Quorum Quenching by an N -Acyl-Homoserine Lactone Acylase from Pseudomonas aeruginosa PAO1

Abstract

The virulence of the opportunistic human pathogen Pseudomonas aeruginosa PAO1 is controlled by an N-acyl-homoserine lactone (AHL)-dependent quorum-sensing system. During functional analysis of putative acylase genes in the P. aeruginosa PAO1 genome, the PA2385 gene was found to encode an acylase that removes the fatty acid side chain from the homoserine lactone (HSL) nucleus of AHL-dependent quorum-sensing signal molecules. Analysis showed that the posttranslational processing of the acylase and the hydrolysis reaction type are similar to those of the beta-lactam acylases, strongly suggesting that the PA2385 protein is a member of the N-terminal nucleophile hydrolase superfamily. In a bioassay, the purified acylase was shown to degrade AHLs with side chains ranging in length from 11 to 14 carbons at physiologically relevant low concentrations. The substituent at the 3 position of the side chain did not affect activity, indicating broad-range AHL quorum-quenching activity. Of the two main AHL signal molecules of P. aeruginosa PAO1, N-butanoyl-Lhomoserine lactone (C 4 -HSL) and N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C 12 -HSL), only 3-oxo-C 12 -HSL is degraded by the enzyme. Addition of the purified protein to P. aeruginosa PAO1 cultures completely inhibited accumulation of 3-oxo-C 12 -HSL and production of the signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone and reduced production of the virulence factors elastase and pyocyanin. Similar results were obtained when the PA2385 gene was overexpressed in P. aeruginosa. These results demonstrate that the protein has in situ quorum-quenching activity. The quorum-quenching AHL acylase may enable P. aeruginosa PAO1 to modulate its own quorum-sensing-dependent pathogenic potential and, moreover, offers possibilities for novel antipseudomonal therapies.Pseudomonas aeruginosa PAO1 is an opportunistic pathogen which causes disease mainly in individuals who are immunocompromised, have cystic fibrosis, or suffer from serious burn wounds. It utilizes two N-acyl-homoserine lactone (AHL)-dependent quorum-sensing systems, termed las and rhl, which together regulate an extensive set of cell population density and growth-phase-dependent virulence factors (7,22). The las and the rhl quorum-sensing systems employ N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C 12 -HSL) and N-butanoyl-L-homoserine lactone (C 4 -HSL), which function by activating the response regulator proteins LasR and RhlR, respectively. In diverse gram-negative bacteria, many different AHL signal molecules have been characterized. These all consist of a homoserine lactone (HSL) ring or nucleus which is connected via an amide bond to a fatty acid side chain of 4 to 14 carbon atoms in length that may contain an oxo or hydroxyl group at the 3Ј position and unsaturated bonds (Fig. 1).In P. aeruginosa PAO1, swarming motility, biofilm maturation, and the expression of virulence factors such as exoproteases, hemolysins, exotoxin A, exoenzyme S, pyocyanin, cyanide, and the cytotoxic lectins PA-IL and PA-IIL, as w...

Biosynthesis of the pyoverdine siderophore of Pseudomonas aeruginosa involves precursors with a myristic or a myristoleic acid chain

“…This is consistent with the observation in the present study, that a pvdQ mutant produces precursors with myristic or myristoleic acid still bound to the L-Glu residue. The physiological function of PvdQ is therefore not in the deacylation of the quorum sensing molecule 3-oxo-C12 as previously suggested [14] but in the deacylation of the PVDI precursor isolated in the present work.…”

“…This precursor has been purified and was proposed to be a substrate of PvdQ [8], providing a direct link between PvdQ and the mechanism of myristate group removal by PvdQ before Abbreviations: PVDI, pyoverdine I; PVDIq, PVDI precursor produced by PAO1pvdQ; NRPS, non-ribosomal peptide synthetases; CA, collision activation; HPLC-MS, high performance liquid chromatography-mass spectrometry secretion into the extracellular milieu. PvdQ belongs to the NTN hydrolase family [13] and was identified as a periplasmic quorum-quenching protein that cleaves acyl homoserine lactones [14]. Transport of newly synthesized PVDI from the periplasm into the extracellular medium involves PvdRT-OpmQ, an ATP-dependent efflux pump [15].…”

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

“…Interestingly, homologues of AiiD were also found in various other Ralstonia and Pseudomonas spp. (46), and these bacteria were later confirmed to also possess AHL acylase activity (42,44,45,47,51,52). Biochemical analysis of the fate of 3OC10HSL incubated with purified AiiD protein confirmed that AiiD functions as an AHL acylase, releasing HSL and 3-oxodecanoic acid as the major degradation products (46).…”

The N-terminal Nucleophile Serine of Cephalosporin Acylase Executes the Second Autoproteolytic Cleavage and Acylpeptide Hydrolysis

“…Other enzymes exhibited more flexibility in their requirements for substrate side chain structure. The substrate side chain of PGA can be an aromatic or aliphatic carboxyl group with 2-6 carbons (with k cat range from 4 to 15 s Ϫ1 , supplemental Table S1), whereas that of PvdQ can be 4 -14 carbons long (40). Because these enzymes lack the special residue, which controlled the cleavage site shift in CA, covalent binding of the spacer became more important for the second cleavage.…”

“…Specific Binding and Covalent Binding for the Second Cleavage-In the previous studies, besides CA, PGA and PvdQ were confirmed to be Ntn hydrolases that require two-step proteolysis for activation, releasing spacers consisting of 54, 22, or 23 aa, respectively (39,40) (Table 4). The long distances between the second cleavage sites and the Ntn Ser residues were also observed in the respective crystal structures (7,41).…”