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Search for inhibitors of bacterial

quorum sensing among themicrobiota of marine sponges

Marcas O Muineachain

Supervisor: Dr. Teresa Barbosa

12/3/2010

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Quorum sensing is a target for novel therapeutics

N-acyl-homoserine lactone (AHL) mediated quorum sensing (QS) is the

most common form in Gram negative bacteria

QS has a role in the control of the expression of multiple virulence factors

in some bacteria Pseudomonas aeruginosa

Target for novel therapeutic

agents screen for QSIs

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Marine sponge microflora are a potential source of

novel bioactive compounds

Marine sponges: filter feeders, harbour diverse range of

microbes, symbiosis with microbes

production of manybioactive compounds.

Culturable approach

to screening has

major limitations

Metagenomics

approach to

screening

Function based

screen requires a

phenotypic assay

Haliclona simulans

Chromobacterium violaceum Violacein

biosynthesis (purple pigment) mediated

by the AHL 3-hydroxy-C10-HSL. QSIs

produces a zone of non pigmentation

surrounding QSI producer.

PAO1 is a positive

control: its long

chain AHLcompetitively binds

and inhibits the

receptor for 3-

hydroxy-C10-HSL

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Objectives of the project

General aim is to look for QSI producers among a metagenomiclibrary and 30 culturable sporeformers from H. simulans.

Culturable sporeformers have been characterised in detail andPCR screening identified the gene for lactonases (aiiA) inisolates #11 and CH8a.

1. Develop an assay to screen for QSIs using Chromobacteriumviolaceum as an indicator

2. Apply the assay to the sporeformers and the metagenomiclibrary

3. Evaluate the effect of lactonases from sponge associatedsporeformers CH8a and #11 on QS regulated virulencedeterminants ofP. aeruginosa

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Developing the QSI assay

MA Unsuitable due to poor growth / pigmentation production

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Screening of the H. simulans associated sporeformers

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Screening of the H. simulans associated sporeformers

B. cereus group: #11, #12, #51, #52 and CH8a

B. aquimaris / B. vietnamensis: #1 and #23

B. pumilis / B. altidunis/ B. aerophilus : #3 and #56

9 potential quorum sensing inhibitor producers:

#1

#3

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Screening of the metagenomic library

27,648 clones: Q plates with 6x 384 well microtitre plates

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Screening of the metagenomic library

#1002

#1001

Clone O3

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Screening of the metagenomic library

Future work:> Repeat to confirm

> Sequence putative positive clones to find source of QSI activity

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Cloning ofaiiA gene into PA01

1 2

aiiA aiiA

pBBR1MCS5

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Effect ofaiiA on pyocyanin production and swarming

motility of PA01

NOImpact on pyocyanin production?Impact on swarming motility?Why not?? BLAST analysis of insert

13 nt missing from start of coding region of gene. Insert was inert How did this

happen?

First 20 nt were missed from the sequencing of the PCR product obtained in the initial

screen only explanation is that there was a HindIII site here.

Analysis of other lactonase sequences shows HindIII site is common (not known before).

Repeat of experiment would have to use a different restriction site.

................

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Conclusion

Developed an assay to screen for QSI producers

Identified 9 of the 30 H. simulans associated sporeformers as

potential QSI producers

Screened metagenomic library

Identified 13 clones issues with reproducibility

Successfully cloned aiiA gene into PA01 no conclusion because the

coding sequence was incomplete

Future perspectives: Ecological significance of sponge-microbesymbiosis better understood and issues due to non culturability

being overcome by modern molecular approaches.

Screen for / Development of QSIs as novel therapeutic agents

attractive approach to treat infections.

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Acknowledgements

Dr. Teresa Barbosa

Robert Phelan

Marc McCarthy

Questions?