Quorum sensing inhibitors Quorum sensing inhibitors ––a new way to control a new way to control

biofoulingbiofouling

Sergey DobretsovSergey Dobretsov

Dep. Marine Science and FisheriesDep. Marine Science and Fisheries

Sultan Qaboos UniversitySultan Qaboos University20 November 2009

Gothenburg, Sweden

sergey@squ.edu.om

Oman is a country of contrastsOman is a country of contrasts

Sultan Sultan QaboosQaboos UniversityUniversity

�� Organized in 1986Organized in 1986

�� 12,000 students12,000 students

�� 9 colleges9 colleges�� 9 colleges9 colleges

�� College of Agricultural and Marine Sciences College of Agricultural and Marine Sciences include departments:include departments:�� Marine Science & fisheriesMarine Science & fisheries

�� Animal & veterinary scienceAnimal & veterinary science

�� Crop SciencesCrop Sciences

�� Food science and nutritionFood science and nutrition

�� Natural resource economicsNatural resource economics

�� Soils, water & agricultural engineering Soils, water & agricultural engineering

Outline of the presentationOutline of the presentation

��Biofouling and biofilmsBiofouling and biofilms

��Antifouling coatings and biofilmsAntifouling coatings and biofilms

��Mechanisms of quorum sensingMechanisms of quorum sensing

����Mechanisms of quorum sensingMechanisms of quorum sensing

��Quorum sensing in marine environmentQuorum sensing in marine environment

��QS inhibitors from marine organismsQS inhibitors from marine organisms

��Summary and future directionsSummary and future directions

�Biofouling or biological fouling is the undesirable accumulation of is the undesirable accumulation of microorganisms, algae and invertebrates on submerged surfacesmicroorganisms, algae and invertebrates on submerged surfaces

10 um 1 cm

Marine Biofouling

Macrofoulers (barnacles, sponges)Microfoulers (bacteria, diatoms)

Microbial biofilms

� Three dimensional

� Highly heterogenic

� Highly dynamic� Highly dynamic

� Multi-species

� Dominated by bacteria (Gram-negative) and diatoms (Navicula, Amphora)

SEM, biofilm on a glass

CSLM, 3D biofilm structure

Biofilm formation and architectureCenter for Biofilm engineering, MSU

� Microbial aggregates with channels

� Channels used for transport (O2, nutrients, biocides)

� Architecture depend on current, species, etc.

Antifouling coatings and biofilms

� Specific species of bacteria and diatoms on antifouling paints (Casse & Swain 2006). Pseudomonas elyakovii, diatoms Pseudomonas elyakovii, diatoms - Amphora & Navicula

� Biofilms and coatings performance� Increase drag and shear stress (Mihm et al. 1988)

� Biocide release and transport (Yebra et al. 2006)

Biofilm on the surface of a paintCasse & Swain 2006

Bacteria on IntersleekMolino et al. 2009

Negative impact of microbial biofilms

� Increase corrosion (produce hydrogen sulfate, sodium chloride, depolarization)

� Contain bacterial species that induce larval settlement

Need to regulate density and composition of biofilmssettlement

� Affect performance of a/f coatings (biocide release rate, nano-coatings, low surface energy coatings)

� Increase drag force� Increase fuel consumption and decrease vessel speed (10um biofilm – 1% less fuel efficiency)

Need to regulate density and composition of biofilms

Quorum sensing is a new method to suppress microbes

Accumulate in a biofilm

Bacterial QS

Quorumsensing moleculesAHLs(acyl-homoserinelactones) R

LuxR transcription factor

1. Signal synthesized

2. Signal diffuses3. Signal binds to

receptor

Gram-negative bacterial cell

ILuxI synthase R R

transcription factor

5. Expressionof target genes

R

Attachment, toxin production, biofilm formation

4. Receptor binds to promoter

Quorum sensing in marine environment

Quorum sensing was first discovered in the marine

http://www.che.caltech.edu/groups/fha/quorum.html

discovered in the marine luminescent bacterium Vibrio fisheri – a symbiont of the squid Euprymna scolopes

QS signals are present in subtidal biofilms

Short chain AHLs

Huang et al. 2007 Microbial Ecology

Long chain AHLs

Temporal production of QS? Difference in bacterial species?

QS signals are widely present

Grains – blue, sulfate reducing bacteria – green, cyanobacteria – red;

Cyanobacterial mats produce C4-, C6-, C7-, C8-, C10-, C12-HSLConcentration of AHLs varied between day and night

cyanobacteria – red;

Bacteria associated with marine snow (Gram et al. 2002) andsponges (Taylor et al. 2004; Mohamed et al. 2008) produce AHLs

Fecho et al. 2009 Microb Ecol

Effect of QS signals on larval and spores settlement

E.coli strains with plasmids producing C6HSL and 3OH-C6HSL induce Ulvazoospore settlement

Joint et al. 2007 Science

C6HSL, 3OH-C6HSL and C10HSL induce zoospore settlement

C6HSL and C12HSL reduce larval speed and induce crawling. No effect on larval settlement

Huang et al. 2007 Microbial Ecology

Bacterial QS is important for macrofouling

Inhibition of QS

Quorumsensing moleculesAHLs(acyl-homoserinelactones) R

LuxR transcription factor

Inhibition:Inhibition:AHL signals(AHL-acylase)

Signal generation

Gram-negative bacterial cell

ILuxI synthase R R

transcription factor

Expressionof target genes

Attachment, toxin production, growth biofilm formation

R R receptor(furanones)

Larval settlement

Signal generation(triclosan)

Known QS inhibitors

Dobretsov et al. 2009 Biofouling

QS inhibitors from marine organisms

QS bioassays

ExtractsNo inhibition

Extract concentrations

After 24h incubation:

AHL Biosensor bacteria + soft agarChromobacterium violaceum CV017

No inhibition(color)

Inhibition of genesQS inhibitionGrowth inhibition

Disk diffusion bioassay

Bioassays with other strainsBiosensor bacteriacultivation

Isolation and purification

Screening of marine organisms for production of QS inhibitorsSpeciesSpecies GroupGroup MIC MIC

Concentration Concentration mg/mlmg/ml

Lyngbya sp. 1Lyngbya sp. 1 CyanobacteriaCyanobacteria 66 66

Lyngbya majusculaLyngbya majuscula CyanobacteriaCyanobacteria 9 9

Lyngbya polychroaLyngbya polychroa CyanobacteriaCyanobacteria 5050

Lyngbya sp. 2Lyngbya sp. 2 CyanobacteriaCyanobacteria 1.81.8

Laurencia filliformisLaurencia filliformis Red algaRed alga 270270

SpatoglossumSpatoglossum sp.sp. Brown algaBrown alga 210210

SarcophytonSarcophyton sp.sp. Soft coralSoft coral 55 55

AsparagopsisAsparagopsis sp.sp. Red algaRed alga 21 21 -- toxictoxic

Overall 83 extracts of 51 species were tested. Cyanobacteria was the most effective

Isolation of the QS inhibitor from Lyngbya majuscula

Separation by C18 column

Fr1 Fr2 Fr3 Fr4 Fr5 Fr6 Fr7 Fr8

30:70% MeOH

Separation by HPLC

HPLC C18 grad 60% MeOH

Fr8

HPLC YMC column iso 90% MeOH

Fr2

HPLC YMC column iso 85% MeOH

Pure active compound was effective at 141 nM

Separation by HPLC

Separation by HPLC

Identification of QS inhibitor 1H NMR

Malyngolide

Dobretsov et al. submitted

How malyngolide inhibits QS?

�� H1: Inhibits general bacterial metabolism H1: Inhibits general bacterial metabolism (tested with pTIM2442 reporter with promoterless (tested with pTIM2442 reporter with promoterless luxCDABEluxCDABE cassette)cassette)

H2: Compete for AHL binding site H2: Compete for AHL binding site �� H2: Compete for AHL binding site H2: Compete for AHL binding site (tested with (tested with

luxCDABE luxCDABE reporter pTIM505 reporter pTIM505 –– luxI regulation)luxI regulation)

�� H3: Inhibit transcription of the genes H3: Inhibit transcription of the genes (tested (tested

with Pwith PlasRlasR--luxCDABEluxCDABE pTIM84 pTIM84 –– lasR genes)lasR genes)

Malyngolide did not affect production of violacein

Malyngolide 3X dilution of 3 mg/ml

Controls

No effect of malyngolide on violacein production by non-QS bacterium

How malyngolide inhibits QS?How malyngolide inhibits QS?

�� H1: Inhibits general bacterial metabolism H1: Inhibits general bacterial metabolism (tested with pTIM2442 reporter with promoterless (tested with pTIM2442 reporter with promoterless luxCDABEluxCDABE cassette)cassette)

H2: Compete for AHL binding site H2: Compete for AHL binding site

XXXX�� H2: Compete for AHL binding site H2: Compete for AHL binding site (tested with (tested with

luxCDABE luxCDABE reporter pTIM505 reporter pTIM505 –– luxI regulation)luxI regulation)

�� H3: Inhibit transcription of the genes H3: Inhibit transcription of the genes (tested (tested

with Pwith PlasRlasR--luxCDABEluxCDABE pTIM84 pTIM84 –– lasR genes)lasR genes)

XXVV

How malyngolide works?

Quorumsensing moleculesAHLs(acyl-homoserinelactones) R

LuxR transcription factor

Inhibition:Inhibition:AHL signals(AHL-acylase)X

Signal generationX

Malyngolide

ILuxI synthase R R

transcription factor

Expressionof target genes

R

Signal generation(triclosan)X

R receptor(furanones)

X

Can we use QS inhibitors to modify multispecies biofilms and multispecies biofilms and suppress larval settlement?

Experimental design

Quorum sensing inhibitorsat 10-3-10-5 M

Biofilms after 24h Larval settlement

5-hydroxy-3[(1R)-1-hydroxypropyl]-4-

methylfuran-2(5H)-one FUR1(5R)-3,4-dihydroxy-5-[(1S)-1,2-

dihydroxyethyl]furan-2(5H)-one FUR2triclosan TRIMicrobial biofilms

after 24h

Cell suspension

Larval settlement bioassay

Bacterial count (DAPI)

Analysis of bacterial composition (T-RFLP, FISH)

Dobretsov et al. 2007 FEMS Microb Ecol

QS inhibitors decrease bacterial density

Bacterial density in multi-species biofilms

3 c

ell

mm

-2

20

25 10-3

M

10-4

M

10-5

M

Biofilm EPS under SCLM

P<0.05Dunnet test

TRI FUR1 FUR2 Control

Bacte

rial density x

103

0

5

10

15

*

*

**

Bacterial density was low in the presence of QS inhibitors at 10-3- 10-4 M

*

*

**

*

Composition of bacterial communities. T-RFLP

10-3 M

10-4 M

10-5 M

Stress = 0.13

FUR1

FUR2

TRI

Control

Both QS inhibitors and their concentration affect bacterial communities composition

Composition of bacterial communities. FISH

%D

AP

I co

unt

60

80

100Alpha

Beta

Gamma

CF

LG

The effect of QS inhibitors at 10-3 M

P<0.05

Treatment

TRI FUR1 FUR2 Control

%D

AP

I co

unt

0

20

40

60

*

*

*

*

QS inhibitors affect different bacterial groups

P<0.05Dunnet test

Modified biofilms inhibit larval settlement

Attachm

ent o

f H

.ele

gan

s %

60

80

10010

-3 M

10-4

M

10-5

M

**

* P<0.05Dunnet test

TRI FUR1 FUR2 Control

Attachm

ent o

f

0

20

40

*

*

Bacterial films formed in the presence of QS inhibitors decreased larval settlement

Dunnet test

Dobretsov et al. 2007 FEMS Microb Ecol

Summary

�QS is widely used for communication among marine bacteria

�QS inhibitors can prevent biofilm formation �QS inhibitors can prevent biofilm formation and inhibit larval settlement

�QS inhibitors can open a new way to control biofouling

Future research directions

�� Need to understand the role of biofilms on Need to understand the role of biofilms on antifouling coatings (antifouling coatings (dynamics, biocide release, QSdynamics, biocide release, QS))

Future research directions

�� Need to evaluate effectiveness, stability Need to evaluate effectiveness, stability and degradability of QS inhibitors in the and degradability of QS inhibitors in the field experimentsfield experimentsfield experimentsfield experiments

http://www.authorsden.com/adstorage/12108/ToxicEarth.gif

Future research directions

�� Search and identify universal mechanisms Search and identify universal mechanisms of QS inhibition of QS inhibition

�� target genes GacS/GacA target genes GacS/GacA –– All GammaAll Gamma--�� target genes GacS/GacA target genes GacS/GacA –– All GammaAll Gamma--Proteobacteria Proteobacteria

�� AIAI--2 system both Gram2 system both Gram--positive and Grampositive and Gram--negative negative

http://www.mentoringinmotion.info/images/MasterKey.jpg

Future research directions

�� Evaluate cost of treatment and method of Evaluate cost of treatment and method of application of QS inhibitorsapplication of QS inhibitors

http://s2.largeimagehost.com/HL/XUhQhQF/ReformMadness.JPG

Thank you!Special thanks to: Prof. Valerie Paul (SMS)Prof. Pei Yuan Qian (HKUST)Dr. Huang Yi Li (HKUST)Dr. Max Teplitski (UFL)Dr. Sarath Gunasekera (SMS)Prof. Bassam Soussi (SQU)Ms. Aisha Wahaibi (SQU)

Smithsonian Marine Station at Fort Pierce

Ms. Aisha Wahaibi (SQU)Mr. Jamal Al-Sabahi (SQU)Mr. Sultan Al-Maskari (SQU)

SQU

HKUST

George E. Burch Fellowship in Theoretical Medicine and Affiliated Sciences

Terminal-Restriction Fragment Length Polymorphism (t-RFLP)

PCR with fluorecentently labeled primer

15’ 3’3’ 5’5’ 3’3’ 5’

2

BiofilmsExtract DNA labeled primer

Restriction digestof PCR product

Fragment separationin sequencing gel

F05 20050627-101 F05-Jun-B1R1

0 100 200 300 400 500 6000

1000

2000

3000

4000

Recognition of labeled fragments

3’ 5’

3

5’ 3’

3’ 5’5’ 3’3’ 5’X

X

45

Fluorescent in situhybridization (FISH)

Fixation

1 2Oligonucleotides probes

16S or 23S rRNA

Sample

Washing

Watching

3

4

Hybridization

Epifluorescentmicroscope

16S or 23S rRNA

Composition of bacterial communities. FISH

DAPI

Alfa

DAPI

Gamma Non-EUB

DAPI

Biocides modify microbial communities

Bacteria Bacteria Bacteria

Macro- Diatoms

No biocide CuNon toxic biocidepositive

negative

Macro-fouling

Diatoms

Flagellates Flagellates Flagellates

Composition of paints change correlations between abundance of different biofouling components

Dobretsov & Railkin 1994 Russ J Mar Biol

How prevent microfouling?

� Physical methods

� Low surface energy coatings

� Micro-topography� Micro-topography

� Chemical methods

� Enzymes

No solution - cells attached to the paint will grow and reproduce