Quorum Sensing as a Potential Antimicrobial Target

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Quorum Sensing as a Potential Antimicrobial
Target
iGEM 2007 International Genetically Engineered
Machine Competition

•  
•  
•  
•  
• By
• Navneet Rai
• Research Scholar
•  
•  School of Biosciences and Bioengineering
• Indian Institute of Technology, Bombay
• Powai, Mumbai 400 076
•  

National Centre for Biological Sciences,
Bangalore, India
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Organization

• 1 Introduction
• 2 Quorum sensing controlled processes
• 3 Quorum sensing molecules
• 4 Quorum sensing in bacterial pathogenesis
• 5 Inhibition of quorum sensing
• 5.1 Strategies for quorum sensing inhibition
• 6 Conclusion and future perspectives

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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 R protein I
protein
AHL diffuse in
Cell density
R gene I gene R protein I
protein

AHL diffuse out
AHL diffuse out
Time
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Quorum sensing controlled processes

• It occurs in various marine bacteria
• such as Vibrio harveyi and Vibrio fischeri.
• Takes place at high cell density.

• Bioluminescence
• Biofilm formation
• Virulence gene expression
• Sporulation
• Competence

• 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

• 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
Christopher et al.,2005
Figure ComX and CSF pathway in Bacillus subtilis
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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) contAI-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 contQuorum 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

RNA polymerase
C4-HSL(AI)
RhlI

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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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Inhibition of quorum sensing

• Inhibition of quorum sensing has been proved to
  be very potent method
• for bacterial virulence inhibition.
• Several QS inhibitors molecules has been
  discovered.
• QS inhibitors have been synthesized and have
  been isolated from several
• natural extracts such as garlic extract.
• QS inhibitors have shown to be potent virulence
  inhibitor both in in-vitro
• and in-vivo,using infection animal models.

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What is the need for Quorum sensing inhibitors ?
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Antibiotic resistance

• Now a days most of bacteria are antibiotic
  resistant
• Penicillin resistant bacteria developed in 1942,
  just after 2 years of its introduction

Antibiotic
Antibiotic sensitive bacteria
Antibiotic
Antibiotic resistant bacteria
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Strategies for quorum sensing inhibition
3 strategies can be applied
Targeting AHL signal dissemination
Targeting the signal receptor
Targeting signal generation
Signal precursor
Signal precursor
Signal precursor
X
Signal
Signal
Signal
X
X
Signal receptor
Signal receptor
Signal receptor
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Targeting signal generation

• Signal generation can be inhibited by using
  analogue of precursor of
• signal molecule.
• AHL signals are generated from precursors acyl
  ACP and SAM.
• Analogues of acyl-ACP and SAM can be used to
  reduce synthesis of
• quorum sensing signals.
• Several analogues of SAM are S-
  adenosylhomocysteine, S-
• adenosylcysteine, sinefungin and butyryl-SAM.

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Effect of substrate analogues on RhlI activity in
P. aeruginosa

• In P. aeruginosa RhlI acts as autoinducer
  synthase

Inhibitors
Inhibition,
Parsek et al., 1999
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Targeting AHL signal dissemination

• QS molecules can be degraded by
• Increasing pH (gt7) as at higher pH AHL
  molecules undergo lactonolysis
• in which its biological activity is lost.
• At higher temperature AHL undergoes
  lactonolysis.
• Some plants infected by pathogenic bacteria E.
  carotovora, increase the
• pH at the site of infection, resulting in
  lactonolysis of AHL molecules.
• Some bacteria produces lactonolysing enzymes,
  such as AiiA.
• Eg Bacillus cereus, B. thuriengiensis.

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AiiA as antipathogenic agent
Potato Tobacco
Tobacco lines expressing AiiA
Corresponding Wild- type Tobacco sps.
Potato lines expressing AiiA
Corresponding Wild- type Tobacco sps.

• Transgenic plants have lesser maceration areas
  than corresponding
• wild types.

(Dong et al., 2001)
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Targeting the signal receptor

• Targeting QS signal receptor by the QS
  antagonists is highly
• investigated and promising strategy.
• Several AHL analogues have been synthesized
  which binds with
• receptor/DNA transactivator, LuxR, but this
  complex is not activated,
• which can not activate virulence genes
  expression.
• Some analogues have been synthesized by
  substitutions in HSL ring or
• in acyl side chain and in some analogues HSL
  ring has been replaced by
• alternative rings.

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Targeting the signal receptor cont.

• Rasmussen et al. (2005), screened several QSIs
  among natural and synthetic compound libraries.
• The two most active were garlic extract and
  4-nitro-pyridine-N-oxide (4-NPO).
• Microarrays analysis revealed that garlic
  extract and 4-NPO reduced QS-controlled virulence
  genes in Pseudomonas aeruginosa.
• These two QSIs also significantly reduced P.
  aeruginosa biofilm tolerance to tobramycin
  treatment as well as virulence in a
  Caenorhabditis elegans pathogenesis model.

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Conclusions and future perspectives

• Q S inhibitors have provided evidence of
  alternative method for fighting
• bacterial infections.
• QS inhibitors can be isolated from the huge
  natural pool of chemicals.
• Most compounds are unsuitable for human use.
• We are lacking in selection of human compatible
  QS inhibitors.
• Further research in this area and isolation of
  proper QS inhibitors, may
• replace the antibiotics.

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Thank You