TY - JOUR
T1 - Mycobacterium tuberculosis requires SufT for Fe-S cluster maturation, metabolism, and survival in vivo
AU - Tripathi, Ashutosh
AU - Anand, Kushi
AU - Das, Mayashree
AU - O’Niel, Ruchika Annie
AU - Sabarinath, P. S.
AU - Thakur, Chandrani
AU - Reddy, Raghunatha R.L.
AU - Rajmani, Raju S.
AU - Chandra, Nagasuma
AU - Laxman, Sunil
AU - Singh, Amit
N1 - Funding Information:
Funding:TheMtbworkwassupportedbythe followingWellcomeTrust/DBTIndiaAlliance Grants,IA/S/16/2/502700(AS),andinpartby DepartmentofBiotechnology(DBT)Grant(BT/ PR13522/COE/34/27/2015,BT/PR29098/Med/29/ 1324/2018,BT/HRD/NBA/39/07/2018-19,andBT/ PR39308/DRUG/134/86/2021)(AS),DBT-IISc PartnershipProgram(22-0905-0006-05-987-436), DST-FISTgrant,andtheRevatiandSatyaNadham AtluriChairProfessorship(AS).Thefundershadno roleinstudydesign,datacollectionandanalysis, decisiontopublish,orpreparationofthe manuscript.
Funding Information:
The Mtb work was supported by the following Wellcome Trust/DBT India Alliance Grants, IA/S/16/2/502700 (AS), and in part by Department of Biotechnology (DBT) Grant (BT/ PR13522/COE/34/27/2015,BT/PR29098/Med/29/ 1324/2018, BT/HRD/NBA/39/07/2018-19, and BT/ PR39308/DRUG/134/86/2021) (AS), DBT-IISc Partnership Program (22-0905-0006-05-987-436), DST-FIST grant, and the Revati and Satya Nadham Atluri Chair Professorship (AS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We are thankful to BSL3 facilities at CIDR, IISc Bangalore for conducting all experiments related to Mtb. We thank Mass spectrometry facility at NCBS/inStem/CCAMP, Bangalore, for the access to, and use of mass spectrometers.
Publisher Copyright:
© 2022 Tripathi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2022/4
Y1 - 2022/4
N2 - Iron-sulfur (Fe-S) cluster proteins carry out essential cellular functions in diverse organisms, including the human pathogen Mycobacterium tuberculosis (Mtb). The mechanisms underlying Fe-S cluster biogenesis are poorly defined in Mtb. Here, we show that Mtb SufT (Rv1466), a DUF59 domain-containing essential protein, is required for the Fe-S cluster maturation. Mtb SufT homodimerizes and interacts with Fe-S cluster biogenesis proteins; SufS and SufU. SufT also interacts with the 4Fe-4S cluster containing proteins; aconitase and SufR. Importantly, a hyperactive cysteine in the DUF59 domain mediates interaction of SufT with SufS, SufU, aconitase, and SufR. We efficiently repressed the expression of SufT to generate a SufT knock-down strain in Mtb (SufT-KD) using CRISPR interference. Depleting SufT reduces aconitase’s enzymatic activity under standard growth conditions and in response to oxidative stress and iron limitation. The SufT-KD strain exhibited defective growth and an altered pool of tricarboxylic acid cycle intermediates, amino acids, and sulfur metabolites. Using Seahorse Extracellular Flux analyzer, we demonstrated that SufT depletion diminishes glycolytic rate and oxidative phosphorylation in Mtb. The SufT-KD strain showed defective survival upon exposure to oxidative stress and nitric oxide. Lastly, SufT depletion reduced the survival of Mtb in macrophages and attenuated the ability of Mtb to persist in mice. Altogether, SufT assists in Fe-S cluster maturation and couples this process to bioenergetics of Mtb for survival under low and high demand for Fe-S clusters.
AB - Iron-sulfur (Fe-S) cluster proteins carry out essential cellular functions in diverse organisms, including the human pathogen Mycobacterium tuberculosis (Mtb). The mechanisms underlying Fe-S cluster biogenesis are poorly defined in Mtb. Here, we show that Mtb SufT (Rv1466), a DUF59 domain-containing essential protein, is required for the Fe-S cluster maturation. Mtb SufT homodimerizes and interacts with Fe-S cluster biogenesis proteins; SufS and SufU. SufT also interacts with the 4Fe-4S cluster containing proteins; aconitase and SufR. Importantly, a hyperactive cysteine in the DUF59 domain mediates interaction of SufT with SufS, SufU, aconitase, and SufR. We efficiently repressed the expression of SufT to generate a SufT knock-down strain in Mtb (SufT-KD) using CRISPR interference. Depleting SufT reduces aconitase’s enzymatic activity under standard growth conditions and in response to oxidative stress and iron limitation. The SufT-KD strain exhibited defective growth and an altered pool of tricarboxylic acid cycle intermediates, amino acids, and sulfur metabolites. Using Seahorse Extracellular Flux analyzer, we demonstrated that SufT depletion diminishes glycolytic rate and oxidative phosphorylation in Mtb. The SufT-KD strain showed defective survival upon exposure to oxidative stress and nitric oxide. Lastly, SufT depletion reduced the survival of Mtb in macrophages and attenuated the ability of Mtb to persist in mice. Altogether, SufT assists in Fe-S cluster maturation and couples this process to bioenergetics of Mtb for survival under low and high demand for Fe-S clusters.
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U2 - 10.1371/journal.ppat.1010475
DO - 10.1371/journal.ppat.1010475
M3 - Article
C2 - 35427399
AN - SCOPUS:85129540472
SN - 1553-7366
VL - 18
JO - PLoS pathogens
JF - PLoS pathogens
IS - 4
M1 - e1010475
ER -