Transcript (BTS 801) Quorum Sensing as a Potential Antimicrobial Target

Biofilm and Quarum Sensing
M. Sc (P) Biotechnology
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Biofilms
-Biofilms are organised microbial systems
consisting of cells associated with surfaces
- Likely the most wide-spread mode of growth for
bacteria in nature
Anton van Leeuwenhook
used a primitive microscope
to look at biofilms in 1684.
Biofilms
In the natural world most bacteria aggregate as biofilms - they
form when bacteria adhere to surfaces in aqueous environments
and begin to excrete a polysaccharide that can anchor them to all
kinds of material.
The biofilm is held together and protected by the polysaccharide
matrix. This matrix protects the cells within it and facilitates
communication among them through biochemical signals.
Bacteria living in a biofilm usually have significantly different
properties from free-floating bacteria of the same species, as the
dense and protected environment of the film allows them to
cooperate and interact in various ways.
Biofilms
• A biofilm can be formed by a single bacterial species, but more
often biofilms consist of many species of bacteria, as well as
fungi, algae, protozoa, debris and corrosion products.
• Once anchored to a surface, biofilm microorganisms carry out a
variety of detrimental or beneficial reactions (by human
standards), depending on the surrounding environmental
conditions.
Advantages for Bacteria
♦ Creation of habitable niches
♦ Protection against:
- Physical forces (e.g. in flowing systems)
- Phagocytosis by immune cells
- Grazers (e.g. ciliates, amoeba)
- Viruses
♦ Barrier against toxic substances
♦ Facilitates intercellular communication
♦ Close proximity of cells enables genetic exchange
Disadvantages for mankind:
♦ Immune system can not attack biofilms
♦ Antibiotics/antimicrobial agents fail
♦ Slow the flow of liquids or clog pipelines
♦ Accelerate corrosion of pipelines
♦ Risk for drinking water supply via pipes
Effects of Biofilms
• Microbial biofilms on surfaces result in billions of dollars in losses
yearly due to equipment damage, product contamination, energy
losses and medical infections.
• Conventional methods of killing bacteria (such as antibiotics, and
disinfection) are often ineffective with biofilm bacteria.
• The huge doses of antimicrobials required to rid systems of biofilm
bacteria are environmentally undesirable and medically impractical.
• Conversely, microbial processes at surfaces also offer
opportunities for positive industrial and environmental
effects, such as bioremediating hazardous waste sites,
biofiltering industrial water, and forming biobarriers to
protect soil and groundwater from contamination.
Pseudomonas strain S61 biofilms on glass slides
Staining technique (Congo red) in which the bacterial cells
stain dark red and the exopolysaccharide stains orange-pink
Biofilms grown in soil
7 day old biofilm
14 day old biofilm
Structure of Biofilms
• Although bacteria can grow in a free-living or “planktonic
state” it is common for them to adhere to surface by producing
extracellular polysaccharides.
• The adherent bacteria produce microcolonies leading to an
intricate three-dimensional structure.
• Biofilms survive so well because they have channels, like
aqueducts, that transport water, oxygen, and nutrients to all
the bacteria of the community.
• These channels also get rid of the bacterial wastes, making
these biofilms seem almost as complex as a city.
• The complexity goes so deep that even the different regions of
the biofilm have bacterial cells with different genetic
information, physical characteristics, and “duties” for the
community.
Close-up of a microcolony
Stages in biofilm formation
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Attachment of bacteria
Irreversible binding by bacteria
Formation of microcolonies
Maturation of microcolonies
Dispersal
biofilm attachment
Maturation
Biofilm-forming Bacteria
Campylobacter jejuni
• Gram-negative bacteria
• genus was first discovered in the
1970’s
• most common cause of
gastroenteritis
• Microbial coloniser of surface
waters
• Incidence in the U.S. is estimated
at 30 to 60% per 100,000 of the
population.
Legionella pneumophila
• Gram-negative bacteria
• Causes legionellosis,
commonly known as
Legionnaire’s disease
• Transmitted to host via
aerosolisation and ingestion.
• Found in domestic households
and large municipal buildings:
plumbing, air conditioning
systems, etc.
• Occurs in biofilms where
symbiotic relationships with
other heterotrophs are
evident
Salmonella enteritidis
• Gram-negative bacteria
• Anaerobic
• Present predominantly in raw
water and occssionally in potable
water.
• Chlorine is effective in destroying
this organism in the planktonic
state.
• Forms dense, metabolically active
biofilms.
• Often develop on stainless steel.
Biofilm Interactions
• Quorum sensing
• Interspecies interactions
– Symbiosis
– Population relationships
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Spatial
Temporal
Metabolic
Genetic
Warning: Biofilms present!
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Biofilms may be 50 to 500 times more
resistant to chemotherapy than planktonic
bacteria of the same strain.
Cause cosmetic degradation in toilet bowls
Are the cause of flawed prints and
malfunctioning machines during photo
processing
Infect implanted devices such as: contact
lens, catheters, prosthetic heart valves, and
cardiac pacemakers.
Cause such chronic infections as: cystic
fibrosis, pneumonia, biliary tract infections,
osteomyelitis, and bacterial prostatitis.
They are also the cause of dental plaque!!!
Biofilms to the Rescue!
• Play an essential role in
the processing of sewage
water prior to its
discharge into rivers
• Bioremediation
– Clean up groundwater
– Oil recovery
– Mine remediation
How do biofilms form?
• The formation of a biofilm requires coordinated
chemical signalling between cells.
• Unless an adequate number of neighbouring cells are
present, the costs of biofilm production to an
individual bacterium outweigh the benefits.
• A signalling process benefits the bacteria by allowing
them to sense the presence of neighbouring bacteria
and respond to varying conditions.
• The process by which a bacterium does this is called
quorum sensing.
Quorum sensing
Biofilm formation involves more than just bacteria
attaching to a solid surface; individual organisms
aggregate with their kin and often congregate with
members of other species.
Bacteria accomplish this through chemical signaling
mechanisms. When the local extracellular
concentration of the chemical signal reaches a
threshold level, indicating that the population of
microbes has reached a minimum density—a quorum—
the community of organisms undergoes phenotypic
changes.
The process of chemically sensing the population
density is called quorum sensing.
Signal molecules produced by individual cells do not have an effect
until the bacterial population density is sufficient to provide a
concentration of molecules, which then cross cell membranes and
activate the manufacture of such cellular products as toxins, enzymes
or surfactants.
Quorum sensing
• The term 'Quorum Sensing' (QS) is used to
describe the phenomenon whereby the
accumulation of signalling molecules enable a
single cell to sense the number of bacteria (cell
density).
• In the natural environment, there are many
different bacteria living together which use various
classes of signalling molecules.
Introduction
 Quorum sensing is cell to cell signaling mechanism that enables the
bacteria to collectively control gene expression.
 This type of bacterial communication is achieved only at higher cell
densities.
 Bacteria release various types of molecules called as autoinducers in
the extracellular medium, these molecules are mediators of quorum
sensing.
 When concentration of these signaling molecules exceed a particular
threshold value, these molecules are internalized in the cell and activate
particular set of genes in all bacterial population, such as genes
responsible for virulence, competence, stationary phase etc .
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Cell density and quorum sensing
R gene
I gene
AHL diffuse in
R protein
I protein
Cell
density
R gene
R protein
+
I gene
I protein
AHL diffuse out
AHL diffuse
out
Time
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Quorum sensing controlled processes
 Bioluminescence
 Biofilm formation
Virulence
expression
  Virulence
genegene
expression

Sporulation
 Competence
It occurs in various marine bacteria
such as Vibrio harveyi and Vibrio fischeri.
Takes place at high cell density.
It iscompact mass of differentiated microbial cells, enclosed
in a matrix of polysaccharides. Biofilm resident bacteria
are antibiotic resistant. Quorum sensing is responsible for
development of thick layered biofilm.
QS upregulates virulence gene expression
QS upregulates spore-forming genes in
Bacillus subtilis
It is ability to take up exogenous DNA
QS Increase competence in Bacillus subtilis
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Quorum sensing molecules
Three types of molecules :
1: Acyl-homoserine lactones (AHLs)
2: Autoinducer peptides (AIPs)
3: Autoinducer-2 (AI-2)
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Acyl-homoserine lactones (AHLs)
 Mediate quorum sensing in Gram-negative bacteria.
 Mediate exclusively intracellular communication.
 These are of several types depending on their length of acyl side chain.
 Able to diffuse through membrane.
 These are synthesized by an autoinducer synthase LuxI and recognized by a
autoinducer receptor/DNA binding transcriptional activator protein LuxR.
AHL core molecule
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Acyl-homoserine lactones (AHLs) cont….
AHL mediated quorum sensing cycle
LuxI
AI
AI
LuxR
+
RNA
polymerase
Transcription
promoter
target genes
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Autoinducer peptides
 These are small peptides, regulate gene expression in Gram-positive
bacteria such as Bacillus subtilis, Staphylococcus aureuas etc.
 Recognized by membrane bound histidine kinase as receptor.
 Regulates competence and sporulating gene expressions.
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Autoinducer peptides cont…
AIPs signaling mechanism in Bacillus subtilis
In Bacillus subtilis QS is mediated by two AIPs :
1: ComX: involve in competence development
2: CSF (competence and sporulation factor): regulates spore
formation
Figure: ComX and CSF pathway in Bacillus subtilis
Christopher et al.,2005
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Autoinducer-2 (AI-2)
 Involve in interspecies communication among bacteria.
 Present in both Gram (+) and Gram (-) bacteria.
 Chemically these are furanosylborate diester.
S-ribosyl-homocysteine (SRH)
LuxS
4,5-dihydroxyl-2,3 pentanedione (DPD)
Cyclization
Autoinducer-2 (AI-2)
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Autoinducer-2 (AI-2) cont…
AI-2 controlled processes
 Induces mini cell formation
 Induces expression of stationary phase genes
 Inhibition of initiation of DNA replication
Figure: AI-2 signaling in E. coli
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Quorum sensing in bacterial pathogenesis
 QS is involved in expression of virulence genes in various bacteria,
indicating the possible role of quorum sensing as a drug target.
 Several QS system mutant bacteria show the heavily reduced pathogenicity.
 Pseudomonas aeruginosa mutant in synthesis of autoinducer molecules
shows heavy reduction in pathogenesis.
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Quorum sensing in bacterial pathogenesis cont…
Quorum sensing in P. aeruginosa
 In P. aeruginosa QS molecules are synthesized by two autoinducer
synthase; LasI and RhlI
LasI
3-O-C12
-HSL (AI)
AI
LasR
+
Transcription
RNA
polymerase
promoter
target virulence genes
AI
RhIR
+
RhlI
C4-HSL(AI)
RNA
polymerase
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Quorum sensing in P. aeruginosa cont..
 In an in-vivo study, using two strains P. aeruginosa; PAO1
(virulent), and PAOR (lasI and rhII double mutant, avirulent), it was
seen that rats infected with PAOR are much immunologically active
and number of P. aeruginosa also reduced.
POA1
POAR
Wu et al., 2001
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Quorum Sensing
AIs are sensed by two major mechanisms:
1. AI diffuses into cytosol and is bound by a cytosolic
regulatory proteins (eg. LuxR); once bound the regulator changes
conformation and either activates or represses genes (eg., Vibrio
fischeri)
2. Sensor kinase on cytoplasmic membrane senses AI and
transmits the signal to a response regulator through a
phosphorylation cascade (Two-component signal transduction
system) (eg., Vibrio harveyi)
Quorum Sensing Mechanisms
1. Autoinducer diffusion and binding to LuxR
homolog directly
Quorum sensing in bioluminescent bacteria
Bioluminescence: first quorum sensing system discovered
light
Vibrio fischeri lux operon
Luciferase
Divergent transcription
Homoserine lactone
(Autoinducer)
Provided by J. Foster
C4
RhlI
C12
LasI
http://www.apsnet.org/education/AdvancedPlantPath/
Quorum Sensing Mechanisms
2. Autoinducer sensed by sensor kinase and signal
relayed to response regulator by phosphorylation
(two-component system)
Two-component signal
transduction systems can
activate or repress gene
transcription
Two-component systems
Transmitter
Sensor kinases often exist as dimers and are often also
phosphatases
No recognition
of AI in cytoplasm
(phosp form
represses)
From B. Bassler
Low Cell Density
LuxQ
LuxN
P
LuxU
P
P
LuxO
σ5
4
sRNAs/H
fq
LuxR
No Light Production
Provided by J. Nordstrom
High Cell Density
AI-1
LuxQ
LuxN
P
LuxU
P
LuxO
LuxR
luxCDABE
Luminescence
Provided by J. Nordstrom
AI2
The End