Validation for high-throughput screening of a VanRS-based reporter gene assay for bacterial cell wall inhibitors.
OBJECTIVE
The present study was undertaken to validate, for antibiotic discovery, a reporter gene assay based on a Bacillus subtilis strain expressing the Enterococcusfaecium vanRS genes and a vanH-lacZ fusion, which produced beta-galactosidase activity in the presence of cell wall inhibitors (CWI) and lysozyme.


RESULTS
The reporter assay was miniaturized, automated and validated 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 bacterial 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 beta-galactosidase activity brought about by the membrane-interfering compounds.


CONCLUSIONS
At least two distinct mechanisms appear to lead to enzymatic activity in the reporter strain. Effective counterscreens can be designed to discard the undesired classes of compounds.


CONCLUSIONS
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.
De Pascale
G
G
Vicuron Pharmaceuticals, Gerenzano, Italy.
Grigoriadou
C
C
Losi
D
D
Ciciliato
I
I
Sosio
M
M
Donadio
S
S
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001412:Bacillus subtilis / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D002473:Cell Wall / Q000187:drug effects; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005798:Genes, Bacterial; D017930:Genes, Reporter; D008826:Microbial Sensitivity Tests / Q000379:methods*; D009994:Osmolar Concentration; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000378:metabolism
D000900:Anti-Bacterial Agents; D003902:Detergents; D001616:beta-Galactosidase