Frontiers in microbiology
High-throughput LacZ/CPRG assay for identifying Gram-negative envelope-targeting antibiotics
By Ghobakhlou F, Mokhtari L, Pfeifer TA and 1 others.
Abstract
Objectives
We aimed to develop a liquid-based high-throughput screening (HTS) assay to identify small molecules that target GN bacterial envelope integrity and to assess their antibacterial potential, distinct from traditional viability-based screens.
Methods
Envelope disruption in Escherichia coli and Pseudomonas aeruginosa was assessed using a β-galactosidase (LacZ)/CPRG reporter assay in LB at 37 °C. The assay was validated by screening the LOPAC1280 and KD2 4761 compound libraries. Hit compounds were further characterized by concentration-response analysis, determination of permeabilization constants (K50), chloramphenicol combination permeabilization assays, minimal inhibitory concentration (MIC) measurements, and microscopy at sub-MICs. Confocal microscopy served as an orthogonal validation method.