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 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.
Bacterial Cell Biology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherland. renevanderploeg@gmail.com.
René
R
van der Ploeg
Bacterial Cell Biology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherland. sthgoudelis@gmail.com.
Spyridon Theodoros
ST
Goudelis
Bacterial Cell Biology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherland. t.denblaauwen@uva.nl.
Tanneke
T
den Blaauwen
D003598:Cytoskeletal Proteins / Q000096:biosynthesis / Q000737:chemistry; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D031541:Fluorescence Resonance Energy Transfer; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D006131:Growth Inhibitors / Q000008:administration & dosage / Q000737:chemistry; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry; D046915:Penicillin-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry; D013379:Substrate Specificity; D013890:Thiourea / Q000008:administration & dosage / Q000031:analogs & derivatives
D003598:Cytoskeletal Proteins; D029968:Escherichia coli Proteins; D006131:Growth Inhibitors; D008565:Membrane Proteins; D046915:Penicillin-Binding Proteins; C538795:RodZ protein, E coli; C061683:mrdB protein, E coli; C078473:MreB protein, E coli; C015799:2,6-dichlorobenzylthiopseudourea; C582982:MrdA protein, E coli; D013890:Thiourea
A22; FRET; Gram-negative; MreB; PBP2; RodA; RodZ; antibiotics; drug targets; growth inhibitors