditions

A
  1. Stress:
    • Decreased Energy
    • Decreased Nutrients
    • Increased Alarmone levels
  2. Alarmone interacts with Obg
  3. Increases HokB mRNA transcription – forming pool of HokB mRNA
  4. Accumulation of HokB overrides inhibtion by Sok RNA
  5. Allows increase in HokB protein
  6. HokB targets membrane
    • Decrease in Membrane potential through ATP/proton leakage
  7. Membrane damage is ‘sensed’ by RNAse E – which detaches from membrane
  8. Decreases RNAse E activity
  9. Decreases Sok degradation -> Sok increases
  10. Increased Sok inhibits HokB translation
  11. Decreased HokB limits the membrane damage by HokB
  12. Prevents the cell from death -> rather it enters state of Persistence
69
Q

How does HokB lead to Persister cell induction?

A

[Dimerization & Pore formation]

  1. HokB mRNA translated -> peptide inserts into membrane
  2. DsbA forms disulphide bond between cysteine residues between 2 HokB peptides
  3. Dimerization stabilizes peptides and forms a pore in the membrane
  4. Pore allows ATP to leak out of the cells and decreases membrane potential -> persistence
70
Q

Process of HokB-mediated Persister Awakening

A
  1. DsbC reduces disulphide bridge of HokB dimer leaving HokB monomers - destabilizes the pore
  2. Disassembled pores allow e- transport chain to repolarize the membrane and produce ATP pool
  3. Repolarization lowers pH - activates DegQ to cleave the periplasmic domain of HokB monomers – thereby preventing re-dimerization & eventually leads to regrowth of HokB-induced persister cells
71
Q

What are the environmental cues for Persister awakening?

A
  • Seems to be random
  • Did proteomic analysis of tisB system – found some proteins are upregulated for recovery
    • Proteins involved in DNA repair are required for awakening
    • Certain antitoxins of some TA systems are involved
72
Q

What is the importance of understanding TA-induced Persistence & Awakening?

A
  1. Understanding how TA systems induce persistence can help us identify possible targets that could inhibit persister induction & thereby decrease persister frequency
  2. Understanding persistence awakening could help in development of therapeutics that prevent awakening and thereby chronic infection
  3. Targets of TA toxins could be targets for developing new antibiotics or improving existing antibiotics
  4. Multiple TA systems shown to influence biofilm production & therefore bacterial pathogenesis
  5. Need to understand role of TA systems in evolution of antimicrobial resistance especially in clinical settings

MCB3026F (6 decks)

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