News & Events
Inactivation, Inhibition and Dysregulation of Alkylquinolone-Signalling in Pseudomonas aeruginosa
Speaker(s): Dr Eliza Soh (SCELSE)
When: 30 July 2015 (9:30)
Where: SBS Classroom 4 (Level 1)
Type: Seminars

Abstract:

Pseudomonas aeruginosa is an opportunistic pathogen that causes severe acute and chronic infections due to the production of an extensive arsenal of virulence determinants which are regulated by quorum sensing (QS). Two signaling systems that are driven by either N-acylhomoserine lactone (AHL) or 2-alkyl-4-quinolone (AQ) molecules form an intricate QS regulatory network. The central AQs in P. aeruginosa are 2-heptyl-3-hydroxy-4(1H)-quinolone, termed the “Pseudomonas quinolone signal” (PQS) and its precursor 2-heptyl-4(1H)-quinolone (HHQ). Biosynthesis of PQS and HHQ rely on PqsR-dependent transcription of the pqsABCDE operon. AQ signalling plays a pivotal role in biofilm development and in controlling the expression of a battery of virulence factors, thus making it a promising target for antimicrobial drugs. With a primary focus on the AQ system, three inter-related topics were investigated; (i) degradation of PQS signal, (ii) development of a new bioassay for activators and inhibitors of AQ signaling and (iii) the association between extracellular DNA in biofilms and AQ signaling. 

To explore topic (i), a chemically defined medium with PQS as the sole carbon source was developed and used in the successful enrichment of a bacterial consortium capable of inactivating PQS. From this consortium, A. xyloxosidans Q19 was shown to inactivate PQS axenically with concomitant formation of an inactivated form of PQS (I-PQS) which does not induce the PAO1 ΔpqsA::(pMiniCTX::PpqsA-lux) bioreporter. Structural elucidation by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy revealed I-PQS to be 2-heptyl-2-hydroxy-1,2-dihydroquinoline-3,4-dione (HHQD). 

(ii) A novel bioreporter (termed PQSIS) for the identification of activators and inhibitors of the AQ system, which uses a novel cell death counter-selection method was developed as follows - Firstly, an AQ-negative P. aeruginosa PAO1 strain was constructed by incorporating a ΔpqsAΔpyrF double knockout and an IPTG-inducible pqsR gene, into which a plasmid with the pqsA promoter fused to pyrF was introduced. The pyrF gene, encoding orotidine-5’-phosphate decarboxylase is required for uracil prototrophy and mediates fluoroorotic acid (FOA) sensitivity. Thus, in the presence of PQS (or any agonist) and FOA, the pqsA’-pyrF fusion is induced resulting in growth inhibition, while an inhibitor of PqsR will block pyrF expression and therefore ‘rescue’ the bioreporter cells. PQSIS responds sensitively to PQS, HHQ and was also successfully used to evaluate different chemical classes of AQ agonists and antagonists. 

(iii) Extracellular DNA is a major constituent of the extracellular matrix in P. aeruginosa biofilms and its release is regulated by PQS signaling.  By screening a PAO1 transposon insertion library to identify additional factors required for DNA release, mutants with insertions in tatA were identified as exhibiting reduced DNA release and defective biofilm architecture. The Tat system encodes components of the twin-arginine translocation pathway and inactivation of this secretion system exerted a profound effect on both AQ and AHL-dependent QS in P. aeruginosa and consequently upon virulence gene expression. Given the pleiotropic impact of Tat mutations on both biofilms and virulence, this work provides new insights into the unexpected association between the Tat system and QS in P. aeruginosa.