ORIGINAL ARTICLE Validation for high-throughput screening of a VanRS-based reporter gene assay for bacterial cell wall inhibitors G. De Pascale, C. Grigoriadou, D. Losi, I. Ciciliato, M. Sosio and S. Donadio Vicuron Pharmaceuticals, Gerenzano, Italy Introduction The increased frequency of multidrug-resistant pathogens has prompted the search for novel antibacterial agents effective against the prevailing mechanisms of resistance. Substantial progress in laboratory robotics, together with the availability of large collections of chemical entities, have made high-throughput screening (HTS) with target- based assays the obvious choice for discovering novel drug candidates. Several asssays suitable for HTS have been recently described (e.g. Miesel et al. 2003; Donadio et al. 2005). The realization that the bacterial cells react to various stresses, including antibiotics, through specific transcriptional responses (Goh et al. 2002; Fischer et al. 2004), has resulted in a renewed interest in reporter gene assays for HTS (Lai and Kirsch 1996; Ulijasz et al. 1996; Mani et al. 1998; Bianchi and Baneyx 1999; Alksne et al. 2002; Hong et al. 2002; Shapiro and Baneyx 2002; Schuj- man et al. 2003; Fischer et al. 2004; Hutter et al. 2004; Mascher et al. 2004). These assays potentially combine the target specificity of cell-free systems with the presence of an intact bacterial cell. However, as the reporter assays do not measure the inhibition of a given pathway directly, an understanding of the actual mechanism(s) leading to expression of the reporter gene is an important validation step before entering a HTS programme. The bacterial cell wall is a recognized target for antibi- otics and reporter assays represent a simple and attractive tool for discovering novel classes of cell wall inhibitors (CWI). Indeed, many reporter strains have been described by employing different hosts and CWI-responsive pro- moters (Lai and Kirsch 1996; Ulijasz et al. 1996; Mani et al. 1998; Cao et al. 2002; Hong et al. 2002; Mascher et al. 2004). Of these, Ulijasz et al. (1996) reported that, Keywords antibiotics, beta-galactosidase, cell wall, HTS, reporter assay. Correspondence Stefano Donadio, KtedoGen, via Cav. Brusa 43, 21046 Malnate, Italy. E-mail: stefano_donadio@libero.it Present addresses Gianfranco De Pascale, Department of Chemistry, University of Warwick, UK Christina Grigoriadou, Department of Biology, University of Camerino, Italy Stefano Donadio, KtedoGen, Malnate, Italy 2006/0052: received 17 January 2006, revised 28 August 2006 and accepted 29 September 2006 doi:10.1111/j.1365-2672.2006.03231.x Abstract Aims: The present study was undertaken to validate, for antibiotic discovery, a reporter gene assay based on a Bacillus subtilis strain expressing the Enterococ- cus faecium vanRS genes and a vanH–lacZ fusion, which produced b-galactosi- dase activity in the presence of cell wall inhibitors (CWI) and lysozyme. Methods and Results: The reporter assay was miniaturized, automated and val- idated with antibiotics and tested against portions of chemical and microbial extract libraries. The assay is simple, fast and reproducible and can detect all CWI, sometimes at concentrations lower than those necessary to inhibit bacter- ial growth. However, some membrane-interfering compounds also generate comparable signals. While most CWI elicit a signal that is transcription- dependent and abolished in an osmoprotective medium, transcription is not required for b-galactosidase activity brought about by the membrane-interfer- ing compounds. Conclusions: At least two distinct mechanisms appear to lead to enzymatic activity in the reporter strain. Effective counterscreens can be designed to dis- card the undesired classes of compounds. Significance and Impact of the Study: Extensive validation is required before introducing a reporter assay in high-throughput screening. However, the ease of operation and manipulation makes the reporter assays powerful tools for antibiotic discovery. Journal of Applied Microbiology ISSN 1364-5072 ª 2006 The Authors Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 133–140 133