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Fundamentals and Applications of BiofilmsBacterial Biofilm Control: Cell-to-Cell CommunicationChing-Tsan Huang (黃慶璨)Office: Agronomy Building, Room 111Tel: (02) 33664454E-mail: cthuang@ntu.edu.tw

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The Discovery of Cell-Cell CommunicationStage 1 (1970s): Discovery and ecological explanation

Quorum sensingLuminous bacteria (e.g. Vibrio fischeri ) contain high levels of luciferase only when cultures reach the late-log phase of growth.

AutoinductionMicroorganisms producing pheromones and communicating with one another, was proposed.

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The Discovery of Cell-Cell CommunicationStage 2 (1980s): Proof of the Autoinduction

Physiological, chemical and genetic evidences were foundThe structure of the autoinducer singal, N-3-(oxohexanoyl) homoserine lactone was solved. Genes required to enable autoinduction of luminescence were identified.

luxR and luxI model was provedluxR: encodes an autoinducer-responsive transcriptional activator. luxI: encodes a protein required for autoinducer synthesis.

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Stage 3 (1990s): Discovery of luxR-luxI-type systems in other bacteria.

The Discovery of Cell-Cell Communication

Bacterium Phenotype Molecule

Photobacterium fischeri Bioluminescence OHHL

Vibrio harveyi Bioluminescence HBHL

Erwinia carotovoraMultiple exoenzymesvirulence, antibiotic

OHHL

Pseudomonas aeruginosaMultiple exoenzymes, virulence OdDHLVirulence, secondary metabolites BHL & HHL

Agrobacterium tumefaciens Ti plasmid conjugal transfer OOHL

Streptomyces spp. Antibiotic, differentiation -butyrolactones

& more

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Quorum Sensing Signal MoleculesAcylated homoserine lactones

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Mechanism of Autoinduction

+

More detailed

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I Gene and R Gene LuxI-type proteins recruit substrates from general

metabolismAt least three LuxI-type proteins (LuxI, RhlI, TraI), the substrates are S-adenosylmethionine (AdoMet) and the appropriate fatty acid substrate conjugated to acyl carrier protein (ACP). (more)

Substrate interactions of acyl-HSL synthetasesThe active site of an acyl-HSL synthetase must accommodate AdoMet and acyl chain carried by ACP.

Regulation of acyl-HSL synthetaseRegulation of enzyme activity Control of acyl-HSL synthetase

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Pseudomonas aeruginosa lasI/rhlI Regulation

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Microbiology. 2008 Aug;154(Pt 8):2184-94.

PMN: polymorphonuclearleukocyte

Quorum Sensing in Acute or Chronic Infection

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Cell-to-Cell Communication & Biofilm Formation

Biofilms are perfect situation for cell-to-cell communicationHigh cell density may be in the region of 1010 cell per cm3

Increased physical proximity

Evidence of cell-to-cell communication within biofilms

Davies et al. (1998) Science 280: 295-298.

& more

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Bio

film

den

sity

log(

cfu/

cm2 )

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7

8

9

10

Time (h)0 12 24 36 48 60 72

Bio

film

den

sity

log

(cel

ls/c

m2 )

6

7

8

9

A

B

WT

QS mutants

Time(h)24 36 48 60 72

g p

olys

acch

arid

e /

g pr

otei

n

0.0

0.1

0.2

0.3

0.4

0.5

0.6 PAO1 PDO100 JP1 JP2

* * *WT

QS mutants

Shih and Huang (2002) J. Antimicrobiol. Chemother. 49: 309-315.

Biofilm Formation: Wild Type vs Mutants

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WT

rhlI

lasI

Cell/EPS Distribution: Wild Type vs QS MutantsEtBr Calcofluor white

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Cell-Cell Communication & Biofilm Resistance

Little was known

Cell-to-cell communication

Biofilm formation

Polysaccharide production

Biofilm resistance

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0.0

0.2

0.4

0.6

0.8

1.0

Time (min)0 30 60 90 120

Sur

vivi

ng fr

actio

n

0.0

0.2

0.4

0.6

0.8

0 30 60 90 120

PAO1 JP1

PDO100 JP2

Shih and Huang (2002) J. Antimicrobiol. Chemother. 49: 309-315.

Biofilm Resistance : Wild type vs Mutants

& others

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Appl Microbiol Biotechnol. 86(5):1267-79, 2010.

Bacterial QS systems

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Quorum Sensing in Gram Positives

Two-component signal transduction

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AIP Structure-Function

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Three canonical quorum-sensing circuits in bacteria

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Interspecies Quorum Sensing by AI-2

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Vibrio harveyi AI-1 & AI-2 Quorum Sensing

Auto-phosphorylate De-

phosphoralate

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Cell Microbiol. 11(3):363-9, 2009.

Three autoinducing strategies in Gram-negatives.

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Trends Microbiol. 18(2):73-80, 2010.

Signaling interactions between different groups of bacteria

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Biofilm ControlCell-to-Cell Communication approach

Center for Biofilm Engineering, MSU-Bozeman

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Potential targets and intervention strategies disrupting quorum-sensing (QS) in bacteria

FRAY R G Ann Bot 2002;89:245-253

AHL

AHL analog

Cyclic dipeptides

activate or repress AHL receptor

Scanning electron micrographs of biofilms

Beighton D ADR 2009;21:69-73

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Quorum Sensing

Engineered QS Systems

Biosensors

Biofouling

Production of biochemicals

Tissue Engineering

Mixed species fermentation

Pathogen diagnostics

Cancer detection

Pathogen therapeutics

Biocontrol

Surgical implants

Membrane bioreactors

Heat exchangers, tanks, pipes

Quorum Sensing on Biotechnology

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Appl Microbiol Biotechnol. 86(5):1267-79, 2010.

Engineered QS systems