BIOL 2044 - bacterial stress response

bacterial resilience - defining resistance, tolerance, persistance

RESISTANCE

• implies genetically encodes systems

• includes mutation, modification

• E.g: active efflux —> mechanism to ensure that AB doesn’t see target, bacteria produces a target for the AB so that it doesn’t bind to it

TOLERANCE

• bacteria grows in biofilm to produce a physcial barrier so AB cant penetrate

PERSISTANCE

• bateria can eneter a dormant state so that they can survive stressful conditions

gene regulation in bacteria

occurs at 3 levels:

1. transcriptional regulation

   • expression of activators, repressors, TFs

2. post transcriptional regulation

   • at the mRNA level

   • mRNA cleavage, sRNA, RNA binding proteins

   • can downregulate protein translation by changing RNA stability

3. post translational regulation

   • at the protein level

   • protein-protein interaction, protein modification

   • can target proteins for degradation

ensure that bacteria can respond quickly at the appropriate level

E.g: if bacteria changes environments and requires a diff set of proteins, then will respond at the transcriptional level

E.g: if toxins are present then intervention at the proetin level best to modify the protein-protein interactions

sigma factors

• sigma factors not all the same

  • housekeeping sigma

  • other stress sigmas which can downregulate certain programmes and upregulate others

RpoS - general stress response sigmas

• nutrient depletion/starvation

• co-ordinated many stress responses

  • elimination of toxins

  • protection

  • repair

  • scavenging

• activated at gene transcription, mRNA transcription and protein level by:

  • N-

  • Mg-

  • P-

  • C-

  • difference in pH, temp, oxidative stress, osmolarity

• genes controlled by the RpoS regulon containing over 400 genes

stringent response

• mechanism of RpoS regulation

• when uncharged tRNAs (tRNAs with no amino acids attached) theyre detected by RelA which becomes active

• Rel A synthesises ppGpp and pppGpp from ATP and GTP

• SpoT helps to regulate the (p)ppGpp levels

• (p)ppGpp then binds DskA TF which binds RpoS sigma which sequesters core RNAP to transcribe stress resistance genes

• nutreint depletion causes sigma 70—>RpoS

• SpoT and RelA kinases become active in response to low carbon, iron, phosphate, fatty acids

accessory genes

• sigma factors general/specific stress response are core genes - all E. coli have them

• some mechanism in accessory genes

• located in plasmids, transposons, genomic islands

• transferred by horizontal gene transfer

• can encode:

  • AB resistance

  • virulence factor

  • metabolic adaptations

  • stress response and survival mechanisms

  • phage defence

  • toxin/antitoxin systems - genetic elements involved in carrying these functional traits

roles of toxin/antitoxin systems

• dormancy/lysis

• antiphage defence

• stress tolerance

• persister cell function

• some require this for infection

• important for understanding AB resistance

• each bacterial species has their own toxin/antitoxin sequences in chromosomes

• varies among bacterial strains belonging to the same species —> can be problematic as when studied in the lab the genes wont be the same as in the patient

• mainly found on chromosomes but also found in plasmids having a structural role

• antitoxin is usually less atble —> if antitoxin degraded the toxin may kill the cell

  • if T/A plasmid in the cell is not present and not actively producing T/A the antitoxin remaining in the cell will degrade faster leaving the toxin to do its job and kill the cell (has a structural role)

  • if bacteria has a plasmid without T/A genes then progeny can survive with or without but this ensures that only bacteria with T/A system survive

classification

• 8 types

• 3 party antitoxin/toxin systems

• based on nature and mode of action

TYPE 1

• antitoxin mRNA

• antitoxin prevents toxin translation

TYPE 2

• antitoxin protein that binds toxin

TYPE 3

• RNA antitoxin

• binds toxin

TYPE 4

• protein antitoxin that counteracts toxin

• intermediate players

Type 2 example

• antitoxin/toxin in operon, transcribed at once

• stress upregulates proteases which degrade antitoxin —> leaves toxin to do its role

• an interfere with DNA replication, impede cell wall synthesis, degrade RNA

• when theres no stress antitoxin binds to toxin which acts as a repressor and downregulates transcription of T/A

• many diff toxin failies

complex network

• dont undergo complex regulation

• undergo crosstalk with one another and regulate the expression of stress response gene

• E.g: all have diff affinities for tRNA so the network is very complex

• no bacterial genome that doesnt have toxin/antitoxin system (some have more than 1) —> even found in plants and higher organisms

• degrading mRNAs not needed in diff environment, can be good for cells to adapt to new conditions quickly

type 2 A/T system —> Maz E/F system in E. coli

• MazF= toxin, MazE= antitoxin

• when stress is present antitoxin is degraded

• toxin then degrades RNA t the ACA sites —> doesnt sirectly induce lysis

• bacteriostatic not bacteriocidal

• in no stress MazE binds MazF —> no downstream effects and acts as repressor of transcriptional unit

• ACA motif not rare —> MazF cleaves its own RNA —> causes dynamic regulation of bacteril cellular processes

• causes oscillating MazF levels during period of stress

• fast regulatiom of MazEF underlies growth heterogeneity (due to RNA degradation)

• beneficial for bacteria as at any point a fraction of the stressed population in the ready to exit state (bet hedging)

role in phage defence

endonucleases such as Ma2F are involved in phage defence

• cleave mRNA of DNA phage genomes and RNA phage genomes

• abortive infection (toxin induced cell lysis/metabolic arrest)

  • with no Abi can produce progeny phage particles

  • with Abi interupts the spread of infection - phage adsorbs to cell and is taken in, interrupts replication cycle

  • cell either lysed or enters metabolic arrest so tht the phage cant spread

• only works when phage DNA/RNA already there although there are other mechanisms to prevent phage entry

mechanisms of defence

1. DarTG

2. ToxIN

3. Retron-sen2

1. DarTG

• type 2 system - toxin and antitoxin proteins

• DarG antitoxin, DarT toxin

• DarT is an enzyme which decorates phage DNA with ribose —> pol stalls and phages cant replicate

• phage evasion: mutates polymerase so it can replicate DNA when ribose still there

2. ToxIN

• type 3 system

• toxin is a protein, antitoxin is a repeat of small RNAs

• toxN is toxin

• bacteria shuts down trancription - prvenets phage propogation and bacterial propogation

• when transcription shut down antitoxin degraded and no more is made —> activates toxin

• ToxN degrades phage trancript

• phage counteracts this only allowing partial transcription

3. Retron Sen 2

• 3 player system

• toxin is RcAT, antitoxin is reverse trancriptase that produces multicopy starnd ssDNA made up of Retron RT and msDNA

• onlly stable in one form - when msDNA bound to retron RT

• during pahge infection diff degradation mechanisms take place which effect stability of antitoxin

• msDNA degraded so that it cant neutralise the toxin

• during phage infection diff degradation mechanisms take place which affect stability of antitoxin

  • msDNA can also be methylated to have the same effect

• toxin doesnt degrade RNA but nuclease hydrolase interferes with DNA and RNA so cant produce progeny

• as a countermeasure pahge has antitoxin neutralise protein (RaCC) which binds the toxin and renders it inactive

phages vs antibiotics

PHAGES

• can be very specific (can have broad host range though)

• replicate at the site of infection

• have no side effects

• development of resistance

• our body has response to phage but not immune response

• finding new pahges is fast

ANTIBIOTICS

• non specific

• not concentrated at the site of infection

• multiple side effects

• developing new ABs is slow

best therapies are now thought to be combination therapies of pahges and antibiotics

phage therapy

• if patient infected with AB resistant bacteria —> isolate and identify strain

• in vitro screening of specific lytic phages isolated from various environmental and clinical samples or sources from pahge banks

• safety/efficay trials

  • inhaled

  • applied to wounds

  • bladder irrigation

  • orally

how do we screen phage collection

1. add 180ul LB to 10ul of bacteria

2. add 10ul of pahge for each (and the control)

3. measure absorbance at 600nm every 10 min for 12h (allow bacteria to grow 2-3 hours before adding phage

• if theres a decrease in the phage line OD600 with bacteria and phage then its able to supress bacterial growth

• sometimes when challenged with phages different modes of action ensures that the bacteria cannot develop a counter defence mechanism —> killed before then

• also want to ensure phage only kills strain of interest

• can be deemed costly especially due to the personalised treatment for phage