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.

[1]  Jung-Kee Lee,et al.  Identification of Extracellular N-Acylhomoserine Lactone Acylase from a Streptomyces sp. and Its Application to Quorum Quenching , 2005, Applied and Environmental Microbiology.

[2]  W. Quax,et al.  Improved beta-lactam acylases and their use as industrial biocatalysts. , 2004, Current opinion in biotechnology.

[3]  L. Otten,et al.  Mutational Analysis of a Key Residue in the Substrate Specificity of a Cephalosporin Acylase , 2004, Chembiochem : a European journal of chemical biology.

[4]  Lian-Hui Zhang,et al.  The quormone degradation system of Agrobacterium tumefaciens is regulated by starvation signal and stress alarmone (p)ppGpp , 2004, Molecular microbiology.

[5]  R. Tompkins,et al.  Analysis of Pseudomonas aeruginosa 4-hydroxy-2-alkylquinolines (HAQs) reveals a role for 4-hydroxy-2-heptylquinoline in cell-to-cell communication. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[6]  Z. Dauter,et al.  Crystal structures of glutaryl 7-aminocephalosporanic acid acylase: insight into autoproteolytic activation. , 2004, Biochemistry.

[7]  A. Görg,et al.  Identification of quorum-sensing regulated proteins in the opportunistic pathogen Pseudomonas aeruginosa by proteomics. , 2003, Environmental microbiology.

[8]  Jared R. Leadbetter,et al.  Utilization of Acyl-Homoserine Lactone Quorum Signals for Growth by a Soil Pseudomonad and Pseudomonas aeruginosa PAO1 , 2003, Applied and Environmental Microbiology.

[9]  S. Diggle,et al.  The Pseudomonas aeruginosa quinolone signal molecule overcomes the cell density‐dependency of the quorum sensing hierarchy, regulates rhl‐dependent genes at the onset of stationary phase and can be produced in the absence of LasR , 2003, Molecular microbiology.

[10]  S. Kjelleberg,et al.  Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors , 2003, The EMBO journal.

[11]  Sang Jun Lee,et al.  AhlD, an N-acylhomoserine lactonase in Arthrobacter sp., and predicted homologues in other bacteria. , 2003, Microbiology.

[12]  J. Mattick,et al.  Proteome analysis of extracellular proteins regulated by the las and rhl quorum sensing systems in Pseudomonas aeruginosa PAO1. , 2003, Microbiology.

[13]  E. Greenberg,et al.  Identification, Timing, and Signal Specificity of Pseudomonas aeruginosa Quorum-Controlled Genes: a Transcriptome Analysis , 2003, Journal of bacteriology.

[14]  A. Brooks,et al.  Microarray Analysis of Pseudomonas aeruginosa Quorum-Sensing Regulons: Effects of Growth Phase and Environment , 2003, Journal of bacteriology.

[15]  I. Lamont,et al.  Identification and characterization of novel pyoverdine synthesis genes in Pseudomonas aeruginosa. , 2003, Microbiology.

[16]  Feng Xu,et al.  Degradation of N-acylhomoserine lactones, the bacterial quorum-sensing molecules, by acylase. , 2003, Journal of biotechnology.

[17]  Say Leong Ong,et al.  Acyl‐homoserine lactone acylase from Ralstonia strain XJ12B represents a novel and potent class of quorum‐quenching enzymes , 2003, Molecular microbiology.

[18]  Wim J. Quax,et al.  Altering the Substrate Specificity of Cephalosporin Acylase by Directed Evolution of the β-Subunit* , 2002, The Journal of Biological Chemistry.

[19]  W. Quax,et al.  Directed evolution of a glutaryl acylase into an adipyl acylase. , 2002, European journal of biochemistry.

[20]  M. Vasil,et al.  GeneChip® expression analysis of the iron starvation response in Pseudomonas aeruginosa: identification of novel pyoverdine biosynthesis genes , 2002, Molecular microbiology.

[21]  Anne K Camper,et al.  Molecular interactions in biofilms. , 2002, Chemistry & biology.

[22]  P. Williams,et al.  Quorum sensing: an emerging target for antibacterial chemotherapy? , 2002, Expert opinion on therapeutic targets.

[23]  E. Greenberg,et al.  A component of innate immunity prevents bacterial biofilm development , 2002, Nature.

[24]  M. Vasil,et al.  Siderophore-mediated signaling regulates virulence factor production in Pseudomonas aeruginosa , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Lian-Hui Zhang,et al.  Genetic control of quorum-sensing signal turnover in Agrobacterium tumefaciens , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[26]  C. Blumer,et al.  Regulatory RNA as Mediator in GacA/RsmA-Dependent Global Control of Exoproduct Formation in Pseudomonas fluorescens CHA0 , 2002, Journal of bacteriology.

[27]  Sanggu Kim,et al.  Active Site Residues of Cephalosporin Acylase Are Critical Not Only for Enzymatic Catalysis but Also for Post-translational Modification* , 2001, The Journal of Biological Chemistry.

[28]  N. A. Whitehead,et al.  Quorum-sensing in Gram-negative bacteria. , 2001, FEMS microbiology reviews.

[29]  M. Schäfer,et al.  Siderotyping--a powerful tool for the characterization of pyoverdines. , 2001, Current topics in medicinal chemistry.

[30]  E. P. Greenberg,et al.  Metabolism of Acyl-Homoserine Lactone Quorum-Sensing Signals by Variovorax paradoxus , 2000, Journal of bacteriology.

[31]  S. Diggle,et al.  The Pseudomonas aeruginosa Lectins PA-IL and PA-IIL Are Controlled by Quorum Sensing and by RpoS , 2000, Journal of bacteriology.

[32]  W. Hol,et al.  The 2.0 A crystal structure of cephalosporin acylase. , 2000, Structure.

[33]  B. Iglewski,et al.  Bacterial Quorum Sensing in Pathogenic Relationships , 2000, Infection and Immunity.

[34]  S. Bron,et al.  Signal Peptide-Dependent Protein Transport inBacillus subtilis: a Genome-Based Survey of the Secretome , 2000, Microbiology and Molecular Biology Reviews.

[35]  S. Lory,et al.  Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen , 2000, Nature.

[36]  E. Greenberg,et al.  Acyl-homoserine lactone quorum sensing in gram-negative bacteria: a signaling mechanism involved in associations with higher organisms. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[37]  S. Bron,et al.  Signal peptide-dependent protein transport in Bacillus subtilis , 2000 .

[38]  Y. Dong,et al.  AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[39]  K. M. Lee,et al.  Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[40]  E. Greenberg,et al.  Quinolone signaling in the cell-to-cell communication system of Pseudomonas aeruginosa. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[41]  W. Jiang,et al.  In vivo post-translational processing and subunit reconstitution of cephalosporin acylase from Pseudomonas sp. 130. , 1999, European journal of biochemistry.

[42]  Young Sik Lee,et al.  Two-Step Autocatalytic Processing of the Glutaryl 7-Aminocephalosporanic Acid Acylase from Pseudomonas sp. Strain GK16 , 1998, Journal of bacteriology.

[43]  M K Winson,et al.  Construction and analysis of luxCDABE-based plasmid sensors for investigating N-acyl homoserine lactone-mediated quorum sensing. , 1998, FEMS microbiology letters.

[44]  A. Roos,et al.  Penicillin Acylase in the Industrial Production of β-Lactam Antibiotics , 1998 .

[45]  J H Lamb,et al.  Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones. , 1997, Microbiology.

[46]  B. Iglewski,et al.  Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes , 1997, Journal of bacteriology.

[47]  M K Winson,et al.  Quorum sensing in Aeromonas hydrophila and Aeromonas salmonicida: identification of the LuxRI homologs AhyRI and AsaRI and their cognate N-acylhomoserine lactone signal molecules , 1997, Journal of bacteriology.

[48]  S. Brunak,et al.  SHORT COMMUNICATION Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites , 1997 .

[49]  K. Tanaka,et al.  A hierarchical quorum‐sensing cascade in Pseudomonas aeruginosa links the transcriptional activators LasR and RhIR (VsmR) to expression of the stationary‐phase sigma factor RpoS , 1996, Molecular microbiology.

[50]  Eleanor J. Dodson,et al.  Penicillin acylase has a single-amino-acid catalytic centre , 1996, Nature.

[51]  A. Murzin,et al.  A protein catalytic framework with an N-terminal nucleophile is capable of self-activation , 1995, Nature.

[52]  T. Kuo,et al.  A simple and rapid method for the preparation of gram-negative bacterial genomic DNA. , 1993, Nucleic acids research.

[53]  G. Salmond,et al.  Autoregulation of carbapenem biosynthesis in Erwinia carotovora by analogues of N-(3-oxohexanoyl)-L-homoserine lactone. , 1993, The Journal of antibiotics.

[54]  N. Koedam,et al.  Stability, frequency and multiplicity of transposon insertions in the pyoverdine region in the chromosomes of different fluorescent pseudomonads. , 1992, Journal of general microbiology.

[55]  I. Crawford,et al.  Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications , 1990, Journal of bacteriology.

[56]  B. Iglewski,et al.  Transformation of Pseudomonas aeruginosa by electroporation. , 1989, Nucleic acids research.

[57]  J. Soliveri,et al.  Determination of cephalosporin‐C amidohydrolase activity with fluorescamine , 1989, The Journal of pharmacy and pharmacology.

[58]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[59]  S. Cryz,et al.  Isolation and characterization of Pseudomonas aeruginosa PAO mutant that produces altered elastase , 1980, Journal of bacteriology.