Mycobacterium tuberculosis SufR responds to nitric oxide via its 4Fe–4S cluster and regulates Fe–S cluster biogenesis for persistence in mice
Kushi Anand,
Ashutosh Tripathi,
Kaustubh Shukla,
Nitish Malhotra,
Anil Kumar Jamithireddy,
Rajiv Kumar Jha,
Susmit Narayan Chaudhury,
Raju S. Rajmani,
Arati Ramesh,
Valakunja Nagaraja,
Balasubramanian Gopal,
Ganesh Nagaraju,
Aswin Sai Narain Seshayee,
Amit Singh
Affiliations
Kushi Anand
Centre for Infectious Disease Research, Indian Institute of Science, Bangalore, 560012, India
Ashutosh Tripathi
Centre for Infectious Disease Research, Indian Institute of Science, Bangalore, 560012, India
Kaustubh Shukla
Department of Biochemistry, Indian Institute of Science, Bangalore, 560012, India
Nitish Malhotra
National Centre for Biological Science, Bangalore, 560065, India
Anil Kumar Jamithireddy
Molecular Biophysics Unit, Indian Institute of Science, Bangalore, 560012, India
Rajiv Kumar Jha
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India
Susmit Narayan Chaudhury
National Centre for Biological Science, Bangalore, 560065, India
Raju S. Rajmani
Centre for Infectious Disease Research, Indian Institute of Science, Bangalore, 560012, India
Arati Ramesh
National Centre for Biological Science, Bangalore, 560065, India
Valakunja Nagaraja
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India
Balasubramanian Gopal
Molecular Biophysics Unit, Indian Institute of Science, Bangalore, 560012, India
Ganesh Nagaraju
Department of Biochemistry, Indian Institute of Science, Bangalore, 560012, India
Aswin Sai Narain Seshayee
National Centre for Biological Science, Bangalore, 560065, India
Amit Singh
Centre for Infectious Disease Research, Indian Institute of Science, Bangalore, 560012, India; Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India; Corresponding author. Wellcome Trust-India Alliance Senior Fellow, Department of Microbiology and Cell Biology (MCBL), Centre for Infectious Disease Research (CIDR), Indian Institute of Science (IISc), Bangalore, 560012, India.
The persistence of Mycobacterium tuberculosis (Mtb) is a major problem in managing tuberculosis (TB). Host-generated nitric oxide (NO) is perceived as one of the signals by Mtb to reprogram metabolism and respiration for persistence. However, the mechanisms involved in NO sensing and reorganizing Mtb's physiology are not fully understood. Since NO damages iron-sulfur (Fe–S) clusters of essential enzymes, the mechanism(s) involved in regulating Fe–S cluster biogenesis could help Mtb persist in host tissues. Here, we show that a transcription factor SufR (Rv1460) senses NO via its 4Fe–4S cluster and promotes persistence of Mtb by mobilizing the Fe–S cluster biogenesis system; suf operon (Rv1460-Rv1466). Analysis of anaerobically purified SufR by UV–visible spectroscopy, circular dichroism, and iron-sulfide estimation confirms the presence of a 4Fe–4S cluster. Atmospheric O2 and H2O2 gradually degrade the 4Fe–4S cluster of SufR. Furthermore, electron paramagnetic resonance (EPR) analysis demonstrates that NO directly targets SufR 4Fe–4S cluster by forming a protein-bound dinitrosyl-iron-dithiol complex. DNase I footprinting, gel-shift, and in vitro transcription assays confirm that SufR directly regulates the expression of the suf operon in response to NO. Consistent with this, RNA-sequencing of MtbΔsufR demonstrates deregulation of the suf operon under NO stress. Strikingly, NO inflicted irreversible damage upon Fe–S clusters to exhaust respiratory and redox buffering capacity of MtbΔsufR. Lastly, MtbΔsufR failed to recover from a NO-induced non-growing state and displayed persistence defect inside immune-activated macrophages and murine lungs in a NO-dependent manner. Data suggest that SufR is a sensor of NO that supports persistence by reprogramming Fe–S cluster metabolism and bioenergetics.
