Download Quorum Sensing as a Potential Antimicrobial Target

iGEM 2007
International Genetically Engineered Machine Competition
Quorum Sensing as a Potential Antimicrobial Target
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
1
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
2
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
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.
Virulence gene
expression
 Virulence
gene
expression
QS upregulates virulence gene expression

QS upregulates spore-forming genes in
Bacillus subtilis
Sporulation
 Competence
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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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 signal
generation
Signal precursor
Targeting AHL signal
dissemination
Targeting the signal
receptor
Signal precursor
Signal precursor
Signal
X
Signal
Signal receptor
Signal receptor
X
Signal
Signal receptor
X
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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, Sadenosylcysteine, 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 (>7): 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 Wildtype Tobacco sps.
Potato lines
expressing AiiA
Corresponding Wildtype 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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