The emergence of drug-resistant tuberculosis (TB) constitutes a major challenge to TB control programmes. There is an urgent need to develop effective anti-TB drugs with novel mechanisms of action. Aspartate-semialdehyde dehydrogenase (ASADH) is the second enzyme in the aspartate metabolic pathway. The absence of the pathway in humans and the absolute requirement of aspartate in bacteria make ASADH a highly attractive drug target. In this study, we used ASADH coupled with Escherichia coli type III aspartate kinase (LysC) to establish a high-throughput screening method to find new anti-TB inhibitors. IMB-XMA0038 was identified as an inhibitor of MtASADH with an IC₅₀ value of 0.59 μg/mL through screening. The interaction between IMB-XMA0038 and MtASADH was confirmed by surface plasmon resonance (SPR) assay and molecular docking analysis. Furthermore, IMB-XMA0038 was found to inhibit various drug-resistant MTB strains potently with minimal inhibitory concentrations (MICs) of 0.25–0.5 μg/mL. The conditional mutant strain MTB::asadh cultured with different concentrations of inducer (10⁻⁵ or 10⁻¹ μg/mL pristinamycin) resulted in a maximal 16 times difference in MICs. At the same time, IMB-XMA0038 showed low cytotoxicity in vitro and vivo. In mouse model, it encouragingly declined the MTB colony forming units (CFU) in lung by 1.67 log10 dosed at 25 mg/kg for 15 days. In conclusion, our data demonstrate that IMB-XMA0038 is a promising lead compound against drug-resistant tuberculosis.

Aspartic acid semialdehyde dehydrogenase (ASADH) lies at the first branch point in the essential aspartic acid biosynthetic pathway that is found in bacteria and plants but is absent from animals. Mutations in the asadh gene encoding ASADH produce an inactive enzyme, which is lethal. Therefore, in this study, we investigated the hypothesis that ASADH represents a new anti-Mycobacterium tuberculosis (MTB) target. An asadh promoter-replacement mutant MTB, designated MTB::asadh, in which asadh gene expression is regulated by pristinamycin, was constructed to investigate the physiological functions of ASADH in the host bacteria. Bacterial growth was evaluated by monitoring OD₆₀₀ and ASADH expression was analyzed by Western blotting. The results showed that the growth and survival of MTB::asadh was completely inhibited in the absence of the inducer pristinamycin. Furthermore, the growth of the mutant was rigorously dependent on the presence of the inducer in the medium. The starved mutant exhibited a marked reduction (approximately 80%) in the cell wall materials compared to the wild-type, in addition to obvious morphological differences that were apparent in scanning electron microscopy studies; however, with the addition of pristinamycin, the cell wall contents and morphology similar to those of the wild-type strain were recovered. The starved mutant also exhibited almost no pathogenicity in an in vitro model of infection using mouse macrophage J774A.1 cells. The mutant showed a concentration-dependent recovery of pathogenicity with the addition of the inducer. These findings implicate ASADH as a promising target for the development of novel anti-MTB drugs.

The generation of conditional mutants has been an effective approach to studying bacteria and validating drug targets, and mutants of Mycobacteria are no exception. However unlike other bacteria, there is still a paucity of available tools for Mycobacteria. We constructed a new plasmid containing tetracycline-repressive expression system (TetRr1.7) and Xer Site-Specific recombinase system to generate label-free controllable expression strains. The plasmid was subsequently used to construct a strain of M. tuberculosis expressing the only copy of d-alanine:d-alanine ligase under the control of the tetracycline-repressive promoter. The results showed that the mutant strain lost the ability of colony formation, became more sensitive to d-cycloserine and the cell wall of the mutant strain was disrupted when anhydrotetracycline was added to the medium. Taken together these observations, confirmed that the expression of d-alanine:d-alanine ligase was tightly controlled by the promoter. In conclusion, the new plasmid is a convenient tool for constructing stable conditional mutant strains in Mycobacteria and can be used for future target identification.