FILE06 prokaryotic communication Iman

Prokaryotic cell

Communication

Overview of cell-to-cell communication

or quorum sensing Iman Rusmana Department of Biology Bogor Agricultural University

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,

Quorum Sensing

Tomasz (1965) – Gram-positive Streptococcus pneumoniae

produce a “competence factor” that controlled factors for uptake of DNA (natural transformation)

Nealson et al. (1970) – luminescence in the marine Gram-

negative bacterium Vibrio fischeri controlled by self-produced chemical signal termed autoinducer

Eberhard et al. (1981) identified the V. fischeri autoinducer

signal to be N-3-oxo-hexanoyl-L-homoserine lactone

Engebrecht et al. (1983) cloned the genes for the signal

generating enzyme, the signal receptor and the lux genes

Quorum Sensing

Fuqua et al. (1994) introduced the term “quorum

sensing” to describe cell-cell signaling in bacteria

• Early 1990’s – homologs of LuxI were discovered in
different bacterial species
• V. fischeri LuxI-LuxR signaling system becomes the
paradigm for bacterial cell-cell communication

Symbiosis between

Euprymna scolopes and Vibrio fischeri

Quorum Sensing PubMed Citations

250 s n o 200 ti a it C 150 f o r 100 e b m u

TABLE 1 Organisms possessing LuxI/LuxR homologues: the regulatory

a proteins, the HSL autoinducers, and the regulated functions

[Fuqua C. et al., 2002. Nature Rev./Molecular Cell Biol. 3:685-695]

LuxI/LuxR Target Genes and

Rhizobium (a) RhiI/RhiR (a) N-hexanoyl-HSL (a) rhiABC (rhizosphere

90 ) Rhodobacter CerI/CerR 7,8-cis-N- Prevents bacterial

(

(b) CinI/CinR (b) N-(3-hydroxy-7- (b) Quorum sensing cis-tetradecenoyl)- regulatory cascade HSL

18 , 51 , 133 )

phase (

leguminosarum genes) and stationary

number ( 134 )

aggregation ( 130 )

Rhizobium etli RaiI/RaiR Multiple, unconfirmed Restriction of nodule

solanacearum N-octanoyl-HSL

34 )

Unknown (

82 , 115 ) Ralstonia SolI/SolR N-hexanoyl-HSL,

therein;

22 and references

sphaeroides (tetradecanoyl)-HSL

Salmonella ?/SdiA ? rck (resistance to

(b) RhlI/RhlR (b) N-butyryl-HSL (b) lasB, rhlAB (rhamnoli-pid), rpoS (stationary phase)

Yersinia YenI/YenR N-hexanoyl-HSL,

motility (

(b) YtbI/YtbR (b) N-octanoyl-HSL regulating bacterial aggregation and

pseudotuberculosis HSL sensing cascade

Yersinia (a) YpsI/YpsR (a) N-(3-oxohexanoyl)- Hierarchical quorum

HSL

N-(3-oxohexanoyl)-

Unknown ( 157 ) enterocolitica

HSL

typhimurium competence killing), ORF on Salmonella

97 )

Unknown (

30 , 47 ) Vibrio anguillarum VanI/VanR N-(3-oxodecanoyl)-

(

liquefaciens tiation, exoprotease

1 ) Serratia SwrI/? N-butanoyl-HSL Swarmer cell differen-

virulence plasmid (

(

114 )

Organism Homologue(s) Autoinducer Identity Functions Vibrio fischeri LuxI/LuxR N-(3-oxohexanoyl)- luxICDABE (biolumin-

tumefaciens HSL

lytic enzymes (

violaceum gen cyanide, antibiotics, exoproteases and chitino-

87 ) Chromobacterium CviI/CviR N-hexanoyl-HSL Violacein pigment, hydro-

cepacia production (

Burkholderia CepI/CepR N-octanoyl-HSL Protease and siderophore

gal transfer) ( 124 , 174 )

Agrobacterium TraI/TraR N-(3-oxooctanoyl)- tra, trb (Ti plasmid conju-

Unknown ( 156 ) agglomerans

salmonicida

Aeromonas AsaI/AsaR N-butanoyl-HSL aspA (exoprotease) ( 155 )

( 154 )

hydrophila loprotease production

Aeromonas AhyI/AhyR N-butanoyl-HSL Serine protease and metal-

28 , 31 )

HSL escence) (

14 , 96 ) Enterobacter EagI/EagR N-(3-oxohexanoyl)-

HSL

references therein;

10 ) Escherichia coli ?/SdiA ? ftsQAZ (cell division),

19 , 22 and

factors), biofilm formation (

aeruginosa canoyl)-HSL (exoprotease virulence

123 , 171 ) Pseudomonas (a) LasI/LasR (a) N-(3-oxodode- (a) lasA, lasB, aprA, toxA

aereofaciens biosynthesis) (

44 , 144 , 170 ) Pseudomonas PhzI/PhzR N-hexanoyl-HSL phz (phenazine antibiotic

chromosome replication (

HSL biosynthesis, virulence (

Erwinia (a) ExpI/ExpR N-(3-oxohexanoyl)- (a) Exoenzyme synthesis,

Erwinia stewartii EsaI/EsaR N-(3-oxohexanoyl)- Capsular polysaccharide

( 103 , 132 )

chrysanthemi HSL pectinase synthesis)

4 ) Erwinia ExpI/ExpR N-(3-oxohexanoyl)- pecS (regulator of

(b) Carbapenem antibiotic synthesis (

72 , 125 )

carotovora (b) CarI/CarR HSL (

3 ) _a TABLE 1 Organisms possessing LuxI/LuxR homologues: the regulatory proteins, the HSL autoinducers, and the regulated functions a

Vast array of molecules are used as chemical

signals – enabling bacteria to talk to each other, and in many cases, to be multilingual Quorum Sensing

Gram-negative bacteria Gram-positive bacteria universal language

O N O O O N O O

O N O O O

N O O N N O O OH N O OH O O OH

1 R

3 R

2 QS signals - Autoinducers acyl homoserine lactones N-butanoyl-L-homoserine lactone (BHL) N-(3-hydroxybutanoyl)- L-homoserine lactone

(HBHL) N-(3-oxohexanoyl)- L-homoserine lactone (OHHL) diketopiperazines cyclo(L-Pro-L-Tyr) cyclo-(vAla-L- Val)

YSTCDFIM S C O ERGMT ERGMT Oligopeptides Furanones 3-Hydroxypalmitic acid methyl ester (3OH PAME) 2-Heptyl-3-hydroxy-4-quinolone (PQS) butyrolactone 4-bromo-5-(bromomethylene)-3-(1 P -hydroxybutyl)-2(5H)-furanone Others

Br H Br O O

O OH O O O R

The three general classes of quorum-sensing systems

Class Autoinducer Strain

Modified oligopeptides Processin g and secreatio n

S H K

A R A T PAD P

O O R ₁ H N O R ₂ P. aeruginisa V. fisheri

E. carotovora

A. tumefaciens Y. enterocolitica E. coli O157:H7 V. harveyi V. cholerae V. vulnificus S. Typhimurium

B. subtilis S. aureus S. pneumoniae S. epidermidis

1 G(+ ) QS

2 AI1 PAI AI2

L. lactis

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

 Bioluminescence

 Biofilm formation

 Virulence gene expression

QS upregulates virulence gene expression Quorum sensing controlled processes

 Sporulation

 Competence

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

How quorum sensing works? Signalling compounds, autoinducers

AI synthases ( luxI gene products) cell density indicators

non-essential aa, acyl homoserine lactones lactone ring part - binding to a receptor site acyl chain tail – determining the species specificity
oligopeptides
diketopiperazines
quinolone
furanones

Recognition systems

LuxR transcriptional regulator specific binding sites for AHL and DNA (sensor/transducer)

Genetic basis regulatory circuit involving both regulatory genes accumulation of AHL - activating gene transcription

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

AHL diffuse in

out

Cell density Time

A Vibrio fischeri Growth Curve Lays Out Quorum Sensing Hastings and

co-workers

25 ) m n

6 l) D

20 )

/m m l/O U n

L /m R

0.1

1 U (

66 L D

15 ce R O n ( ( ll y ce e it s /c s e n in ce e

10 n m l d lu ce el

0.01

0.1 io es C

B in m

5 lu io B

60 120 180 240 300

0.0

0.1

0.2

0.3

0.4 Time (min) Cell density (OD660nm)

In V. fisheri, bioluminsecence only occurs when V.

fischeri is at high cell density Quorum Sensing

N-3-oxo-hexanoyl-L-homoserine lactone Quorum Sensing in Pseudomonas aeruginosa

• P. aeruginosa uses a hierarchical quorum sensing

circuit to regulate expression of virulence factors and biofilm formation Quorum Sensing in Gram-Positive Bacteria

Gram-positive bacteria utilizes modified

oligopeptides as signaling molecules – secreted via an ATP-binding cassette (ABC) transporter complex

Detectors for these signals are two-component

signal transduction systems sensor kinase

binding of autoinducer leads to autophosphorylation at conserved histidine residue

response regulator

- phosphorylation at conserved

aspartate by sensor kinase leads to binding of regulator to specific target promoters

The Bacillus subtilis comP/ComA Competence/Sporulation

System

Quorum sensing control of competence and sporulation in Bacillus subtilis. B. subtilis employs two processed peptide

autoinducers, ComX (gray circles) and CSF (white diamonds), to regulate the competence and sporulation processes.

Accumulation of the processed ComX peptide enables it to interact with the ComP sensor kinase. ComP autophosphorylates on a histidine residue (H), and subsequently phosphate is transferred to an aspartate residue (D) on

the ComA response regulator. Phospho-ComA activates the transcription of comS. The ComS protein increases the

level of ComK protein (+) by inhibiting ComK proteolysis. ComK is a transcription factor that activates the expression of genes required for development of the competent state. The second peptide autoinducer, competence and sporulation factor (CSF), while accumulating extracellularly in a density-dependent manner, has an intracellular role. CSF is transported into the cell via the Opp transporter (gray protein complex).

At low internal concentrations CSF inhibits the ComA-specific phosphatase RapC . Inhibition of RapC increases the

level of phospho-ComA, which leads to competence (dashed lines).

At high internal CSF concentrations, CSF inhibits competence and promotes spore development (black lines).

Specifically, CSF inhibits ComS. CSF inhibition of ComS activity reduces transcription of competence genes, promoting sporulation instead. Additionally, CSF inhibits the RapB phosphatase . The role of RapB is to

dephosphorylate the response regulator Spo0A. Phospho-Spo0A induces sporulation. Therefore, CSF inhibition of the

Hybrid quorum sensing circuit in Vibrio harveyi

V. harveyi – marine bacterium, but unlike V. fischeri,

does not live in symbiotic associations with higher organisms, but is free-living

Similar to V. fischeri, V. harveyi uses quorum sensing

to control bioluminescence

• Unlike V. fischeri and other gram-negative bacteria, V. harveyi has evolved a quorum sensing circuit that has characteristics typical of both Gram-negative and Gram-positive systems Hybrid quorum sensing circuit in Vibrio harveyi

V. harveyi uses acyl-HSL similar to other Gram-

negatives but signal detection and relay apparatus consists of two-component proteins similar to Gram-positives

V. harveyi also responds to AI-2 that is designed

for interspecies communication AI-1

AI-2

LuxN and LuxQ – autophosphorylating kinases at low cell densities Accumulation of autoinducers – LuxN and LuxQ  phosphatases draining phosphate from LuxO via LuxU Dephosphorylated LuxO is inactive

X = transcriptional repressor

Quorum-sensing in V. harveyi: a model for a new

language?

Quorum-sensing and the regulation of

bioluminescence in V. harveyi.

A: At low cell density, in the absence of HBHL and AI-2,

LuxN and LuxQ autophosphorylate. A multistep phosphorelay continues through the shared

phosphotransfer protein, LuxU, ultimately phosphorylating

the response regulator, LuxO. Phosphorylated LuxO, in

54 conjunction with , is thought to indirectly repress transcription of the genes required for bioluminescence

by activating the transcription of an unidentified negative

regulator (repressor X).

B: At high cell density, corresponding to a critical

concentration of signal molecules, LuxN and LuxQ/P sense

their cognate signals and switch from kinases to phosphatases. Consequently, dephosphorylation of LuxO results in its inactivation thereby preventing the up- regulation of repressor X activity. Such de-repression

LuxQ LuxN

H1 D1 H1 D1

p H2 D2 HTH



54 LuxO

Repressor LuxCDABE

AI-2 Lux P

H1 D1 H1 D1 p H2 D2

HTH

LuxO LuxCDABE LuxR LuxS

LuxM AI-1 Low Cell density High Cell density QS mechanisms in V. harveyi LuxU p p LuxS and interspecies communication

• LuxS homologs found in both Gram-negative and

Gram-positive bacteria; AI-2 production detected in bacteria such as E. coli, Salmonella typhimurium, H. pylori, V. cholerae, S.aureus, B

subtilis using engineered V. harveyi biosensor

• Biosynthetic pathway, chemical intermediates in

AI-2 production, and possibly AI-2 itself, are

identical in all AI-2 producing bacteria to date –

reinforces the proposal of AI-2 as a “universal”

language

LuxS quorum sensing: more than just a

[Fuqua C. et al., 2002. Nature Rev./Molecular Cell Biol. numbers game 3:685-695] 'Bacterial esperanto' — a universal language? The initial description of Vibrio fischeri quorum sensing was paralleled by a similar description in the related

103 luminescent marine bacterium Vibrio harveyi . Before we had any mechanistic understanding of acyl-homoserine lactone (acyl-HSL) signalling, it was shown that many other marine bacteria made something that signalled V.

104 harveyi to induce its luminescence genes .

It seemed that V. harveyi might measure the total bacterial load in its local environment rather than simply its own

104 population size .

There are, in fact, two integrated quorum-controlled circuits that govern the V. harveyi lux genes, either of which

43 105 can induce luminescence independently . The signal for one is the acyl-HSL 3-OH-C4-HSL . The second quorum-

sensing system is based on a signal originally described as autoinducer-2 (AI-2), and it is this system that responds

106, 107

to interspecies bacterial signals . There is an increasing amount of evidence that bacteria other than V. harveyi

respond to AI-2-type signals and that, by analogy with V. harveyi, these microbes might also monitor the

108 abundance of other AI-2-synthesizing bacteria in their local environment .

A gene called luxS, which is conserved in a diverse range of bacteria, is responsible for the production of AI-2 by 109

Escherichia coli . LuxS is an enzyme that can synthesize a molecule derived from S-ribosylhomocysteine, an 110, 111 intermediate in methionine recycling . Despite this information and tremendous efforts, the true nature of the AI-2 signal remained elusive. Only recently have Bonnie Bassler and colleagues identified the enigmatic signal,

112

associated with its receptor protein: receptor-bound AI-2 is a furanosyl borate diester . Apparently, the sugar from

S-ribosylhomocysteine is cyclized and an atom of boron is incorporated to form the diester. Not only does this work provide at least one view of the interspecies signal, but it also suggests an unexpected role for elemental boron in the signalling pathway. Infect Immun. 2000 Jun;68(6):3193-9.

Alignment of the deduced H. pylori LuxS sequence with deduced LuxS sequences from four other bacterial species.

LuxS sequences from H. pylori 26695 (GenBank accession no. AE000532), S. aureus (preliminary sequence data

obtained from The Institute for Genomic Research website at http://www.tigr.org/), B. subtilis (accession no. Z9919),

C. perfringens (accession no. AB028629), and V. harveyi (accession no. AAD 17292) were aligned using the ClustalW

algorithm. H. pylori LuxS is most closely related to LuxS from S. aureus (67% amino acid identity; 15% similarity).

Positions of amino acid identity are indicated by asterisks.

Genes and functions controlled by LuxS in bacteria Xavier K. B. et al., 2003. Curr. Opin. Microbiol. 6:191-197.

The molecular basis of bioluminescence regulation

The regulation of bioluminescence in V. fischeri: the quorum-sensing paradigm. A: At low cell density, transcription

of the genes for bioluminescence (luxICDABEG) is weak and insufficient for light emission due to low levels of OHHL.

The LuxI family of acyl HSL synthase proteins

A putative scheme for HHL synthesis, catalysed by LuxI. SAM binds to the active site on LuxI, and

the hexanoyl group is transferred from the appropriately charged ACP. The hexanoyl group forms

an amide bond with the amino group of SAM. 5 -Methylthioadenosine is released, and a ′ lactonisation reaction results in the synthesis of HHL [78].

R O

1 O N R

2 O H

The acyl HSL molecules

N- The quorum-sensing molecules. A–H: Some of the more common microbial acyl HSLs: (A)

butanoyl-L-homoserine lactone (BHL); (B) N-(3-hydroxybutanoyl)-L-homoserine lactone (HBHL); (C)

N-hexanoyl-L-homoserine lactone (HHL); (D) N-(3-oxohexanoyl)-L-homoserine lactone (OHHL); (E)

N-octanoyl-L-homoserine lactone (OHL); (F) N-(3-oxooctanoyl)-L-homoserine lactone (OOHL); (G)

N-(3-hydroxy-7-cis-tetradecenoyl)-L-homoserine lactone (HtdeDHL); (H) N-(3-oxododecanoyl)-L- homoserine lactone (OdDHL). I,J: Two microbial diketopiperazines: (I) cyclo( -Pro-L-Tyr); (J) cyclo(ΔAla-L-Val). K: 2-Heptyl-3-hydroxy-4-quinolone (PQS). L: A furanone of Delisea pulchra, 4-

H)-furanone. M: The butyrolactone putatively bromo-5-(bromomethylene)-3-(1′-hydroxybutyl)-2(5 Xanthomonas campestris. N: 3-Hydroxypalmitic acid methyl ester (3OH PAME). produced by

Structural Insights

Structure and function of LuxI-type acyl-homoserine-lactone (acyl-HSL) synthases. Residues conserved

in all LuxI-type proteins are labelled with an asterisk. Residues whose mutation in LuxI and RhlI results in significant

loss of activity are shown in red; residues for which inactivating mutations have been isolated in LuxI only are shown in blue; residues for which an inactivating mutation has been isolated in RhlI only are shown in green. The threonine residue that is conserved in LuxI homologues that synthesize 3-oxo-acyl-HSL derivatives is shown in grey. Numbering is relative the LuxI sequence. Blue and red bars define the areas that are proposed to be involved in catalysis and specificity, respectively . [Fuqua C. et al., 2002. Nature Rev./Molecular Cell Biol. 3:685-695]

Homoserine Aspartyl Aspartate semialdehyde phosphate

Aspartate phosphate Homoserine Threonine

H H

H H H H H N C CO H H N C CO H H N C CO H H N C CO H H N C CO H H N C CO H

2 CH CH CH CH CH CH

2 CO H COPO CHO CH OH CH OPO CH

3 O

Isoleucine Lysine

(?) Methionine ATP

Pi + PPi

2 O

_ N O N O .. .. H N

N + O S H N O

2 H C

3 OH OH

Homoserine S-adenosyl methionine lactone acyl-ACP LuxI or acyl-CoA Acylation

? Methylthioadenosine

LuxI-directed biosynthesis of acylated homoserine lactone autoinducers. The LuxI family of proteins uses S-adenosylmethionine (SAM)

and specific acyl-acyl carrier proteins (acyl-ACP) as substrates for HSL autoinducer biosynthesis. The LuxI-type proteins direct the formation of an amide linkage between SAM and the acyl moiety of the acyl-ACP (denoted 1). Subsequent lactonization of the ligated intermediate with the concomitant release of methylthioadenosine occurs (denoted 2). This step results in the formation of the acylated

homoserine lactone (denoted 3). Shown in the figure is the HSL autoinducer N-(3-oxooctanoyl)-homoserine lactone, which is synthesized

[Fuqua C. et al. 2002, 3:685-695]

Model of acyl-homoserine-lactone (acyl-HSL) quorum sensing in a single generalized bacterial cell.

Tentative mechanisms for acyl-HSL synthesis and acyl-HSL interaction with LuxR-type proteins are shown. Double arrows with filled yellow

circles at the cell envelope indicate the potential two-way diffusion of acyl-HSLs into and out of the cell. The proposed dimerization of LuxR

(red) is based on genetic evidence and biochemical analysis of TraR; other LuxR-type proteins might form higher-order multimers. Binding

of the acyl-HSL to LuxR and multimerization are represented as distinct events, although they might occur simultaneously. The LuxI label

indicates LuxI-type proteins. 5'-MTA, 5'-methylthioadenosine; ACP, acyl carrier protein; SAM, S-adenosylmethionine. Modified with permission from Ref. 22 © (2001) Annual Reviews.

Stereo view of the structure of the TraR–OOHL–DNA complex. Domains in the two monomers

are shown in different colours (light/dark orange and light/dark green), whereas the DNA is

coloured blue and the OOHL is coloured red. Note that the two-fold dyad axis of the DNA and

DNA-binding domains lies in the plane of the page (horizontal red line), whereas that relating

to the pheromone-binding domains is swiveled by approximately 90° (short red line). Side

chains of residues in the upper monomer (light/dark green) that mediate interaction between

DNA-binding and pheromone-binding domains are shown in red and residues that affect transcription activation are shown in light blue. The N terminus and C terminus of the lower subunit are labelled. [Zhang R.-G. et al., 2002. Nature 417:971-974.]

1 252 N-terminus

C-terminus Modular structure of Vibrio fischeri LuxR protein

A (2-20th a.a.) region for the negative autoregulation of LuxR

B (79-127th a.a.) binding region for the acylated homoserine lactone

C (116-161st a.a.) multimerization site of 2 LuxR proteins

D (193-197th a.a.) putative transcriptional activation element

E (200-220th a.a.) helix-turn-helix DNA-binding motif

F (240-250th a.a.) region for LuxR-dependent transcription of

lux operon

C N A R C S T T G G T

V A A G

X G G A A T T C N G

X T T A R C C A A G S G R T T V. fischeri MJ1 lux box lux box-like consensus sequence

Organization of LuxR The pheromone-binding site. a, Surface around the pheromone, which is coloured by the pK

(red for acidic and blue for basic residues) of the residues of the pheromone-binding cavity. b,

Four hydrogen bonds between the pheromone and TraR. The hydrogen bond between the 3-

keto group and protein is water-mediated. The distance between interacting atoms is shown in

Å . [Zhang R.-G. et al., 2002. Nature 417:971-974.]

Vibrio fischeri lux-gene organization and symbiotic bioluminescence

[Fuqua C. et al., 2002. Nature Rev./Molecular Cell Biol. 3:685-695]

Signaling molecules and types of

[Podbielski A. et al., 2004. Int J Infect Dis. regulation 8(2):81-95.]

Examples of signaling molecules used for bacterial quorum sensing regulation. The figure shows the names and

Introduction– three steps in cell-cell- signaling

The three steps in quorum sensing regulation. (1) In the first step, the signaling molecules are produced either by

employing the intracellular machinery and subsequent outward-bound transport or by secreting a protease and

subsequent cleavage from bacterial or even adjacent host structures. The signaling molecules may stay bound to

the bacterial surface or could be secreted to the environment. (2) In the second step, the signaling molecules accumulate outside the bacteria either due to the continuous production of a growing number of bacteria, a decrease of available space even without further production of signaling molecules, or due to the vicinity of an impermeable structure in combination with a low level production of the molecules. (3) In the third step, the

signaling molecules reach a threshold level, at which it is sensed at the bacterial surface or after passive or active

passage through the cell membrane by intracellular receptors. As a consequence, specific regulators will be activated and start their quorum sensing control of gene expression. [Podbielski A. et al., 2004. Int J Infect Dis.

8(2):81-95.]

Quorum-sensing vs. central metabolism AI 2; furanosyl borate diester bioluminescence (V. harveyi) ABC transporter (S. typhimurium) type III secretion (EHEC) virulence factor, VirB (S. flexneri) protease (S. pyogenes)

in vivo fitness (N. meningitidis) Fe-acquisition (Actinobacillus sp.)

Chen et al., 2002. Nature 415: 545 - 549

The autoinducer AI-2, synthesized by LuxS, is bound by the sensor protein LuxP. a, Biosynthesis of the AI-2 precursor

9–13 4,5-dihydroxy-2,3-pentanedione (DPD) from S-adenosylmethionine . b, Induction of bioluminescence in the V.

13 harveyi bioassay was measured following the addition of the products of an in vitro reaction of S-

13 adenosylhomocysteine with Pfs and LuxS proteins , reaction buffer, or AI-2 released from LuxP overproduced in

LuxS or LuxS

E. coli BL21. Concentrations of AI-2 in the Pfs/LuxS and LuxP (BL21) reactions were estimated to be 20 µM (see Methods).

Chen et al., 2002. Nature 415: 545 - 549

Structure of LuxP-AI-2 complex. a, Overview. b–d, F - F difference electron density (contoured at 4 ) calculated o c

using phases derived from the model before AI-2 addition. The final refined model for AI-2 is shown superimposed on

this density. Boron, oxygen, nitrogen and carbon are coloured yellow, red, blue and grey, respectively. In the

28 stereoviews shown in c–d, hydrogen bonds are shown as dashed red lines. Figure prepared using O , Molscript /

Quorum-Sensing in (Eu)bacterial Systems

Bioluminescence : Vibrio fischeri, V. harveyi Symbioses : V. fischeri Biofilm architecture : Pseudomonas aeruginosa Virulence : Erwinia stewartii, P. aeruginosa Antibiotics/exoenzyme release : Chromobacterium violaceum,

Erwinia carotovora, Pseudomonas aurefaciens, Streptomyces spp

Conjugation : Agrobacterium tumefaciens Cell division : E. coli (Social gliding) Motility : Serratia liquifaciens Stationary phase-related : Rhizobium leguminosarum Lag phase-related : Nitrosomonas europea Competence : Streptomyces spp. Bacillus spp.

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 AHL signal dissemination Targeting the signal receptor Targeting signal generation

Signal precursor Signal Signal receptor Signal precursor Signal precursor

X X

Signal Signal 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

Inhibitors

Inhibition,%

 In P. aeruginosa RhlI acts as autoinducer synthase 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

Tobacco lines Potato Tobacco expressing AiiA Corresponding Wild- type Tobacco sps.

Potato lines expressing AiiA Corresponding Wild- type Tobacco sps.

(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.

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.