Qualitative and quantitative determination of quorum sensing inhibition in vitro

TY - JOUR

T1 - Qualitative and quantitative determination of quorum sensing inhibition in vitro

AU - Jakobsen, Tim Holm

AU - van Gennip, Maria

AU - Christensen, Louise Dahl

AU - Bjarnsholt, Thomas

AU - Givskov, Michael

PY - 2011/1/1

Y1 - 2011/1/1

N2 - The formation of biofilms in conjunction with quorum sensing (QS)-regulated expression of virulence by opportunistic pathogens contributes significantly to immune evasion and tolerance to a variety of antimicrobial treatments. The present protocol describes methods to determine the in vitro efficacy of potential quorum sensing inhibitors (QSIs). Work on Pseudomonas aeruginosa has shown that chemical blockage of QS is a promising new antimicrobial strategy. Several live bacterial reporter systems been developed to screen extracts and pure compounds for QSI activity. Here we describe the usage of reporter strains consisting of a lasB-gfp or rhlA-gfp fusion in P. aeruginosa for qualitative and quantitative evaluation of the inhibition of the two major QS pathways, monitored as reduced expression of green fluorescence. By the use of an in vitro flow cell system it is possible to study the QSI activity by monitoring its ability to interfere with the protective functions of bacterial biofilm. For evaluation of the global effects of QSI compounds, we present a protocol for the DNA microarray-based transcriptomics. Using these in vitro methods it is possible to evaluate the potential of various QSI compounds.

AB - The formation of biofilms in conjunction with quorum sensing (QS)-regulated expression of virulence by opportunistic pathogens contributes significantly to immune evasion and tolerance to a variety of antimicrobial treatments. The present protocol describes methods to determine the in vitro efficacy of potential quorum sensing inhibitors (QSIs). Work on Pseudomonas aeruginosa has shown that chemical blockage of QS is a promising new antimicrobial strategy. Several live bacterial reporter systems been developed to screen extracts and pure compounds for QSI activity. Here we describe the usage of reporter strains consisting of a lasB-gfp or rhlA-gfp fusion in P. aeruginosa for qualitative and quantitative evaluation of the inhibition of the two major QS pathways, monitored as reduced expression of green fluorescence. By the use of an in vitro flow cell system it is possible to study the QSI activity by monitoring its ability to interfere with the protective functions of bacterial biofilm. For evaluation of the global effects of QSI compounds, we present a protocol for the DNA microarray-based transcriptomics. Using these in vitro methods it is possible to evaluate the potential of various QSI compounds.

U2 - 10.1007/978-1-60761-971-0_18

DO - 10.1007/978-1-60761-971-0_18

M3 - Journal article

C2 - 21031317

SN - 1064-3745

VL - 692

SP - 253

EP - 263

JO - Methods in Molecular Biology

JF - Methods in Molecular Biology

IS - 3

ER -