Acyl‐homoserine lactone acylase from Ralstonia strain XJ12B represents a novel and potent class of quorum‐quenching enzymes

Abstract

SummaryN -acylhomoserine lactones (AHLs) are used as signal molecules by many quorum-sensing Proteobacteria. Diverse plant and animal pathogens use AHLs to regulate infection and virulence functions. These signals are subject to biological inactivation by AHLlactonases and AHL-acylases. Previously, little was known about the molecular details underlying the latter mechanism. An AHL signal-inactivating bacterium, identified as a Ralstonia sp., was isolated from a mixed-species biofilm. The signal inactivation encoding gene from this organism, which we call aiiD , was cloned and successfully expressed in Escherichia coli and inactivated three AHLs tested. The predicted 794-amino-acid polypeptide was most similar to the aculeacin A acylase (AAC) from Actinoplanes utahensis and also shared significant similarities with cephalosporin acylases and other N-terminal (Ntn) hydrolases. However, the most similar homologues of AiiD are deduced proteins of undemonstrated function from available Ralstonia , Deinococcus and Pseudomonas genomes. LC-MS analyses demonstrated that AiiD hydrolyses the AHL amide, releasing homoserine lactone and the corresponding fatty acid. Expression of AiiD in Pseudomonas aeruginosa PAO1 quenched quorum sensing by this bacterium, decreasing its ability to swarm, produce elastase and pyocyanin and to paralyse nematodes. Thus, AHLacylases have fundamental implications and hold biotechnological promise in quenching quorum sensing.

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

“…Following this initial report, acylase activity was demonstrated in another betaproteobacterium, a Ralstonia isolate, and the responsible AHL inactivation gene was named aiiD (46). The AiiD protein was most similar to aculeacin A acylase (AAC) from Actinoplanes utahensis but also shared similarity with other acylases responsible for hydrolysis of penicillin and cephalosporin (46). Interestingly, homologues of AiiD were also found in various other Ralstonia and Pseudomonas spp.…”

“…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).…”

“…In contrast, AHL acylases hydrolyze the acyl-amide bond between the acyl tail and latone ring of AHLs in a nonreversible manner, resulting in the release of a fatty acid chain and a homoserine lactone moiety ( Fig. 3) (46,53). Unlike lactonases, acylases can exhibit substrate specificity based on the acyl chain length of the AHL and substitution on the third position of the chain (Table 2) due to a constrained binding pocket in the enzyme that must adapt structurally upon interaction with its ligand (41).…”

“…This enzymatic activity was not fully defined at the time but was predicted to be the result of an acylase due to HSL accumulation in the culture medium (53). Following this initial report, acylase activity was demonstrated in another betaproteobacterium, a Ralstonia isolate, and the responsible AHL inactivation gene was named aiiD (46). The AiiD protein was most similar to aculeacin A acylase (AAC) from Actinoplanes utahensis but also shared similarity with other acylases responsible for hydrolysis of penicillin and cephalosporin (46).…”

“…(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). It was also demonstrated that AiiD does not degrade penicillin G or ampicillin and that porcine kidney aclyase and penicillin acylase are unable to degrade AHLs, indicating that certain acylase enzymes with unique specificity for AHL molecules exist (46).…”