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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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 |
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1716747246100283392 |