Bacterial cell lysis and death

1
Bacterial cell lysis and death

• 11-11-2009

2
Intrinsic control of lysis

• Autolysis by autolysins
• The self-destructive end stage of the bacterial
  life cycle by a unique family of murein (PG)
  hydrolases that specifically cleave structural
  components of PG
• Autolysin- the murein hydrolases that lead to the
  destruction of the cell wall and subsequent cell
  lysis

3
Autolysis in Neisseria gonorrhoeae

• prone to undergo autolysis
• provision of nutrients to a starving population
• modulation of the host immune response by
  released cell components
• HSP60
• donation of DNA for natural transformation

JB (2006)1887211-
4
Induction of autolysin activity

• Influenced by a variety of different factors
  including NaCl, pH, growth phase, proteases
  (oxidative stresses), teichoic acids (Gram
  positive cell wall recycle), antibiotics and
  phage infection

5
Endolysin- a murein hydrolase

• Bacteriophage-induced lysis
• Upon holin activation, the membrane becomes leaky
  ? Endolysin produced by bacteriophage accumulate
  in an active form in the cytoplasm? activated
  holin? allows the escape of the endolysin to the
  PG

6
Activation of murein hydrolase

• Holin and anti-holin control the PG turnover in
  the fibrous layer
• CidA- holin
• LrgA- anti-holin
• ?the murein hydrolases activated in a depolarized
  cell? the PG degraded? compromising the
  structural integrity of the cell wall? resulting
  in cell lysis

Periplasmic space
Micro Mol Biol Rev (2008) Mar
7
Regulation of autolysis

• lrgAB
• anti-holin expression
• cidABC
• CidAB? holin
• CidC- pyruvate oxidase
• CidR transcription factor
• LytSR
• AlsSD

Anti-holin
Pyruvate oxidase
holin
8
Balance between CidC and AlsSD impact cell
viability

• In the presence of excess glucose ? acetic acid
  accumulation ? induce the transcription of
  cidABC and lrgAB ? enhanced cell death
• The alsSD mutant ? rapid cell death (RCD)

9
A key determinant of life or death

• The fate of pyruvate in bacteria? converted to
  acetic acid or acetoin?
• In both prokaryotes and eukaryotes, rapid growth
  is fueled by aerobic glycolysis to supply the
  building blocks needed for macromolecular
  synthesis and pyruvate metabolism plays a
  critical role in the control of cell death

Micro Mol Biol Rev (2008) Mar
10
LytSR 2CS

• The LytSR sense viral infection- a membrane
  depolarization upon bacteriophage attachment ?
  inducing lrgAB transcription ?inhibit cell
  Lysis?? allow bacteriophage replication and
  assembly

11
Balance between life and death within biofilm
The control of this balance via the differential
expression of cid and lrg ? contribute to the
tolerance of biofilm cells to antibiotic treatment
12
Cid/Lrg regulatory system
- for bacterial apoptosis?

• Cid/Lrg regulatory system is functionally
  analogous to the eukaryotic Bax/Bcl-2 regulatory
  system for apoptosis
• Apoptosis
• A programmed cell death (self-destruct process
  with orderly morphologic disintegration)

13
History of TA system

• Initially characterized as plasmid-borne
  mediators of plasmid stability (1985)
• When bacteria lose the plasmids (or
  extrachromosomal element- prophage), the cured
  cells are selectively killed because the unstable
  antitoxin is degraded (by protease) faster than
  is the stable and lethal toxin
• The toxin-antitoxin pair is also called addiction
  module

Post-segregation killing effect
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Suicidal modules on chromosome?

• MazEF, a homolog of the addiction module on E.
  coli K-12 chromosome
• Can not prevent the loss of chromosome
• Under nutrient stress led to PCD? suicidal module
  
• MazF is a stable toxin? inhibits translation by
  cleaving mRNA
• MazE, a labile protein degraded by ClpAP
  protease? counteracts MazF function
• MazE and MazF interact and are coexpressed
• Hanna Engelberg-Kulka et
  al., 1996. PNAS

15
TA system induce PCD?

• MazFE, a toxin-antitoxin (TA) system, induces
  apoptosis? allowing a subset of cells to be
  sacrificed to benefit the population as a whole
• JB (2006) 1883420-

16
MazEF and bactericidal antibiotics

• Any stressful condition that prevents the
  expression of mazEF module will lead to the
  reduction of MazE in the cell, permitting toxin
  MazF to act freely.
• antibiotics inhibiting transcription and/or
  translation like rifampicin, chloramphenicol, and
  spectinomycin
• antibiotics causing DNA damage like trimethiprim
  or nalidixic acid

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TAs are stress response elements

• In addition to be involved in plasmid maintenance
  or PCD, TA proteins modulate translation and DNA
  replication under nutrient stress
• When cells encounter nutritional or environmental
  stresses, cellular proteases (Lon or Clp)
  activate toxins by degrading the antitoxins
• The toxin activity does not necessary lead to
  cell death provided that within a certain window
  of time synthesis of the antitoxin is resumed
• Kenn Gerdes and colleagues JMB 2003
  332809-19

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E. coli RelBE TA system

• The relB and relE genes encode a TA module
• The RelE toxin acts as a inhibitor of
  translation
• The overexpression of RelE severely inhibited
  translation
• The RelB is degraded by Lon protease
• A relBE mutant achieved a higher steady-state
  level of translation after amino acid starvation

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Antibiotic resistance in biofilms

• Bacteria growing in biofilms resist killing by
  antibiotics?
• Antibiotics fail to fully penetrate biofilms?
• Persisters
• Some of the bacteria enter a slow- or nongrowing
  state (and hence the bacteria are less
  susceptible to killing)?

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Persisters

• Refers to the emergence of a small population of
  cells (10-6) that survive treatment with lethal
  concentrations of bactericidal antibiotics yet
  are genetically identical to the original culture
  
• The hipA-hipB linked to the persister state in E.
  coli
• Persistence is linked not only to HipA expression
  but also to expression of other unrelated
  proteins
• JB
  2006. 1883494-7.

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HipBA TA-like module

• Obtained by screening of E. coli mutants for a
  high frequency of persister cell formation
• HipB- a small DNA-binding protein that negatively
  regulates the expression of hipBA and interacts
  with the HipA protein
• HipA appears to be toxic in the absence of HipB
• The hipA mutation increased persister cell
  formation
• HipA is a protein kinases and is capable of
  autophosphorylation
• The expression of HipA effectively protected
  cells from antibiotic-induced killing

JB (2006) 1888360-
22
Multiple TA systems in a genome

• Multiple locations within a genome
• Comparative genome analysis revealed that
  obligate host-associated organisms are largely
  free of TA loci, while such loci are present in
  the majority of sequenced free-living bacteria
• TA systems move through horizontal gene transfer
• At least 5 toxin-antitoxin systems in E. coli
  K-12 genome
• 13 TA loci in Vibrio cholerae genome
• 
  

JB 2007. 189491-500
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Multiple and polymorphic TA systems

• Bioinformatic surveys also indicate that the TA
  systems are frequently polymorphic (they are
  found in some but not all isolates of a given
  species)
• Stable polymorphisms may be attributed to
  niche-specific adaptations

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TA in Mycobacterium tuberculosis

• The TA loci have now been shown to be abundant in
  free-living bacteria but are absent from most
  obligate intracellular bacteria
• M. leprae has no complete TA loci, yet M.
  tuberculosis carries 38 chromosomal TA loci
• 3 encode RelBE and 9 encode MazEF homologues
• All are toxins that cleave mRNA in response to
  nutrient stress or starvation

25
A differential effect of E. coli TA systems on
cell death

• 5 TA systems
• only the TA system mazEF mediated cell death both
  in liquid media and during biofilm formation

PLoS ONE August 2009 Volume 4 Issue 8 e6785
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Different effects on E. coli cell survival
following treatment with different antibiotics

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TA as drug target?

• How?
• An inhibitor that mimics the most relevant toxin
  residues and their interactions with the
  antitoxin, leading to a new complex
  (inhibitor-antitoxin) that frees the toxin to
  cause bacterial damage
• 
  J Bacteriol (2007) 1891266-78

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Life, death, differentiation, and the
multicellualrity

• MazF RNase? degrade most cellular mRNAs ? rapid
  loss of most proteins
• Some mRNAs (encode less than 20 kDa small
  proteins) are protected
• some of the small proteins promote the death of
  most of the cells in the population
• whereas others promote the survival of a small
  cell subpopulation

PLoS Genetics March 2009 Volume 5 Issue 3
e1000418
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Survival proteins or death proteins

• 2D proteome analysis for the proteins synthesized
  after the induction of MazF

PLoS Genetics March 2009 /5 e1000390.
doi10.1371/journal.pgen.1000390.
30

• Escherichia coli MazF
• - Leads to the simultaneous selective
  synthesis of both Death Proteins and
  Survival Proteins

mazEF-mediated cell death survival induced by
brief inhibition of translation
mazEF-mediated cell death (occurs following
either DNA damage or brief inhibition of
translation)
mazEF-mediated death proteins induced by DNA
damage
PLoS Genetics March 2009 /5 e1000390.
doi10.1371/journal.pgen.1000390.
31
Diversity and abundance of TAs

PLoS Genetics March 2009 Volume 5 Issue 3
e1000437
32
Why so many?

• What is the benefit? Are they involved in
  general stress responses?
• E. coli K-12 (MG1655)
• mazEF, relBE, yefM-yoeB, chpB, and dinJ-yafQ
• All five toxins are protein synthesis inhibitors
• Unable to detect TA-dependent PCD under various
  stress conditions
• J
  Bacteriol 2007. 1896101-8.

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Hypothetical functions of TA systems

• Junk, stabilization of genomic parasites, selfish
  alleles, gene regulation, growth control,
  persisters, programmed cell arrest, anti-phage
• 
  JB 2007. 1896089-92

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Triggers of the MazEF-mediated cell death

• High level of ppGpp inhibited mazEF transcription
  leading to cell death
• Various stressful conditions
• in the presence of antibiotics
• high temperature, DNA damage, and oxidative
  stress also induced mazEF-mediated cell death
• 
  J Bacteriol 2004. 1863663-

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ppGpp- a stringent response molecule

• Nutritional stress signal molecule produced by
  RelA (ppGpp synthase) and SpoT (ppGpp hysrolase)
  proteins

cessation of transcription of stable RNAs (rRNA
and tRNA)
May 2005
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Regulatory function of (p)ppGpp

• Regulation of many aspects of microbial cell
  biology that are sensitive to changing nutrient
  availability growth, adaptation, secondary
  metabolism, survival, persistence, cell division,
  motility, biofilms, development, competence, and
  virulence.

Annu Rev Microbiol (2008) 6235-51
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Escherichia coli stringent responses

• The adjustment of gene expression program allows
  for prolonged survival in stationary phase

Annu Rev Microbiol (2008) 6235-51
38
Gene expression effected by ppGpp

• A vital role of ppGpp in relaying information
  about the translational status of the cell not
  only to genes involved in translation and amino
  acid biosynthesis, but also to genes involved in
  intermediary metabolism and macromolecule
  synthesis by comparative analysis of E. coli K12
  and the relAspoT mutant

Mol Microbiol (2008)
39
RSH (Rel Spo homolog)
ACP binding

• E. coli (and all beta- and gamma-proteobacteria)
  synthesizes (p)ppGpp with RelA and SpoT
• Other bacteria and plants contain one or more
  Rel/Spo homolog gene rsh genes
• Regulating the balance of the opposing activities
  of RSH enzymes is crucial
• Equally active, unregulated hydrolase and
  synthase activities would catalyze a futile cycle
  of (p)ppGpp synthesis and hydrolysis
• Too much synthase ? provokes a stringent
  response? inhibits growth
• Too little (p)ppGpp from excess hydrolase? makes
  cells less able to respond appropriately to
  nutritional stress