Biochemical and Biophysical Research Communications 363 (2007) 71–76 www.elsevier.com/locate/ybbrc Comparative analysis of mutants in the mycothiol biosynthesis pathway in Mycobacterium smegmatis Mamta Rawat a, Chantale Johnson a, Vanessa Cadiz a, Yossef Av-Gay b,* a Department of Biology, California State University-Fresno, Fresno, CA 937401, USA b Department of Medicine, Division of Infectious Diseases, University of British Columbia, Vancouver, BC, Canada V5Z 3J5 Received 17 August 2007 Available online 31 August 2007 Abstract The role of mycothiol in mycobacteria was examined by comparative analysis of mutants disrupted in the four known genes encoding the protein machinery needed for mycothiol biosynthesis. These mutants were sensitive to acid stress, antibiotic stress, alkylating stress, and oxidative stress indicating that mycothiol and mycothiol-dependent enzymes protect the mycobacterial cell against attack from various different types of stresses and toxic agents. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Mycothiol; Mycothiol deacetylase; Mycothiol ligase; Mycothiol synthase; Oxidative stress; Toxins; Xenobiotics Mycobacteria, like most other Gram-positive bacteria do We have previously reported that Mycobacterium not make glutathione but produce another low molecular smegmatis mutants disrupted in the four known genes weight thiol, mycothiol (MSH) (Fig. 1), 1-D-myoinosityl- [3,9–11] involved in mycothiol biosynthesis are resistant to 2-(n-acetyl-L-cysteinyl)-amido-2-deoxy-a-D-glucopyranoside. isoniazid, a front-line drug used in the treatment of tubercu- Since MSH is unique to actinomycetales [1], enzymes losis. We have also reported that M. smegmatis mutants involved in MSH biosynthesis and metabolism are poten- lacking mycothiol ligase activity and thus mycothiol are tial targets for drugs directed against pathogenic mycobac- sensitive to a wide range of antibiotics, alkylating agents, teria like Mycobacterium tuberculosis. and oxidative stress [11]. Lastly, we have demonstrated that Mycothiol biosynthesis proceeds through a five-step a direct depletion of mycothiol pool occurs upon exposure pathway [2]. The initial substrates, 1L-inositol-1-phosphate to oxidative stress [12] and nitrosative stress [13]. This study and UDP-N-acetylglucosamine react to form N-acetylglu- further examines the role of mycothiol in mycobacteria by cosaminylinositol phosphate, a reaction catalyzed by the comparative analysis of mutants disrupted in four of the N-acetylglucosamine transferase, MshA [3,4]. An unidenti- known genes involved in mycothiol biosynthesis. fied phosphatase dephosphorylates this molecule to yield N-acetylglucosaminylinositol [4], which is deacetylated by Materials and methods an MshB deacetylase [5,6]. The resulting glucosaminylinos- itol (GI) is ligated with a cysteine in a reaction catalyzed by Growth and culture conditions. Mycobacterium smegmatis mc2155 was a ligase, MshC [7]. The cysteinylglucosaminylinositol the parent wild-type strain used for construction of the knockout mutants (CGI) is then acetylated to form mycothiol in a reaction and transposon mutants and thus served as the control for the sensitivity catalyzed by MshD acetyltransferase [8]. studies. Initially, M. smegmatis and the mutants, A1 (DmshA), Myco504 (DmshB), I64 (DmshC), and mshD::Tn5 (DmshD) disrupted in genes involved in mycothiol biosynthesis were grown in Middlebrook 7H9 broth (DIFCO) with 0.05% Tween and OADC supplement. Media for mutants, * Corresponding author. Fax: +1 604 875 4013. DmshA, DmshB, and DmshD, were also supplemented with 25 lgmlÀ1 E-mail address: [email protected] (Y. Av-Gay). kanamycin. After the cultures were in log phase, wild-type and the 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.08.142 72 M. Rawat et al. / Biochemical and Biophysical Research Communications 363 (2007) 71–76 1.0×1009 Results and discussion Mycothiol and mycothiol precursors levels in * Mycobacterium smegmatis * * 08 In M. smegmatis, a nonpathogenic surrogate of 1.0×10 * CFU/ml M. tuberculosis, mutants disrupted in mshA, mshB, mshC, and mshD, have been isolated (Table 1); the gene(s) coding for the enzyme catalyzing the phosphatase activity, MshA2, has not been identified. The four biosynthetic 1.0×1007 pathway mutants differ in their mycothiol content ranging Wild-type ΔmshA ΔmshBΔ mshC ΔmshD from <0.1% of the wild-type mycothiol level in DmshA strains [3,9], A1 and 49, to 5–10% of the wild-type mycoth- Strains iol level in DmshB [10]. M. tuberculosis mutants disrupted Fig. 1. Viability of MSH mutants under acidic stress; blank bars: the in mshB [15] and mshD [16] have also been constructed number of colony forming units per ml (cfu/ml) for untreated samples, and characterized. However, attempts to construct mutants filled bars: the number of cfu/ml for samples incubated in acidified media for 2 h, *P 6 0.005 using Student’s t-test. Strains were grown to mid-log disrupted in mshA [17] and mshC [18] by homologous phase in Middlebrook 7H9 liquid media supplemented with 0.05% Tween recombination were unsuccessful until a second copy of and 1% glucose at 37 °C. One set of cultures in quadruplicates was diluted the genes was inserted into the M. tuberculosis genome sug- to 0.5 OD600 in normal media and another set, also in quadruplicates, was gesting that these genes are essential in M. tuberculosis, diluted in media acidified to pH 4.0 by adding 20 mM HCl. After although recently an M. tuberculosis mshA mutant was gen- incubation for 2 h at 37 °C, cfus were determined on 7H10 1% glucose agar. erated using allelic exchange (W.R. Jacobs, personal com- munication). Interestingly, the M. smegmatis mshD mutant produces two novel thiols, N-formyl CGI and N-succinyl- mutants were diluted and transferred to Middlebrook 7H9 broth with CGI [16,19]. 0.05% Tween containing 1% glucose as the carbon source. When these diluted cultures reached mid-log phase, the cultures were diluted to 0.5 optical density (600) for sensitivity assays. Antibiotic sensitivity MSH determination. Derivitization of cell extracts with monobromo- bimane (mBBr) and HPLC analysis of the derivitized samples to determine In this study, we examined mutants disrupted in mshA the mycothiol content was performed essentially as described earlier [14]. (A1), mshB (myco504), and in mshC (I64), in mshD N Control samples, treated with -ethylmalemide and then with mBBr, were mshD also analyzed. Results are reported as lmol per gram of residual dry ( ::Tn5) for sensitivity to a wide variety of drugs that weight of the pellet obtained from the 50% acetonitrile–water extraction or are used in the treatment of tuberculosis as well as other as nmol per OD (600). drugs that are known to be ineffective against tuberculosis. Sensitivity assays. Sensitivity was assessed by a number of different We had already demonstrated that all M. smegmatis assays. First, E-test strips (AB Biodisk) were used according to the man- mutants disrupted in mycothiol are sensitive to streptomy- ufacturer’s directions to determine the minimal inhibitory concentration DmshB (MIC) of the different mycobacterial strains for the antibiotics, rifampin, cin [20]. Also, in our previous work with the ,we erythromycin, azithromycin, and vancomycin. Second, disk diffusion had reported that we did not see any difference in sensitiv- assays were performed for various antibiotics, oxidants, reductants, and ity to the antibiotics rifampin, vancomycin, and erythro- toxins according to Rawat et al. [11]. Lastly, cell viability assays were mycin based on disk assays [10]. In this report, we performed by dividing cultures diluted to 0.5 OD600 into two sets; one set determined antibiotic sensitivity using E tests, which was left untreated and the second set was treated with various stresses for proved to be more sensitive determinant of susceptibility 2 h at 37 °C. Colony forming units (cfus) were determined on 7H10 1% glucose agar and MSH content was analyzed. These assays were per- (Table 2). For the antibiotic rifampin, DmshA proved to formed with either five or four replicates. be most sensitive, DmshD intermediate in its sensitivity, Statistical analysis. Student’s t-test was applied to statistically evaluate and DmshB and DmshC similar to wild-type. In contrast, the results and P values were calculated to show significant differences. DmshA is least sensitive and DmshD most sensitive to van- Table 1 Definition, annotation and mycothiol levels in M. smegmatis mutants in the mycothiol biosynthetic pathway Enzyme Gene ORF ORF M. smegmatis Type of mutant Relative mycothiol (M. tuberculosis) (M. smegmatis) mutant strain content (%) Mycothiol glycosyl mshA Rv0486 MSMEG0924 A1 Transposon <0.1 transferase 49 Chemically induced <0.1 Phosphatase mshA2 ———— — Mycothiol deacetylase mshB Rv1170 MSMEG5117 Myco504 Homologous 5–10 recombination Mycothiol ligase mshC Rv2130c MSMEG4193 I64 Chemically induced 1–5 Mycothiol synthase mshD Rv0819 MSMEG5754 mshD::Tn5 Transposon 0.65–1.2 M. Rawat et al. / Biochemical and Biophysical Research Communications 363 (2007) 71–76 73 Table 2 mide confirming that mycothiol plays a role in the activa- Sensitivity of mycothiol deficient mutants to antibiotics as determined by tion of ethioniamide (Table 3). In line with earlier E-test studies, wild-type M. smegmatis does not show any zone Cell strain Minimum inhibitory concentration (lg) of clearings with fosfomycin (100 lg), pyrazinamide Rifampin Erythromycin Azithromycin Vancomycin (100 lg), cycloserine (100 lg), penicillin (200 lg), and sul- mc2155 (wild- 30–32 24–16 6–4 6–12 fonamide (25 lg), and thus is resistant to these agents as type) are mutants disrupted in mycothiol. DmshA 3 0.5–1.5 0.5–1.0 6 DmshB 32 0.5–1.5 0.38–0.5 4 Sensitivity to alkylating agents DmshC 32 0.75–2.0 0.75–1.0 1.9–6 DmshD 8 0.38–1.5 0.38–0.50 1 We have reported the construction and characteriza- tion of a mutant in the mca gene lacking mycothiol ami- comycin. The E-test values for erythromycin and azithro- dase hydrolase, a mycothiol-dependent detoxification mycin demonstrate that all mutants are sensitive to these enzyme [20].