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

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

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 .

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

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

Quorum sensing molecules Three types of molecules : 1: Acyl-homoserine lactones (AHLs) 2: Autoinducer peptides (AIPs) 3: Autoinducer-2 (AI-2)

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

LuxI + AI Transcription Acyl-homoserine lactones (AHLs) cont…. AHL mediated quorum sensing cycle LuxI AI AI LuxR + RNA polymerase Transcription promoter target genes

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.

In Bacillus subtilis QS is mediated by two AIPs : 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

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)

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

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.

Quorum sensing in bacterial pathogenesis cont… Quorum sensing in P 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 + RNA polymerase C4-HSL(AI) RhlI

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

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.

What is the need for Quorum sensing inhibitors ?

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

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

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.

Effect of substrate analogues on RhlI activity in P. aeruginosa In P. aeruginosa RhlI acts as autoinducer synthase Inhibitors Inhibition,% Parsek et al., 1999

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.

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)

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.

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.

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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