Abstract
There is a pressing need to develop novel anti-tubercular drugs. High-throughput phenotypic screening yields chemical series that inhibit bacterial growth. Target identification for such series is challenging, but necessary for optimization of target engagement and the development of series into clinical drugs. We constructed a library of recombinant Mycobacterium tuberculosis strains each expressing a single protein from an inducible promoter as a tool for target identification. The library of 1733 clones was arrayed in 96-well plates for rapid screening and monitoring growth. The library contains the majority of the annotated essential genes as well as genes involved in cell wall and fatty acid biosynthesis, virulence factors, regulatory proteins, efflux, and respiration pathways. We evaluated the growth kinetics and plasmid stability over three passages for each clone in the library. We determined expression levels (mRNA and/or protein) in 396 selected clones. We screened the entire library and identified the Alr-expressing clone as the only recombinant strain, which grew in the presence of d-cycloserine (DCS). We confirmed that the Alr-expressing clone was resistant to DCS (7-fold shift in minimum inhibitory concentration). The library represents a new tool that can be used to screen for compound resistance and other phenotypes.
Highlights
One-quarter of the global population is infected withMycobacterium tuberculosis, the causative agent of tuberculosis (TB) [1, 2]
The majority of the clones were constructed by polymerase chain reaction (PCR) amplification from H37Rv genomic deoxyribonucleic acid (DNA) using oligonucleotides incorporating the Gateway recombination sequences; products were cloned into pDONR221 using BP clonase, and subsequently into pDTNF and/or pDTCF vectors using LR clonase (Thermo Fisher)
Since overexpression of a drug target often leads to resistance, we wanted to construct a library of overexpression strains that could be used to identify the targets, or mechanisms of resistance, to novel chemical agents
Summary
One-quarter of the global population is infected withMycobacterium tuberculosis, the causative agent of tuberculosis (TB) [1, 2]. Due to the increasing prevalence of drug-resistant tuberculosis and an ageing arsenal of anti-tuberculosis drugs, there is a pressing and continuing need to develop novel, welltolerated anti-tubercular drugs. Current TB drug discovery has focused on phenotypic screens against chemical libraries to identify compound scaffolds that inhibit bacterial growth [3, 4]. Treated as series, these compounds are optimized to minimize mammalian cytotoxicity while maintaining antibacterial activity and improving bioavailability. Identification of the target or metabolic pathway is crucial to drug discovery and development to improve on-target activity and specificity. Additional methods for target identification will increase the potential to identify relevant targets or metabolic pathways, enhancing drug development.
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