Analysis of Pseudomonas aeruginosa 4-hydroxy-2-alkylquinolines (HAQs) reveals a role for 4-hydroxy-2-heptylquinoline in cell-to-cell communication

Abstract

Bacterial communities use "quorum sensing" (QS) to coordinate their population behavior through the action of extracellular signal molecules, such as the N-acyl-l-homoserine lactones (AHLs). The versatile and ubiquitous opportunistic pathogen Pseudomonas aeruginosa is a well-studied model for AHL-mediated QS. This species also produces an intercellular signal distinct from AHLs, 3,4-dihydroxy-2-heptylquinoline (PQS), which belongs to a family of poorly characterized 4-hydroxy-2-alkylquinolines (HAQs) previously identified for their antimicrobial activity. Here we use liquid chromatography (LC)/MS, genetics, and whole-genome expression to investigate the structure, biosynthesis, regulation, and activity of HAQs. We show that the pqsA-E operon encodes enzymes that catalyze the biosynthesis of five distinct classes of HAQs, and establish the sequence of synthesis of these compounds, which include potent cytochrome inhibitors and antibiotics active against human commensal and pathogenic bacteria. We find that anthranilic acid, the product of the PhnAB synthase, is the primary precursor of HAQs and that the HAQ congener 4-hydroxy-2-heptylquinoline (HHQ) is the direct precursor of the PQS signaling molecule. Significantly, whereas phnAB and pqsA-E are positively regulated by the virulence-associated transcription factor MvfR, which is also required for the expression of several QS-regulated genes, the conversion of HHQ to PQS is instead controlled by LasR. Finally, our results reveal that HHQ is itself both released from, and taken up by, bacterial cells where it is converted into PQS, suggesting that it functions as a messenger molecule in a cell-to-cell communication pathway. HAQ signaling represents a potential target for the pharmacological intervention of P. aeruginosa-mediated infections.

Fig. 1.

LC/MS analysis of PA14 and mvfR mutant culture supernatants. Shown are MS chromatograms of PA14 (Upper) and the mvfR mutant (Lower). The numbers above the peaks represent the m/z values of the most intense ions. Intensities are normalized to the most abundant ion in Upper.

Fig. 2.

Chemical structures of five distinct series of HAQ compounds isolated from the PA14 culture supernatant. Shown is a detailed MS analysis of the peaks in Fig. 1. R, Alkyl side chain length.

Fig. 3.

Expression profiles of pqsA and phnA in PA14 vs. the mvfR mutant using the GeneChip P. aeruginosa array. •, PA14; □, the mvfR mutant. Shown are signal intensity values calculated by dchip software. OD, optical density at 600 nm of the cultures when harvested.

Fig. 4.

HAQ production kinetics in PA14 vs. the isogenic pqsE mutant. Bacteria were grown in LB at 37°C, and their extracellular HAQ concentrations were analyzed by MS at regular time intervals. Filled symbols, PA14; open symbols, pqsE mutant. ▪ and □, optical density of the culture (OD₆₀₀); • and ○, PQS; ▴ and ▵, HQNO; ♦ and ⋄, HHQ.

Fig. 5.

Proposed HAQ biosynthetic pathway in P. aeruginosa. The sequence of synthesis was determined by supplementing cultures of PA14 and various pqs/mvfR mutants with deuterium-labeled intermediates. Bracketed structures are hypothetical.

Fig. 6.

HHQ/PQS cell-to-cell communication model. 1, HHQ is synthesized and released by bacterial cells. 2, Extracellular HHQ is taken up by adjacent bacteria and converted into PQS, possibly in the periplasm. 3, PQS is released to act as a signaling molecule for other cells. 4, PQS activates target gene expression, such as the phz1 operon. Note that both HHQ availability and PqsH activity determine the final PQS concentration. In the experimental paradigm used to test the model, cell A and cell B were a lasR mutant and an mvfR mutant, respectively. In the case of a wild-type population, both the A and B cells are producing HHQ and PQS.

Fig. 7.

Pyocyanin production in a mixed-mutant culture illustrates the HAQ cell-to-cell communication pathway. Culture suspensions of the mvfR mutant, the lasR mutant, or a 1:1 mixture of both were spotted onto an LB plate and incubated for 18 h at 37°C. Only the mixed culture produces detectable amounts of pyocyanin, presumably from the lasR^– cells because mvfR^– cells are deficient for phz1 operon expression. This result demonstrates that HHQ, produced by the lasR^– cells, is released and taken up by the mvfR^– cells and converted into PQS. This PQS signal is then released by the mvfR^– cells and taken up by the lasR^– cells, where it signals phz1 expression and pyocyanin production.