screening for quorum sensing inhibitors...marine sponges (pres 1)

8/14/2019 Screening for quorum sensing inhibitors...marine sponges (Pres 1)

1/10

Marcas O Muineachan


2/10

Quorum sensing (QS): Cell density gene expression.

Release, detect and respond to a signalling molecule.

Signalling molecule in G- bacteria is an N-acylatedhomoserine lactone (AHL).


3/10

QS systems are key regulators for the expression of virulencefactors in certain pathogens Pseudomonas aeruginosa.

P. aeruginosaQS system is highly complex - two interdependentLuxIR type QS systems, LasIR and Rh1R interacting withquinolone signal and numerous regulators and sigma factors.


4/10

Marine sponges (Porifera) are filter feeders which harbour adiverse 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 coastalwaters, good source of new bioactive natural products.

Some QS inhibitors (QSIs) discovered among the microbiota ofmarine sponges more to be found.

How do QSIs work? Target AHL synthesis, target receptor,enzymatic degradation of AHLs.


5/10

We have a metagenomic library of organisms from HaliclonaE.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 metagenomiclibrary.

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


6/10

Chromobacterium violaceum(DSM30191) used as an indicator

Regulates pigment production (purple) by different AHLs (3-hydroxy-C10-HSL is dominant) which is inhibited by AHLanalogues and other antagonists.

Positive result indicated by a lack of pigmentation surroundingthe QSI.

PAO1 is a positive control: its long chain AHL competitivelybinds and inhibits the receptor for 3-hydroxy-C10-HSL

CV026 is a mutant of a wild type C. violaceum: violaceinproduction is inducible by the short-chain AHLs (C4C8), and thelong-chain AHLs (C10C14) were detected by their ability toinhibit violacein production


7/10

grew CH8A, #11, #49, PAO1 separately and overlayed withDSM30191 .

PAO1

#11CH8A

#49


8/10

2nd assay: multiple strains per plate overlayed with DSM30191


9/10

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 #12

PAO1CH8A

#11#12

PAO1

CH8A

#11#12

PA01

CH8A


10/10

Optimisation of the QSI assay.

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

screening for quorum sensing inhibitors...marine sponges (pres 1)

Documents