Marcas O Muineachan

 Quorum sensing (QS): Cell – density gene expression.

 Release, detect and respond to a signalling molecule.
 Signalling molecule in G- bacteria is an N-acylated
homoserine lactone (AHL).

 QS systems are key regulators for the expression of virulence
factors in certain pathogens – Pseudomonas aeruginosa.
 P. aeruginosa QS system is highly complex - two interdependent
LuxIR – type QS systems, LasIR and Rh1R interacting with
quinolone signal and numerous regulators and sigma factors.
 Marine sponges (Porifera) are filter feeders which harbour a
diverse range of microbes. ~50% of sponge tissue volume =
microbes.
 Sponges form symbiosis with microbes – production of many
bioactive natural compounds.
 Haliclona simulans: abundant, found throughout Irish coastal
waters, good source of new bioactive natural products.
 Some QS inhibitors (QSIs) discovered among the microbiota of
marine sponges – more to be found.
 How do QSIs work? Target AHL synthesis, target receptor,
enzymatic degradation of AHLs.
 We have a metagenomic library of organisms from Haliclona –
E.coli/pCC1phos.
 We have 30 sporeformers which have been characterised in detail.
Of these 30, PCR screening has shown that two have genes (aiiA)
for lactonases: CH8A and #11.

1. Devolop an assay to screen for QSIs using Chromobacterium

violaceum DSM30191 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.
 Chromobacterium violaceum (DSM30191) used as an indicator

 Regulates pigment production (purple) by different AHLs (3-

hydroxy-C10-HSL is dominant) which is inhibited by AHL
analogues and other antagonists.

 Positive result indicated by a lack of pigmentation surrounding

the QSI.

 PAO1 is a “positive control”: its long chain AHL competitively

binds and inhibits the receptor for 3-hydroxy-C10-HSL

 CV026 is a mutant of a wild type C. violaceum: violacein

production is inducible by the short-chain AHLs (C4–C8), and the
long-chain AHLs (C10–C14) were detected by their ability to
inhibit violacein production
 grew CH8A, #11, #49, PAO1 separately and overlayed with
DSM30191 .
PAO1

CH8A
#11

#49
 2nd assay: multiple strains per plate overlayed with DSM30191
 3rd assay: testing #1, #2, #3, #4, CH8A, #11 and #12 and PAO1.
Four streaks per plate and controls (overlaying with DSM30191)
for 3 different media: LB, MA, and MA

#11 #11
#12 #11 #12 #12

PA01 PAO1
CH8A
CH8A
PAO1
CH8A
 Optimisation of the QSI assay.

 Clone the lactonase encoding gene aiiA from #11 and CH8A,
into pBBR1MCS5 which can be transferred to P. aeruginosa
and evaluation of the effect on the different virulence factors
(such as biofilm formation, swarming, etc).