Antibiotics pp 237-245 | Cite as

Cell-Based Fluorescent Screen to Identify Inhibitors of Bacterial Translation Initiation

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1520)

Abstract

A strategy that can be applied to the research of new molecules with antibacterial activity is to look for inhibitors of essential bacterial processes within large collections of chemically heterogeneous compounds. The implementation of this approach requires the development of proper assays aimed at the identification of molecules interfering with specific cell pathways and potentially applicable to the high throughput analysis of large chemical library. Here, I describe a fluorescence-based whole-cell assay in Escherichia coli devised to find inhibitors of the translation initiation pathway. Translation is a complex and essential mechanism. It involves numerous sub-steps performed by factors that are in many cases sufficiently dissimilar in bacterial and eukaryotic cells to be targetable with domain-specific drugs. As a matter of fact, translation has been proven as one of the few bacterial mechanisms pharmacologically tractable with specific antibiotics. The assay described in this chapter is tailored to the identification of molecules affecting the first stage of translation initiation, which is the most dissimilar step in bacteria vs. mammals. The effect of the compounds under analysis is assayed in living cells, thus allowing evaluating their in vivo performance as inhibitors of translation initiation. Compared with other assays for antibacterials, the major advantages of this screen are its simplicity and high mechanism specificity.

Key words

Translation initiation Whole-cell assay Gram-negative bacteria Leaderless mRNA S1 ribosomal protein Ribosome Antibacterial compounds 

Notes

Acknowledgments

I thank lab members who were involved in the assay setup and in the screening of the Prestwick library, and in particular Matteo Raneri. The development of the assay was supported by the Italian Cystic Fibrosis Research Foundation (grant FFC#8/2013 sponsored by the FFC Delegation of Montebelluna “La bottega delle donne”).

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Dipartimento di BioscienzeUniversità degli Studi di MilanoMilanItaly

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