Quinolone signaling in the cell-to-cell communication system of Pseudomonas aeruginosa

Abstract

Numerous species of bacteria use an elegant regulatory mechanism known as quorum sensing to control the expression of specific genes in a cell-density dependent manner. In Gram-negative bacteria, quorum sensing systems function through a cell-to-cell signal molecule (autoinducer) that consists of a homoserine lactone with a fatty acid side chain. Such is the case in the opportunistic human pathogen Pseudomonas aeruginosa, which contains two quorum sensing systems (las and rhl) that operate via the autoinducers, N-(3-oxododecanoyl)-L-homoserine lactone and N-butyryl-L-homoserine lactone. The study of these signal molecules has shown that they bind to and activate transcriptional activator proteins that specifically induce numerous P. aeruginosa virulence genes. We report here that P. aeruginosa produces another signal molecule, 2-heptyl-3-hydroxy-4-quinolone, which has been designated as the Pseudomonas quinolone signal. It was found that this unique cell-to-cell signal controlled the expression of lasB, which encodes for the major virulence factor, LasB elastase. We also show that the synthesis and bioactivity of Pseudomonas quinolone signal were mediated by the P. aeruginosa las and rhl quorum sensing systems, respectively. The demonstration that 2-heptyl-3-hydroxy-4-quinolone can function as an intercellular signal sheds light on the role of secondary metabolites and shows that P. aeruginosa cell-to-cell signaling is not restricted to acyl-homoserine lactones.

Figure 1

Chemical structures of relevant compounds. (A) 3-oxo-C₁₂-HSL. (B) C₄-HSL. (C) 2-heptyl-3-hydroxy-4-quinolone (PQS). (D) 2-hydroxy-3-heptyl-4-quinolone. (E) 2-heptyl-4-hydroxy-quinoline-N-oxide.

Figure 2

A novel signal induces lasB in P. aeruginosa. Strain PAO-R1 (lasR⁻) (pTS400) (A) or strain PAO-JP3 (lasR⁻, rhlR⁻) (pTS400) (B) was grown in the presence of culture supernatant extracts from the following P. aeruginosa strains: PAO1 (wild type), PAO-JP2 (lasI⁻, rhlI⁻), PAO-JP2 (pECP39), or PAO-JP2 (pUCP22). After 18 h of growth, β-gal activity was assayed and is presented in Miller units +/− SDⁿ⁻¹. (Note: A control culture with no extract added produced 8 Miller units of β-gal activity.) Data represent the means of duplicate assays performed during three separate experiments.

Figure 3

HPLC analysis of a novel signal extracted from P. aeruginosa culture medium. A culture medium extract from strain PAO-JP2 (lasI⁻, rhlI⁻) (pECP39) was separated by reverse-phase HPLC, and fractions were collected between the times indicated by each point on the graph. The extract was loaded onto the column at time 0. Fractions were assayed for our bioactive signal, and results are presented as β-gal activity in Miller units. These data are the average of duplicate β-gal assays from one experiment that was representative of multiple repeated experiments. The dashed line (----) indicates acetonitrile concentration.

Figure 4

Electron impact mass spectra of purified natural PQS (A) and synthetic PQS (B). Natural PQS was purified from strain PAO-JP2 (lasI⁻, rhlI⁻) (pECP39). Comparable peaks are labeled with their respective m/z.

Figure 5

¹H NMR spectra of purified, natural PQS (A) and synthetic PQS (B) in DMSO-d₆. ¹H NMR spectra for natural and synthetic PQS are identical and can be described as follows: 11.42 (br s, 1H), 8.08 (d, J 8.0, 1H), 8.05 (br s, 1H), 7.55–7.48 (m, 2H), 7.23–7.17 (m, 1H), 2.72 (t, J 7.5, 2H), 1.65 (quin., J 6.4, 2H), 1.35–1.18 (m, 8H), 0.82 (t, J 6.7, 3H).

Figure 6

PQS bioassay with synthetic or natural PQS. Synthetic PQS (open squares), natural PQS (closed squares), 2-hydroxy-3-heptyl-4-quinolone (circles), or 2-heptyl-4-hydroxy-quinoline-N-oxide (triangles) were added to bioassay cultures at the indicated concentrations. Data represent the means of duplicate assays performed during three separate experiments and are presented as β-gal activity in Miller units +/− SDⁿ⁻¹.