QUORUM SENSINGHow Bacteria Talk to Each Other ?

1

Bacteria exhibit complex cooperative behaviours, such as conjugalplasmid transfer, biofilm maturation and virulence.

Many of these behaviours are regulated by a process known asquorum sensing.

Each individual bacterium is capable of producing a signalingmolecule (inducer) and each bacterium also has a receptor for theinducer.

When the inducer binds to the receptor, it activates thetranscription of certain genes, including those responsible for thesynthesis of the inducer itself.

Imagine that only a few bacteria of the same kind are nearby….. Diffusion reduces the concentration of the inducer in the

surrounding medium to a negligible amount, so each bacteriumproduces a very small amount of the inducer. 2

3

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

Time4

When concentration of these signaling molecules exceed aparticular threshold value, these molecules are internalized in thecell and activate particular set of genes in all bacterial population,such as genes responsible for virulence, competence, stationaryphase etc .

Quorum sensing thus enables bacteria to co-ordinate and respondquickly to environmental changes, such as the availability ofnutrients, other microbes or toxins in their environment.

5

6

QUORUM SENSING MOLECULES

Three types of molecules :

1: Acyl-homoserine lactones (AHLs)

2: Autoinducer peptides (AIPs)

3: Autoinducer-2 (AI-2)

7

SIGNAL MOLECULES INVOLVED IN QUORUM SENSING

8

ACYL-HOMOSERINE LACTONES (AHLS) AHL MEDIATED QUORUM SENSING CYCLE

AI

LuxI

+

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 aureus etc.

Recognized by membrane bound histidine kinase as receptor.

Regulates competence and sporulating gene expressions.

10

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

11

Gram negative bacteriaQuorum sensing was originally discovered in the luminescentbacterium Vibrio fischeri.These bacteria exist as free-living cells or as symbionts in thelight-producing organ of an animal host, such as the Hawaiianbobtail squid.The host provides a nutrient-rich environment for thebacterium and the bacterium provides light for the host.It was observed that liquid cultures of V. fischeri producedlight only when large numbers of Bacteria were present.The initial explanation for this was that the culture mediumcontained an inhibitor of luminescence, which was removedwhen large numbers of bacteria were present.

12

When a V. fischeri cell is alone, the autoinducer (3-oxo-C6-HSL, an AHL) is at a low concentration.At high cell concentrations, the level of the autoinducerbecomes sufficient to induce transcription of the genes thatproduce the enzyme luciferase, leading to bioluminescence.On reflection, this system is clearly a sensible one. Asingle cellis not capable of producing enough luciferase to cause visibleluminescence.Using quorum sensing, the cell can save its effort for the timewhen sufficient similar cells are around, so that their combinedaction produces a visible glow.The bacteria thus behave differently in the free-living andsymbiotic states.

13

The pathogen Pseudomonas aeruginosa uses quorum sensingto coordinate behaviours such as biofilm formation, swarmingmotility, and aggregation.These bacteria grow inside a host organism without harmingit, until they reach a threshold concentration.Then, having detected that their number is sufficient toovercome the host’s immune system, they become aggressiveand form a biofilm, causing disease.This pathogen uses AHL-mediated quorum sensing to regulatethe production of many factors needed for virulence.

14

15

16

Gram-positive bacteriaThey communicate using modified oligopeptides as signals and“two component”- type membrane-bound sensor histidine kinasesas receptors.Signaling is mediated by a phosphorylation cascade thatinfluences the activity of a DNA-binding transcriptional regulatoryprotein termed a response regulator.Each Gram-positive bacterium uses a signal different

from that used by other bacteria and the cognate receptors

are exquisitely sensitive to the signals’ structures.

Peptide signals are not diffusible across the membrane,

hence signal release is mediated by dedicated

oligopeptide exporters.

it is known that most peptide quorum-sensing signals are

cleaved from larger precursor peptides, which then are

modified to contain lactone and thiolactone rings,lanthionines, and isoprenyl groups 17

S. aureus uses a biphasic strategy to cause disease:At low cell density, the bacteria express protein factors thatpromote attachment and colonization,whereas at high cell density, the bacteria repress these traitsand initiate secretion of toxins and proteases that are presumablyrequired for disseminationThe system consists of an autoinducing peptide of

Staphylococcus aureus (AIP) encoded by agrD and a

two-component sensor kinase-response regulator pair,

AgrC and AgrA, respectively.

Activated AgrA induces expression of the agrBDCA.

results in increased AIP levels, which ensures that the

entire population switches from the low-cell-density to thehigh-cell-density

18

19

20

21

INHIBITION OF QUORUM SENSING

Inhibition of quorum sensing has been proved to be very potent methodfor bacterial virulence inhibition.

Several QS inhibitors molecules has been discovered.

QS inhibitors have been synthesized and have been isolated from severalnatural extracts such as garlic extract.

QS inhibitors have shown to be potent virulence inhibitor both in in-vitroand in-vivo, using infection animal models.

22

QUORUM QUENCHINGThe ability to disrupt quorum sensing may give one

bacterial species an advantage over another thatrelies on quorum sensing.

Likewise, a host’s ability to interfere with bacterialcell-cell communication may be crucial inpreventing colonization by pathogenic bacteria thatuse quorum sensing to coordinate virulence.

Thus, mechanisms that have evolved to interferewith bacterial cell-cell communication in processestermed quorum quenching.

23

Biotechnological Applications of

Quorum Quenching

Naturally occurring quorum-quenching processes are being tested as

novel antimicrobial therapies. Over expression of aiiA in tobacco and

potato plants confers resistance to E. carotovora, which requires AHL-

controlled virulence factor expression to cause disease.

Likewise, co culture of Bacillus thuringiensis decreased

E. carotovora–mediated plant disease in an aii A-dependent manner.

Mice treated with synthetic antagonists of S. aureus AIP show

resistance to infection.

Similarly, purified halogenated furanones appear to attenuate

virulence of bacteria in mouse models.

These and other examples predict that inhibition of quorum sensing

which offers an attractive alternative to traditional antibiotics because

these strategies are not bactericidal and the occurrence of bacterial

resistance therefore could be reduced.

Likewise, approaches aimed at promoting beneficial quorum sensing

associations may enhance industrial scale production of natural or

engineered bacterial products.24

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

25

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

X26

QUESTIONS ?

27