Indole Propionic Acid Disturbs the Normal Function of Tryptophanyl-tRNA Synthetase in Mycobacterium tuberculosis.

Tuberculosis (TB) is the leading infectious disease caused by Mycobacterium tuberculosis and the second-most contagious killer after COVID-19. The emergence of drug-resistant TB has caused a great need to identify and develop new anti-TB drugs with novel targets. Indole propionic acid (IPA), a structural analog of tryptophan (Trp), is active against M. tuberculosis in vitro and in vivo. It has been verified that IPA exerts its antimicrobial effect by mimicking Trp as an allosteric inhibitor of TrpE, which is the first enzyme in the Trp synthesis pathway of M. tuberculosis. However, other Trp structural analogs, such as indolmycin, also target tryptophanyl-tRNA synthetase (TrpRS), which has two functions in bacteria: synthesis of tryptophanyl-AMP by catalyzing ATP + Trp and producing Trp-tRNATrp by transferring Trp to tRNATrp. So, we speculate that IPA may also target TrpRS. In this study, we found that IPA can dock into the Trp binding pocket of M. tuberculosis TrpRS (TrpRSMtb), which was further confirmed by isothermal titration calorimetry (ITC) assay. The biochemical analysis proved that TrpRS can catalyze the reaction between IPA and ATP to generate pyrophosphate (PPi) without Trp as a substrate. Overexpression of wild-type trpS in M. tuberculosis increased the MIC of IPA to 32-fold, and knock-down trpS in Mycolicibacterium smegmatis made it more sensitive to IPA. The supplementation of Trp in the medium abrogated the inhibition of M. tuberculosis by IPA. We demonstrated that IPA can interfere with the function of TrpRS by mimicking Trp, thereby impeding protein synthesis and exerting its anti-TB effect.

Amino acid 17 in QRDR of Gyrase A plays a key role in fluoroquinolones susceptibility in mycobacteria.

IMPORTANCE: Fluoroquinolones (FQs) play a key role in the treatment regimens against tuberculosis and non-tuberculous mycobacterial infections. However, there are significant differences in the sensitivities of different mycobacteria to FQs. In this study, we proved that this is associated with the polymorphism at amino acid 17 of quinolone resistance-determining region of Gyrase A by gene editing. This is the first study using CRISPR-associated recombination for gene editing in Mycobacterium abscessus to underscore the contribution of the amino acid substitutions in GyrA to FQ susceptibilities in mycobacteria.

Comparative genome analysis reveals high-level drug resistance markers in a clinical isolate of Mycobacterium fortuitum subsp. fortuitum MF GZ001.

Introduction: Infections caused by non-tuberculosis mycobacteria are significantly worsening across the globe. M. fortuitum complex is a rapidly growing pathogenic species that is of clinical relevance to both humans and animals. This pathogen has the potential to create adverse effects on human healthcare. Methods: The MF GZ001 clinical strain was collected from the sputum of a 45-year-old male patient with a pulmonary infection. The morphological studies, comparative genomic analysis, and drug resistance profiles along with variants detection were performed in this study. In addition, comparative analysis of virulence genes led us to understand the pathogenicity of this organism. Results: Bacterial growth kinetics and morphology confirmed that MF GZ001 is a rapidly growing species with a rough morphotype. The MF GZ001 contains 6413573 bp genome size with 66.18 % high G+C content. MF GZ001 possesses a larger genome than other related mycobacteria and included 6156 protein-coding genes. Molecular phylogenetic tree, collinearity, and comparative genomic analysis suggested that MF GZ001 is a novel member of the M. fortuitum complex. We carried out the drug resistance profile analysis and found single nucleotide polymorphism (SNP) mutations in key drug resistance genes such as rpoB, katG, AAC(2')-Ib, gyrA, gyrB, embB, pncA, blaF, thyA, embC, embR, and iniA. In addition, the MF GZ001strain contains mutations in iniA, iniC, pncA, and ribD which conferred resistance to isoniazid, ethambutol, pyrazinamide, and para-aminosalicylic acid respectively, which are not frequently observed in rapidly growing mycobacteria. A wide variety of predicted putative potential virulence genes were found in MF GZ001, most of which are shared with well-recognized mycobacterial species with high pathogenic profiles such as M. tuberculosis and M. abscessus. Discussion: Our identified novel features of a pathogenic member of the M. fortuitum complex will provide the foundation for further investigation of mycobacterial pathogenicity and effective treatment.