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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
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