Identification of Mutations Conferring Tryptanthrin Resistance to Mycobacterium smegmatis

Abstract

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a global burden, responsible for over 1 million deaths annually. The emergence and spread of drug-resistant M. tuberculosis strains (MDR-, XDR- and TDR-TB) is the main challenge in global TB-control, requiring the development of novel drugs acting on new biotargets, thus able to overcome the drug-resistance. Tryptanthrin is a natural alkaloid, with great therapeutic potential due to its simple way of synthesis and wide spectrum of biological activities including high bactericidal activity on both drug-susceptible and MDR M. tuberculosis strains. InhA was suggested as the target of tryptanthrins by in silico modeling, making it a promising alternative to isoniazid, able to overcome drug resistance provided by katG mutations. However, neither the mechanism of action of tryptanthrin nor the mechanism of resistance to tryptanthrins was ever confirmed in vitro. We show that the MmpS5-MmpL5 efflux system is able to provide resistance to tryptanthrins using an in-house test-system. Comparative genomic analysis of spontaneous tryptanthrin-resistant M. smegmatis mutants showed that mutations in MSMEG_1963 (EmbR transcriptional regulator) lead to a high-level resistance, while those in MSMEG_5597 (TetR transcriptional regulator) to a low-level one. Mutations in an MFS transporter gene (MSMEG_4427) were also observed, which might be involved in providing a basal level of tryptanthrins-resistance.

Transcriptomic Profile of Mycobacterium smegmatis in Response to an Imidazo[1,2-b][1,2,4,5]tetrazine Reveals Its Possible Impact on Iron Metabolism

“…These mutations result in mmpS5–mmpL5 overexpression and increased efflux of the compounds from the cell ( Maslov et al, 2020 ). A similar mechanism of resistance was also shown for bedaquiline and clofazimine ( Hartkoorn et al, 2014 ), azoles ( Milano et al, 2009 ), thiacetazone derivatives ( Halloum et al, 2017 ), and tryptanthrins ( Frolova et al, 2021 ) in various mycobacterial species. However, the mode of action, as well as biotarget(s) of imidazo[1,2- b ][1,2,4,5]tetrazines have not yet been confirmed in vitro .…”

Repurposing Based Identification of Novel Inhibitors against MmpS5-MmpL5 Efflux Pump of Mycobacterium smegmatis: A Combined In Silico and In Vitro Study

“…Moreover, we were able to show that BDE_26593610 and BDD_27860195 can be considered active MmpS5-MmpL5 inhibitors, as they were able to sensitize in vitro mmpS5-mmpL5 overexpressing M. smegmatis atr9c strain to the tested imidazo[1,2-b][1,2,4,5]tetrazine and the tryptanthrins, while M. smegmatis mc2 155 (with a w.t. expression level of the mmpS5-mmpL5 operon) was also sensitized by these compounds to PK31, confirming that the MmpS5-MmpL5 efflux specifically provides a basal level of resistance to this tryptanthrin derivative [ 15 ].…”

“…Briefly, M. smegmatis cultures were grown overnight in Middlebrook 7H9 broth to mid-exponential phase (OD 600 = 1.2), and afterwards were diluted in the proportion of 1:9:10 (culture:water:soyabean-casein digest agar) and 5 mL were poured as the top layer on Petri dishes with soyabean-casein digest agar. After the plates have dried for at least 30 min, paper discs with antimycobacterial agents (imidazo[1,2-b][1,2,4,5]tetrazine 3a [ 35 ], tryptanthrin (TRP) and 8-fluorotryptanthrin (PK31) [ 15 ]) subjected to MmpS5-MmpL5 efflux alone, paper discs with tested inhibitors alone, and paper discs with the combination of an antimycobacterial agent and an inhibitor were plated on the agar. Plates were incubated for 2 days at 37 °C, until the bacterial lawn was fully grown, and the growth inhibition halos were clearly visible.…”

“…The mycobacterial MmpS5-MmpL5 efflux system was found to have a role in the development of tolerance against anti-TB drugs and drug candidates [ 11 ] in different mycobacterial species, including bedaquiline, clofazimine, and azoles in M. tuberculosis [ 7 , 12 ], thiacetazone derivatives in M. abscessus [ 13 ], and imidazo[1,2-b][1,2,4,5]tetrazines, and tryptanthrins in M. smegmatis [ 14 , 15 ]. In M. tuberculosis H37Rv strain, there are 14 genes expressed to form MmpL proteins which showed structural similarities to the members of HAE family of efflux proteins which belong to the RND superfamily of transporter proteins [ 11 ].…”

Genetic interaction between F‐box motif encoding YDR131C and retrograde signaling‐related RTG1 regulates the stress response and apoptosis in Saccharomyces cerevisiae

“…We analyzed the cellular growth response of the WT, ydr131cΔ, rtg1Δ, and ydr131cΔrtg1Δ cells in presence of chemical agent 8fluorotryptanthrin (a small molecule synthesized and named as RK64). [57,58] RK64 as well as other related tryptanthrin analogs are known to act as immunomodulatory, antineoplastic agents and inhibit indole amine 2,3-dioxygenase-1 (IDOI) enzyme. Tryptanthrin has attracted the attention of both synthetic chemists and biological researchers due to its vast spectrum of biological activities and synthetic possibilities around the core structure.…”