1

Quorum Sensing as a Potential Antimicrobial Target  

 

 

 By

Navneet RaiResearch Scholar

  School of Biosciences and Bioengineering

Indian Institute of Technology, BombayPowai, Mumbai 400 076

 

iGEM 2007 International Genetically Engineered Machine Competition

National Centre for Biological Sciences, Bangalore, India

2

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

3

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 .

4

Cell density and quorum sensing

R gene I gene

R protein I protein

AHL diffuse out

R gene I gene

R protein I protein

AHL diffuse out

+

AHL diffuse in

Cell density

Time

5

QS upregulates virulence gene expression

Quorum sensing controlled processes

Bioluminescence

Biofilm formation

Virulence gene expression

Sporulation

Competence

Virulence gene expression

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 spore-forming genes in Bacillus subtilis

It is ability to take up exogenous DNAQS Increase competence in Bacillus subtilis

6

Quorum sensing molecules

Three types of molecules :

1: Acyl-homoserine lactones (AHLs)

2: Autoinducer peptides (AIPs)

3: Autoinducer-2 (AI-2)

7

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

8

Acyl-homoserine lactones (AHLs) cont….

AHL mediated quorum sensing cycle

AILuxI

+

promoter target genes

LuxR

RNA polymerase

Transcription

AI

9

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.

10

Autoinducer peptides cont…

AIPs signaling mechanism in Bacillus subtilisIn 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.,2005Figure: ComX and CSF pathway in Bacillus subtilis

11

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)

4,5-dihydroxyl-2,3 pentanedione (DPD)

Autoinducer-2 (AI-2)

LuxS

Cyclization

12

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

13

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.

14

Quorum sensing in bacterial pathogenesis cont…

Quorum sensing in P. aeruginosa

3-O-C12-HSL (AI)

LasI

+

promoter target virulence genes

LasR

RNA polymerase

Transcription

RhlI

AI

AI

RNA polymerase

RhIR

C4-HSL(AI)

+

In P. aeruginosa QS molecules are synthesized by two autoinducer

synthase; LasI and RhlI

15

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

16

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.

17

What is the need for Quorum sensing inhibitors ?

18

Antibiotic resistance

Antibiotic

Antibiotic

Antibiotic sensitive bacteria

Antibiotic resistant bacteria

Now a days most of bacteria are antibiotic resistant

Penicillin resistant bacteria developed in 1942, just after 2 years of its introduction

19

Strategies for quorum sensing inhibition

3 strategies can be applied

Targeting AHL signal

dissemination

Targeting the signal

receptor

Targeting signal

generation

Signal precursor

Signal

Signal receptor

Signal precursor Signal precursor

Signal Signal

Signal receptor Signal receptor

X

X

X

20

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.

21

Effect of substrate analogues on RhlI activity in P. aeruginosa

Inhibitors Inhibition,%

In P. aeruginosa RhlI acts as autoinducer synthase

Parsek et al., 1999

22

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.

23

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)

24

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.

25

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.

Targeting the signal receptor cont….

26

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.

27