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 approximately 1% 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-GlcN-Ins 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.

FIG. 1.

Pathway for mycothiol biosynthesis. Ac, acetyl.

FIG. 2.

Growth (OD₆₀₀) of wild-type (▴) and ΔmshD (○) M. tuberculosis cells in Middlebrook 7H9 medium plus ADS versus time.

FIG. 3.

Total thiol (▴ and ▵) and disulfide (▪ and □) content of wild-type (▴ and ▪) and ΔmshD M. tuberculosis cells grown in Middlebrook 7H9 medium as a function of time. CoA data were not included.

FIG. 4.

Colony appearance of wild-type (A and B) and ΔmshD M. tuberculosis (C and D) cells grown on Middlebrook 7H9 plates supplemented with ADS (A and C) or OADC (B and D). Cells were plated at dilutions of 10⁻¹ (upper right quadrant), 10⁻² (lower right quadrant), 10⁻³ (lower left quadrant), and 10⁻⁴ (upper left quadrant). Plates were photographed after 4 weeks of incubation.

FIG. 5.

Survival (percentage of control) for wild-type and ΔmshD M. tuberculosis cells incubated at 37°C in 5 mM H₂O₂ as a function of time.

FIG. 6.

Growth of wild-type (▪ and □) and ΔmshD (• and ○) M. tuberculosis cells at pH 7.0 (▪ and •) and pH 5.5 (□ and ○) in Middlebrook 7H9 medium. Error bars show standard errors of the means for triplicate determinations (not shown where smaller than symbol).