The end of an old hypothesis: the pseudomonas signaling molecules 4-hydroxy-2-alkylquinolines derive from fatty acids, not 3-ketofatty acids

Abstract

Groups of pathogenic bacteria use diffusible signals to regulate their virulence in a concerted manner. Pseudomonas aeruginosa uses 4-hydroxy-2-alkylquinolines (HAQs), including 4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS), as unique signals. We demonstrate that octanoic acid is directly incorporated into HHQ. This finding rules out the long-standing hypothesis that 3-ketofatty acids are the precursors of HAQs. We found that HAQ biosynthesis, which requires the PqsABCD enzymes, proceeds by a two-step pathway: (1) PqsD mediates the synthesis of 2-aminobenzoylacetate (2-ABA) from anthraniloyl-coenzyme A (CoA) and malonyl-CoA, then (2) the decarboxylating coupling of 2-ABA to an octanoate group linked to PqsC produces HHQ, the direct precursor of PQS. PqsB is tightly associated with PqsC and required for the second step. This finding uncovers promising targets for the development of specific antivirulence drugs to combat this opportunistic pathogen.

Figure 1. Proposed pathway for the biosynthesis of 2-ABA, 2-AA, HHQ and HQNO

The * and + symbols refer to the source of the carbon as originating from the carbon 1 and 2 of acetate, respectively. Pathway in the box corresponds to the biosynthesis of DHQ as published by Zhang et al. (Zhang et al., 2008). AA, anthranilic acid; 2-ABA, 2-aminobenzoylacetate; 2-AA, 2-aminoacetophenone; HHQ, 4-hydroxy-2-heptylquinoline; HQNO, 4-hydroxy-2-heptylquinoline N-oxide; DHQ, 2,4-dihydroxyquinoline; PQS (Pseudomonas Quinolone Signal), 3,4-dihydroxy-2-heptylquinoline.

Figure 2. Positive electrospray mass spectra of HHQ obtained after feeding P. aeruginosa with isotope-labelled precursors

Addition of (a) 1 mM decanoic-9,9,10,10,10-d₅ acid, or b) 1 mM decanoic-1,2-¹³C₂ acid to a P. aeruginosa PA14 culture in TSB. See also Fig. S1, where we are showing that feeding dodecanoic acid is similarly increasing HAQ production.

Figure 3. Supernatant cross-feeding experiments

Total HAQ production in the WT and in the supernatant (S) of nonpolar mutants of genes from pqsABCDE operon involved in HAQ synthesis. In a second step (B) the supernatants of each mutant were added to cultures of all the other nonpolar mutants. As shown in Fig. S2, the active intermediate present in the culture supernatant of a nonpolar pqsB- or pqsC- mutant was ultimately identified as 2-ABA.

Figure 4. Positive electrospray spectra of HHQ produced by a double pqsA- pqsH- mutant fed with various culture supernatants providing 2-ABA

When a double pqsB⁻pqsL⁻ mutant was cultivated in M9 mineral medium with (a) acetate labelled at position 1 or (b) at position 2 with ¹³C as carbon source and the supernatant added to a nonpolar pqsA- pqsH- mutant culture in TSB. (c) pqsB⁻ pqsL⁻ mutant cultivated in M9 medium with unlabelled acetate and the supernatant fed to a double pqsA- pqsH- mutant mutant grown in fully ¹³C acetate, (d) supernatant of a pqsC- mutant cultivated in TSB and fed to a pqsA- pqsH- mutant grown in TSB and supplemented with fully ¹³C-labelled octanoic acid.

Figure 5. Malonyl-CoA and octanoyl-CoA are two precursors of HHQ

(a) Dose-response relationship between added malonyl-CoA and HHQ-d₄ production of a cytoplasmic extract of PA14 fed anthranilic acid-d₄, CoA, and octanoyl-CoA. (b) Dose-response relationship between added octanoyl-CoA and in vitro HHQ production when copurified His-tagged PqsC and PqsB are provided with 2-ABA. Data are represented as mean +/− SD.

Figure 6. Polyacrylamide gels showing purified PqsB and PqsC

(a) Native gel of copurified His-tagged PqsC and PqsB showing comigration (lane 1). (b) Denaturing gel showing purified PqsB and PqsC proteins (lane 1) and reanalysis of the complex from the native gel (a) after that the band was excised, showing two bands corresponding to PqsC and PqsB (lane 2). M= molecular weight ladder.