Robust High Throughput Real-time Monitoring Assay for the Specific Screening of Bacterial Cell Envelope Inhibitors

Abstract

Fluorescent dyes have been widely used for the assessment of the microbial and eukaryotic cell viability. However, the available methods require special protocols to be used in specific methods such as fluorescence microscopy and fluorescenceactivated cell sorting (FACS). Although simpler methods have been developed using these fluorescent dyes that directly provide a “read-out” for immediate estimation of cell survival, these require constant monitoring and manual intervention. To circumvent all of these problems, we have developed a robust automated high-throughput real-time cell viability monitoring assay using Sytox green dye. Our assay requires no manual intervention during bacterial cell growth. Furthermore, the assay is very specific for the cell membrane perturbing agents. As a proof-of-concept, we show E. coli growth in the presence of three different antibiotics that inhibit three different processes in the cell – Ampicillin, for cell wall; Kanamycin, for protein translation; Ofloxacin, for DNA replication; Nisin, for cell membrane. We show that the assay is very specific for Ampicillin and Nisin and does not respond to Kanamycin and Ofloxacin. This assay is performed in a 96-well microtitre plate which makes it possible to analyze, at the same time, several antibiotics and chemical compounds that have the potential to specifically disrupt the cell envelope. Our method is thus very rapid and specific, and can be efficiently used to screen a library of compounds. The assay was further tested on the D29 mycobacteriophage holin protein and its mutant HolG28D. The assay provided very distinct functional differences between the two proteins.