Search for inhibitors of bacterial

quorum sensing among the

microbiota of marine sponges
Marcas O Muineachain
Supervisor: Dr. Teresa Barbosa
12/3/2010
 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
 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 many
bioactive compounds.
Culturable approach PAO1 is a positive
to screening has control: its long
major limitations chain AHL
competitively binds
and inhibits the
Metagenomics receptor for 3-
approach to Haliclona simulans hydroxy-C10-HSL
screening
Chromobacterium violaceum – Violacein
biosynthesis (purple pigment) mediated
Function based by the AHL 3-hydroxy-C10-HSL. QSIs
screen requires a produces a zone of non – pigmentation
phenotypic assay surrounding QSI producer.
 Objectives of the project
General aim is to look for QSI producers among a metagenomic
library and 30 culturable sporeformers from H. simulans.
Culturable sporeformers have been characterised in detail and
PCR screening identified the gene for lactonases (aiiA) in
isolates #11 and CH8a.

1. Develop an assay to screen for QSIs using Chromobacterium

violaceum as an indicator

2. Apply the assay to the sporeformers and the metagenomic

library

3. Evaluate the effect of lactonases from sponge associated

sporeformers CH8a and #11 on QS regulated virulence
determinants of P. aeruginosa
 Developing the QSI assay

MA Unsuitable due to poor growth / pigmentation production

Screening of the H. simulans associated sporeformers
 Screening of the H. simulans associated sporeformers
#1

#3

9 potential quorum sensing inhibitor producers:

 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
 Screening of the metagenomic library
• 27,648 clones: Q plates with 6x 384 well microtitre plates
 Screening of the metagenomic library

#1002

#1001
Clone O3
 Screening of the metagenomic library

Future work:
> Repeat to confirm
> Sequence putative positive clones to find source of QSI activity
 Cloning of aiiA gene into PA01

pBBR1MCS5

aiiA aiiA

1 2
 Effect of aiiA on pyocyanin production and swarming
motility of PA01
Impact on pyocyanin production?
Impact on swarming motility? NO
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.
 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-microbe

symbiosis 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.
Acknowledgements

Dr. Teresa Barbosa

Robert Phelan

Marc McCarthy

Questions?