Construction of an overexpression library for

Area of Science:

• Microbiology
• Drug Discovery
• Genetics

Background:

• Developing novel anti-tubercular drugs is critical to combat tuberculosis.
• High-throughput screening identifies compounds inhibiting bacterial growth, but target identification remains a challenge.
• Understanding drug targets is essential for optimizing drug engagement and clinical development.

Purpose of the Study:

• To construct a comprehensive library of recombinant Mycobacterium tuberculosis strains for target identification.
• To create a tool for screening compound resistance and other phenotypes.
• To facilitate the development of new anti-tubercular therapies.

Main Methods:

• A library of 1733 recombinant Mycobacterium tuberculosis strains was created, each expressing a single protein from an inducible promoter.
• The library was arrayed in 96-well plates for efficient screening and growth monitoring.
• Growth kinetics, plasmid stability, and expression levels were evaluated; the library was screened for resistance to d-cycloserine (DCS).

Main Results:

• The library encompasses essential genes and those involved in key metabolic and virulence pathways.
• The Alr-expressing clone was identified as the only strain resistant to DCS, exhibiting a 7-fold increase in minimum inhibitory concentration.
• The developed library proved effective in identifying a specific drug resistance phenotype.

Conclusions:

• The constructed Mycobacterium tuberculosis library is a valuable new tool for drug target identification.
• This resource can accelerate the discovery and development of novel anti-tubercular agents.
• The library enables screening for various phenotypes, aiding in understanding drug mechanisms and resistance.