Towards establishing a method to screen for inhibitors of essential genes in mycobacteria: evaluation of the acetamidase promoter.
As a successful pathogen, Mycobacterium tuberculosis has effectively infected one-third of the world's population. Despite the existence of compound libraries developed by recent advances in combinatorial chemistry, few compounds have been screened against M. tuberculosis. The use of a regulable promoter to control the level of expression of a drug target in living organisms has been shown to be advantageous compared with targetless whole-cell-based or in vitro biochemical screening approaches towards antibiotic discovery. In this study, we demonstrate that the acetamidase promoter from Mycobacterium smegmatis responds in a dose-dependent manner to different concentrations of its inducer acetamide. Using this promoter to regulate expression of a zeocin resistance gene in M. smegmatis, we show that the test strain exhibits increased sensitivity to zeocin at a low concentration of acetamide compared with a fully resistant phenotype at high doses of the inducer. This model system has indicated the feasibility of using a regulable promoter in designing a whole-cell-based high throughput screen for specific inhibitors against potential drug targets of M. tuberculosis.
Raghunand
Tirumalai R
TR
Center for Tuberculosis Research, Department of Medicine, The Johns Hopkins University, School of Medicine, Baltimore, MD 21231-1001, USA.
Bishai
William R
WR
Chen
Ping
P
D000581:Amidohydrolases / Q000235:genetics*; D001483:Base Sequence; D001761:Bleomycin / Q000494:pharmacology; D017931:DNA Primers; D004352:Drug Resistance, Microbial; D005798:Genes, Bacterial / Q000187:drug effects*; D009161:Mycobacterium / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D011401:Promoter Regions, Genetic
D017931:DNA Primers; D001761:Bleomycin; C105427:Zeocin; D000581:Amidohydrolases; C021445:acetamidase