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Detection and Analysis of Quorum‐Quenching Enzymes Against Acyl Homoserine Lactone Quorum‐Sensing Signals

Hai‐Bao Zhang1,  Lian‐Hui Wang1,  Lian‐Hui Zhang1

1Institute of Molecular and Cell Biology, Proteos, Singapore

Publication Name: 
Current Protocols in Microbiology
Unit Number: 
Unit 1C.3
DOI: 
10.1002/9780471729259.mc01c03s05
Online Posting Date: 
June, 2007
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Abstract

Many Gram-negative bacterial pathogens employ N-acyl homoserine lactones (AHLs) quorum-sensing signals for regulation of various biological functions. Several types of AHL-inactivating enzymes, also known as quorum-quenching enzymes, have been unveiled in recent years. These enzymes seem to play important roles in microbial physiology and ecology and hold promising potential in biotechnology. This unit describes methods based on a simple diffusion plate assay for qualitative detection and quantitative measurement of AHL-inactivating enzyme activity. The qualitative detection method is suitable for rapid and large-scale screening and identification of quorum-quenching enzymes. Furthermore, HPLC methods are provided for accurate determination of whether the quorum-quenching enzyme is a lactonase or an acylase. The unit also presents concise background information on the quorum-quenching enzymes identified from various sources, including bacterial and mammalian species.

Keywords: AHL-lactonase; AHL-acylase; paraoxonase; quorum sensing; quorum-quenching enzyme; acyl homoserine lactone; diffusion plate assay

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol: Qualitative Detection of AHL-Inactivating Enzymes
  • Support Protocol 1: Confirm Enzymatic AHL Inactivation
  • Support Protocol 2: Determine Whether the AHL-Inactivating Enzyme is a Lactonase
  • Support Protocol 3: Quantitative Analysis of Quorum-Quenching Enzyme Activity
  • Support Protocol 4: Assaying Enzyme Activity Using Cell-Free Total Protein Extracts
  • Support Protocol 5: HPLC Identification of AHL-Lactonase Hydrolysis Products
  • Support Protocol 6: HPLC Identification of AHL-Acylase Hydrolysis Products
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol: Qualitative Detection of AHL-Inactivating Enzymes

 Materials
  • Biosensor organism: Agrobacterium tumefaciens NT1(traR, tra::lacZ749; Piper et al., 1993)
  • YEB medium with and without 50 µg/ml kanamycin and 10 µg/ml tetracycline (see recipe)
  • Glycerol (optional)
  • Test organisms (e.g., bacteria from soil, water, and plant or animal tissues)
  • 10 µM N-3-oxo-octanoyl-l-homoserine lactone (3OC8HSL) and other AHLs (see Zhang et al., 1993; Sigma-Aldrich; Cayman Chemical): dilute in YEB medium
  • Minimal medium agar plates supplemented with 50 µg/ml X-gal (see recipe)
  • 250-ml culture flasks
  • 28°C incubator
  • Rotary shaker
  • 96-well tissue-culture plates
  • UV chamber (Stratagene)
  • Marker pen
  • Ruler
  • Scalpel, sterile
  • Microcentrifuge with (optional) 96-well plate rotor
  • Plastic film (for sealing 96-well plates)

Support Protocol 1: Confirm Enzymatic AHL Inactivation

 Additional Materials (also see Basic Protocol)
  • Bacterial isolate showing positive AHL-inactivating activity (identified using the Basic Protocol)

Support Protocol 2: Determine Whether the AHL-Inactivating Enzyme is a Lactonase

 Additional Materials (also see Basic Protocol)
  • Bacterial isolate with confirmed AHL-inactivating enzyme (Support Protocol 1)
  • 1 M HCl solution

Support Protocol 3: Quantitative Analysis of Quorum-Quenching Enzyme Activity

 Additional Materials (also see Basic Protocol)
  • Methanol
  • Equation-fitting program (e.g., Microsoft Excel)

Support Protocol 4: Assaying Enzyme Activity Using Cell-Free Total Protein Extracts

 Additional Materials (also see Basic Protocol)
  • Phosphate-buffered saline containing potassium (KPBS; appendix 2A)
  • Sonicator (e.g., Digital Sonifier; Branson Ultrasonics)

Support Protocol 5: HPLC Identification of AHL-Lactonase Hydrolysis Products

 Additional Materials (also see Support Protocol 4)
  • 50:50 methanol (HPLC grade, Merck)/water (HPLC grade or Milli-Q-purified)
  • 50 mM N-3-oxo-octanoyl-l-homoserine lactone (3OC8HSL; Sigma-Aldrich) stock solution in methanol
  • 10 mM N-3-oxo-octanoyl-l-homoserine (3OC8HS) stock solution: prepared by incubating 10 mM 3OC8HSL (Sigma-Aldrich) in 100 µl 200 mM NaOH and 100 µl DMSO for 4 hr at 25°C
  • HPLC column: C18 reverse-phase column, 4.6 × 250 mm, 5 µm (Symmetry)
  • HPLC system: Alliance 2690 separations module, and 996 photodiode array detector (Waters)
  • 0.45-µm PTFE filter (Waters)
  • Additional reagents and materials for preparing bacterial cell-free extracts (Support Protocol 4)

Support Protocol 6: HPLC Identification of AHL-Acylase Hydrolysis Products

 Materials
  • N-3-oxo-octanoyl-l-homoserine lactone (3OC8HSL; Sigma-Aldrich) in KPBS (see appendix 2A for KPBS; for purified enzyme assay)
  • Purified enzyme (Lin et al., 2003)
  • 3OC8HSL (solid; for cell debris assay)
  • Cell debris from 2 ml bacterial cultures (Support Protocol 4, step 4 pellet)
  • Homoserine lactone (HSL; Sigma-Aldrich) standard
  • 2.5 mg/ml DANSYL chloride (Sigma-Aldrich) in acetone
  • 0.2 M HCl
  • Additional reagents and equipment for HPLC (see Support Protocol 5)
Looking for materials?  Suppliers Guide
     
 
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Figures

  • Figure 1C.3.1
    The AHL bioassay plate. Blue spot indicates that the diffusible AHL signals were detected by the biosensor cells. Bar 1: the positive control (5 µM 3OC8HSL in YEB medium) was added. Bars 2 to 6: the reaction supernatants of five bacterial isolates were added, separately. The shorter distance of blue spots from the sample loading area in bars 2, 4, and 6 suggests that the bacterial samples may have AHL-inactivating activity, whereas no blue spot in bar 5 indicates a strong AHL-inactivating activity.

    View Image
  • Figure 1C.3.2
    Establishment of a standard curve for quantification of AHL. (A) Bioassay results for 3OC8HSL dilutions. (B) The standard curve for 3OC8HSL concentrations (µM) versus their corresponding diffusion distances in an agar bar (cm) generated by the Microsoft Excel program.

    View Image
  • Figure 1C.3.3
    Enzymatic inactivation of AHL by AHL-lactonase and AHL-acylase. Unlike the AHL-acylase hydrolysis products, the hydrolysis product of AHL-lactonase or paraoxonases (PONs) can reform AHL by acidification.

    View Image

Literature Cited

Literature Cited
    Byers, J.T., Lucas, C., Salmond, G.P.C., and Welch, M. 2002. Nonenzymatic turnover of an Erwinia carotovora quorum sensing signaling molecule. J. Bacteriol. 184:1163-1171.
    Chun, C.K., Ozer, E.A., Welsh, M.J., Zabner, J., and Greenberg, E.P. 2004. Inactivation of a Pseudomonas aeruginosa quorum-sensing signal by human airway epithelia. Proc. Natl. Acad. Sci. U.S.A. 101:3587-3590.
    Collins, C.H., Lyne, P.M., and Grange, J.M. 1989. Collins and Lyne's Microbiological Methods, 6th ed. Butterworth, London.
    Dong, Y.H. and Zhang, L.H. 2005. Quorum sensing and quorum-quenching enzymes. J. Microbiol. 43:101-109.
    Dong, Y.H., Xu, J.L., Li, X.Z., and Zhang, L.H. 2000. AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora. Proc. Natl. Acad. Sci. U.S.A. 97:3526-3531.
    Dong, Y.H., Wang, L.H., Xu, J.L., Zhang, H.B., Zhang, X.F., and Zhang, L.H. 2001. Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase. Nature 411:813-817.
    Dong, Y.H., Gusti, A.R., Zhang, Q., Xu, J.L., and Zhang, L.H. 2002. Identification of quorum-quenching N-acyl homoserine lactonases from Bacillus species. Appl. Environ. Microbiol. 68:1754-1759.
    Dong, Y.H., Zhang, X.F., Xu, J.L., and Zhang, L.H. 2004. Insecticidal Bacillus thuringiensis silences Erwinia carotovora virulence by a new form of microbial antagonism – signal interference. Appl. Environ. Microbiol. 70:954-960.
    Draganov, D.I. and La Du, B.N. 2004. Pharmacogenetics of paraoxonases: A brief review. Naunyn Schmiedebergs Arch. Pharmacol. 369:78-88.
    Draganov, D.I., Teiber, J.F., Speelman, A., Osawa, Y., Sunahara, R., and La Du, B.N. 2005. Human paraoxonases (PON1, PON2, PON3) are lactonases with overlapping and distinct substrate specificities. J. Lipid Res. 46:1239-1247.
    Leadbetter, J.R. and Greenberg, E.P. 2000. Metabolism of acyl-homoserine lactone quorum-sensing signals by Variovorax paradoxus. J. Bacteriol. 182:6921-6926.
    Lin, Y.H., Xu, J.L., Hu, J., Wang, L.H., Ong, S.L., Leadbetter, J.R., and Zhang, L.H. 2003. Acyl-homoserine lactone acylase from Ralstonia strain XJ12B represents a novel and potent class of quorum-quenching enzymes. Mol. Microbiol. 47:849-860.
    McClean, K.H., Winson, M.K., Fish, L., Taylor, A., Chhabra, S.R., Camara, M., Daykin, M., Lamb, J.H., Swift, S., Bycroft, B.W., Stewart, G.S., and Williams, P. 1997. Quorum-sensing and Chromobacterium violaceum: Exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones. Microbiology 143:3703-3711.
    Molina, L., Rezzonico, F., Défago, G., and Duffy, B. 2005. Autoindution in Erwinia amylovora: Evidence of an acyl-homoserine lactone signal in the fire blight pathogen. J. Bacteriol. 187:3206-3213.
    Piper, K.R., von Bodman, S.B., and Farrand, S.K. 1993. Conjugation factor of Agrobacterium tumefaciens regulates Ti plasmid transfer by autoinduction. Nature 362:448-450.
    Wang, L.H, Weng, L.X., Dong, Y.H., and Zhang, L.H. 2004. Specificity and enzyme kinetics of the quorum-quenching N-Acyl homoserine lactone lactonase (AHL-lactonase). J. Biol. Chem. 279:13645-13651.
    Yang, F., Wang, L.H., Wang, J., Dong, Y.H., Hu, J.Y., and Zhang, L.H. 2005. Quorum-quenching enzyme activity is widely conserved in the sera of mammalian species. FEBS Lett. 579:3713-3717.
    Zhang, L.H. 2003. Quorum quenching and proactive host defense. Trends Plant Sci. 8:238-244.
    Zhang, L.H. and Kerr, A. 1991. A diffusible compound can enhance conjugal transfer of the Ti plasmid in Agrobacterium tumefaciens. J. Bacteriol. 173:1867-1872.
    Zhang, L.H. and Dong, Y.H. 2004. Quorum sensing and signal interference: diverse implications. Mol. Microbiol. 53:1563-1571.
    Zhang, L.H., Murphy, P.J., Kerr, A., and Tate, M.E. 1993. Agrobacterium conjugation and gene regulation by N-acyl-L-homoserine lactones. Nature 362:446-447.
    Zhang, H.B., Wang, L.H., and Zhang, L.H. 2002. Genetic control of quorum-sensing signal turnover in Agrobacterium tumefaciens. Proc. Natl. Acad. Sci. U.S.A. 99:4638-4643.
    Zhang, H.B., Wang, C., and Zhang, L.H. 2004. Quormone turnover in Agrobacterium tumefaciens is regulated by starvation signal and stress alarmone (p)ppGpp. Mol. Microbiol. 52:1389-1401.
 Key Reference
    Dong and Zhang, 2005. See above.

A comprehensive review on quorum-quenching enzymes.

     
 
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Namita Das (not verified)
Posted on July 22, 2010 - 9:58am
Sir, The quantification process is quite nice. with regards Namita

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