Heterologous Expression of ethA and katG in Mycobacterium marinum Enables the Rapid Identification of New Prodrugs Active against Mycobacterium tuberculosis

Abstract

Screening strategies for anti-tuberculosis compounds using Mycobacterium tuberculosis are time-consuming and require BSL-3 facilities, which makes the development of high-throughput assays difficult and expensive. Mycobacterium marinum, a close genetic relative of M. tuberculosis, possesses several advantages as a suitable model for tuberculosis drug screening. However, despite the high genetic similarity, there are some obvious differences in susceptibility to some tuberculosis drugs between these two species, especially for the pro-drugs ethionamide and isoniazid. In this study, we aimed to improve M. marinum as a model for anti-tuberculosis drugs identification by heterologous expression of two common drug activators, EthA and KatG. These two activators were overexpressed in M. marinum and the strains were tested against ethionamide, isoniazid and a library of established antimycobacterial compounds from TbAlliance to compare drug susceptibility. Both in vitro and in vivo using zebrafish larvae, these genetically-modified M. marinum strains showed significantly higher susceptibility against ethionamide and isoniazid, which require activation by EthA and KatG. More importantly, a strain overexpressing both ethA and katG was potentially more susceptible to approximately 20% of the anti-tuberculosis hit compounds from the TB Alliance library. Most of these compounds were activated by EthA in M. marinum. Four of these compounds were selected for further analysis and three of them showed obvious EthA-dependent activity against M. tuberculosis. Overall, our developed M. marinum strains are valuable tools for high-throughput discovery of potential novel anti-tuberculosis pro-drugs.

An anti-tuberculosis compound screen using a zebrafish infection model identifies an aspartyl-tRNA synthetase inhibitor

“…After the setup of the in vivo screening approach, the platform was used to screen compounds from an anti-Mtb hit library [provided by TB Alliance (TBA)] for their in vivo activity. This library comprises 1392 compounds previously shown to inhibit Mtb viability in vitro ( Ho et al, 2021 ) . All compounds that showed ≥80% inhibition of M. marinum viability in vitro at 10 µM (240 compounds) were selected and tested further in the zebrafish- M. marinum infection model via automated yolk injection and waterborne treatment using a single dose (10 µM) ( Fig.…”

“…Furthermore, zebrafish embryos were used to evaluate the efficacy and toxicity of several anti-TB drugs ( Ho et al, 2021 ; Makarov et al, 2014 ; Ordas et al, 2015 ), including PBTZ169 (macozinone), which is currently in phase 2 of a clinical trial. The M. marinum -zebrafish model can be established by injecting bacteria via the caudal vein or by injection in the yolk.…”

Diving into drug-screening: zebrafish embryos as an in vivo platform for antimicrobial drug discovery and assessment

“…Different studies have employed varying numbers of embryos per experimental group. For instance, studies using a single bacterial strain with different compound treatments have utilized 5–10 embryos per treatment group (Ho et al 2021 , Winters et al 2022 ), 12–15 embryos (Habjan et al 2021 , Schouten et al 2022 ), as well as 20–30 (Ordas et al 2015 , Dalton et al 2017 , Goh et al 2023 ) or even more than 30 zebrafish embryos per experimental group (Wambaugh et al 2017 ). The optimal number may vary depending on experimental requirements and constraints.…”

“…Besides showing the activity of known antibiotics like delamanid, pretonamid, and rifampicin, they also showed the activity of two novel pretonamid analogues SN30527 and SN30488. Another study described several zebrafish-active prodrugs (Ho et al 2021 ), which were identified by using the M. marinum strain overexpressing katG and ethA , two common prodrug-activating enzymes. Furthermore, the efficacy of several nitronaphthofuran derivatives was investigated in a zebrafish– M. marinum model, where compounds were injected into the zebrafish posterior cardinal vein, which is the vein that follows the upper side of the yolk extension and leads to the caudal vein (Thisse et al 2001 ).…”

“…However, it should be noted that experiments with negative outcomes are rarely published; thus, the positive outcomes might be over-represented. The differences between in vitro and in vivo activity can steer both ways; potential antimicrobials may show better in vitro activity than in vivo efficacy, but they can also appear more active in vivo compared to in vitro (Ordas et al 2015 , Habjan et al 2021 , Ho et al 2021 , Schouten et al 2022 ). The former can likely be attributed to in vivo ADME properties (Diekman and Hill 2013 ), the latter, however, is not as easily explained.…”