Validation of FRET Assay for the Screening of Growth Inhibitors of Escherichia coli Reveals Elongasome Assembly Dynamics

The increase in antibiotic resistant bacteria demands the development of new antibiotics against preferably new targets. The common approach is to test compounds for their ability to kill bacteria or to design molecules that inhibit essential protein activities in vitro. In the first case, the mode...

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Main Authors: René van der Ploeg, Spyridon Theodoros Goudelis, Tanneke den Blaauwen
Format: Article
Language:English
Published: MDPI AG 2015-07-01
Series:International Journal of Molecular Sciences
Subjects:
A22
Online Access:http://www.mdpi.com/1422-0067/16/8/17637
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spelling doaj-1dadf8f46e0e4f7bbda113f33c512a042020-11-24T21:14:26ZengMDPI AGInternational Journal of Molecular Sciences1422-00672015-07-01168176371765410.3390/ijms160817637ijms160817637Validation of FRET Assay for the Screening of Growth Inhibitors of Escherichia coli Reveals Elongasome Assembly DynamicsRené van der Ploeg0Spyridon Theodoros Goudelis1Tanneke den Blaauwen2Bacterial Cell Biology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The NetherlandBacterial Cell Biology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The NetherlandBacterial Cell Biology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The NetherlandThe increase in antibiotic resistant bacteria demands the development of new antibiotics against preferably new targets. The common approach is to test compounds for their ability to kill bacteria or to design molecules that inhibit essential protein activities in vitro. In the first case, the mode of action of the drug is unknown and in the second case, it is not known whether the compound will pass the impermeable barrier of the bacterial envelope. We developed an assay that detects the target of a compound, as well as its ability to pass the membrane(s) simultaneously. The Escherichia coli cytoskeletal protein MreB recruits protein complexes (elongasomes) that are essential for cell envelope growth. An in cell Förster Resonance Energy Transfer (FRET) assay was developed to detect the interaction between MreB molecules and between MreB and the elongasome proteins RodZ, RodA and PBP2. Inhibition of the polymerization of MreB by S-(3,4-dichlorobenzyl) isothiourea (A22) or of the activity of PBP2 by mecilinam resulted in loss or reduction of all measured interactions. This suggests that the interactions between the elongasome proteins are governed by a combination of weak affinities and substrate availability. This validated in cell FRET assay can be used to screen for cell envelope growth inhibitors.http://www.mdpi.com/1422-0067/16/8/17637antibioticsA22drug targetsFRETGram-negativegrowth inhibitorsMreBPBP2RodARodZ
collection DOAJ
language English
format Article
sources DOAJ
author René van der Ploeg
Spyridon Theodoros Goudelis
Tanneke den Blaauwen
spellingShingle René van der Ploeg
Spyridon Theodoros Goudelis
Tanneke den Blaauwen
Validation of FRET Assay for the Screening of Growth Inhibitors of Escherichia coli Reveals Elongasome Assembly Dynamics
International Journal of Molecular Sciences
antibiotics
A22
drug targets
FRET
Gram-negative
growth inhibitors
MreB
PBP2
RodA
RodZ
author_facet René van der Ploeg
Spyridon Theodoros Goudelis
Tanneke den Blaauwen
author_sort René van der Ploeg
title Validation of FRET Assay for the Screening of Growth Inhibitors of Escherichia coli Reveals Elongasome Assembly Dynamics
title_short Validation of FRET Assay for the Screening of Growth Inhibitors of Escherichia coli Reveals Elongasome Assembly Dynamics
title_full Validation of FRET Assay for the Screening of Growth Inhibitors of Escherichia coli Reveals Elongasome Assembly Dynamics
title_fullStr Validation of FRET Assay for the Screening of Growth Inhibitors of Escherichia coli Reveals Elongasome Assembly Dynamics
title_full_unstemmed Validation of FRET Assay for the Screening of Growth Inhibitors of Escherichia coli Reveals Elongasome Assembly Dynamics
title_sort validation of fret assay for the screening of growth inhibitors of escherichia coli reveals elongasome assembly dynamics
publisher MDPI AG
series International Journal of Molecular Sciences
issn 1422-0067
publishDate 2015-07-01
description The increase in antibiotic resistant bacteria demands the development of new antibiotics against preferably new targets. The common approach is to test compounds for their ability to kill bacteria or to design molecules that inhibit essential protein activities in vitro. In the first case, the mode of action of the drug is unknown and in the second case, it is not known whether the compound will pass the impermeable barrier of the bacterial envelope. We developed an assay that detects the target of a compound, as well as its ability to pass the membrane(s) simultaneously. The Escherichia coli cytoskeletal protein MreB recruits protein complexes (elongasomes) that are essential for cell envelope growth. An in cell Förster Resonance Energy Transfer (FRET) assay was developed to detect the interaction between MreB molecules and between MreB and the elongasome proteins RodZ, RodA and PBP2. Inhibition of the polymerization of MreB by S-(3,4-dichlorobenzyl) isothiourea (A22) or of the activity of PBP2 by mecilinam resulted in loss or reduction of all measured interactions. This suggests that the interactions between the elongasome proteins are governed by a combination of weak affinities and substrate availability. This validated in cell FRET assay can be used to screen for cell envelope growth inhibitors.
topic antibiotics
A22
drug targets
FRET
Gram-negative
growth inhibitors
MreB
PBP2
RodA
RodZ
url http://www.mdpi.com/1422-0067/16/8/17637
work_keys_str_mv AT renevanderploeg validationoffretassayforthescreeningofgrowthinhibitorsofescherichiacolirevealselongasomeassemblydynamics
AT spyridontheodorosgoudelis validationoffretassayforthescreeningofgrowthinhibitorsofescherichiacolirevealselongasomeassemblydynamics
AT tannekedenblaauwen validationoffretassayforthescreeningofgrowthinhibitorsofescherichiacolirevealselongasomeassemblydynamics
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