Abstract
BackgroundThe infection and virulence functions of diverse plant and animal pathogens that possess quorum sensing systems are regulated by N-acylhomoserine lactones (AHLs) acting as signal molecules. AHL-acylase is a quorum quenching enzyme and degrades AHLs by removing the fatty acid side chain from the homoserine lactone ring of AHLs. This blocks AHL accumulation and pathogenic phenotypes in quorum sensing bacteria.ResultsAn aac gene of undemonstrated function from Ralstonia solanacearum GMI1000 was cloned, expressed in Escherichia coli; it inactivated four AHLs that were tested. The sequence of the 795 amino acid polypeptide was considerably similar to the AHL-acylase from Ralstonia sp. XJ12B with 83% identity match and shared 39% identity with an aculeacin A acylase precursor from the gram-positive actinomycete Actinoplanes utahensis. Aculeacin A is a neutral lipopeptide antibiotic and an antifungal drug. An electrospray ionisation mass spectrometry (ESI-MS) analysis verified that Aac hydrolysed the amide bond of AHL, releasing homoserine lactone and the corresponding fatty acids. However, ESI-MS analysis demonstrated that the Aac could not catalyze the hydrolysis of the palmitoyl moiety of the aculeacin A. Moreover, the results of MIC test of aculeacin A suggest that Aac could not deacylate aculeacin A. The specificity of Aac for AHLs showed a greater preference for long acyl chains than for short acyl chains. Heterologous expression of the aac gene in Chromobacterium violaceum CV026 effectively inhibited violacein and chitinase activity, both of which were regulated by the quorum-sensing mechanism. These results indicated that Aac could control AHL-dependent pathogenicity.ConclusionThis is the first study to find an AHL-acylase in a phytopathogen. Our data provide direct evidence that the functioning of the aac gene (NP520668) of R. solanacearum GMI1000 is via AHL-acylase and not via aculeacin A acylase. Since Aac is a therapeutic potential quorum-quenching agent, its further biotechnological applications in agriculture, clinical and bio-industrial fields should be evaluated in the near future.
Highlights
The infection and virulence functions of diverse plant and animal pathogens that possess quorum sensing systems are regulated by N-acylhomoserine lactones (AHLs) acting as signal molecules
Identification of candidate AHL-degrading enzymes encoded by R. solanacearumGMI1000 BLASTN and BLASTP searches of the annotated R. solanacearumGMI1000 genome sequence (NC 003295) and megaplasmid pGMI1000MP (NC003296) [28] identified a single 2,388-bp aac gene (Locus tag RSc2547) with an 83% identity match when interrogated with the AHLacylase aiiD sequence [14]
Cloning and expression of the aac gene of R. solanacearumGMI1000 The aac gene was PCR amplified and the 2,405 bp product was cloned in pBBR1MCS-3 to yield plasmid pS3aac
Summary
The infection and virulence functions of diverse plant and animal pathogens that possess quorum sensing systems are regulated by N-acylhomoserine lactones (AHLs) acting as signal molecules. Diverse gram-negative bacteria can synthesise Nacyl-homoserine lactones (AHLs) as quorum-sensing signal molecules by means of LuxI-type AHL synthases [3]. These quorum-sensing signal molecules share identical homoserine lactone moieties but vary in length or the carbon substitution on the third position on the acyl side chain. The AHL-mediated quorum sensing mechanisms are highly conserved and could regulate infections and virulence factors in several human and plant pathogenic bacteria, such as Chromobacterium violaceum, Burkholderia cepacia, Erwinia carotovora, Brucella melitensis, and Pseudomonas aeruginosa [3,4,5]. Quorum quenching has the potential to overcome drug related toxicities, complicating superinfections, and antibiotic resistance in antibiotic therapy [4,6,7,8]
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