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-l-homoserine lactone (C4-HSL) and N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL), only 3-oxo-C12-HSL is degraded by the enzyme. Addition of the purified protein to P. aeruginosa PAO1 cultures completely inhibited accumulation of 3-oxo-C12-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.

FIG. 1.

Hydrolysis of AHL and β-lactam substrates by acylase enzymes. (A) Deacylation of C₄-HSL (R1 = O) and C₄-thio-HSL (R1 = S). (B) Hydrolysis of other AHL compounds (R2 = H, OH, or O; n = 1, 2, 3, 5, 7, or 9). (C) Hydrolysis of the cephalosporins glutaryl-7-aminoacetoxycephalosporanic acid (R3 = glutaric acid; R4 = methylacetoxy), adipyl-7-aminodesacetoxycephalosporanic acid (R3 = adipic acid; R4 = methylacetoxy), cephalosporin C (R3 = 5-aminoadipic acid, R4 = methylacetoxy), cephalexin (R3 = phenylglycine; R4 = methylacetoxy), and cefadroxil (R3 = 4-hydroxyphenylglycine; R4 = methylacetoxy). (D) Hydrolysis of the penicillins penicillin G (R5 = phenylacetic acid), penicillin V (R5 = phenoxyacetic acid), ampicillin (R5 = phenylglycine), and amoxicillin (R5 = 4-hydroxyphenylglycine).

FIG. 2.

The PA2385 protein in cell extracts and as purified protein. The P. aeruginosa AHL acylase consists of a small α-subunit and a large β-subunit. Lane A1: cell extract of E. coli DH10B::pMcTNde (control). Lane A2: cell extract of E. coli DH10B::pMc-PA2385 showing additional bands. Lanes A3 and B1: marker proteins (broad range; Bio-Rad). Lane B2: purified PA2385 protein showing two subunits.

FIG. 3.

Schematic diagram of the structure of the P. aeruginosa PAO1 PA2385 gene product. The gene product is comprised of four sections: a signal sequence, the α-subunit, a spacer peptide, and the β-subunit. The first and last residues in each section are given, and the sequence of the spacer peptide is given in full. Ala194 may be a part of the α-subunit or a part of the spacer peptide.

FIG. 4.

GC analysis of C₁₂-HSL reaction. The incubation of the PA2385 protein with C₁₂-HSL results in the formation of lauric acid, whereas no lauric acid is formed if C₁₂-HSL is incubated without enzyme. (A) Reaction mixture at t = 0 h. (B) Reaction mixture at t = 20 h. (C) Control reaction mixture without enzyme at t = 0 h. (D) Control reaction mixture at t = 20 h. The retention time of C₁₂-HSL is 21.1 min, and that of lauric acid is 12.7 min.

FIG. 5.

Effect of exogenous addition of the AHL acylase on quorum-sensing signal molecules and virulence factors of P. aeruginosa PAO1. The acylase degrades 3-oxo-C₁₂-HSL (A) in P. aeruginosa PAO1 cultures and inhibits PQS accumulation (B), elastolytic activity (C), and pyocyanin production (D). Lanes: A1, 3-oxo-C₁₂-HSL; B1, PQS; A2 and B2, control cultures without added acylase (6 h postinoculation); A3 and B3, cultures to which acylase is added (6 h postinoculation); C1 and D1, control cultures without added acylase; C2 and D2, cultures to which acylase is added, 6 h postinoculation. OD600, optical density at 600 nm.

FIG. 6.

Effect of overexpression of the AHL acylase gene on quorum-sensing signal molecules and virulence factors of P. aeruginosa PAO1. The acylase degrades 3-oxo-C₁₂-HSL (A) in P. aeruginosa PAO1 cultures and inhibits PQS accumulation (B), elastolytic activity (C), and lecA expression (D). Lanes: A1, 3-oxo-C₁₂-HSL; B1, PQS; A2 and B2, control cultures in which the gene is not induced, 5 h postinoculation; A4 and B4, as A2 and B2 but 8 h postinoculation; A3 and B3, cultures in which expression of the acylase is induced by IPTG, 5 h postinoculation; A5 and B5, as A3 and B3 but 8 h postinoculation; C1, control culture in which the gene is not induced; C2, culture in which expression of the acylase is induced by IPTG. In panel D, closed circles indicate control culture, in which the gene is not induced, and open circles indicate culture in which expression of the acylase is induced by IPTG. OD495, optical density at 495 nm; RLU, relative light units.