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4-Quinolone signalling in Pseudomonas aeruginosa: Old molecules, new perspectives

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Abstract

In Pseudomonas aeruginosa, diverse virulence determinants and secondary metabolites are regulated via the action of a hierarchical quorum-sensing system which integrates two chemically distinct classes of signal molecules, the N-acylhomoserine lactones (AHLs) and the 4-quinolones (4Qs). Synthesis of the pseudomonas quinolone signal, 2-heptyl-3-hydroxy-4-quinolone (PQS) depends on the pqsABCDE locus which is responsible for generating multiple 4Qs including 2-heptyl-4-quinolone (HHQ), the immediate PQS precursor. Exported HHQ is taken up by adjacent bacterial cells and converted into PQS by PqsH, a putative mono-oxygenase. In addition, PQS regulates its own production by driving the expression of pqsABCDE through a direct interaction with PqsR (MvfR). PQS regulates diverse target genes including those coding for elastase, rhamnolipid, the PA-IL lectin and pyocyanin via the action of PqsE as well as influencing biofilm development and impacting on cellular fitness. Furthermore, 4Q signalling is not restricted to P. aeruginosa raising the possibility of cross-talk with other related bacterial species which occupy similar ecological niches.

Introduction

In Pseudomonas aeruginosa, many virulence determinants and secondary metabolites are regulated in a cell population density-dependent manner via cell-to-cell communication or “quorum sensing” (QS) (Swift et al., 2001). P. aeruginosa possesses two N-acylhomoserine lactone (AHL)-dependent QS systems. These are termed the las and rhl systems, consisting of the LuxRI homologues, LasRI (Gambello and Iglewski, 1991; Passador et al., 1993) and RhlRI (Latifi et al., 1995), respectively. LasI directs the synthesis of primarily N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL) and together with the transcriptional regulator LasR regulates the production of virulence factors including elastase, the LasA protease, alkaline protease, and exotoxin A (Gambello and Iglewski, 1991; Toder et al., 1991). RhlI directs the synthesis of N-butanoyl-l-homoserine lactone (C4-HSL) (Winson et al., 1995) which activates RhlR, and in turn RhlR/C4-HSL induces the production of rhamnolipid, elastase, LasA protease, hydrogen cyanide, pyocyanin, siderophores, and the cytotoxic lectins PA-I and PA-II (Brint and Ohman, 1995; Diggle et al., 2002; Latifi et al., 1995, Latifi et al., 1996; Winson et al., 1995; Winzer et al., 2000). The las and the rhl systems are organised hierarchically such that the las system exerts transcriptional control over both rhlR and rhlI (Latifi et al., 1996).

Pesci et al. (1999) discovered the presence of a chemically distinct class of QS signal molecule present in spent P. aeruginosa culture supernatants, which belongs to the 4-quinolone (4Q) family. Previously it had been demonstrated that the lasB gene was not expressed in a lasR mutant even when supplied with exogenous 3-oxo-C12-HSL (Pearson et al., 1995). However, spent culture supernatant from the P. aeruginosa PAO1 wild type activated a lasB-lacZ fusion in a LasR null mutant (Pesci et al., 1999). The molecule responsible was purified, chemically characterised as 2-heptyl-3-hydroxy-4-quinolone and termed the Pseudomonas quinolone signal (PQS). PQS belongs to a family of compounds called the 2-alkyl-4-quinolones (4Qs) (Déziel et al., 2004; Lépine et al., 2004) which were chemically characterised more than 60 years ago although their history dates back to 1889 when Bouchard observed that injecting small quantities of P. aeruginosa (Bacillus pyocyaneus) cultures prevented the development of anthrax in rabbits (Bouchard, 1889). It was later demonstrated that sterile P. aeruginosa cultures had the same effect suggesting the culture medium itself contained antibacterial properties. The compounds responsible for this activity were first purified in 1945 and identified as a series of four structurally related “Pyo” compounds termed pyoI-IV (Hays et al., 1945). These compounds were reported to be biologically active primarily towards Gram-positive bacteria (Hays et al., 1945). PQS itself was first chemically characterised by Takeda (1959).

Section snippets

Biosynthesis of 4Qs in P. aeruginosa

“Pyo” compound synthesis was originally shown to be dependent on the condensation of anthranilate and a β-keto-fatty acid (Cornforth and James, 1956) and so it seemed likely that anthranilate is involved in the biosynthesis of PQS. Anthranilate is an intermediate in the tryptophan biosynthetic pathway, and recently it was demonstrated that 14C-ring-labelled anthranilate was modified and converted to PQS (Calfee et al., 2001). Furthermore, addition of increasing concentrations of the

Regulation of 4Q biosynthesis

The role of PqsR (PA1003) in the regulation of 4Qs and PQS production has been intensively investigated. PqsR, originally termed MvfR, was first identified as a membrane-associated LysR-type transcriptional activator which regulated the production of elastase, phospholipase, 3-oxo-C12-HSL, PQS and controlled the expression of the phnAB genes (Cao et al., 2001). It was established that PqsR is membrane associated and acts as a transcriptional activator until the cells reach stationary phase when

Quorum sensing and 4Q signalling

The discovery that PQS regulates the expression of lasB and that the synthesis and bioactivity of PQS are mediated by both the las and rhl QS systems, respectively, suggests that 4Q and AHL-dependent QS must be interlinked (Pesci et al., 1999). Since the addition of exogenous PQS causes a major induction of a rhlI′-lacZ fusion but lesser effects on lasR′-lacZ and rhlR′-lacZ fusions suggests that PQS regulates QS in P. aeruginosa at the level of the rhl system (McKnight et al., 2000).

PQS is produced during infection and is required for P. aeruginosa virulence

P. aeruginosa is a major cause of morbidity and mortality in cystic fibrosis (CF) patients (Govan and Deretic, 1996). PQS has been detected in sputum, bronchoalveolar lavage fluid and mucopurulent fluid from P. aeruginosa-infected CF patients (Collier et al., 2002). The levels of PQS estimated to be present correlated with the population density of P. aeruginosa in the sample (Collier et al., 2002). Adaptation to the CF airway occurs during the first 3 years of life and interestingly, P.

PQS solubility, export and the fitness of P. aeruginosa

Although PQS is a hydrophobic molecule with low water solubility and a high affinity for the lipid-rich membranes of bacterial cells (Lépine et al., 2003), its presence was first noted in P. aeruginosa culture supernatants (Pesci et al., 1999) suggesting that it is exported by the bacterial cell. PQS can be solublised by rhamnolipid biosurfactants which increase the solubility of molecules such as PQS which incorporate long chain hydrocarbons (Calfee et al., 2005). As rhamnolipid production is

4Q signalling in other bacteria

Many QS signal molecules are derived from key cellular metabolites such as S-adenosyl methionine (SAM) which is required for both AHL and autoinducer-2 (AI-2) synthesis while fatty acids contribute to both AHL and 4Q production. The 4Qs also depend on aromatic amino acid biosynthesis via tryptophan metabolism. The production of PQS by P. aeruginosa from the common tryptophan biosynthetic pathway suggests that other bacterial species may be capable of making similar molecules. Previously, Lépine

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