Construction of an overexpression library for Mycobacterium tuberculosis

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.

Comparative fitness analysis of D-cycloserine resistant mutants reveals both fitness-neutral and high-fitness cost genotypes

“…This mutation has not yet been found in the clinic. In agreement with our results, Alr overexpression was shown to be sufficient to cause DCS resistance in M. tuberculosis , where overexpression of Alr caused a sevenfold increase in the MIC of DCS 47 . The most common DCS resistance mechanism involves Alr modification (D322’N).…”

Identification of Enolase as the Target of 2-Aminothiazoles in Mycobacterium tuberculosis

“…To validate enolase as a target of 2-AT compounds in M. tuberculosis , we generated a strain which overexpressed Eno (Rv1023) (Table 1 ). We used an ATc-inducible promoter to control eno expression ( Melief et al, 2018 ). If Eno is the target of 2-AT compounds, we expect that overexpression of Eno would make the bacteria more resistant to these compounds.…”

Biosynthesis of Galactan in Mycobacterium tuberculosis as a Viable TB Drug Target?

“…Despite their commonality, whole-cell screens are limited by the chemical diversity available in the commercial or proprietary libraries and, in the case of UGM Mtb , this shortcoming can be exemplified by the results of experimental and in silico screening of available compound collections. Nevertheless, we imagine that recent technological advances, both computational and experimental, in the field of tuberculosis drug development [4,12,[109][110][111][112][113] provide a promising avenue for therapeutic innovation. Moreover, critical information concerning the galactan biosynthesis pathway has been delineated, including the structures of UGM Mtb [53] and GlfT2 Mtb [63].…”