Mycobacterium tuberculosis requires SufT for Fe-S cluster maturation, metabolism, and survival in vivo

Abstract

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.

Effects of colonization-associated gene yqiC on global transcriptome, cellular respiration, and oxidative stress in Salmonella Typhimurium

“…By contrast, in our study, sublethal ampicillin did not have a considerable effect on the OCR and ECFR of S. Typhimurium or the phenotype of yqiC. A study that used the Seahorse Analyzer revealed that the iron-sulfur cluster biosynthesis protein SufT is required for glycolysis, oxidative phosphorylation, and survival in Mycobacterium tuberculosis after exposure to oxidative stress and nitric oxide [61]; this finding echoes our findings regarding the association of yqiC with electron transfer activity, iron-sulfur cluster assembly, and glycolysis in S. Typhimurium. Our RNA-seq analysis revealed the involvement of yqiC in energy and carbohydrate metabolism, and a series of experiments in the Seahorse XFp Analyzer further clarified how yqiC influences cellular respiration and glycolysis.…”

“…Typhimurium or the phenotype of yqiC . A study that used the Seahorse Analyzer revealed that the iron–sulfur cluster biosynthesis protein SufT is required for glycolysis, oxidative phosphorylation, and survival in Mycobacterium tuberculosis after exposure to oxidative stress and nitric oxide [ 61 ]; this finding echoes our findings regarding the association of yqiC with electron transfer activity, iron–sulfur cluster assembly, and glycolysis in S . Typhimurium.…”

“…The Seahorse XFp Analyzer was used to measure glycolysis and mitochondria respiration in the mammalian cells [ 55 ], and eukaryotic cells, including Caenorhabditis elegans (nematode) [ 56 ], Dictyostelium discoideum (amoeba) [ 57 ], Candida albicans [ 58 ], and Cryptococcus neoformans [ 59 ]. Cellular respiration plays a similar role in mitochondrial respiration, and several studies have used the Seahorse XFp Analyzer to investigate mitochondria-absent prokaryotic bacteria such as E. coli , Staphylococcus aureus [ 24 , 60 ] and Mycobacterium tuberculosis [ 61 ]; however, in this context, the research on Salmonella is limited. In the Seahorse Analyzer, ampicillin at a dose of 5 × MIC or 50 × MIC accelerates cellular respiration by increasing OCR, indicating the association of antibiotic efficacy and phenotypic resistance with cellular respiration in E. coli [ 24 , 60 ].…”

Cysteine desulfurase (IscS)–mediated fine-tuning of bioenergetics and SUF expression prevents Mycobacterium tuberculosis hypervirulence

“…The E. coli Isc system is generally poisoned with as low as 1 μM H 2 O 2 , whereas the Suf system remains resistant to oxidative stress (6). Similar differences in sensitivity to oxidants are expected for Mtb IscS and Suf systems (10,11,17). However, the artificial overproduction of IscS suppresses the growth defect of MtbΔiscS and renders the mutant more resistant to H 2 O 2 than WT Mtb (17).…”

Iron–Sulfur Clusters toward Stresses: Implication for Understanding and Fighting Tuberculosis

“…SufT is proposed to act as an accessory factor in Fe-S biogenesis in Mtb, since it is dispensable for growth of Mtb under standard culture conditions, required under conditions of iron limitation [170], and SufT loss does not increase susceptibility to oxidative stress [170]. Very recently, Mtb SufT protein was shown to interact with SufS and SufU and to maintain the activity of Fe-S cluster proteins during normal growth conditions, and under environmental settings that enforce a high demand of Fe-S clusters [171]. Characterizations of Mtb SufS and SufU will certainly provide additional information and interesting insights into the sulfur production.…”