Figure 1.
Diagrammatic representation of the AQ-dependent QS network in Pseudomonas aeruginosa.
The PqsABCD proteins synthesize HHQ, which is converted to PQS by PqsH. Autoinduction occurs when either HHQ or PQS binds to PqsR and amplifies expression of the pqsABCDE operon. The terminal output of this regulatory network is PqsE, a putative metallohydrolase protein of unknown enzymatic function which positively regulates virulence genes, secondary metabolites and biofilm development when expressed in the absence of HHQ and PQS. The PqsE regulatory pathway also downregulates pqsA and AQ biosynthesis. The conversion of HHQ to PQS confers additional functionalities since PQS unlike HHQ induces microvesicle formation and is a potent iron chelator which induces expression of the pyoverdin and pyochelin high affinity iron transport systems. AQ-dependent QS is closely linked to the AHL-dependent las and rhl QS systems. The las system positively regulates the transcription of pqsR, pqsABCDE and pqsH while rhl exerts a negative effect on the AQ system, although it is itself positively regulated by AQs. Filled arrows and blunted lines represent positive and negative regulation, respectively.
Figure 2.
Activation and inhibition of PqsR in P. aeruginosa by the 2-alkyl-4(1H)-quinolones.
*EC50 determined in a P. aeruginosa ΔpqsA CTX::pqsA'-lux strain; **EC50 determined in a P. aeruginosa ΔpqsAH CTX::pqsA'-lux strain; – no activity.
Figure 3.
Crystal Structure of the PqsRCBD.
(A) Diagrammatic representation of the linear PqsR protein showing the positions of the DNA-binding (DBD) and co-inducer-binding (CBD) domains; (B) Topology diagram of the PqsRCBD monomer consisting of two sub-domains and a hinge region; (C) PqsRCBD hydrophobic ligand-binding pocket with two bound MPD molecules shown as purple sticks; (D) Charged surface representation of the A and B pockets shown as two views related by a 180° rotation. Hydrogen bonds are shown as dotted lines and water molecules as red spheres; (E) The PqsRCBD dimer shown as a topology diagram with bound MPD molecules shown as sticks. Loops forming a lid are coloured red and blue. The Lys266 and Glu259 residues are shown as sticks with the salt bridge shown as a purple dotted line. The pocket shape is shown as a white mesh. (F) The PqsRCBD dimer shown as solid spheres with the same colour scheme.
Table 1.
Crystallographic data collection and refinement statistics.
Figure 4.
2-Alkyl-4-quinolone interactions with PqsR.
(A) Structures of 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), 2-heptyl-4(1H)-quinolone (2-heptyl-4-hydroxyquinoline, HHQ) and 2-nonyl-4(1H)-quinolone (2-nonyl-4-hydroxyquinoline, NHQ); (B) A stereo diagram of the topology of the PqsRCBD-NHQ complex showing the ligand as stick (yellow). The quinolone ring is buried within the B pocket and the alkyl chain bound to the more surface accessible A pocket; (C) Charge surface representation of the PqsRCBD showing the hydrophobic pocket occupied by NHQ. The colours represent acidic (red) and basic (blue) residues; (D) Ligplot schematic diagram showing PqsR hydrophobic contacts with NHQ (within a 3.9 Å radius); (E) Superposed PqsRCBD-NHQ (yellow) and PqsRCBD-MPD (purple) structures shown as stick.
Figure 5.
Response of PqsRCBD ligand binding site mutants to AQs.
(A) PqsR-6His is functional in P. aeruginosa. The pqsR gene with or without a 6His tag on pME6032 was introduced into a P. aeruginosa pqsR deletion mutant (ΔpqsR) containing a chromosomal miniCTX::pqsA'-lux reporter gene fusion. Relative PqsR activity was determined as a % of the maximum bioluminescence produced by the miniCTX::pqsA'-lux reporter fusion in a wild type P. aeruginosa PAO1 background carrying the empty pME6032 vector; (B) Light output from the P. aeruginosa ΔpqsR miniCTX::pqsA'-lux strain transformed with one of each of the 13 site-specific mutations introduced into the PqsRCBD ligand-binding pocket. Bioluminescence is presented as % of pqsA promoter activity with respect to PAO1 ΔpqsR miniCTX::pqsA'-lux expressing the PqsR-6His protein (WT); (C) Western blot analysis confirming expression of each of the PqsR-6His mutant proteins; (D) Light output from the P. aeruginosa ΔpqsA ΔpqsH ΔpqsR miniCTX::pqsA'-lux strain transformed with either the gene coding for PqsR-6His or one of the 13 site-specific mutants and supplemented with either HHQ or PQS (40 µM). Bioluminescence is presented as % of pqsA promoter activity with respect to the P. aeruginosa ΔpqsA ΔpqsH ΔpqsR miniCTX::pqsA'-lux strain expressing PqsR-6His.
Figure 6.
Activation and inhibition of PqsR in P. aeruginosa by the 2-alkyl-4(3H)-quinazolinones.
*EC50 determined in a P. aeruginosa ΔpqsA miniCTX::pqsA'-lux strain; **IC50 determined in a P. aeruginosa wild type strain incorporating a miniCTX::pqsA'-lux fusion; – no activity; ‡compounds exhibited growth inhibition; X compounds did not dissolve in MeOH at a workable concentration; 3-NH2-7Cl-PhOBn-QZN (46) is barely soluble.
Figure 7.
3-NH2-7Cl-C9-QZN is a competitive inhibitor of PqsR.
(A) PqsR activity as reflected by the maximum bioluminescence produced by a miniCTX::pqsA'-lux fusion in a ΔpqsA mutant in the presence of 100 µM QZN (inset) and increasing concentrations of PQS; (B) Topology diagram of the PqsRCBD ligand-binding site occupied by the QZN shown in stick (orange), with hydrogen bonds coloured purple; (C) Orientation of 4-quinolone ring (right panel) and 7Cl-substituted QZN ring (left panel) within the PqsR ligand-binding pocket. The 7Cl is accommodated within a crevice forming a hydrogen bond (dotted line) with Thr265 (left panel); (D) Superposed PqsRCBD-3-NH2-7Cl-C9-QZN and PqsRCBD-NHQ structures with residues shown as stick (orange and yellow respectively).
Figure 8.
3-NH2-7Cl-C9-QZN inhibits AQ-dependent P. aeruginosa virulence factor production and biofilm development.
(A) expression of lecA in P. aeruginosa PAO1 is inhibited by 3-NH2-7Cl-C9-QZN as reflected by a lecA'-lux chromosomal reporter fusion; (B) production of pyocyanin by P. aeruginosa PAO1 grown in the presence of 0, 50 or 100 µM 3-NH2-7Cl-C9-QZN and in a corresponding ΔpqsA mutant. The inhibition of pyocyanin production by 3-NH2-7Cl-C9-QZN is observed as an absence of green pigmentation in culture supernatants (inset; numbers correspond to columns); (C) Semi-quantitative analysis by LC-MS/MS of HHQ, NHQ, NQNO and PQS production by P. aeruginosa PAO1 grown in the absence or presence of 200 µM 3-NH2-7Cl-C9-QZN. The ΔpqsA mutant was used as a negative control; (D) Biofilm development is reduced in P. aeruginosa by a ΔpqsA mutation and following treatment of the wild type PAO1 strain with 3-NH2-7Cl-C9-QZN.