A Novel Quorum-Quenching N -Acylhomoserine Lactone Acylase from Acidovorax sp. Strain MR-S7 Mediates Antibiotic Resistance

Abstract

N-Acylhomoserine lactone acylase (AHL acylase) is a well-known enzyme responsible for disrupting cell-cell communication (quorum sensing) in bacteria. Here, we isolated and characterized a novel and unique AHL acylase (designated MacQ) from a multidrug-resistant bacterium, Acidovorax sp. strain MR-S7. The purified MacQ protein heterologously expressed in Escherichia coli degraded a wide variety of AHLs, ranging from C6 to C14 side chains with or without 3-oxo substitutions. We also observed that AHL-mediated virulence factor production in a plant pathogen, Pectobacterium carotovorum, was dramatically attenuated by coculture with MacQ-overexpressing Escherichia coli, whereas E. coli with an empty vector was unable to quench the pathogenicity, which strongly indicates that MacQ can act in vivo as a quorum-quenching enzyme and interfere with the quorum-sensing system in the pathogen. In addition, this enzyme was found to be capable of degrading a wide spectrum of β-lactams (penicillin G, ampicillin, amoxicillin, carbenicillin, cephalexin, and cefadroxil) by deacylation, clearly indicating that MacQ is a bifunctional enzyme that confers both quorum quenching and antibiotic resistance on strain MR-S7. MacQ has relatively low amino acid sequence identity to any of the known acylases (<39%) and has among the broadest substrate range. Our findings provide the possibility that AHL acylase genes can be an alternative source of antibiotic resistance genes posing a threat to human health if they migrate and transfer to pathogenic bacteria.IMPORTANCE N-Acylhomoserine lactones (AHLs) are well-known signal molecules for bacterial cell-cell communication (quorum sensing), and AHL acylase, which is able to degrade AHLs, has been recognized as a major target for quorum-sensing interference (quorum quenching) in pathogens. In this work, we succeeded in isolating a novel AHL acylase (MacQ) from a multidrug-resistant bacterium and demonstrated that the MacQ enzyme could confer multidrug resistance as well as quorum quenching on the host organism. Indeed, the purified MacQ protein was found to be bifunctional and capable of degrading not only various AHL derivatives but also multiple β-lactam antibiotics by deacylation activities. Although quorum quenching and antibiotic resistance have been recognized to be distinct biological functions, our findings clearly link the two functions by discovering the novel bifunctional enzyme and further providing the possibility that a hitherto-overlooked antibiotic resistance mechanism mediated by the quorum-quenching enzyme may exist in natural environments and perhaps in clinical settings.

Bifunctional quorum-quenching and antibiotic-acylase MacQ forms a 170-kDa capsule-shaped molecule containing spacer polypeptides

“…The expression of macQ isolated from Acidovorax sp. strain MR-S7 was performed using Escherichia coli cells, as previously described 3 . The precursors of AHL-acylases and related enzymes are cleaved into three polypeptide chains (α-chain, internal SP, and β-chain) and the catalytically active mature enzymes are generated as a heterodimer consisting of α- and β-chains, with the SP being released 30 .…”

“…Acidovorax sp. strain MR-S7 is a gram-negative bacterium that was isolated from activated sludge in a treatment system for penicillin G-polluted wastewater 1 – 3 . We previously reported that Acidovorax sp.…”

“…We previously reported that Acidovorax sp. strain MR-S7 is resistant to various β-lactam antibiotics and is able to degrade a broad range of N -acylhomoserine lactones (AHLs) 3 . AHLs are a class of signalling compounds involved in the bacterial intercellular communication known as quorum sensing 4 .…”

“…Thus, quorum quenching could be a key strategy to interfere with bacterial infectious diseases 23 – 26 . AHL-acylase is one of the enzymes capable of inactivating AHLs via hydrolysis of the amide bond of the acyl side-chain to produce homoserine lactone (HSL) and fatty acid 3 , 22 . Several enzymes exhibiting AHL-acylase activity have been reported.…”

“…The recombinant MacQ protein exhibited bifunctional acylase activity against various ranges of AHLs and against multiple β-lactam antibiotics, including penicillin G, ampicillin, and amoxicillin (Fig. 1 ) 3 . The MacQ protein is comprised of 806 amino acid residues, including an N-terminal signal peptide (Fig.…”