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

Screening strategies for antituberculosis compounds using are time consuming and require biosafety level 3 (BSL3) facilities, which makes the development of high-throughput assays difficult and expensive. , a close genetic relative of , 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 prodrugs ethionamide and isoniazid. In this study, we aimed to improve as a model for antituberculosis drug identification by heterologous expression of two common drug activators, EthA and KatG. These two activators were overexpressed in , and the strains were tested against ethionamide, isoniazid, and a library of established antimycobacterial compounds from TB Alliance to compare drug susceptibility. Both and using zebrafish larvae, these genetically modified strains showed significantly higher susceptibility against ethionamide and isoniazid, which require activation by EthA and KatG. More importantly, a strain overexpressing both and was potentially more susceptible to approximately 20% of the antituberculosis hit compounds from the TB Alliance library. Most of these compounds were activated by EthA in Four of these compounds were selected for further analysis, and three of them showed obvious EthA-dependent activity against Overall, our developed strains are valuable tools for high-throughput discovery of potential novel antituberculosis prodrugs.