Orphan response regulator NnaR is critical for nitrate and nitrite assimilation in Mycobacterium abscessus

Abstract

Mycobacterium abscessus (Mab) is an opportunistic pathogen afflicting individuals with underlying lung disease such as Cystic Fibrosis (CF) or immunodeficiencies. Current treatment strategies for Mab infections are limited by its inherent antibiotic resistance and limited drug access to Mab in its in vivo niches resulting in poor cure rates of 30-50%. Mab's ability to survive within macrophages, granulomas and the mucus laden airways of the CF lung requires adaptation via transcriptional remodeling to counteract stresses like hypoxia, increased levels of nitrate, nitrite, and reactive nitrogen intermediates. Mycobacterium tuberculosis (Mtb) is known to coordinate hypoxic adaptation via induction of respiratory nitrate assimilation through the nitrate reductase narGHJI. Mab, on the other hand, does not encode a respiratory nitrate reductase. In addition, our recent study of the transcriptional responses of Mab to hypoxia revealed marked down-regulation of a locus containing putative nitrate assimilation genes, including the orphan response regulator nnaR (nitrate/nitrite assimilation regulator). These putative nitrate assimilation genes, narK3 (nitrate/nitrite transporter), nirBD (nitrite reductase), nnaR, and sirB (ferrochelatase) are arranged contiguously while nasN (assimilatory nitrate reductase identified in this work) is encoded in a different locus. Absence of a respiratory nitrate reductase in Mab and down-regulation of nitrogen metabolism genes in hypoxia suggest interplay between hypoxia adaptation and nitrate assimilation are distinct from what was previously documented in Mtb. The mechanisms used by Mab to fine-tune the transcriptional regulation of nitrogen metabolism in the context of stresses e.g. hypoxia, particularly the role of NnaR, remain poorly understood. To evaluate the role of NnaR in nitrate metabolism we constructed a Mab nnaR knockout strain (Mab_ΔnnaR ) and complement (Mab_ΔnnaR+C ) to investigate transcriptional regulation and phenotypes. qRT-PCR revealed NnaR is necessary for regulating nitrate and nitrite reductases along with a putative nitrate transporter. Loss of NnaR compromised the ability of Mab to assimilate nitrate or nitrite as sole nitrogen sources highlighting its necessity. This work provides the first insights into the role of Mab NnaR setting a foundation for future work investigating NnaR's contribution to pathogenesis.

Keywords: Mycobacterium abscessus; NnaR; nitrate; nitrite; nitrogen metabolism; orphan response regulator.

Figure 1

Bioinformatic analysis of NnaR domain structure and sequence alignment. Uniprot Bioanalysis Online Tool was used to generate the (A) Alpha-fold model of 3-D structure of Mab NnaR HemD-ORR protein. and (B) sequence alignment of Mab NnaR (MAB_3520c) compared to Msm NnaR (MSMEG_0432), Mtb (Rv0260c) and S. coelicolor NnaR (SCO2958). The N-terminal HemD domain is outlined in green, and C-terminal DNA-binding domain in red. Sequences for Mab, Msm and Mtb were obtained from the online data base Mycobrowser. S. coelicolor sequence was obtained from the online KEGG data base.

Figure 2

Genetic organization of the NnaR operon and regulon. (A) Schematic depicting nitrogen assimilation gene arrangement and NnaR binding sites discovered using DNA Pattern Find. Yellow highlighted nucleotides coincide with motifs used to find promoter region. P1-P4 indicates intergenic amplicons for RT-PCR. (B) Agarose gel of RT-PCR products P1-P4 amplified from DNA (positive control), RNA (negative control for DNA contamination), or cDNA.

Figure 3

The NnaR regulon is induced when nitrate or nitrite are the sole sources of nitrogen. qRT-PCR was performed to confirm induction of nnaR regulon when nitrate (A) or nitrite (B) are the sole nitrogen sources. Mab390S supplemented with 2.5 mM ammonium sulfate (black bars), 2.5 mM nitrate (pink bars) and 0.5mM nitrite (orange bars). qRT-PCR data is representative of 3 experiments performed in duplicate. P values were calculated via t-test using GraphPad. *P-value <0.05, **P-value <0.01 ****P-value <0.0001.

Figure 4

NnaR regulates the nark3-sirB operon and nasN. qRT-PCR was performed to confirm the deletion and restoration of nnaR in Mab _ΔnnaR  and Mab _ΔnnaR+C , respectively, and effect on predicted regulon in 7H9 media (A), in minimal media supplemented with nitrate (B) or nitrite (C). WT Mab 390S (black), Mab _ΔnnaR (red) and Mab _ΔnnaR+C (blue). Data is representative of 3 experiments performed in duplicate. P values were calculated via one-way ANOVA using GraphPad. Red stars indicate C_t values were not detected for Mab nnaR in the mutant strain. *P-value <0.05, **P-value <0.01 ****P-value <0.0001. ns denotes samples that were not significantly different than WT.

Figure 5

NnaR is responsible for nitrite utilization and growth when nitrate and nitrite are sole sources of nitrogen. Growth kinetics in minimal media supplemented with various nitrogen sources was assessed via serial dilutions, spot plating and enumeration of CFU/ml on days 0-4. Cultures supplemented with (A) ammonium sulfate, (B) nitrate or (C) nitrite. WT Mab (black circle), Mab_ΔnnaR  (red square), Mab_ΔnnaR+C  (blue triangle), WT Mab-Minimal Media (serving as negative control-no nitrogen supplementation, black upside down triangle). Griess assay (D) was performed to measure nitrite accumulation on day 2 and 4. Mab 390S (black bars), Mab_ΔnnaR  (red bars) and Mab_ΔnnaR+C  (blue bars). Data reflects 3 independent experiments performed in triplicate. ** P-value <0.01, **** P-value <0.001.