Pseudomonas aeruginosaPqsA Is an Anthranilate-Coenzyme A Ligase

Abstract

Pseudomonas aeruginosa is an opportunistic human pathogen which relies on several intercellular signaling systems for optimum population density-dependent regulation of virulence genes. The Pseudomonas quinolone signal (PQS) is a 3-hydroxy-4-quinolone with a 2-alkyl substitution which is synthesized by the condensation of anthranilic acid with a 3-keto-fatty acid. The pqsABCDE operon has been identified as being necessary for PQS production, and the pqsA gene encodes a predicted protein with homology to acyl coenzyme A (acyl-CoA) ligases. In order to elucidate the first step of the 4-quinolone synthesis pathway in P. aeruginosa, we have characterized the function of the pqsA gene product. Extracts prepared from Escherichia coli expressing PqsA were shown to catalyze the formation of anthraniloyl-CoA from anthranilate, ATP, and CoA. The PqsA protein was purified as a recombinant His-tagged polypeptide, and this protein was shown to have anthranilate-CoA ligase activity. The enzyme was active on a variety of aromatic substrates, including benzoate and chloro and fluoro derivatives of anthranilate. Inhibition of PQS formation in vivo was observed for the chloro-and fluoroanthranilate derivatives, as well as for several analogs which were not PqsA enzymatic substrates. These results indicate that the PqsA protein is responsible for priming anthranilate for entry into the PQS biosynthetic pathway and that this enzyme may serve as a useful in vitro indicator for potential agents to disrupt quinolone signaling in P. aeruginosa.Pseudomonas aeruginosa is a common environmental organism and also an opportunistic human pathogen. A large number of the virulence factors which it produces during infection are regulated by low-molecular-weight molecules which are part of the cell-to-cell signaling systems of this organism. One class of signals, which are common in gram-negative bacteria, is the acyl homoserine lactone molecules. In the case of P. aeruginosa, the molecules in this class are N-(3-oxo-dodecanoyl)-L-homoserine lactone, which is the signal of the las quorum-sensing system, and N-butanoyl-homoserine lactone, which is the signal of the rhl quorum-sensing system (28,40,(43)(44)(45). The second signal class, the members of which thus far appear to be synthesized only by P. aeruginosa and certain Burkholderia species (22), is based on the 2-alkyl-4-quinolone structure. The compound 2-heptyl-3-hydroxy-4-quinolone, referred to as the Pseudomonas quinolone signal (PQS), has been shown to control the expression of multiple virulence factors in P. aeruginosa (20,37,47). PQS has been shown to act as a ligand for PqsR, the transcriptional activator which controls the pqsABCDE operon (57). The biosynthesis of PQS appears to involve the condensation of anthranilate with a 10-carbon fatty acid and requires the expression of multiple genetic operons (Fig. 1) (10,12,19,27). Based on protein similarities, the pqsABCDE operon is thought to code for the activating and condensation enzymes (except for pqsE, which is not required for bi...

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

“…Interestingly, the halogenated anthrnilate analogues were also shown to increase the osmosensitivity of a range of bacterial pathogens in a QS-independent manner (158), suggesting that these compounds may have broader-range therapeutic potential than originally anticipated. The identification of PqsA as the carboxylic acid-CoA ligase responsible for activation of anthranilate has now provided an additional means by which to screen for inhibitors of PQS production (159). Initial experiments have shown that PQS synthesis inhibitors can be either substrates or nonsubstrates of PqsA, and thus it appears that either competitive inhibition of anthranilate binding or inhibition of downstream enzymes may be a successful approach for PQS quorum quenching (159).…”

The Multiple Signaling Systems Regulating Virulence in Pseudomonas aeruginosa

“…Farnesol, a common sesquiterpene produced by many organisms, was found to inhibit PQS production in P. aeruginosa, although the high concentrations required may hamper in vivo applications (39). Synthetic anthranilate derivatives (23,36,116) have been developed and have shown promising results in a P. aeruginosa mouse infection model (116). In parallel, Fetzner and coworkers reported PQS-degrading activity by conversion of PQS to N-octanoylanthranilic acid by the dioxygenase Hod (1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase) of Arthrobacter nitroguajacolicus (162).…”

Iron Depletion Enhances Production of Antimicrobials by Pseudomonas aeruginosa

“…PqsA was previously reported to be required for lysis of Staphylococcus aureus, resulting in increased iron availability to P. aeruginosa (30). The specific mechanism for this activity remains unclear and is likely to be multifactorial, as anthraniloyl-CoA can serve as the precursor for at least 55 unique alkylquinolone (AQ) molecules (29,31). One of these, 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), is an N-oxide ubiquinone that inhibits growth of Gram-positive bacteria, including S. aureus (32)(33)(34)(35)(36).…”

“…PQS production is initiated by PqsA, a coenzyme ligase that converts anthranilate to anthraniloyl coenzyme A (anthraniloylCoA) (29). PqsA was previously reported to be required for lysis of Staphylococcus aureus, resulting in increased iron availability to P. aeruginosa (30).…”