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Detection of quorum sensing molecules in Burkholderia cepacia culture supernatants with enzyme-linked immunosorbent assays

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Abstract

The Burkholderia cepacia complex (Bcc) employs a quorum sensing (QS) mechanism which is a cell density-dependent bacterial communication system to regulate certain gene expressions. As with many other Gram-negative bacteria, Burkholderia cepacia species use (N-acyl-)homoserine lactones (AHLs or HSLs) as signalling molecules. Because of the essential role of QS in bacterial behavior, the aim of this study was to demonstrate the applicability of our in-house-developed enzyme-linked immunosorbent assays (ELISAs) for the detection of bacterial activities via HSLs in B. cepacia strain LA3 culture supernatants. For this purpose the previously developed monoclonal antibodies (mAbs) HSL1/2-2C10 and HSL1/2-4H5 were exploited. N-3-Oxo-decanoyl-L-homoserine lactone (3-oxo-C10-HSL) was used as main analyte throughout all experiments. With the bacterial culture medium (named ABC medium) a matrix effect in both ELISAs was visible (slight increase in optical density, shift in test midpoints (IC50) and working ranges). For example, ELISA with mAb HSL1/2-2C10 and enzyme tracer HSL3-HRP (HSL derivative conjugated to horseradish peroxidase) had an IC50 of 120 μg L−1 for 3-oxo-C10-HSL in phosphate-buffered saline versus 372 μg L−1 in ABC medium. A significant increase of HSLs in B. cepacia strain LA3 culture supernatants after 12 h to 48 h of growth was observed. Although the analytical result of these immunoassays cannot distinguish HSLs from homoserines (HSs), the appearance of these compounds can be easily followed. Hydrolysis and spiking experiments were carried out with these biological samples. According to our knowledge, these are the first immunoassays for the detection of quorum sensing molecules in biological culture supernatants. This study provides a cost-effective, fast, and sensitive analytical method for detection of HSLs/HSs in biological samples without complex sample preparation and will offer a quick idea about B. cepacia activities. The low sample amount requirement (less than 1 mL) constitutes a tremendous advantage for many analytical questions with biological samples.

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Abbreviations

AHL or HSL:

(N-acyl-)homoserine lactone

ACN:

acetonitrile

AIs:

autoinducers

Bcc:

Burkholderia cepacia complex

CF:

cystic fibrosis

CR:

cross reactivity

ELISA:

enzyme-linked immunosorbent assay

HRP:

horseradish peroxidase

HS:

N-acyl-homoserine

IC50 :

inhibitory concentration 50%, test midpoint of the standard curve

LOD:

limit of detection

mAb:

monoclonal antibody

NaOH:

sodium hydroxide

OD:

optical density

3-oxo-C10-HSL:

N-3-oxo-decanoyl-l-homoserine lactone

PBS:

phosphate-buffered saline

PBST:

phosphate-buffered saline with Tween 20

Protein G:

Protein G from Streptococcus

RT:

room temperature

QS:

quorum sensing

TMB:

3,3′,5,5′-tetramethylbenzidine

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Acknowledgements

The authors acknowledge Dr. Elisabeth Kremmer and her team (Institute of Immunology, Helmholtz Zentrum München) for excellent collaboration and for providing us with the purified mAbs HSL1/2-2C10 and HSL1/2-4H5. We thank Ms. Mandy Starke (formally Institute of Ecological Chemistry, Helmholtz Zentrum München) for very helpful discussion and suggestions. We acknowledge Dr. Agnes Fekete, formerly Institute of Ecological Chemistry, Helmholtz Zentrum München, for the UPLC-MS analysis of the B. cepacia culture supernatant samples.

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Authors and Affiliations

  1. Institute of Ecological Chemistry, Helmholtz Zentrum München – German Research Center for Environmental Health (GmbH), Ingolstädter Landstrasse 1, 85764, Neuherberg, Germany

    Xiao Chen & Petra M. Krämer

  2. Department Microbe–Plant Interactions, Helmholtz Zentrum München – German Research Center for Environmental Health (GmbH), Ingolstädter Landstrasse 1, 85764, Neuherberg, Germany

    Katharina Buddrus-Schiemann, Michael Rothballer & Anton Hartmann

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  1. Xiao Chen
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  2. Katharina Buddrus-Schiemann
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  3. Michael Rothballer
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  4. Petra M. Krämer
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  5. Anton Hartmann
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Correspondence to Petra M. Krämer.

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Published in the special issue on Focus on Bioanalysis with Guest Editors Antje J. Baeumner, Günter Gauglitz, and Frieder W. Scheller.

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Chen, X., Buddrus-Schiemann, K., Rothballer, M. et al. Detection of quorum sensing molecules in Burkholderia cepacia culture supernatants with enzyme-linked immunosorbent assays. Anal Bioanal Chem 398, 2669–2676 (2010). https://doi.org/10.1007/s00216-010-4045-5

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