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The stringent response enzyme is a promising new target for Mycobacterium tuberculosis persisters., The stringent response enables Mycobacterium tuberculosis (Mtb) to shut down its replication and metabolism under various stresses. Here we show that Mtb lacking the stringent response enzyme RelMtb was unable to slow its replication rate during nutrient starvation. Metabolomics analysis revealed that the nutrient-starved relMtb-deficient strain had increased metabolism similar to that of exponentially growing wild-type bacteria in nutrient-rich broth, consistent with an inability to enter quiescence. Deficiency of relMtb increased the susceptibility of mutant bacteria to killing by isoniazid during nutrient starvation and in the lungs of chronically infected mice. We screened a pharmaceutical library of over 2 million compounds for inhibitors of RelMtb and showed that the lead compound X9 was able to directly kill nutrient-starved M. tuberculosis and enhanced the killing activity of isoniazid. Inhibition of RelMtb is a promising approach to target M. tuberculosis persisters, with the potential to shorten the duration of TB treatment.

Here we describe the development and validation of an intracellular high-throughput screening assay for finding new antituberculosis compounds active in human macrophages. The assay consists of a luciferase-based primary identification assay, followed by a green fluorescent protein-based secondary profiling assay. Standard tuberculosis drugs and 158 previously recognized active antimycobacterial compounds were used to evaluate assay robustness. Data show that the assay developed is a short and valuable tool for the discovery of new antimycobacterial compounds.