Identification of a Mycothiol-Dependent Nitroreductase from Mycobacterium tuberculosis

Abstract

The success of Mycobacterium tuberculosis (Mtb) as a pathogen depends on the redundant and complex mechanisms it has evolved for resisting nitrosative and oxidative stresses inflicted by host immunity. Improving our understanding of these defense pathways can reveal vulnerable points in Mtb pathogenesis. In this study, we combined genetic, structural, computational, biochemical, and biophysical approaches to identify a novel enzyme class represented by Rv2466c. We show that Rv2466c is a mycothiol-dependent nitroreductase of Mtb and can reduce the nitro group of a novel mycobactericidal compound using mycothiol as a cofactor. In addition to its function as a nitroreductase, Rv2466c confers partial protection to menadione stress.

Keywords: Mycobacterium tuberculosis; Rv2466c; menadione; mrx-2; mycothiol; nitrofuranylcalanolide; nitroreductase; oxidoreductase.

Figure 1

Rv2466c is essential for activity of NFCs. (A) Structure of NFCs 30a and 18, which kill both replicating and nonreplicating Mtb. (B) Mtb (for mutants 1–16) or M. bovis BCG (mutants 17–19) were exposed to 5 μg/mL of 30a or its analog 18 on 7H11-OADC agar plates for 3 weeks, after which surviving colonies were selected. The PCR products of the rv2466c coding region of these clones were sequenced and listed here. *Mutant 17 was observed twice in M. bovis BCG. fs = frameshift. (C) WT bacteria or Mutant 1 were transformed with either parent plasmid pMV261 or pMV261 containing a WT copy of rv2466c (pmv261-rv2466c) and exposed to 30a for 7 days. % inhibition of growth was calculated in relation to growth in vehicle (DMSO) wells. Data are means and SDs of triplicate wells and representative of two similar experiments. (D–F) Semiquantitative analysis of Rv2466c by immunblot of lysates from log phase bacteria normalized by total protein amount. Rv2215 (dihydrolipoamide acyltransferase, DlaT) served as loading control. In E and F, log phase Mtb were exposed to 5 mM diamide for 24 h at 37 °C prior to lysate preparation for immunoblotting.

Figure 2

C19 and C22 are critical residues for the activity of Rv2466c. (A) Δrv2466c was transformed with pMV261 plasmid carrying either a WT copy of rv2466c or rv2466c expressing the following mutations: C19S, C22S, or both C19S and C22S. Log phase bacteria were exposed to varying concentrations of 30a for 7 days and % inhibition was calculated on the basis of growth in vehicle (DMSO) wells. Data are means ± SDs of triplicate wells. (B) Structural superposition of one subunit of Rv2466c C22S mutant (pink, current publication) with that of the published structure of Rv2466c (blue, PDB ID: 4NXI; green, PDB ID: 4ZIL) and the M. leprae homologue (gray, PDB ID: 4WKW) represented in ribbon format. Subunits are superimposed and shown as ribbon. Dashed circles denote regions that undergo significant conformational changes. Side chains of C19 and C22S residues of the C22S mutant are depicted as spheres with yellow for sulfur, red for oxygen, and pink for carbon. (C) Conformational rearrangement of the α2−α3 region to generate a putative substrate pocket around C19. The pocket is shown by a black arrow; side chain of C19 is depicted as spheres. The “open state” α2−α3 region of wild-type Rv2466c is shown in green ribbon form.

Figure 3

With the exception of resisting menadione stress, Rv2466c has a predominantly redundant role in bacterial pathogenesis. (A) Mutant 1 (W181C) was transformed with pMV261 or pMV261 with a WT copy of rv2466c, and all strains were exposed to 0.5 mM menadione. Bacterial survival was assessed by CFU enumeration. Data are means and SDs of duplicate wells for each time point. Evaluation of the last time point using an unpaired t test shows significance with a p value of 0.0035 when comparing WT and mutant 1. (B) C57BL/6 mice were infected with Mtb strains via the aerosol route. Lungs of mice were harvested at the indicated time points and plated for CFU enumeration. The data show the means ± SDs of 5 mice per strain at each time point.

Figure 4

Rv2466c reduces 30a into its amine. (A) 400 μg/mL WT or mutant rRv2466c was coincubated with 1 mg/mL M. smegmatis lysate in the presence of DTT and 30a for 4.5 h and OD₃₉₀ was measured at indicated time points in relation to the absorbance at t = 0. (B, C) 400 μg/mL WT or mutant rRv2466c was coincubated with 1 mg/mL M. smegmatis lysate in the presence of DTT and 30a, and the reaction mixture was analyzed by LC-MS. (D) LC/MS/MS analysis of synthetic AN1 and isolated reaction product. (E) Synthetic AN1 was tested against replicating and nonreplicating WT Mtb at varying concentrations, and growth was assessed using absorbance at 580 nm.

Figure 5

Mycothiol is required for activity of Rv2466c. (A) M. smegmatis protein lysates were parted into two fractions by size cutoff spin columns and coincubated with 400 μg/mL of Rv2466c, 50 μg/mL 30a, and 1 mM DTT. Values are means ± SD of duplicate wells, representing two similar experiments. (B) Bacteria were coincubated with varying doses of 30a or PA-824, and growth was evaluated at the end of 7 days by absorbance at 580 nm. Values are means ± SD of triplicate wells. (C) 400 μg/mL WT was coincubated with 1 mg/mL lysate from WT or ΔmshA M. smegmatis in the presence of DTT and 30a, and the reaction mixture was analyzed by LC-MS. (D) Bacteria were coincubated with varying concentrations of 30a, and growth was evaluated at the end of 7 days by absorbance at 580 nm. Values are means ± SDs of triplicate wells. (E) Rv2466c amount was determined by immunoblot of Mtb lysates from log phase bacteria which were normalized to protein content. Mtb Dihydrolipoamide acyltransferase (DlaT) served as loading control. (F) rRv2466c and 30a were coincubated with synthetic reduced mycothiol (MSH) or mycothione (oxidized MSH) (MSSM), with or without DTT, and fluorescence (Ex/Em 370/470) was measured at the end of 90 min. Results are mean ± SD of duplicate wells. (G) 30a, 0.5 mM MSH, and 1 mM DTT were coincubated with or without rRv2466c and analyzed by LC-MS.

Figure 6

Intrabacterial detection of 30a conversion products. (A) Log phase bacteria were diluted to OD 0.1 and exposed to 1.25 μg/mL of 30a in triplicate. Fluorescence (Ex 370/Em 470) was measured at indicated time points, and DMSO background controls subtracted from measured values. (B) Bacteria were grown in media without tyloxopol for 1–2 weeks, after which they were diluted to OD 0.2 and exposed to 5 μg/mL of 30a. Four hours after exposure, they were harvested, washed once with PBS, and lysed by bead beating for LC-MS analysis. Samples were analyzed with Profinder software.

Figure 7

Mycothiol binds Rv2466c through interactions with the C19 of the active site. (A) Details of the substrate-binding site of the Rv2466c–mycothiol complex after mycothiol was docked into the proposed binding site of Rv2466c. The ligand shown represents the best-scoring pose from the CovDock algorithm. C atoms of mycothiol are labeled green, and Rv2466c is displayed as a ribbon in pink. Only relevant residues discussed in the text are shown as sticks and labeled. The black dashes represent the proposed hydrogen bonds involved in the binding of mycothiol. The disulfide bond formed between mycothiol and C19 is shown in yellow. (B) The impact of point mutations on the function of Rv2466c’s ability to convert 30a to AN1 in the presence of mycothiol and DTT. (C) WT or C19S mutants of rRv2466c were incubated with reduced mycothiol (MSH), oxidized mycothiol (MSSM), glutathione (GSH), or dithiothreitol (DTT); and the effect of this interaction was measured by change in peak melting temperature via differential fluorimetry.

Figure 8

Rv2466c is a phylogenetically divergent mycothiol-dependent nitrooxidoreductase. (A) Unrooted phylogenetic tree for the sequences of proteins functionally or structurally related to Rv2466c, including HCCAs (in blue) and NAD(P)H nitroreductases (in green), as well as diverse oxidoreductases including thioredoxins (in brown), short-chain dehydrogenases (in gray), NAD(P)/FAD-dependent oxidoreductases (in purple), ferredoxin reductases (in pink), and other proteins annotated as DsbA in other Actinobacteria (in red) or Gram positive or negative bacteria (in orange). The references of the sequences used to generate the tree are reported in the Experimental Methods section. (B) The model of how Rv2466c alters NFCs. The left side of the reaction emphasizing the role of the mtr/NADPH system, and its kinetics were worked out by Rosado et al. *In the majority of our experiments, we utilized DTT as a surrogate reductant.