Nitrite reductase NirBD is induced and plays an important role during in vitro dormancy of Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis is one of the strongest reducers of nitrate among all mycobacteria. Reduction of nitrate to nitrite, mediated by nitrate reductase (NarGHJI) of M. tuberculosis, is induced during the dormant stage, and the enzyme has a respiratory function in the absence of oxygen. Nitrite reductase (NirBD) is also functional during aerobic growth when nitrite is the sole nitrogen source. However, the role of NirBD-mediated nitrite reduction during the dormancy is not yet characterized. Here, we analyzed nitrite reduction during aerobic growth as well as in a hypoxic dormancy model of M. tuberculosis in vitro. When nitrite was used as the sole nitrogen source in the medium, the organism grew and the reduction of nitrite was evident in both hypoxic and aerobic cultures of M. tuberculosis. Remarkably, the hypoxic culture of M. tuberculosis, compared to the aerobic culture, showed 32- and 4-fold-increased expression of nitrite reductase (NirBD) at the transcription and protein levels, respectively. More importantly, a nirBD mutant of M. tuberculosis was unable to reduce nitrite and compared to the wild-type (WT) strain had a >2-log reduction in viability after 240 h in the Wayne model of hypoxic dormancy. Dependence of M. tuberculosis on nitrite reductase (NirBD) was also seen in a human macrophage-based dormancy model where the nirBD mutant was impaired for survival compared to the WT strain. Overall, the increased expression and essentiality of nitrite reductase in the in vitro dormancy models suggested that NirBD-mediated nitrite reduction could be critical during the persistent stage of M. tuberculosis.

Fig 1

Nitrite reduction and CFU of M. tuberculosis H37Ra during aerobic growth and in the Wayne model. Bacteria were cultured with 1 mM nitrite as the sole nitrogen source. (A) Nitrite reduction was determined by testing aliquots at the indicated time points for depletion of nitrite from the medium. (B) Growth of the organism was determined by testing aliquots at the indicated time points by plating on the 7H11 agar plates to enumerate CFU. Circles represent the aerobic cultures, whereas rectangles represent the hypoxic cultures. The results are averages ± SD from three identical experiments.

Fig 2

(A) Expression of M. tuberculosis genes involved in nitrate/nitrite metabolism under aerobic and Wayne hypoxic conditions with different nitrogen sources. cDNA was prepared from total RNA isolated from bacilli grown in the presence of nitrate, nitrite, ammonium, or asparagine either aerobically (+O₂) or in the Wayne model (−O₂). cDNA was used as the template for PCR amplification using gene-specific primers for narG (nitrate reductase), nirB (nitrite reductase), and glnA1 (glutamine synthetase), and the amplified products are shown after electrophoresis on agarose gels. Lane (-), negative control without cDNA; lane M, 100-bp marker (Invitrogen). (B) Quantitative PCR analysis of nirB, narG, and narK2 expression in bacilli grown in the presence of nitrite as the nitrogen source under different conditions. cDNA was prepared from the M. tuberculosis culture grown under aerobic (+O₂) or hypoxic (−O₂) conditions. The relative levels of the transcripts are shown as the difference from that obtained under aerobic conditions, while the 16S gene was used as an internal control. (C) Specific activity of nitrite reductase enzymes in the cytoplasm and membrane fractions of M. tuberculosis cells grown in the presence of nitrite as the nitrogen source. Specific enzyme activity was measured during mid-logarithmic phase for aerobic cultures (+O₂) and on the 7th day in Wayne model cultures (−O₂). White bars represent the Nir activity measured in cytoplasmic fractions, whereas gray bars represent Nir activity measured in membrane fractions. One unit of specific activity of enzyme was defined as 1 μM NO₂⁻ depleted per min/mg of total protein during the in vitro Nir enzyme assay.

Fig 3

Viability and nitrite reduction by the nirBD mutant and WT M. tuberculosis in the Wayne model with different nitrogen sources. Viability (A, C, and E) and nitrite levels (B and D) in M. tuberculosis H37Rv (○) and the nirBD mutant (□) when nitrite (A and B) or nitrite and asparagine (C and D) or asparagine only (E) was the nitrogen source. The results are averages ± SD from three identical experiments.

Fig 4

(A) Survival of the nirBD mutant and WT M. tuberculosis H37Rv in THP-1 macrophages treated with retinoic acid and vitamin D (RAVD). Macrophages were lysed at the indicated time points and plated on 7H11 agar to determine the intracellular burden of bacilli. *, P < 0.05. ↓, MNGC formation, which indicates the induction of dormancy in intracellular M. tuberculosis. (B) Presence of nitrite within THP-1 macrophages infected with the nirBD mutant (□) and WT M. tuberculosis H37Rv (○). The results are averages ± SD from three identical experiments.