Mycobacterium tuberculosis Reporter Strains as Tools for Drug Discovery and Development

Abstract

Reporter strains have proven to be powerful tools to study Mycobacterium tuberculosis (Mtb) physiology. Transcriptional and translational reporter strains are engineered by fusing a readout gene, encoding a fluorescent, luminescent or enzymatic protein, downstream of a promoter or in-frame with a gene of interest. When the reporter is expressed, it generates a signal that acts as a synthetic phenotype, enabling the study of physiologies that might have otherwise been hidden. This review will discuss approaches for generating reporter strains in Mtb and how they can be used as tools for high-throughput genetic and small molecule screening and as biomarkers for examining Mtb responses to drug or immune stresses during animal infections. Fluorescent reporter strains have an added benefit in that they can be used for single-cell studies both in vitro and in vivo, thus enabling the study of mechanisms underlying phenotypic heterogeneity. Recent examples of the use of Mtb reporter strains will be presented with a focus on how they can be used as tools for drug discovery and development. © 2018 IUBMB Life, 70(9):818-825, 2018.

Inhibition of Mycobacterium tuberculosis DosRST two-component regulatory system signaling by targeting response regulator DNA binding and sensor kinase heme

“…Fluorescent reporter strains can be used as tools in drug discovery to conduct targeted, whole cell phenotypic screens for pathways that play a role during pathogenesis, but have limited impact when bacteria are grown in vitro (25, 26). In an effort to discover the new chemical probes that inhibit Mtb persistence, we previously performed a whole cell phenotypic high throughput screening (HTS) of a >540,000 compound library using the DosRST regulon reporter strain CDC1551 ( hspX’:: GFP) (27).…”

Development and Optimization of Chromosomally-Integrated Fluorescent Mycobacterium tuberculosis Reporter Constructs

“…One method to accomplish the brightness of the new strain was to exploit the variety of available FPs. Although several green and red FPs are described to have higher molecular brightness than eGFP and mCherry, those two FPs are still most frequently used for the development of Mtb reporter strains (Abramovitch, 2018;MacGilvary and Tan, 2018). In this study, our green FP of choice was mWasabi, an FP derived from mTFP1 that exhibits high photostability, preferable narrow excitation and emission spectra, and improved molecular brightness relative to eGFP (Ai et al, 2008).…”

“…To date, several fluorescent mycobacterial reporter strains have been successfully applied in mice and have revealed novel insights about the local microenvironment and bacteriahost interactions (Abramovitch, 2018;MacGilvary and Tan, 2018). For instance, Abramovitch et al (2011) and Tan et al (2013) developed reporter strains that monitor pH, an environmental cue known to be important for Mtb in vitro growth (Supplementary Figure 1A) and intracellular survival (Vandal et al, 2009).…”

“…The drawback of current reporter strains is the use of replicating plasmids, which are not suitable for long term mouse infections or studies in non-human primates due to the loss of the reporter construct over time (our unpublished data; Abramovitch, 2018). Furthermore, relying on a single plasmid limits the number of responsive elements that can be introduced into one reporter strain, thereby restricting the ability to correlate different environmental factors and gain deeper insights into the complexity of the microenvironment.…”

Tuberculosis: Today's researches—tomorrow's therapies

“…This heterogeneity in bacterial physiology is recognized as an important bottleneck in developing new drugs as a subset of bacteria inside host niches may adopt a drug-tolerant physiological state. In this context, the fourth review by Robert Abramovitch (7) discusses approaches for generating reporter strains in Mtb to uncover physiological heterogeneity during infection, setup high-throughput genetic and small molecule screens, and to measure response to current/experimental drug pressures. The reporter strains were constructed by expressing transcriptional (promoter-based) or translational fusion of stress-responsive genes with the reporter proteins exhibiting fluorescence, luminescence or enzymatic activity.…”