A Mycothiol Synthase Mutant of Mycobacterium tuberculosis Has an Altered Thiol-Disulfide Content and Limited Tolerance to Stress

Abstract

Mycothiol (MSH) (acetyl-Cys-GlcN-Ins) is the major low-molecular-mass thiol in Mycobacterium tuberculosis. MSH has antioxidant activity, can detoxify a variety of toxic compounds, and helps to maintain the reducing environment of the cell. The production of MSH provides a potential novel target for tuberculosis treatment. Biosynthesis of MSH requires at least four genes. To determine which of these genes is essential in M. tuberculosis, we have been constructing targeted gene disruptions. Disruption in the mshC gene is lethal to M. tuberculosis, while disruption in the mshB gene results in MSH levels 20 to 100% of those of the wild type. For this study, we have constructed a targeted gene disruption in the mshD gene that encodes mycothiol synthase, the final enzyme in MSH biosynthesis. The mshD mutant produced ϳ1% of normal MSH levels but high levels of the MshD substrate Cys-GlcN-Ins and the novel thiol N-formyl-Cys-GlcN-Ins. Although N-formyl-Cys-GlcNIns was maintained in a highly reduced state, Cys-GlcN-Ins was substantially oxidized. In both the wild type and the mshD mutant, cysteine was predominantly oxidized. The M. tuberculosis mshD mutant grew poorly on agar plates lacking catalase and oleic acid and in low-pH media and had heightened sensitivity to hydrogen peroxide. The inability of the mshD mutant to survive and grow in macrophages may be associated with its altered thiol-disulfide status. It appears that N-formyl-Cys-GlcN-Ins serves as a weak surrogate for MSH but is not sufficient to support normal growth of M. tuberculosis under stress conditions such as those found within the macrophage.Mycothiol (MSH) (acetyl-Cys-GlcN-Ins) ( Fig. 1) is the major thiol in mycobacteria (13) and contains the core amino acid cysteine similar to the predominant thiol in eukaryotes, glutathione (6). MSH is a unique product of the actinomycetes and is present at millimolar levels in Mycobacterium tuberculosis (10). Small thiols such as MSH play an important role in a number of essential functions, which include maintaining the reducing environment within the cell, protecting the cellular contents from oxidants, and detoxifying thiol-reactive compounds (4,13,18,20,25). In addition, MSH has the ability to detoxify antibiotics (4,13,18,25). Failure to recover M. tuberculosis mutants in the MSH biosynthetic gene mshC has indicated that MSH is essential for the growth of M. tuberculosis (21,23). Thus, the MSH biosynthetic pathway provides a novel antimycobacterial target, and the enzymes essential for MSH biosynthesis are potential drug targets for the treatment of tuberculosis.Four genes involved in the biosynthesis of MSH have been identified in studies of the model organism Mycobacterium smegmatis. A glycosyltransferase (MshA, encoded by Rv0486 in the M. tuberculosis genome) is involved in the production of GlcNAc-Ins, but the detailed biochemistry of this step is still being elucidated (Fig. 1). The deacetylase MshB (Rv1170) produces GlcN-Ins (11), which is ligated to Cys under catalysis by MshC (Rv2130c) (22). Mycothio...

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