Functional studies of multiple thioredoxins from Mycobacterium tuberculosis

Abstract

Cytoplasmic protein reduction via generalized thiol/disulfide exchange reactions and maintenance of cellular redox homeostasis is mediated by the thioredoxin superfamily of proteins. Here, we describe the characterization of the thioredoxin system from Mycobacterium tuberculosis, whose genome bears the potential to encode three putative thioredoxins from the open reading frames designated trxAMtb, trxBMtb, and trxCMtb. We show that all three thioredoxins, overproduced in Escherichia coli, are able to reduce insulin, a model substrate, in the presence of dithiothreitol. However, we observe that thioredoxin reductase is not capable of reducing TrxAMtb in an NADPH-dependent manner, indicating that only TrxBMtb and TrxCMtb are the biologically active disulfide reductases. The absence of detectable mRNA transcripts of trxAMtb observed when M. tuberculosis strain H37Rv was cultivated under different growth conditions suggests that trxAMtb expression may be cryptic. The measured redox potentials of TrxBMtb and TrxCMtb (-262+/-2 mV and -269+/-2 mV, respectively) render these proteins somewhat more oxidizing than E. coli thioredoxin 1 (TrxA). In E. coli strains lacking components of cytoplasmic protein reduction pathways, heterologous expression of the mycobacterial thioredoxins was able to effectively substitute for their function.

FIG. 1.

Multiple sequence alignment of M. tuberculosis thioredoxins and E. coli TrxA. (A) Trp 32 and Asp 27 are highly conserved in the Trx superfamily but are replaced by Leu and Tyr in TrxA_Mtb at positions 29 and 24, respectively. A histogram depicting amino acid conservation among the indicated thioredoxins was generated with CLC Protein Workbench 2.0.2. (B) Structure of the redox-active region of TrxC_Mtb (Protein Data Bank accession no. 2I1U) (20). Tryptophan residues are depicted in magenta, aspartic acid residues are depicted in red, and the disulfide between two cysteine residues is depicted in yellow. The amino acid variation in TrxA_Mtb is marked with arrows.

FIG. 2.

Thioredoxin-catalyzed reduction of insulin by DTT. Insulin turbidity due to thioredoxin-promoted precipitation of the insulin β chain by DTT was measured at 650 nm and is plotted as a function of time. The assay mixture contained 130 μM insulin, 1 mM DTT in 100 mM potassium phosphate, 2 mM EDTA, (pH 7.5), and 8 μM of thioredoxins. OD, optical density; •, control lacking thioredoxins; ▴, TrxA_Mtb; ▾, TrxB_Mtb; ♦, TrxC_Mtb; and ▪, TrxA_Ec.

FIG. 3.

TrxR-dependent M. tuberculosis thioredoxin activity. The activities of thioredoxins as measured by insulin β-chain precipitation in the presence of TrxR and NADPH are depicted as a histogram. The assay mixture contained 130 μM insulin, 200 μM NADPH and 2 μM of M. tuberculosis TrxA/B/C or TrxA_Ec in 100 mM potassium phosphate, 1 mM EDTA (pH 6.5), and 2 μM bovine serum albumin.

FIG. 4.

Redox equilibrium curves of M. tuberculosis thioredoxins and TrxA_Ec with GSH. The plots show the fractions of reduced protein (R) at equilibrium under various concentrations of GSH-GSSG buffer, obtained by recording the changes in the redox state-dependent fluorescence emission spectra at the appropriate emission maxima, for the oxidized versions of indicated thioredoxins. ▪, TrxA_Mtb; •, TrxB_Mtb; ▵, TrxC_Mtb; and ▾, TrxA_Ec.

FIG. 5.

RT-PCR analysis of M. tuberculosis trx transcripts under various growth regimens. (A) Cultures of M. tuberculosis H37Rv were grown aerobically to mid-exponential phase (optical density at 600 nm, ∼0.9) and treated individually with the indicated oxidants, each at a concentration of 2 mM, for 2 h. Total RNA was isolated from grown cultures, and RT-PCR was performed with primers specific for the indicated trx transcripts. Total RNA extracted from a nontreated culture was used as a control. A primer pair specific to the sigA transcript was used as an internal control. (B) Control experiment showing that the trxA_Mtb gene is amplified using M. tuberculosis genomic DNA (GD).

FIG. 6.

Rescue of the cysteine auxotrophy of an E. coli trxA grxA double mutant by M. tuberculosis trx expression. (I) Transformants of strain GJ8025 (ΔtrxA grxA::kan) bearing indicated trx genes under the expression control of the P_BAD promoter in plasmids pHYD3058 (trxA_Mtb), pHYD3059 (trxB_Mtb), pHYD3060 (trxC_Mtb), pHYD3064 (trxA_Ec), and pBAD33 (vector) were streaked on minimal agar plates containing 0.2% d-glucose and 40 μg/ml l-cysteine (A), 0.2% d-glucose (B), and 0.2% d-glucose and 0.2% arabinose (C). An appropriate amount of l-leucine-l-isoleucine supplement was added to the indicated plates. (II) Immunodetection of TrxA_Mtb in lysates of DH5α bearing the indicated plasmids pHYD3058 (trxA_Mtb) and pBAD33 (vector). Whole-cell extracts of transformants of DH5α bearing plasmids pHYD3058 (trxA_Mtb) and pBAD33 (vector) after cultivation of the cells in LB broth with 0.2% d-glucose and 0.2% l-arabinose, prepared after normalization for cell number, were probed with a monoclonal antibody directed against hexahistidine, an epitope appended to the carboxy terminus of TrxA_Mtb.