The anti-tuberculosis drug ethionamide (ETH), which is a structural analog of isoniazid (INH), is known to strongly inhibit mycolic acid synthesis in Mycobacterium tuberculosis. Although several targets have been identified for INH, only speculative information is available concerning ETH. Mutations within the promoter and the coding region of enoyl-acyl carrier protein reductase (InhA) were found to confer resistance to both drugs, thus leading to the impression that INH and ETH may share a common mode of action. However, a notable distinction between the two drugs lies in the lack of cross-resistance in clinical isolates. This may be attributed in part to the fact that the pro-drug INH must be activated via KatG, and no activation step for ETH has yet been described. Here we report the identification of an activator for ETH. The ETH activator (Rv3854c), which we have termed EthA, was found to be homologous to various monooxygenases and induced ETH sensitivity when overexpressed in mycobacteria. Interestingly, the neighboring open reading frame (Rv3855), which was found homologous to transcriptional repressors of the tetR family, led to ETH resistance when overexpressed. In addition, chromosomal inactivation of this gene by transposition led to ETH hypersensitivity. These data strongly suggest that Rv3855, which we have termed EthR, regulates the production of EthA, which subsequently activates the pro-drug ETH. This study opens up new avenues of research relating to ETH activation in mycobacteria, possibly leading to an improved efficacy of ETH and to the generation of new anti-mycobacterial agents.

Prevention efforts and control of tuberculosis are seriously hampered by the appearance of multidrug-resistant strains of Mycobacterium tuberculosis, dictating new approaches to the treatment of the disease. Thiolactomycin (TLM) is a unique thiolactone that has been shown to exhibit anti-mycobacterial activity by specifically inhibiting fatty acid and mycolic acid biosynthesis. In this study, we present evidence that TLM targets two beta-ketoacyl-acyl-carrier protein synthases, KasA and KasB, consistent with the fact that both enzymes belong to the fatty-acid synthase type II system involved in fatty acid and mycolic acid biosynthesis. Overexpression of KasA, KasB, and KasAB in Mycobacterium bovis BCG increased in vivo and in vitro resistance against TLM. In addition, a multidrug-resistant clinical isolate was also found to be highly sensitive to TLM, indicating promise in counteracting multidrug-resistant strains of M. tuberculosis. The design and synthesis of several TLM derivatives have led to compounds more potent both in vitro against fatty acid and mycolic acid biosynthesis and in vivo against M. tuberculosis. Finally, a three-dimensional structural model of KasA has also been generated to improve understanding of the catalytic site of mycobacterial Kas proteins and to provide a more rational approach to the design of new drugs.

Summary: Ethionamide (ETH) is an important second‐line antitubercular drug used for the treatment of patients infected with multidrug‐resistant Mycobacterium tuberculosis. Although ETH is a structural analogue of isoniazid, only little cross‐resistance to these two drugs is observed among clinical isolates. Both isoniazid and ETH are pro‐drugs that need to be activated by mycobacterial enzymes to exert their antimicrobial activity. We have recently identified two M. tuberculosis genes, Rv3854c (ethA) and Rv3855 (ethR), involved in resistance to ETH. ethA encodes a protein that belongs to the Flavin‐containing monooxygenase family catalysing the activation of ETH. We show here that ethR, which encodes a repressor belonging to the TetR/CamR family of transcriptional regulators, negatively regulates the expression of ethA. By the insertion of the ethA promoter region upstream of the lacZ reporter gene, overexpression of ethR in trans was found to cause a strong inhibition of ethA expression, independently of the presence of ETH in the culture media. Electrophoretic mobility shift assays indicated that EthR interacts directly with the ethA promoter region. This interaction was confirmed by DNA footprinting analysis, which, in addition, identified the EthR‐binding region. Unlike other TetR/CamR members, which typically bind 15 bp operators, EthR recognises an unusually long 55 bp region suggesting multimerization of the repressor on its operator. Identification by primer‐extension of the ethA transcriptional start site indicated that it is located within the EthR‐binding region. Taken together, bacterial two‐hybrid experiments and gel filtration assays suggested a dimerization of EthR in the absence of its operator. In contrast, surface plasmon resonance analyses showed that eight EthR molecules bind cooperatively to the 55 bp operator, which represents a novel repression mechanism for a TetR/CamR member.

The thiourea isoxyl (thiocarlide; 4,4′-diisoamyloxydiphenylthiourea) is known to be an effective anti-tuberculosis drug, active against a range of multidrug-resistant strains of Mycobacterium tuberculosis and has been used clinically. Little was known of its mode of action. We now demonstrate that isoxyl results in a dose-dependent decrease in the synthesis of oleic and, consequently, tuberculostearic acid in M. tuberculosis with complete inhibition at 3 μg/ml. Synthesis of mycolic acid was also affected. The anti-bacterial effect of isoxyl was partially reversed by supplementing growth medium with oleic acid. The specificity of this inhibition pointed to a Δ9-stearoyl desaturase as the drug target. Development of a cell-free assay for Δ9-desaturase activity allowed direct demonstration of the inhibition of oleic acid synthesis by isoxyl. Interestingly, sterculic acid, a known inhibitor of Δ9-desaturases, emulated the effect of isoxyl on oleic acid synthesis but did not affect mycolic acid synthesis, demonstrating the lack of a relationship between the two effects of the drug. The three putative fatty acid desaturases in the M. tuberculosis genome, desA1, desA2, and desA3, were cloned and expressed in Mycobacterium bovis BCG. Cell-free assays and whole cell labeling demonstrated increased Δ9-desaturase activity and oleic acid synthesis only in the desA3-overexpressing strain and an increase in the minimal inhibitory concentration for isoxyl, indicating that DesA3 is the target of the drug. These results validate membrane-bound Δ9-desaturase, DesA3, as a new therapeutic target, and the thioureas as anti-tuberculosis drugs worthy of further development.