Mycothiol-Deficient Mycobacterium smegmatis Mutants Are Hypersensitive to Alkylating Agents, Free Radicals, and Antibiotics

Abstract

Mycothiol (MSH; 1D-myo-inosityl 2-[N-acetyl-L-cysteinyl]amido-2-deoxy-␣-D-glucopyranoside) is the major low-molecular-weight thiol produced by mycobacteria. Mutants of Mycobacterium smegmatis mc2 155 deficient in MSH production were produced by chemical mutagenesis as well as by transposon mutagenesis. One chemical mutant (mutant I64) and two transposon mutants (mutants Tn1 and Tn2) stably deficient in MSH production were isolated by screening for reduced levels of MSH content. The MSH contents of transposon mutants Tn1 and Tn2 were found to be less than 0.1% that of the parent strain, and the MSH content of I64 was found to be 1 to 5% that of the parent strain. All three strains accumulated 1D-myo-inosityl 2-deoxy-␣-D-glucopyranoside to levels 20-to 25-fold the level found in the parent strain. The cysteine:1D-myo-inosityl 2-amino-2-deoxy-␣-D-glucopyranoside ligase (MshC) activities of the three mutant strains were <2% that of the parent strain. Phenotypic analysis revealed that these MSH-deficient mutants possess increased susceptibilities to free radicals and alkylating agents and to a wide range of antibiotics including erythromycin, azithromycin, vancomycin, penicillin G, rifamycin, and rifampin. Conversely, the mutants possess at least 200-fold higher levels of resistance to isoniazid than the wild type. We mapped the mutation in the chemical mutant by sequencing the mshC gene and showed that a single amino acid substitution (L205P) is responsible for reduced MSH production and its associated phenotype. Our results demonstrate that there is a direct correlation between MSH depletion and enhanced sensitivity to toxins and antibiotics. Fig. 1A) is a low-molecular-weight thiol that serves functions in mycobacteria analogous to those of glutathione in gram-negative bacteria and eukaryotes (7). These functions include protection against oxidative damage and inactivation of electrophilic toxins (15,17). The distribution of MSH is limited to gram-positive actinomycetes, and of all the species tested, mycobacteria appear to generate the highest levels of MSH (14). The absence of MSH in mammalian cells suggests that the enzymes involved in the metabolism of MSH may be attractive drug targets for either rational drug design or screening of libraries of inhibitory compounds (25). Mycothiol (MSH;Although studies of MSH biosynthesis are still at an early stage, as shown in Fig. 1B, elements of the overall pathway have been described (1, 4, 16). Moreover, direct measurements of intermediates in bacterial extracts that support the proposed biosynthetic pathway have been reported (1). The genes for the MSH biosynthesis pathway were designated mshA, mshB, mshC, and mshD (16), with the corresponding enzymes labeled as shown in Fig. 1. It has been shown that 1D-myo-inosityl 2-acetamino-2-deoxy-␣-D-glucopyranoside (GlcNAc-Ins) is an intermediate (16), and we presume that its formation is the first dedicated step in MSH biosynthesis. Glucosaminylinositol is formed by deacetylation of GlcNAc-Ins. We have previously identified m...

Activity of 7-methyljuglone derivatives against Mycobacterium tuberculosis and as subversive substrates for mycothiol disulfide reductase

“…The NADPH-dependent enzyme mycothiol disulfide reductase (Mtr) 30 helps to maintain an intracellular reducing environment by reducing MSSM back to MSH. MSH is essential for the growth of M. tuberculosis 31 and MSH-deficient mycobacteria exhibit increased sensitivity to oxidative stress, [32] and [33] making this redox pathway a potential biological target for novel antitubercular chemotherapies. The catalytic properties of Mtr were originally reported using both MSSM and some commercial naphthoquinones (e.g.…”

Ancestral antibiotic resistance in Mycobacterium tuberculosis

“…However, Jarlier and Nikaido (4) have also pointed out that this permeability barrier is insufficient to fully explain the high levels of drug resistance in Mycobacterium, suggesting that there must be synergistic systems effective against drugs that penetrate this barrier. Indeed, several mycobacterial genes not involved in outer envelope assembly confer resistance to specific, broadspectrum antibiotics (6)(7)(8).…”

“…The expression of some of these antibioticinduced genes is under the control of stress inducible systems that respond to (13) or lead to (15) decreases in growth rate. The underlying control elements that affect antibiotic resistance include general stress-responsive sigma factors (16) and transcriptional activator proteins of the AraC family (MarA, SoxR, and Rob) (17), as well as genetic systems that provide for more specific adaptation to DNA damage (15) or oxidative stress (8). Such systems are commonly found in diverse bacterial groups and typically modulate antibiotic resistance within a rather narrow concentration range (8,17).…”

Structural and Enzymatic Analysis of MshA from Corynebacterium glutamicum

“…GlcNAc-Ins is then deacetylated (MshB) and subsequently cysteinylated (MshC) at the amino group to produce Cys-GlcN-Ins, which then is acetylated (MshD) to produce AcCys-GlcN-Ins or MSH (9 -13). In Mycobacterium smegmatis, mutants blocked in MSH biosynthesis exhibit enhanced sensitivity to cellular stress reagents, including hydrogen peroxide and rifampicin (14,15), whereas in Mycobacterium tuberculosis (Erdman strain) mutants blocked in MSH production were not viable (16). Of the four genes, mshA and mshC were found to be critical to the production of MSH and therefore viability of the organism (16,17).…”