Molecular Microbiology (2008) 69(5), 1316–1329 ᭿ doi:10.1111/j.1365-2958.2008.06365.x First published online 21 July 2008 Mycothiol biosynthesis is essential for ethionamide susceptibility in Mycobacterium tuberculosis ᭿ OnlineOpen: This article is available free online at www.blackwell-synergy.com Catherine Vilchèze,1 Yossef Av-Gay,2 mice. Mutations in mshA demonstrate the non- Rodgoun Attarian,2 Zhen Liu,3 Manzour H. Hazbón,4† essentiality of mycothiol for growth in vitro and in Roberto Colangeli,4 Bing Chen,1 Weijun Liu,1 vivo, and provide a novel mechanism of ethionamide David Alland,4 James C. Sacchettini3 and resistance in M. tuberculosis. William R. Jacobs Jr1* 1 Howard Hughes Medical Institute, Department of Introduction Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA. The increase in drug resistance in Mycobacterium tuber- 2Division of Infectious Diseases, University of British culosis clinical isolates has impeded the full success of Columbia, Vancouver, BC V5Z 3J5, Canada. tuberculosis (TB) control. The WHO estimates that 4.3% 3Department of Biochemistry and Biophysics, Texas of the newly and previously treated TB cases are multi- A&M University, College Station, TX 77843, USA. drug-resistant (MDR) meaning that these strains are 4Division of Infectious Disease, Department of Medicine resistant to at least the two best anti-TB drugs: isoniazid and the Ruy V. Lourenço Center for the Study of (INH) and rifampicin (Zignol et al., 2006). Alarmingly, Emerging and Re-emerging Pathogens, New Jersey there has been an emergence of M. tuberculosis strains Medical School, University of Medicine and Dentistry resistant to four to seven TB drugs (termed XDR-TB for of New Jersey, Newark, NJ 07103, USA. extensively drug-resistant TB) that have been associated with the rapid death of HIV-infected individuals (Gandhi Summary et al., 2006; Shah et al., 2007). A more effective treatment for both MDR- and XDR-TB strains requires rapid detec- Spontaneous mutants of Mycobacterium tuberculo- tion and therefore understanding of all the mechanisms sis that were resistant to the anti-tuberculosis drugs leading to drug resistance. ethionamide and isoniazid were isolated and found Isoniazid, the cornerstone of front-line TB treatment, to map to mshA, a gene encoding the first enzyme shares a common target with the second-line TB drug involved in the biosynthesis of mycothiol, a major ethionamide (ETH). Both INH and ETH are pro-drugs low-molecular-weight thiol in M. tuberculosis. Seven that require activation to form adducts with NAD to sub- independent missense or frameshift mutations sequently inhibit InhA, the NADH-dependent enoyl-ACP within mshA were identified and characterized. reductase (Dessen et al., 1995; Quemard et al., 1995) of Precise null deletion mutations of the mshA gene the fatty acid biosynthesis type II system (Marrakchi et al., were generated by specialized transduction in three 2000). However, activation of INH and of ETH occurs different strains of M. tuberculosis. The mshA dele- through different pathways. INH is activated by the katG- tion mutants were defective in mycothiol biosynthe- encoded catalase peroxidase (Zhang et al., 1992; Wilming sis, were only ethionamide-resistant and required and Johnsson, 1999) to form the INH-NAD adduct catalase to grow. Biochemical studies suggested (Rozwarski et al., 1998). ETH, on the other hand, that the mechanism of ethionamide resistance in is activated by the ethA-encoded mono-oxygenase mshA mutants was likely due to a defect in ethiona- (Baulard et al., 2000; DeBarber et al., 2000; Vannelli et al., mide activation. In vivo, a mycothiol-deficient strain 2002) to yield the ETH-NAD adduct (Wang et al., 2007). grew normally in immunodeficient mice, but was Mutations in either activator confer resistance to INH or slightly defective for growth in immunocompetent ETH respectively (Piatek et al., 2000; Morlock et al., 2003; Ramaswamy et al., 2003; Hazbon et al., 2006). Accepted 5 July, 2008. *For correspondence. E-mail jacobsw@ hhmi.org; Tel. (+1) 718 678 1075; Fax (+1) 718 678 1085. †Present Co-resistance to INH and ETH can be mediated by muta- address: Division of Viral Diseases, Walter Reed Army Institute of tions that alter the InhA target so as to prevent the INH- Research, Silver Spring, MD 20910, USA. NAD or the ETH-NAD adduct from binding (Vilcheze et al., Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial 2006), by mutations that cause InhA overexpression exploitation. (Larsen et al., 2002; Vilcheze et al., 2006) or by mutations © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd Identification of a new ethionamide resistance mechanism 1317 Table 1. M. tuberculosis strains used in this study. mshA allele characterization Strain Genotype Nucleotide Amino acid Mutant generation Source H37Rv mshA1 –– Trudeau Institute mc24931 mshA3 c382t Stop codon AA128 Spontaneous mutant of H37Rv This work mc24932 mshA4 c817t R273C Spontaneous mutant of H37Rv This work mc24933 mshA5 g895t G299C Spontaneous mutant of H37Rv This work mc24934 mshA6 c991t Stop codon AA331 Spontaneous mutant of H37Rv This work mc24935 mshA7 g1067a G356D Spontaneous mutant of H37Rv This work mc24936 mshA8 a1082c E361A Spontaneous mutant of H37Rv This work mc24937 mshA9 a1242del Frameshift Spontaneous mutant of H37Rv This work mc24938 mshA10 DmshA Specialized transduction with phAE222 This work CDC1551 mshA1 –– CSU mc24939 mshA10 DmshA Specialized transduction with phAE222 This work Erdman mshA2 a332g N111S Trudeau Institute mc24942 mshA10 DmshA Specialized transduction with phAE222 This work CSU, Colorado State University. in ndh that increase the intracellular NADH concentration, tions of both INH and ETH [Յ 4-fold the minimum inhibi- thereby competitively inhibiting the binding of the INH-NAD tory concentration (MIC)]. Seven mutants were isolated at and ETH-NAD adducts to InhA (Miesel et al., 1998; low frequencies (1–4 ¥ 10-8). DNA sequence analysis of Vilcheze et al., 2005). While the majority of clinical isolates targeted genes in these seven strains revealed the resistant to INH or ETH have been shown to map to the absence of mutations in the genes known to mediate activator genes (katG, ethA)ortheinhA target, current co-resistance to INH and ETH, namely inhA (the gene or studies still show that up to 22% of the INH-resistant its promoter region) and ndh. This analysis provided the M. tuberculosis clinical isolates have no mutations in the evidence that these strains possessed mutations that genes known to be involved in INH or ETH resistance conferred INH and ETH resistance and had not been (Hazbon et al., 2006). In this study, to identify novel muta- previously identified in M. tuberculosis. The mutants were tions conferring INH and ETH resistance, we isolated transformed with a cosmid genomic library of the drug- spontaneous mutants of M. tuberculosis in vitro and found susceptible M. tuberculosis parent. The frequency of that they map to mshA, a gene encoding a glycosyltrans- transformation was extremely low for most of the mutants ferase involved in mycothiol biosynthesis, suggesting that (less than 100 transformants per transformation), and mshA was non-essential. Additional genetic and biochemi- only one mutant, mc24936, which had the lowest level of cal studies demonstrated that mycothiol biosynthesis is INH resistance, yielded more than 1000 transformants. required for ETH susceptibility in M. tuberculosis. Further- The cosmid transformants were screened for restoration more, in vivo studies showed that mycothiol is not required of INH and ETH susceptibility. One potential comple- for growth in mice. menting cosmid was isolated, sequenced and shown to contain the mshA gene, a gene characterized as mediat- Results and discussion ing the first step in the biosynthesis of mycothiol (Newton et al., 2003; 2006), a key thiol in the family of Actino- Spontaneous mutants of M. tuberculosis, co-resistant to mycetes bacteria (Newton et al., 1996). A link between INH and ETH, map to mshA mycothiol biosynthesis and resistance to INH and Numerous studies have demonstrated that there exist ETH had been previously established in Mycobacterium strains of M. tuberculosis that are resistant to INH and smegmatis when transposon mutants in mshA were do not have mutations in the genes associated with found to be resistant to INH (more than 25-fold) and ETH INH resistance (katG, inhA structural gene and promoter, (sixfold) (Newton et al., 1999; 2003; Rawat et al., 2003). ndh) (Telenti et al., 1997; Piatek et al., 2000; Subsequent sequence analysis of mc24936 and the other Ramaswamy et al., 2003; Cardoso et al., 2004; Hazbon mutants showed that all the M. tuberculosis H37Rv et al., 2006). To eliminate the majority of spontaneous mutants had missense, nonsense or frameshift mutations mutants of M. tuberculosis that are singly resistant to INH in mshA (Table 1). The mshA mutants had various levels and map to katG, we chose to isolate mutants that were of resistance to INH (2- to 16-fold) and ETH (four- to co-resistant to INH and its structural analogue ETH. eightfold) (Table 2). This is the first report that mshA Samples of three independent M. tuberculosis H37Rv mutations confer co-resistance to INH and ETH in cultures were plated on media containing low concentra- M. tuberculosis. © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 69, 1316–1329 1318 C. Vilchèze et al. ᭿ Table 2. INH and ETH minimum inhibitory concentrations (MICs). analyse the effects of the diverse mutations in mshA on the biosynthesis of mycothiol, the levels of mycothiol were MIC (mg l-1) MIC (mg l-1) pMV361::mshA measured in all the mutants using fluorescent high- performance liquid chromatography (HPLC) assay Strain INH ETH INH ETH (Newton et al., 2000a). We found a dramatic reduction H37Rv 0.06 2.5 0.06 2.5 (83% to undetectable levels) in the concentration of myco- mc24931 0.6 20 0.06 2.5 thiol compared with wild type (Fig.