Zinc pyrithione impairs iron-sulfur cluster biogenesis in Mycobacterium tuberculosis

Lu Wang; Xuelin Wang; Lingling Xu; Bin Wang; Deqian Ji; Yangxue Ye; Yu Lu

doi:10.1186/s12866-025-04644-7

. 2025 Dec 20;26:54. doi: 10.1186/s12866-025-04644-7

Zinc pyrithione impairs iron-sulfur cluster biogenesis in Mycobacterium tuberculosis

Lu Wang ¹, Xuelin Wang ¹, Lingling Xu ¹, Bin Wang ¹, Deqian Ji ¹, Yangxue Ye ¹, Yu Lu ^1,^✉

PMCID: PMC12836896 PMID: 41419803

Abstract

Background

Tuberculosis remains a major global health threat, especially with the increasing prevalence of drug-resistant Mycobacterium tuberculosis (Mtb). There is an urgent need to develop new antibiotics with novel mechanisms of action. Zinc pyrithione (ZnPT), a bidentate metal-chelating agent, displays potent in vitro activity against bacteria and fungi. This study aimed to evaluate the antimycobacterial activity of ZnPT and to explore its potential mechanisms.

Methods

The bactericidal activity of ZnPT against Mtb strains was evaluated using minimum inhibitory concentration and minimum bactericidal concentration assays. The role of copper was assessed through metal chelator supplementation studies, while intracellular metal accumulation was quantified using inductively coupled plasma mass spectrometry (ICP-MS). Mechanistic analyses included transcriptomic profiling, enzyme activity assays, and targeted metabolomics to assess effects on iron-sulfur (Fe-S) cluster biogenesis and energy metabolism.

Results

ZnPT demonstrated potent bactericidal activity against both drug-sensitive and drug-resistant Mtb strains. Copper supplementation significantly enhanced the efficacy of ZnPT, and ICP-MS confirmed elevated intracellular copper levels. Transcriptomic analysis revealed disruption of multiple pathways, including the copper ion stress response, sulfur metabolism, Fe-S cluster biogenesis, siderophore biosynthesis, and intermediary metabolism. Notably, ZnPT induced upregulation of the sulfur mobilization (SUF) operon while repressing electron transfer ferredoxins, indicating disturbed Fe-S cluster homeostasis. Enzyme assays showed marked inhibition of cysteine desulfurase activity, a key step in Fe-S cluster assembly. Targeted metabolomics revealed depletion of tricarboxylic acid (TCA) cycle intermediates and accumulation of metabolic bottlenecks, indicating impaired Fe-S enzyme activity. ZnPT treatment further led to dysfunction of the electron transport chain, reduced proton motive force, and ATP depletion.

Conclusions

ZnPT exhibits antimycobacterial activity by disrupting Fe-S cluster biogenesis and impairing energy metabolism in Mtb.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12866-025-04644-7.

Keywords: Zinc pyrithione, Mycobacterium tuberculosis, Antimicrobial, Fe-S cluster biogenesis

Introduction

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a significant global health crisis. In 2023, the World Health Organization (WHO) reported 10.8 million new cases of tuberculosis and over 1.25 million deaths [1]. The lengthy treatment regimens, which range from 6 to 24 months for drug-sensitive and drug-resistant strains, respectively, along with the increasing prevalence of multidrug-resistant (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB), underscore the urgent need for more effective and shorter treatment protocols [2]. Current first-line drugs, which inhibit cell wall synthesis, DNA replication, or transcription, are often evaded by Mtb through genetic mutations or efflux pumps, revealing the vulnerabilities of existing therapeutic strategies [3, 4]. This therapeutic challenge demands a paradigm shift toward novel compounds that can simultaneously disrupt distinct or convergent molecular pathways critical to Mtb survival.

Zinc pyrithione (ZnPT) is a broad-spectrum antimicrobial agent with significant activity against a variety of bacterial and fungal pathogens. It is widely used as an active antifungal ingredient in medicated shampoos [5–7]. Its chemical structure is shown in Figure S1. In this study, we first identified ZnPT as a potent anti-mycobacterial agent against Mtb, exhibiting a remarkably low minimum inhibitory concentration (MIC) through small-molecule library screening. Previous studies have attributed ZnPT’s antibacterial effects to proton pump inhibition or ionophore activity [8, 9], while these mechanisms do not adequately explain its exceptional antitubercular efficacy [5–7]. The underlying mechanisms of its antitubercular activity remain largely unknown, necessitating further investigation.

Iron-sulfur clusters (Fe-S clusters) are inorganic cofactors composed of iron and sulfur atoms which play a crucial role in core metabolic and respiratory processes in Mtb [10, 11]. These clusters are indispensable for enzymes involved in essential metabolic pathways, such as the tricarboxylic acid (TCA) cycle, the electron transport chain, and DNA repair mechanisms [12]. Notably, Fe-S enzymes serve as central nodes in intermediary metabolism and respiration, making their inactivation a multi-target bactericidal strategy with a low potential for resistance. Unlike eukaryotes, which utilize the mitochondrial iron-sulfur cluster (ISC) assembly system [13, 14], Mtb exclusively relies on the sulfur mobilization (SUF) pathway for Fe-S cluster biogenesis [15]. Although Mtb retains the ancestral ISC system component IscS [16, 17], the function of ISC cannot compensate for the SUF pathway. This unique dependence renders the SUF pathway essential for the survival of Mtb.

In this study, we demonstrate that ZnPT disrupts SUF‑mediated Fe‑S cluster biogenesis, thereby inactivating key Fe‑S enzymes involved in energy metabolism and respiration. Metabolic dysregulation arising from Fe-S protein dysfunction is lethal to Mtb and does not exhibit cross‑resistance with existing first‑line or second‑line drugs. Our findings establish the inhibition of Fe-S cluster biogenesis as a novel and promising strategy for combating MDR‑TB and XDR‑TB.

Results

ZnPT exerts potent copper-dependent bactericidal activity against both drug-susceptible and drug-resistant Mtb

We identified ZnPT as a potent anti-mycobacterial agent against Mtb, exhibiting a remarkably low MIC of 0.015 µg/mL (47 nM). Notably, ZnPT maintained strong activity against clinical MDR-TB and XDR-TB isolates (Table 1), with MIC values ranging from 0.008 to 0.060 µg/mL. This demonstrates significant efficacy even against strains resistant to rifampicin (RIF) and isoniazid (INH). In checkerboard assays, ZnPT exhibited predominantly indifferent or additive interactions with the tested anti-tubercular agents, and no antagonism was observed (Table S1). Due to the intracellular nature of Mtb infection, it is essential to assess the intracellular bactericidal activity of candidate compounds. Cytotoxicity of ZnPT in J774A.1 macrophages was assessed using the MTT assay, yielding an IC₅₀ value of 4.6 µg/mL. We treated Mtb-infected J774A.1 macrophages with ZnPT and observed an approximately 1-log₁₀ reduction in bacterial load at 2 µg/mL, demonstrating potent intracellular bactericidal activity comparable to 5 µg/mL RIF (Fig. 1A).

Table 1.

MICs of ZnPT against standard and clinically isolated susceptible and drug-resistant strains of Mtb

Strains	MIC (µg/mL)
Strains	BDQ	CFZ	EMB	INH	LZD	RIF	ZnPT
11,264	0.020	0.312	2.500	0.040	0.313	0.040	0.015
11,492	0.160	2.500	> 10	> 10	0.160	0.160	0.015
12,585	0.040	0.625	> 10	> 10	0.313	> 10	0.015
12,657	0.310	1.250	10.00	> 10	0.310	0.300	0.030
12,897	0.020	0.625	> 10	5.000	0.625	5.000	0.008
13,385	0.125	1.250	> 10	> 10	0.625	0.620	0.030
13,529	0.040	0.310	> 10	> 10	0.310	0.002	0.015
13,946	0.040	0.625	5.000	> 10	0.310	> 10	0.015
14,470	0.310	0.160	1.250	> 10	0.310	> 10	0.060
15,171	0.150	0.625	2.500	> 10	2.500	0.625	0.015
15,771	0.320	2.500	> 10	> 10	0.160	0.160	0.015
16,030	0.040	2.500	2.500	2.500	0.625	> 10	0.030
16,835	0.040	0.080	2.500	0.080	0.310	0.020	0.008
16,995	0.040	0.160	> 10	> 10	0.310	> 10	0.015
17,080	0.040	0.080	5.000	> 10	10	> 10	0.015
28,350	0.040	0.313	> 10	> 10	0.313	> 10	0.015
29,223	0.640	5.000	> 10	> 10	0.625	> 10	0.015
29,928	0.640	5.000	> 10	> 10	0.625	> 10	0.015
29,420	0.039	1.250	> 10	> 10	1.250	> 10	0.030
29,499	0.039	1.250	> 10	> 10	1.250	> 10	0.030
30,044	0.313	1.250	5.000	> 10	2.500	1.250	0.030
30,141	0.040	0.625	> 10	> 10	0.160	> 10	0.015
30,180	0.040	0.625	> 10	> 10	0.313	> 10	0.008
30,306	0.040	0.313	5.000	0.080	0.625	0.040	0.015
30,314	0.040	0.160	5.000	0.080	0.313	0.020	0.015
30,450	0.020	0.156	> 10	> 10	0.156	> 10	0.015
30,459	0.040	1.250	> 10	> 10	0.313	> 10	0.015
30,554	0.010	1.250	> 10	5.000	0.625	> 10	0.015
30,797	0.160	5.000	> 10	> 10	0.625	> 10	0.015
30,819	0.020	0.310	5.000	> 10	0.160	0.002	0.008
81,167	0.040	0.160	2.500	> 10	0.160	> 10	0.015
2-MDR	0.015	0.008	1.250	> 10	2.500	> 10	0.063
11-MDR	0.015	> 10	1.250	> 10	1.250	> 10	0.030
14-MDR	0.015	5	> 10	> 10	0.625	> 10	0.030
31-MDR	0.030	10	> 10	> 10	1.250	> 10	0.063
40-MDR	0.015	5	> 10	1.250	1.250	> 10	0.063
95-MDR	0.015	0.600	> 10	> 10	0.310	> 10	0.063
103-MDR	0.015	> 10	2.500	> 10	1.250	> 10	0.063
152-MDR	0.030	2.5	0.030	> 10	1.250	> 10	0.063
422-MDR	0.030	5	> 10	> 10	2.500	> 10	0.063
H37Rv	0.020	0.160	2.500	0.080	0.313	0.020	0.015

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MICs were determined by broth microdilution in 7H9 medium supplemented with 10% OADC at 37℃, without additional copper supplementation

^a The abbreviation of anti-tuberculosis drugs Bedaquiline, BDQ; clofazimine, CFZ; ethambutol, EMB; isoniazid, INH; linezolid, LZD; rifampicin, RIF; zinc pyrithione, ZnPT

^b Clinical Strains were isolated from patients in Beijing Chest Hospital

Fig. 1 — ZnPT exhibits potent anti-tubercular activity. A Intracellular activity of ZnPT against *Mtb* H37Rv in macrophages. J774A.1 macrophages infected with *Mtb* H37Rv were treated with ZnPT at 0.5, 1.0, and 2.0 µg/mL for 72 h. B Time-kill kinetics of ZnPT in 7H9 medium. C Time-kill kinetics of ZnPT in 7H9 medium supplemented with 50 µg/mL BCS. Conducted in parallel with panel B. D Time-kill kinetics of ZnPT in Sauton medium. The experimental conditions matched panel B, but were conducted in copper-limiting Sauton medium. E Time-kill kinetics of ZnPT in Sauton medium supplemented with 5 µM CuSO₄. Conducted in parallel with panel D. F ICP-MS quantification of intracellular copper levels. H37Rv cultures were treated with ZnPT (0.015 µg/mL; 0.03 µg/mL) or with elesclomol (0.5 µg/mL) for 4 h. Data are presented as the mean ± SD of three biological replicates. Statistical significance was determined using one-way ANOVA with Dunnett’s test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

According to previous studies demonstrating the copper ionophore activity of ZnPT [5], and considering the inherent toxicity of excessive intracellular copper to Mtb, we hypothesized that the anti-tubercular activity of ZnPT may depend on the availability of copper. To validate our hypothesis, we assessed the MIC of ZnPT under metal-chelating conditions. Supplementing Middlebrook 7H9 medium with the copper-specific chelator bathocuproine disulfonate (BCS) at 50 µg/mL resulted in an eightfold increase in the minimum inhibitory concentration (MIC) of ZnPT, while the addition of EDTA (100 µg/mL), a broad-spectrum metal chelator, led to a 16-fold increase in MIC, while the MICs of RIF and INH remained unaffected under the same conditions (Table 2). Consistent with these findings, time-kill kinetics in 7H9 medium demonstrated bactericidal activity with a minimum bactericidal concentration (MBC) of 0.03 µg/mL (2× MIC). However, the supplementation of BCS (50 µg/mL) significantly diminished the bactericidal effects of ZnPT, as even 0.06 µg/mL failed to inhibit bacterial growth (Fig. 1B and C). Sauton medium, a liquid medium routinely used for cultivating Mtb without supplemental copper ions, was employed to ensure a controlled baseline of metal ions during the assessment of ZnPT activity. In contrast, the exogenous addition of CuSO₄ (5 µM) to Sauton medium significantly accelerated bactericidal kinetics, reducing the bacterial eradication time from 3 days to 1 day at concentrations of 0.5 µg/mL and 1 µg/mL (Fig. 1D and E). Furthermore, inductively coupled plasma mass spectrometry (ICP-MS) analysis confirmed substantial intracellular copper accumulation (Fig. 1F), along with a notable increase in zinc levels following treatment with 0.03 µg/mL ZnPT, while levels of iron and manganese exhibited minimal changes (Fig. S2). These results confirm that the antibacterial activity of ZnPT is specifically dependent on copper.

Table 2.

Modulation of ZnPt’s anti-tuberculosis activity by metal chelators

	7H9	7H9 + 50 µg/ml BCS	7H9 + 100 µg/ml EDTA
RIF	0.030	0.030	0.030
INH	0.060	0.060	0.060
ZnPT	0.015	0.125	0.250

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The MIC of ZnPT against H37Rv was determined supplemented with metal chelators EDTA or BCS

ZnPT impacts the SUF iron-sulfur biogenesis and metabolic activities in Mtb

Transcriptomic analysis revealed that ZnPT treatment induced significant differential gene expression in Mtb H37Rv, identifying 97 differentially expressed genes (DEGs) at 4 h (76 upregulated, 21 downregulated) and 35 DEGs at 24 h (22 upregulated, 13 downregulated) (Fig. 2A and B). Functional enrichment analysis indicated that these genes were primarily involved in pathways related to copper ion stress response (Fig. 2C), sulfur metabolism (Fig. 2D), SUF system (Fig. 2E), siderophore biosynthesis (Fig. 2F), intermediary metabolism and respiration (Fig. 2G), transcription, translation and post-translational modification (Fig. 2H). Detailed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment results are presented in Figure S3. Notably, copper-responsive genes were significantly enriched at both time points, comprising 23.3% and 47.4% of upregulated genes at 4 and 24 h, respectively, highlighting the critical role of copper ions in ZnPT-induced stress responses. Additionally, the SUF operon (Rv1460 - Rv1466), which mediates Fe-S cluster biosynthesis [5], was markedly upregulated at 4 h (Fig. 2E), likely due to ZnPT-induced copper accumulation disrupting Fe-S cluster homeostasis (Fig. 2C). In contrast, ferredoxin genes (fdxB and fdxA), which deliver electrons to Fe-S biosynthetic enzymes [18], were repressed (Fig. 2G). Disruption of the Fe-S cluster biogenesis system also coincided with the downregulation of Fe-S cluster-dependent metabolic genes such as udgA, gltB, and ilvD (Fig. 2G). These genes are involved in NAD⁺ regeneration [19], glutamate synthesis [20], and branched-chain amino acid biosynthesis [21, 22], respectively.

At 24 h, the Fe-S cluster-associated gene frdB was significantly upregulated (Fig. 2G), suggesting enhanced succinate production via the reverse the TCA cycle to facilitate NADH oxidation and maintain the proton gradient [23]. Meanwhile, multiple subunits of succinate dehydrogenase (sdhA-sdhD) and the key ferredoxin-NADP⁺ reductase gene fprB were notably downregulated (Fig. 2G), further impairing Fe-S enzyme activity and exacerbating metabolic dysfunction [24]. Collectively, these findings demonstrate that ZnPT profoundly disrupts Fe-S cluster biogenesis and associated metabolic networks in Mtb, ultimately leading to widespread metabolic perturbation.

To validate these transcriptional changes, we performed reverse transcription quantitative polymerase chain reaction (RT-qPCR) on genes involved in Fe-S biogenesis, biogenesis of siderophore, sulfur metabolism, and Fe-S proteins related to energy metabolism. The results confirmed a pronounced upregulation of the SUF operon genes at both 4 and 24 h, with higher expression at 24 h (Fig. 3A). Notably, iscS, which encodes an alternative cysteine desulfurase for Fe-S cluster biogenesis [17], was also upregulated at 24 h (Fig. 3B). A possible explanation is that, despite sustained activation of the SUF system, impairment of the SUF-associated cysteine desulfurase activity causes Mtb to upregulate the ISC homologue in an attempt to compensate for the deficiency in Fe-S cluster biogenesis. Consistent with the transcriptomic findings, RT-qPCR analysis confirmed that furA was upregulated, cysK2 was induced, and acn was downregulated (Fig. 3B), indicating significant metabolic perturbation. Furthermore, the opposing regulation of frdB (upregulated) and acn (downregulated) further supports remodeling of the TCA cycle under ZnPT-induced stress (Fig. 3B).

Fig. 3 — Validation by RT-qPCR of transcriptional changes. A SUF operon induction under ZnPT stress. SUF genes (*Rv1460* - *Rv1466*) were upregulated after 4 and 24 h of exposure to 0.015 and 0.03 µg/mL. Fold-change values are normalized to untreated controls. B Dose-dependent gene expression responses to ZnPT at 24 h. There was a dose-dependent upregulation of *ctpV* (copper efflux), *iscS* (Fe-S biogenesis), *furA* (iron regulation), *cysK2* (cysteine synthesis), and the Fe-S enzyme *frdB* (metabolism). Additionally, *acn* (metabolism) was downregulated. Data are presented herein as the mean ± SD of three biological replicates. Statistical significance was performed using two-way ANOVA with Dunnett’s test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

ZnPT inhibits the activity of cysteine desulfurase and Fe-S enzymes in energy metabolism

During Fe-S cluster biogenesis, desulfurization and sulfur transfer are key steps. SufS (cysteine desulfurase, Rv1464) functions as a cysteine desulfurase that extracts sulfur from L-cysteine to generate a persulfide intermediate, while SufU (Rv1465) acts as a zinc-dependent sulfurtransferase that receives sulfur from SufS and enhances its activity, thereby facilitating Fe-S cluster biogenesis in Mtb [15]. Given the transcriptomic evidence indicating perturbation of sulfur metabolism and SUF pathway by ZnPT, along with the marked upregulation of the ISC-type cysteine desulfurase, we hypothesize that ZnPT may impair the desulfurization step during Fe-S cluster biogenesis. We quantified total cysteine desulfurase activity in bacterial lysates using a methylene blue assay. ZnPT treatment significantly suppressed enzymatic activity (Fig. 4A), mirroring the inhibitory effect observed with the Zn²⁺-specific chelator TPEN, which suggests disruption of sulfur supply for Fe-S cluster assembly. Impaired Fe-S cluster formation subsequently leads to dysfunction of Fe-S proteins. To assess downstream functional consequences, we measured the activity of two Fe-S cluster enzymes related to intermediary metabolism and respiration: aconitase (ACO) and succinate dehydrogenase (SDH). ACO activity, assessed in the presence of 2,2’-bipyridyl (used as an iron chelator control), was significantly reduced at ZnPT concentrations of 0.015 µg/mL (1× MIC) and 0.03 µg/mL (2× MIC) (Fig. 4B). Similarly, SDH activity was suppressed following ZnPT exposure, paralleling the inhibition observed with 3-nitropropionate (3-NP), an SDH-specific inhibitor (Fig. 4C). These results demonstrate that ZnPT-induced defects in Fe-S cluster biogenesis impair the maturation and function of essential Fe-S enzymes involved in energy metabolism.

Fig. 4 — ZnPT inhibits the activities of cysteine desulfurase and Fe-S enzymes. A Cysteine desulfurase activity assay. Enzyme activity was measured by quantifying sulfide release from L-cysteine via methylene blue absorbance (670 nm), 10 µM TPEN serving as a positive control. B Aconitase activity. The iron chelator 2,2’-bipyridyl (BPY, 250 µM) was used as a positive control. C Succinate dehydrogenase activity. The SDH inhibitor 3-nitropropionic acid (3-NP, 100 µM) was employed as a positive control. Data are presented as the mean ± SD of three biological replicates. Statistical significance was determined using two-way ANOVA with Dunnett’s test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. DMSO)

ZnPT induces remodeling of energy metabolism characterized by impaired activity of Fe-S cluster enzymes

To provide additional evidence regarding the effects of ZnPT on the activity of Fe-S enzymes in H37Rv, bacterial cultures were treated with 0.03 µg/mL ZnPT for 24 h, followed by comprehensive profiling of energy metabolism intermediates using LC-MS/MS. Volcano plot (Fig. 5A) analysis revealed significant downregulation of key metabolites involved in central carbon metabolism, including citrate (decreased by 21.7%), cis-aconitate (decreased by 29.7%), isocitrate (decreased by 24.0%), α-ketoglutarate (decreased by 20.6%), and succinate (decreased by 12.4%) (Fig. 5B). KEGG pathway enrichment analysis further demonstrated marked perturbations in carbon metabolism and the TCA cycle (Fig. 5C and D), highlighting ZnPT-mediated disruption of energy metabolism. These findings are consistent with the inhibition of ACO, suggesting Fe-S cluster destabilization. Concurrently, glyoxylate, a key intermediate in the glyoxylate shunt, decreased by 59.3% (p < 0.05), likely due to upstream TCA suppression. Glycolysis and gluconeogenesis intermediates also exhibited broad depletion, as indicated by reductions in glucose-6-phosphate (decreased by 17.8%) and fructose-1,6-bisphosphate (decreased by 26.0%), pointing to systemic attenuation of metabolic flux. Notably, succinate levels remained statistically unchanged (p > 0.2), despite the Fe-S dependence of SDH, possibly reflecting compensatory pathways such as anaplerotic reactions or reductive TCA flux. Collectively, these data demonstrate that ZnPT impairs metabolic flux and disrupts energy metabolism in Mtb.

Fig. 5 — ZnPT disrupts energy metabolism in *Mtb*. A Volcano plot of differential metabolites after ZnPT treatment. Energy metabolism-associated metabolites are highlighted. B Quantification of TCA cycle intermediates and glyoxylate shunt metabolites in response to ZnPT treatment. The metabolites analyzed include citrate, cis-aconitate, isocitrate, α-ketoglutarate, succinate, and glyoxylate (glyoxylate shunt). Data are presented as the mean ± SD of three biological replicates. C KEGG pathway enrichment analysis presented as a bubble plot, emphasizing pathways related to central carbon metabolism. D KEGG pathway classification of the identified metabolites. Statistical significance was determined using Student’s t-test or Welch’s t-test (for heterogeneous variances) (*p < 0.05 and **p < 0.01 vs. DMSO)

ZnPT collapses proton motive force and inhibits ATP synthesis

Iron-sulfur proteins are widely distributed throughout the respiratory chain, the TCA cycle, and other central energy metabolism pathways. These pathways provide reducing equivalents for aerobic respiration, which are transferred through the electron transport chain (ETC) to ultimately generate H₂O and ATP, which are the primary modes of energy production in Mtb. A reduced TCA cycle flux leads to a diminished supply of reducing equivalents to the ETC, resulting in decreased ATP synthesis. Furthermore, the concurrent upregulation of the frdB gene suggests an impaired capacity to maintain the transmembrane proton motive force (PMF), which drives ATP synthesis. We assessed PMF changes using the pH-sensitive fluorescent probe BCECF-AM and measured ATP levels with an ATP Assay Kit. ZnPT exposure significantly elevated BCECF-derived relative fluorescence units (Fig. 6A), a phenomenon attributed to impaired probe efflux resulting from PMF dissipation. In parallel, intracellular ATP levels decreased by approximately 30% following treatment with 0.03 µg/ml ZnPT (Fig. 6B). These findings indicate that ZnPT disrupts cellular energy metabolism in Mtb.

Fig. 6 — ZnPT disrupts the proton motive force and reduces ATP levels in *Mtb*. A ZnPT-induced PMF dissipation. PMF was assessed using the fluorescent probe BCECF-AM (15 µM) after 24 h treatment with ZnPT. The protonophore CCCP (15 µM) served as a positive control. B Dose-dependent reduction of ATP. ATP levels were quantified after 24 h exposure to ZnPT, with bedaquiline (BDQ, 0.24 µg/mL) serving as a positive control. Data normalized to total protein and expressed as a percentage of untreated controls. Results were presented as the mean ± SD of three biological replicates. Statistical significance was determined using one-way ANOVA with Dunnett’s test (****p < 0.0001 vs. DMSO)

Discussion

ZnPT, a broad-spectrum antimicrobial agent, has been extensively documented for its bactericidal activity against Gram-positive bacteria, Gram-negative bacteria, and fungi [8, 25]. A significant finding of the present study is the potent bactericidal efficacy of ZnPT against Mtb. Its MIC values for the drug-sensitive strain H37Rv and multidrug-resistant clinical isolates are as low as 0.015 µg/mL (47 nM), demonstrating a sensitivity that is two orders of magnitude greater than that of previously reported Acinetobacter baumannii (5 µM) and Klebsiella pneumoniae (10 µM) [26, 27]. The exceptional antitubercular potency and unique bactericidal mechanism of ZnPT, which markedly differ from those of conventional antibiotics, underscore the importance of conducting mechanistic studies.

Our results revealed that ZnPT induces a significant overexpression of the SUF operon, an early transcriptional response that aligns with bacterial defense mechanisms against copper toxicity. This is particularly relevant as Fe-S cluster biogenesis is highly susceptible to copper-mediated damage under metal stress conditions [28, 29]. Consistent with this, supplementation with Cu²⁺ significantly enhanced the efficacy of ZnPT, and the persistent upregulation of ctpV (responsible for copper efflux) at 24 h indicated unresolved copper overload and sustained copper stress. We further observed the induction of cysK2, which encodes a PLP-dependent S-sulfocysteine synthase [30, 31]. In conjunction with the observed activation of sulfur metabolism pathways in the transcriptomic data and the inhibition of cysteine desulfurase activity in vitro, we speculate that ZnPT may disrupt the function of the SufS-SufU complex, which is responsible for sulfur mobilization during Fe-S cluster biogenesis. The enzymatic activity of the SufS-SufU complex relies on a conserved zinc-coordinated metallochaperone domain within SufU, where Zn²⁺ ions stabilize the structural integrity of the persulfide transfer interface [32, 33]. Studies in Bacillus subtilis and Mtb have shown that zinc is essential for SufU sulfurtransferase activity, while submicromolar copper concentrations can destabilize this process [33, 34]. Moreover, pyrithione derivatives such as ZnPT are known to form ternary complexes with zinc-containing proteins, potentially impairing their function [35]. Additionally, the increased expression of iscS, which encodes a protein involved in Fe-S cluster biogenesis [16, 17], further suggests a compensatory response to the inhibition of the SUF pathway and reduced cysteine desulfurase activity. Consequently, despite the upregulation of SUF operon expression, the inhibition of desulfurization and sulfur transfer steps during Fe-S cluster assembly may impair the Fe-S cluster biogenesis and repair processes. Furthermore, we observed transcriptional repression of fdxB and fdxA, which encode ferredoxins essential for electron transfer during Fe-S cluster maturation [36]. These events ultimately result in the suppression of Fe-S cluster biogenesis and repair processes.

To evaluate the functional consequences of inhibiting Fe-S biogenesis, we quantified the enzymatic activities of two key Fe-S cluster-dependent metabolic enzymes, ACO and SDH, and observed significant reductions in both. The roles of Fe-S proteins in Mtb have been systematically categorized, with a substantial proportion involved in intermediary metabolism and respiration [10]. The activity of Fe-S proteins is regulated by the occupancy of Fe-S cluster [37, 38], and prior research has linked their function to both the SUF and ISC Fe-S biogenesis systems. Specifically, SufS and SufU interact with SufT to modulate ACO activity in Mtb [39], while the ΔiscS mutant also exhibits reduced ACO activity due to impaired Fe-S cluster delivery [17]. Our findings are consistent with previous reports indicating that ZnPT can influence the activity of Fe-S proteins [5]. Because aconitase requires a [4Fe-4 S] cofactor, repression of acn (Fig. 3B) may represent a resource-sparing, stress-adapted response that conserves limited Fe-S clusters and prioritizes their allocation to essential Fe-S cluster-dependent processes. Notably, ACO catalyzes the isomerization of citrate to isocitrate in the TCA cycle, providing the substrate for downstream reactions that generate reducing equivalents; thus, its impairment disrupts energy production. Similarly, SDH, a key component of the electron transport chain complex II, further disrupts oxidative phosphorylation upon inactivation. The reduced activities of ACO and SDH hinder the flow of reducing equivalents through energy metabolism and the respiratory electron transport chain, thereby triggering a systemic energy crisis in Mtb.

To further investigate the metabolic consequences of Fe-S protein disruption, we quantified energy pathway intermediates using LC-MS/MS. ZnPT significantly disrupted the homeostasis of cellular energy metabolism, as evidenced by widespread reductions in metabolite levels associated with glycolysis, gluconeogenesis, and the TCA cycle. Specifically, the activities of ACO and SDH were selectively inhibited, leading to substantial decreases in TCA intermediates, including cis-aconitate, citrate, and isocitrate. The observed activation of frdB coincided with impaired maintenance of the PMF, while the stable succinate levels may result from compensatory conversion of fumarate to succinate via the frdB-driven reductive TCA cycle. This metabolic adaptation partially restored NAD⁺/NADH homeostasis, alleviating respiratory chain dysfunction. Previous studies have shown that the fumarate reductase-mediated reverse TCA cycle may help preserve membrane potential [23], potentially mitigating respiratory chain dysfunction caused by the inactivation of Fe-S enzymes. Despite ZnPT-induced activation of the reverse TCA cycle as a metabolic compensation, Mtb is still unable to maintain the PMF, leading to a decline in ATP levels. These findings are consistent with Fe-S cluster disruption, which is known to affect redox and energy metabolism and to be associated with broad metabolic and respiratory defects.

Based on our findings, we propose a schematic model in which ZnPT increases intracellular copper levels and disrupts Fe-S cluster biogenesis. This disruption reduces Fe-S enzyme activity, broadly impairs respiration and energy metabolism (Fig. 7).

Fig. 7 — ZnPT induces impairment of Fe-S biogenesis and causes energy metabolism limitation

Fe-S clusters serve as essential cofactors in critical biological processes, including cellular metabolism, DNA repair, and electron transport. Disruption of Fe-S cluster biogenesis represents a strategy that exhibits no cross-resistance to current antitubercular drugs, ensuring efficacy against MDR-TB and XDR-TB strains. Previous studies and BLAST analyses reveal no significant homology between Mtb SufS-SufU/IscS proteins and their human counterparts, indicating a high degree of specificity [40]. Furthermore, disrupting Fe-S cluster biogenesis exerts broad bactericidal effects by impairing essential pathways in energy metabolism, respiratory electron transport, ribosomal transcription, and translation, leading to a systemic metabolic collapse that results in bacterial death. Consequently, the widespread dependence of Mtb on Fe-S clusters significantly increases its vulnerability. Targeting the SUF/IscS machinery induces multi-faceted lethality, presenting a promising avenue for next-generation tuberculosis therapeutics.

While this study elucidates the mechanism underlying the bactericidal action of ZnPT and identifies the Fe-S cluster biogenesis system as a promising therapeutic pathway, several limitations persist. First, we did not evaluate the in vivo efficacy of ZnPT in animal models, primarily due to its high toxicity and the resulting challenges in determining an appropriate dosing regimen. Secondly, although our findings suggest a disruption of the Fe-S cluster biogenesis pathway, the specific small molecules interactions and direct targets necessitate further experimental validation. Third, in preliminary selection assays, we attempted to isolate spontaneous ZnPT-resistant mutants by plating H37Rv on 7H10 agar containing ZnPT at 10× MIC. Rare colonies appeared at apparent frequencies of ~ 7.9 × 10^− 8 to 2.31 × 10^− 7; however, after single-colony purification, drug-free passage, and MIC re-testing, none retained a reproducible ≥ 2-fold increase in MIC relative to the parental strain. Future research should concentrate on developing less toxic ZnPT analogs with enhanced pharmacological profiles and on identifying the specific molecular targets within the Fe-S cluster biogenesis system.

This study has advanced antitubercular drug research by identifying ZnPT as a potent inhibitor of Mtb Fe-S cluster biogenesis, thereby highlighting a previously unexplored therapeutic vulnerability. Furthermore, this research emphasizes the therapeutic potential of compounds that target the unique metabolic and respiratory dependencies of bacterial pathogens by disrupting the SUF Fe-S cluster biogenesis system. This system serves as the sole and essential pathway for Fe-S cluster assembly in Mtb, while in eukaryotes, such as humans, Fe-S cluster biogenesis is mediated by the cytosolic iron-sulfur assembly (CIA) and ISC pathways [13, 14]. Additionally, this novel mechanism circumvents conventional resistance strategies, paving the way for new approaches to combat multidrug-resistant tuberculosis.

Conclusions

ZnPT exhibits nanomolar potency against both drug-susceptible and multidrug-resistant Mtb. By disrupting the Fe-S cluster biogenesis and repair machinery, ZnPT inactivates downstream Fe‑S enzymes, resulting in metabolic paralysis and bioenergetic collapse. Given that the SUF pathway is essential in Mtb but absent and non-redundant in humans, targeting this pathway provides high therapeutic selectivity. Our findings validate Fe-S cluster biogenesis as a promising and valuable therapeutic vulnerability in Mtb.

Materials and methods

Strains and culture conditions

Mtb H37Rv (ATCC 27294) and clinical isolates were cultured in Middlebrook 7H9 medium supplemented with 0.05% Tween 80 and 10% OADC (Oleic Albumin Dextrose Catalase) at 37℃ with 5% CO₂. Sauton’s medium was utilized for ICP-MS and time-kill assays. All strains used for susceptibility testing were maintained within five passages.

Drugs and compounds

Anti-Bacterial Compound Library (TargetMol, batch no. L4520), Zinc pyrithione (TargetMol, ≥ 99%; batch no. 113968), Linezolid (LZD; Ark Pharm, ≥ 99%; batch no. AQX137), Rifampicin (RIF; Aladdin, ≥ 97%; batch no. J2324037), Bedaquiline (BDQ; Biochempartner, batch no. 20210318), Ethambutol (EMB; MCE, batch no. 13585), Isoniazid (INH; Sigma-Aldrich, batch no. MKBQ8553V), Cupric sulfate (ACMEC, ≥ 99%; batch no. C72038536), Bathocuproine disulfonate (Thermo Scientific, ≥ 97%; batch no. 10212995), and Ammonium tetrathiomolybdate (Aladdin, ≥ 99.9%; batch no. G2329020).

Minimum inhibitory concentration determination

The MICs of ZnPT against H37Rv and 40 clinical isolates were determined using the microplate alamarBlue assay (MABA) [41]. Positive controls included INH, RIF, LZD, BDQ, and CFZ. Briefly, serial 2-fold dilutions of the test compounds were prepared in 96-well black-frame microplates. Each well received 100 µL of bacterial suspension (2 × 10⁵ CFU/mL) and 100 µL of the diluted compound. The plates were incubated at 37℃ under 5% CO₂ for 7 days. AlamarBlue reagent mixed with 20% Tween 80 was added to each well, and fluorescence was measured after 24 h using a Tecan Infinite M200 microplate reader (excitation/emission: 530/590 nm). The MIC was defined as the lowest drug concentration that reduced fluorescence by ≥ 90% compared to drug-free controls.

Time-kill kinetics assay

Bacterial cultures (2 × 10⁵ CFU/mL) were treated with ZnPT at concentrations ranging from 1/2× MIC to 8× -16× MIC, while drug-free 7H9 and Sauton’s media served as growth controls. At days 0, 3, 7, 10, and 14, 10-fold serial dilutions of the cultures were plated on 7H10 solid agar. After 3 to 4 weeks of incubation, colony-forming units (CFUs) were enumerated, and bactericidal activity was analyzed by plotting log₁₀ CFU/mL. The copper-dependent time-kill kinetics of ZnPT against Mtb were assessed in 24-well plates containing either 7H9 broth supplemented with 50 µg/mL BCS to chelate endogenous copper or Sauton’s liquid medium amended with 5 µM CuSO₄ to restore copper availability.

Intracellular antituberculosis activity in J774A.1 macrophages

H37Rv was cultured in Middlebrook 7H9 broth for 3 weeks to reach the exponential growth phase. J774A.1 macrophages were then infected with H37Rv at an MOI (Multiplicity of Infection) of 5:1 for 4 h to establish the infection model. Following the infection, extracellular bacteria were removed by extensive washing with phosphate-buffered saline (PBS). Test compounds and control drugs were then added to the culture medium, and the infected macrophages were incubated for an additional 72 h under standard conditions. After treatment, residual compounds were eliminated by washing with PBS, and the macrophages were lysed using a 0.1% SDS lysis buffer (Sigma-Aldrich). Lysates and serial 10-fold dilutions of the suspension were plated on Middlebrook 7H10 agar. CFUs were quantified after 3–4 weeks of incubation.

Intracellular metal ion concentrations

Mid-log phase H37Rv cultures were treated with compounds for 4 h and then extensively washed with metal-free Sauton’s medium (containing 1 mM EDTA) to eliminate extracellular metals. The cultures were lysed using a FastPrep-24 5G homogenizer (MP Biomedicals, USA; Cat. 116005500) and digested with Trace Metal-grade nitric acid (1:1 v/v) for 30 min. Metal ion quantification was conducted using a Thermo iCAP 7200 ICP-OES (axial mode, RF power: 1.15 kW, plasma gas flow: 15 L/min Ar) and ICP-MS. Samples were diluted in a solution of 2% HNO₃ and 0.5% HCl and analyzed against a multi-element calibration series ranging from 0 to 100 ppb (Cu, Zn, Mn, Fe), ensuring sensitivity and precision across biological matrices. The ICP-MS analysis was performed by Zhongke Baice Co., Ltd. (Beijing, China).

RNA extraction

Mid-log phase H37Rv cultures were harvested and washed twice with fresh 7H9 medium. Bacterial suspensions were treated with test compounds for 4 h and 24 h. Following treatment, bacterial cultures were stabilized by adding RNAprotect Bacteria Reagent (QIAGEN, Cat. 76506). Total RNA was extracted using the RNeasy Mini Kit (QIAGEN, Cat. 74104) and Buffer RLT (QIAGEN, Cat. 79216) according to the manufacturer’s protocol. Cultures were lysed using a homogenizer. RNA integrity was confirmed through agarose gel electrophoresis, which allowed for the visualization of distinct rRNA bands.

RNA sequencing and analysis

Total RNA was purified using the Bioyou^® RNA Purification Kit (Column) (Shanghai Biotechnology Corporation, Shanghai, China) according to the manufacturer’s instructions. The RNA integrity was assessed by determining the RNA Integrity Number using an Agilent Bioanalyzer 5300 (Agilent technologies, Santa Clara, CA, US). The RNA sequencing protocol was conducted on the Illumina NovaSeq6000 platform (Illumina, US). Raw reads were quality-filtered, mapped to the reference genome using Bowtie2 (version: 2–2.0.5) [42], and gene counts were generated with HTSeq [43]. DEGs were identified using edgeR (FDR < 0.05, fold-change > 2) [44]. GO and KEGG pathway enrichment analyses were performed in R (version 3. 2. 1), and the results were visualized using ggplot2.

RT-qPCR analysis

RNA concentration was quantified using a DeNovix DS-11FX⁺ spectrophotometer. Genomic DNA was removed from 2000 ng of RNA using the PrimeScript™ RT Kit with gDNA Eraser (Takara), followed by cDNA synthesis. RT-qPCR reactions utilized TB Green^® Premix Ex Taq™ II FAST (Takara) on the StepOnePlus™ system. The primer information is provided in Table S2.

Enzyme activity assays

Mid-log phase H37Rv cultures were treated with ZnPT, 100 µM 3-nitropropionate, 250 µM 2,2′-bipyridyl (positive control), or DMSO (negative control) at 37℃ for either 24 h (SDH) or 72 h (ACO) [39]. Following treatment, bacterial pellets were washed with PBS, homogenized using a homogenizer, and centrifuged to obtain crude lysates. SDH activity was quantified using a commercial kit (Solarbio, BC0955), while ACO activity was measured in UV-transparent plates (Solarbio, YA0602) by absorbance at 240 nm. Protein concentrations were determined using a BCA assay (Beyotime, P0012).

Assay for Cysteine Desulfurase Activity: Bacterial pellets were collected and resuspended in 1.5 ml of MOPS buffer (50 mM, pH 8) containing 2 mM DTT. Samples were prepared using a homogenizer. Bacterial lysates were treated with ZnPT or 10 µM TPEN, followed by the addition of 500 µM L-cysteine. The mixture was incubated at 25℃ in the dark for 15 min. For each 100 µL of treated lysate, 20 µL of freshly prepared color reagent (a solution of FeCl₃ and N, N-diethyl-p-phenylenediamine (DMPN) in a 1:20 ratio) was added. After incubation in the dark for 30 min, the absorbance at 665 nm was measured [33, 45].

Metabolomic profiling

Mid-log phase H37Rv cultures were treated with ZnPT or DMSO for 24 h. Cell pellets were suspended in 1 mL of pre-cooled extraction solvent [acetonitrile: methanol: water (2:2:1, v/v/v) at -30℃] and transferred to screw-cap tubes containing silica beads, followed by cell lysis using bead beating on a homogenizer. The samples were analyzed using an HPLC-MS/MS system (Thermo Scientific Dionex ICS-6000 coupled with an AB SCIEX 6500 QTRAP+). Chromatographic separation was achieved using an IonPac AS11-HC column (2 × 250 mm). Mass spectrometry was conducted in negative ESI mode (-4500 V, 450℃, GS1/GS2 45 psi), with multiple reaction monitoring (MRM) transitions optimized for quantifier and qualifier ion pairs. Metabolite standards (10 mM stock, serially diluted) were used to validate calibration curves (1-1000 nM, R² > 0.99). Data were normalized to protein content, processed using MultiQuan™ 3.0.3, and analyzed with MetaboAnalyst 5.0 (FDR < 0.05). Quality control measures included triplicate injections and pooled quality controls (RSD < 15%).

Proton motive force

PMF disruption was assessed using the fluorescent probe BCECF-AM. Mid-log phase H37Rv cultures were washed three times and resuspended in a 5 mM HEPES buffer containing 5 mM glucose at pH 7.0. Cells were incubated with 15 µM BCECF-AM and 0.01% Pluronic F-127 for 30 min at 37℃ in the dark. Subsequently, the bacteria were treated with ZnPT or the protonophore CCCP in HEPES buffer for 15 min. Fluorescence was measured (excitation/emission: 500/522 nm) to determine relative fluorescence units.

ATP assay

ATP levels were measured using the ATP Assay Kit (Beyotime, S0026) following a 24-hour treatment of Mtb cultures with ZnPT or BDQ as a positive control. After treatment, bacterial pellets were harvested by centrifugation (8,000× g, 10 min), washed twice with PBS, and lysed on ice using the lysis buffer provided in the kit until complete clarification was achieved. The lysates were then boiled at 100℃ for 2 min to release ATP. ATP detection was performed according to the manufacturer’s protocol. Protein concentrations were determined using a bicinchoninic acid (BCA) assay.

Supplementary Information

Supplementary Material 1.^{(189.4KB, docx)}

Acknowledgements

The authors would like to thank the members of the Department of Pharmacology, Beijing Key Laboratory of Drug-Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, for their technical support.

Clinical trial number

Not applicable.

Authors’ contributions

Conceptualization, LW, XLW and YL; methodology, LW, XLW, LLX, BW, YXY, DQJ and YL; bioinformatics analysis, LW, XLW; investigation, LW, and XLW; writing—original draft preparation, LW, XLW, LLX and YL; writing—review and editing, LW, XLW, LLX, and YL; project administration, YL; funding acquisition, YL. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (82173862) and the Beijing Municipal Administration of Hospitals’ Ascent Plan (DFL20221402).

Data availability

Data that support the findings of this study are presented in the main article and Supplementary Information files. Raw data generated from the RNA-Seq experiments of Mycobacterium tuberculosis strains have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject PRJNA1303096.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

1.WHO. Global Tuberculosis Report 2024. 2024; ISBN 978-92-4-010153-1.
2.Lange C, Bothamley G, Günther G, Guglielmetti L, Kontsevaya I, Kuksa L, Lange B, Lorent N, Saluzzo F, Sester M, et al. A year in review on tuberculosis and non-tuberculous mycobacteria disease: a 2025 update for clinicians and scientists. PAI. 2025;10:1–45. 10.20411/pai.v10i2.791. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Coll F, Phelan J, Hill-Cawthorne GA, Nair MB, Mallard K, Ali S, Abdallah AM, Alghamdi S, Alsomali M, Ahmed AO, et al. Genome-Wide analysis of Multi- and extensively Drug-Resistant Mycobacterium tuberculosis. Nat Genet. 2018;50:307–16. 10.1038/s41588-017-0029-0. [DOI] [PubMed] [Google Scholar]
4.Pang Y, Lu J, Wang Y, Song Y, Wang S, Zhao Y. Study of the Rifampin monoresistance mechanism in Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2013;57:893–900. 10.1128/aac.01024-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Reeder NL, Kaplan J, Xu J, Youngquist RS, Wallace J, Hu P, Juhlin KD, Schwartz JR, Grant RA, Fieno A, et al. Zinc pyrithione inhibits yeast growth through copper influx and inactivation of Iron-Sulfur proteins. Antimicrob Agents Chemother. 2011;55:5753–60. 10.1128/AAC.00724-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Park M, Cho Y-J, Lee YW, Jung WH. Understanding the mechanism of action of the Anti-Dandruff agent zinc pyrithione against Malassezia restricta. Sci Rep. 2018;8:12086. 10.1038/s41598-018-30588-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Reeder NL, Xu J, Youngquist RS, Schwartz JR, Rust RC, Saunders CW. The antifungal mechanism of action of zinc pyrithione: the antifungal mechanism of action of zinc pyrithione. Br J Dermatol. 2011;165:9–12. 10.1111/j.1365-2133.2011.10571.x. [DOI] [PubMed] [Google Scholar]
8.Dinning A-A. Austin; collier pyrithione biocides as inhibitors of bacterial ATP synthesis. J Appl Microbiol. 1998;85:141–6. 10.1046/j.1365-2672.1998.00478.x. [DOI] [PubMed] [Google Scholar]
9.Chandler CJ, Segel IH. Mechanism of the antimicrobial action of pyrithione: effects on membrane Transport, ATP Levels, and protein synthesis. Antimicrob Agents Chemother. 1978;14:60–8. 10.1128/AAC.14.1.60. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Elchennawi I, de Ollagnier S. Iron–Sulfur clusters toward stresses: implication for Understanding and fighting tuberculosis. Inorganics. 2022;10:174. 10.3390/inorganics10100174. [Google Scholar]
11.Huet G, Daffé M, Saves I. Identification of the Mycobacterium tuberculosis SUF machinery as the exclusive mycobacterial system of [Fe-S] cluster assembly: evidence for its implication in the pathogen’s survival. J Bacteriol. 2005;187:6137–46. 10.1128/jb.187.17.6137-6146.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Chen T, Ye Z, Huang W, Zhang Q, Jiang F, Yang Z, et al. Fe-S Cluster Deficiency drives small colony variant formation in persistent infections. J Adv Res. 2025. 10.1016/j.jare.2025.05.018 [DOI] [PubMed]
13.Braymer JJ, Stehling O, Stümpfig M, Rösser R, Spantgar F, Blinn CM, Mühlenhoff U, Pierik AJ, Lill R. Requirements for the biogenesis of [2Fe-2S] proteins in the human and yeast cytosol. Proc Natl Acad Sci U S A. 2024;121:e2400740121. 10.1073/pnas.2400740121. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Braymer JJ, Freibert SA, Rakwalska-Bange M, Lill R. Mechanistic concepts of Iron-Sulfur protein biogenesis in biology. Biochim Biophys Acta Mol Cell Res. 2021;1868:118863. 10.1016/j.bbamcr.2020.118863. [DOI] [PubMed] [Google Scholar]
15.Berdal V, Py B, De Choudens SO, Kremer L, Daher W. Iron–Sulfur proteins in mycobacteria: master regulators of physiology and pathogenesis. Trends Microbiol. 2025;S0966842X25002057. 10.1016/j.tim.2025.06.014. [DOI] [PubMed]
16.Das M, Sreedharan S, Shee S, Malhotra N, Nandy M, Banerjee U, Kohli S, Rajmani RS, Chandra N, Seshasayee ASN, et al. Cysteine desulfurase (IscS)–Mediated Fine-Tuning of bioenergetics and SUF expression prevents Mycobacterium tuberculosis hypervirulence. Sci Adv. 2023;9:eadh2858. 10.1126/sciadv.adh2858. [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Rybniker J, Pojer F, Marienhagen J, Kolly GS, Chen JM, van Gumpel E, Hartmann P, Cole ST. The cysteine desulfurase iscS of Mycobacterium tuberculosis is involved in Iron-Sulfur cluster biogenesis and oxidative stress defence. Biochem J. 2014;459:467–78. 10.1042/BJ20130732. [DOI] [PubMed] [Google Scholar]
18.Yan R, Adinolfi S, Pastore A. Ferredoxin, in conjunction with NADPH and Ferredoxin-NADP Reductase, transfers electrons to the IscS/IscU complex to promote Iron–Sulfur cluster assembly. Biochim Et Biophys Acta (BBA) - Proteins Proteom. 2015;1854:1113–7. 10.1016/j.bbapap.2015.02.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Chen J, Yu Y, Gao J, Yang SUDP-G, Dehydrogenase. The First-Step oxidation is an NAD(+)-Dependent bimolecular nucleophilic substitution reaction (S(N)2). Int J Biol Sci. 2019;15:341–50. 10.7150/ijbs.28904. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Lee JJ, Lim J, Gao S, Lawson CP, Odell M, Raheem S, Woo J, Kang S-H, Kang S-S, Jeon B-Y, et al. Glutamate mediated metabolic neutralization mitigates propionate toxicity in intracellular Mycobacterium tuberculosis. Sci Rep. 2018;8:8506. 10.1038/s41598-018-26950-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Shi L, Sohaskey CD, Pheiffer C, Datta P, Parks M, McFadden J, North RJ, Gennaro ML. Carbon flux rerouting during Mycobacterium tuberculosis growth arrest. Mol Microbiol. 2010;78:1199–215. 10.1111/j.1365-2958.2010.07399.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Bashiri G, Grove TL, Hegde SS, Lagautriere T, Gerfen GJ, Almo SC, Squire CJ, Blanchard JS, Baker EN. The active site of the Mycobacterium tuberculosis Branched-Chain amino acid biosynthesis enzyme dihydroxyacid dehydratase contains a 2Fe–2S cluster. J Biol Chem. 2019;294:13158–70. 10.1074/jbc.RA119.009498. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Watanabe S, Zimmermann M, Goodwin MB, Sauer U, Barry CE 3, Boshoff HI. Fumarate reductase activity maintains an energized membrane in anaerobic Mycobacterium tuberculosis. PLoS Pathog. 2011;7:e1002287. 10.1371/journal.ppat.1002287. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Romsang A, Duang-nkern J, Wirathorn W, Vattanaviboon P, Mongkolsuk S. Pseudomonas aeruginosa IscR-Regulated ferredoxin NADP(+) reductase gene (fprB) functions in Iron-Sulfur cluster biogenesis and multiple stress response. PLoS ONE. 2015;10:e0134374. 10.1371/journal.pone.0134374. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Ermolayeva E, Sanders D. Mechanism of Pyrithione-Induced membrane depolarization in Neurospora crassa. Appl Environ Microbiol. 1995;61:3385–90. 10.1128/aem.61.9.3385-3390.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Magallon J, Vu P, Reeves C, Kwan S, Phan K, Oakley-Havens CL, Rocha K, Jimenez V, Ramirez MS, Tolmasky ME. Amikacin potentiator activity of zinc complexed to a pyrithione derivative with enhanced solubility. Sci Rep. 2022;12:285. 10.1038/s41598-021-04724-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Chiem K, Fuentes BA, Lin DL, Tran T, Jackson A, Ramirez MS, Tolmasky ME. Inhibition of aminoglycoside 6’-N-Acetyltransferase type Ib-Mediated Amikacin resistance in Klebsiella pneumoniae by zinc and copper pyrithione. Antimicrob Agents Chemother. 2015;59:5851–3. 10.1128/AAC.01106-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Garcia-Santamarina S, Uzarska MA, Festa RA, Lill R, Thiele DJ. Cryptococcus Neoformans iron-sulfur protein biogenesis machinery is a novel layer of protection against cu stress. mBio. 2017;8:e01742–17. 10.1128/mBio.01742-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Tsvetkov P, Coy S, Petrova B, Dreishpoon M, Verma A, Abdusamad M, Rossen J, Joesch-Cohen L, Humeidi R, Spangler RD, et al. Copper induces cell death by targeting lipoylated TCA cycle proteins. Science. 2022;375:1254–61. 10.1126/science.abf0529. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Sao Emani C, Richter A, Singh A, Bhatt A, Av-Gay Y. The ∆CysK(2) mutant of Mycobacterium tuberculosis is sensitive to Vancomycin associated with changes in cell wall phospholipid profile. Biochem Biophys Res Commun. 2022;624:120–6. 10.1016/j.bbrc.2022.07.096. [DOI] [PubMed] [Google Scholar]
31.Steiner EM, Böth D, Lössl P, Vilaplana F, Schnell R, Schneider G. CysK2 from Mycobacterium tuberculosis is an O-Phospho-L-Serine-Dependent S-Sulfocysteine synthase. J Bacteriol. 2014;196:3410–20. 10.1128/JB.01851-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Terahata T, Shimada Y, Maki C, Muroga S, Sakurai R, Kunichika K, Fujishiro T. Cysteine-Persulfide sulfane Sulfur-Ligated Zn complex of Sulfur-Carrying SufU in the SufCDSUB system for Fe–S cluster biosynthesis. Inorg Chem. 2024;63:19607–18. 10.1021/acs.inorgchem.4c02654. [DOI] [PubMed] [Google Scholar]
33.Elchennawi I, Carpentier P, Caux C, Ponge M, de Ollagnier S. Structural and biochemical characterization of Mycobacterium tuberculosis zinc SufU-SufS complex. Biomolecules. 2023;13:732. 10.3390/biom13050732. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Chillappagari S, Seubert A, Trip H, Kuipers OP, Marahiel MA, Miethke M. Copper stress affects iron homeostasis by destabilizing iron-Sulfur cluster formation in Bacillus subtilis. J Bacteriol. 2010;192:2512–24. 10.1128/JB.00058-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Fatema K, Lund E, Petering DH. Zinc trafficking: 1,10-Phenanthroline, Glutathione, and other metal binding ligands form adducts with proteomic Zn2+. Metallomics. 2023;15(mfad026). 10.1093/mtomcs/mfad026. [DOI] [PubMed]
36.Sushko T, Kavaleuski A, Grabovec I, Kavaleuskaya A, Vakhrameev D, Bukhdruker S, Marin E, Kuzikov A, Masamrekh R, Shumyantseva V, et al. A new twist of rubredoxin function in M. Tuberculosis. Bioorg Chem. 2021;109:104721. 10.1016/j.bioorg.2021.104721. [DOI] [PubMed] [Google Scholar]
37.Bancroft PJ, Turapov O, Jagatia H, Arnvig KB, Mukamolova GV, Green J. Coupling of peptidoglycan synthesis to central metabolism in mycobacteria: Post-Transcriptional control of CwlM by aconitase. Cell Rep. 2020;32:108209. 10.1016/j.celrep.2020.108209. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Adolph C, McNeil MB, Cook GM. Impaired Succinate Oxidation Prevents Growth and Influences Drug Susceptibility in Mycobacterium Tuberculosis. mBio. 2022;13:e01672–22. 10.1128/mbio.01672-22. [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Tripathi A, Anand K, Das M, O’Niel RA, Thakur PSS, Rajmani C;RLRR, Chandra RS, Laxman N. Mycobacterium tuberculosis requires suft for Fe-S cluster Maturation, Metabolism, and survival in vivo. PLoS Pathog. 2022;18:e1010475. 10.1371/journal.ppat.1010475. [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Gorityala N, Baidya AS, Sagurthi SR. Genome mining of Mycobacterium Tuberculosis: targeting SufD as a novel drug candidate through in Silico characterization and inhibitor screening. Front Microbiol. 2024;15:1369645. 10.3389/fmicb.2024.1369645. [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Collins L, Franzblau SG. Microplate Alamar blue assay versus BACTEC 460 system for High-Throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrob Agents Chemother. 1997;41:1004–9. 10.1128/AAC.41.5.1004. [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and Memory-Efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009;10:R25. 10.1186/gb-2009-10-3-r25. [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Anders S, Pyl PT, Huber W. HTSeq—a python framework to work with High-Throughput sequencing data. Bioinformatics. 2015;31:166–9. 10.1093/bioinformatics/btu638. [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Robinson MD, McCarthy DJ, Smyth GK, edgeR. A bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010;26:139–40. 10.1093/bioinformatics/btp616. [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Olivieri P, Crack JC, Lehmann A, Le Brun NE, Leimkühler S. CyaY and TusA regulate ISC- and SUF-Mediated l -Cysteine desulfurase activity. RSC Chem Biol. 2024;5:1165–76. 10.1039/D4CB00225C. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1.^{(189.4KB, docx)}

Data Availability Statement

[CR1] 1.WHO. Global Tuberculosis Report 2024. 2024; ISBN 978-92-4-010153-1.

[CR2] 2.Lange C, Bothamley G, Günther G, Guglielmetti L, Kontsevaya I, Kuksa L, Lange B, Lorent N, Saluzzo F, Sester M, et al. A year in review on tuberculosis and non-tuberculous mycobacteria disease: a 2025 update for clinicians and scientists. PAI. 2025;10:1–45. 10.20411/pai.v10i2.791. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR3] 3.Coll F, Phelan J, Hill-Cawthorne GA, Nair MB, Mallard K, Ali S, Abdallah AM, Alghamdi S, Alsomali M, Ahmed AO, et al. Genome-Wide analysis of Multi- and extensively Drug-Resistant Mycobacterium tuberculosis. Nat Genet. 2018;50:307–16. 10.1038/s41588-017-0029-0. [DOI] [PubMed] [Google Scholar]

[CR4] 4.Pang Y, Lu J, Wang Y, Song Y, Wang S, Zhao Y. Study of the Rifampin monoresistance mechanism in Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2013;57:893–900. 10.1128/aac.01024-12. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR5] 5.Reeder NL, Kaplan J, Xu J, Youngquist RS, Wallace J, Hu P, Juhlin KD, Schwartz JR, Grant RA, Fieno A, et al. Zinc pyrithione inhibits yeast growth through copper influx and inactivation of Iron-Sulfur proteins. Antimicrob Agents Chemother. 2011;55:5753–60. 10.1128/AAC.00724-11. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR6] 6.Park M, Cho Y-J, Lee YW, Jung WH. Understanding the mechanism of action of the Anti-Dandruff agent zinc pyrithione against Malassezia restricta. Sci Rep. 2018;8:12086. 10.1038/s41598-018-30588-2. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR7] 7.Reeder NL, Xu J, Youngquist RS, Schwartz JR, Rust RC, Saunders CW. The antifungal mechanism of action of zinc pyrithione: the antifungal mechanism of action of zinc pyrithione. Br J Dermatol. 2011;165:9–12. 10.1111/j.1365-2133.2011.10571.x. [DOI] [PubMed] [Google Scholar]

[CR8] 8.Dinning A-A. Austin; collier pyrithione biocides as inhibitors of bacterial ATP synthesis. J Appl Microbiol. 1998;85:141–6. 10.1046/j.1365-2672.1998.00478.x. [DOI] [PubMed] [Google Scholar]

[CR9] 9.Chandler CJ, Segel IH. Mechanism of the antimicrobial action of pyrithione: effects on membrane Transport, ATP Levels, and protein synthesis. Antimicrob Agents Chemother. 1978;14:60–8. 10.1128/AAC.14.1.60. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR10] 10.Elchennawi I, de Ollagnier S. Iron–Sulfur clusters toward stresses: implication for Understanding and fighting tuberculosis. Inorganics. 2022;10:174. 10.3390/inorganics10100174. [Google Scholar]

[CR11] 11.Huet G, Daffé M, Saves I. Identification of the Mycobacterium tuberculosis SUF machinery as the exclusive mycobacterial system of [Fe-S] cluster assembly: evidence for its implication in the pathogen’s survival. J Bacteriol. 2005;187:6137–46. 10.1128/jb.187.17.6137-6146.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR12] 12.Chen T, Ye Z, Huang W, Zhang Q, Jiang F, Yang Z, et al. Fe-S Cluster Deficiency drives small colony variant formation in persistent infections. J Adv Res. 2025. 10.1016/j.jare.2025.05.018 [DOI] [PubMed]

[CR13] 13.Braymer JJ, Stehling O, Stümpfig M, Rösser R, Spantgar F, Blinn CM, Mühlenhoff U, Pierik AJ, Lill R. Requirements for the biogenesis of [2Fe-2S] proteins in the human and yeast cytosol. Proc Natl Acad Sci U S A. 2024;121:e2400740121. 10.1073/pnas.2400740121. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR14] 14.Braymer JJ, Freibert SA, Rakwalska-Bange M, Lill R. Mechanistic concepts of Iron-Sulfur protein biogenesis in biology. Biochim Biophys Acta Mol Cell Res. 2021;1868:118863. 10.1016/j.bbamcr.2020.118863. [DOI] [PubMed] [Google Scholar]

[CR15] 15.Berdal V, Py B, De Choudens SO, Kremer L, Daher W. Iron–Sulfur proteins in mycobacteria: master regulators of physiology and pathogenesis. Trends Microbiol. 2025;S0966842X25002057. 10.1016/j.tim.2025.06.014. [DOI] [PubMed]

[CR16] 16.Das M, Sreedharan S, Shee S, Malhotra N, Nandy M, Banerjee U, Kohli S, Rajmani RS, Chandra N, Seshasayee ASN, et al. Cysteine desulfurase (IscS)–Mediated Fine-Tuning of bioenergetics and SUF expression prevents Mycobacterium tuberculosis hypervirulence. Sci Adv. 2023;9:eadh2858. 10.1126/sciadv.adh2858. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR17] 17.Rybniker J, Pojer F, Marienhagen J, Kolly GS, Chen JM, van Gumpel E, Hartmann P, Cole ST. The cysteine desulfurase iscS of Mycobacterium tuberculosis is involved in Iron-Sulfur cluster biogenesis and oxidative stress defence. Biochem J. 2014;459:467–78. 10.1042/BJ20130732. [DOI] [PubMed] [Google Scholar]

[CR18] 18.Yan R, Adinolfi S, Pastore A. Ferredoxin, in conjunction with NADPH and Ferredoxin-NADP Reductase, transfers electrons to the IscS/IscU complex to promote Iron–Sulfur cluster assembly. Biochim Et Biophys Acta (BBA) - Proteins Proteom. 2015;1854:1113–7. 10.1016/j.bbapap.2015.02.002. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR19] 19.Chen J, Yu Y, Gao J, Yang SUDP-G, Dehydrogenase. The First-Step oxidation is an NAD(+)-Dependent bimolecular nucleophilic substitution reaction (S(N)2). Int J Biol Sci. 2019;15:341–50. 10.7150/ijbs.28904. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR20] 20.Lee JJ, Lim J, Gao S, Lawson CP, Odell M, Raheem S, Woo J, Kang S-H, Kang S-S, Jeon B-Y, et al. Glutamate mediated metabolic neutralization mitigates propionate toxicity in intracellular Mycobacterium tuberculosis. Sci Rep. 2018;8:8506. 10.1038/s41598-018-26950-z. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR21] 21.Shi L, Sohaskey CD, Pheiffer C, Datta P, Parks M, McFadden J, North RJ, Gennaro ML. Carbon flux rerouting during Mycobacterium tuberculosis growth arrest. Mol Microbiol. 2010;78:1199–215. 10.1111/j.1365-2958.2010.07399.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR22] 22.Bashiri G, Grove TL, Hegde SS, Lagautriere T, Gerfen GJ, Almo SC, Squire CJ, Blanchard JS, Baker EN. The active site of the Mycobacterium tuberculosis Branched-Chain amino acid biosynthesis enzyme dihydroxyacid dehydratase contains a 2Fe–2S cluster. J Biol Chem. 2019;294:13158–70. 10.1074/jbc.RA119.009498. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR23] 23.Watanabe S, Zimmermann M, Goodwin MB, Sauer U, Barry CE 3, Boshoff HI. Fumarate reductase activity maintains an energized membrane in anaerobic Mycobacterium tuberculosis. PLoS Pathog. 2011;7:e1002287. 10.1371/journal.ppat.1002287. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR24] 24.Romsang A, Duang-nkern J, Wirathorn W, Vattanaviboon P, Mongkolsuk S. Pseudomonas aeruginosa IscR-Regulated ferredoxin NADP(+) reductase gene (fprB) functions in Iron-Sulfur cluster biogenesis and multiple stress response. PLoS ONE. 2015;10:e0134374. 10.1371/journal.pone.0134374. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR25] 25.Ermolayeva E, Sanders D. Mechanism of Pyrithione-Induced membrane depolarization in Neurospora crassa. Appl Environ Microbiol. 1995;61:3385–90. 10.1128/aem.61.9.3385-3390.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR26] 26.Magallon J, Vu P, Reeves C, Kwan S, Phan K, Oakley-Havens CL, Rocha K, Jimenez V, Ramirez MS, Tolmasky ME. Amikacin potentiator activity of zinc complexed to a pyrithione derivative with enhanced solubility. Sci Rep. 2022;12:285. 10.1038/s41598-021-04724-4. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR27] 27.Chiem K, Fuentes BA, Lin DL, Tran T, Jackson A, Ramirez MS, Tolmasky ME. Inhibition of aminoglycoside 6’-N-Acetyltransferase type Ib-Mediated Amikacin resistance in Klebsiella pneumoniae by zinc and copper pyrithione. Antimicrob Agents Chemother. 2015;59:5851–3. 10.1128/AAC.01106-15. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR28] 28.Garcia-Santamarina S, Uzarska MA, Festa RA, Lill R, Thiele DJ. Cryptococcus Neoformans iron-sulfur protein biogenesis machinery is a novel layer of protection against cu stress. mBio. 2017;8:e01742–17. 10.1128/mBio.01742-17. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR29] 29.Tsvetkov P, Coy S, Petrova B, Dreishpoon M, Verma A, Abdusamad M, Rossen J, Joesch-Cohen L, Humeidi R, Spangler RD, et al. Copper induces cell death by targeting lipoylated TCA cycle proteins. Science. 2022;375:1254–61. 10.1126/science.abf0529. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR30] 30.Sao Emani C, Richter A, Singh A, Bhatt A, Av-Gay Y. The ∆CysK(2) mutant of Mycobacterium tuberculosis is sensitive to Vancomycin associated with changes in cell wall phospholipid profile. Biochem Biophys Res Commun. 2022;624:120–6. 10.1016/j.bbrc.2022.07.096. [DOI] [PubMed] [Google Scholar]

[CR31] 31.Steiner EM, Böth D, Lössl P, Vilaplana F, Schnell R, Schneider G. CysK2 from Mycobacterium tuberculosis is an O-Phospho-L-Serine-Dependent S-Sulfocysteine synthase. J Bacteriol. 2014;196:3410–20. 10.1128/JB.01851-14. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR32] 32.Terahata T, Shimada Y, Maki C, Muroga S, Sakurai R, Kunichika K, Fujishiro T. Cysteine-Persulfide sulfane Sulfur-Ligated Zn complex of Sulfur-Carrying SufU in the SufCDSUB system for Fe–S cluster biosynthesis. Inorg Chem. 2024;63:19607–18. 10.1021/acs.inorgchem.4c02654. [DOI] [PubMed] [Google Scholar]

[CR33] 33.Elchennawi I, Carpentier P, Caux C, Ponge M, de Ollagnier S. Structural and biochemical characterization of Mycobacterium tuberculosis zinc SufU-SufS complex. Biomolecules. 2023;13:732. 10.3390/biom13050732. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR34] 34.Chillappagari S, Seubert A, Trip H, Kuipers OP, Marahiel MA, Miethke M. Copper stress affects iron homeostasis by destabilizing iron-Sulfur cluster formation in Bacillus subtilis. J Bacteriol. 2010;192:2512–24. 10.1128/JB.00058-10. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR35] 35.Fatema K, Lund E, Petering DH. Zinc trafficking: 1,10-Phenanthroline, Glutathione, and other metal binding ligands form adducts with proteomic Zn2+. Metallomics. 2023;15(mfad026). 10.1093/mtomcs/mfad026. [DOI] [PubMed]

[CR36] 36.Sushko T, Kavaleuski A, Grabovec I, Kavaleuskaya A, Vakhrameev D, Bukhdruker S, Marin E, Kuzikov A, Masamrekh R, Shumyantseva V, et al. A new twist of rubredoxin function in M. Tuberculosis. Bioorg Chem. 2021;109:104721. 10.1016/j.bioorg.2021.104721. [DOI] [PubMed] [Google Scholar]

[CR37] 37.Bancroft PJ, Turapov O, Jagatia H, Arnvig KB, Mukamolova GV, Green J. Coupling of peptidoglycan synthesis to central metabolism in mycobacteria: Post-Transcriptional control of CwlM by aconitase. Cell Rep. 2020;32:108209. 10.1016/j.celrep.2020.108209. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR38] 38.Adolph C, McNeil MB, Cook GM. Impaired Succinate Oxidation Prevents Growth and Influences Drug Susceptibility in Mycobacterium Tuberculosis. mBio. 2022;13:e01672–22. 10.1128/mbio.01672-22. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR39] 39.Tripathi A, Anand K, Das M, O’Niel RA, Thakur PSS, Rajmani C;RLRR, Chandra RS, Laxman N. Mycobacterium tuberculosis requires suft for Fe-S cluster Maturation, Metabolism, and survival in vivo. PLoS Pathog. 2022;18:e1010475. 10.1371/journal.ppat.1010475. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR40] 40.Gorityala N, Baidya AS, Sagurthi SR. Genome mining of Mycobacterium Tuberculosis: targeting SufD as a novel drug candidate through in Silico characterization and inhibitor screening. Front Microbiol. 2024;15:1369645. 10.3389/fmicb.2024.1369645. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR41] 41.Collins L, Franzblau SG. Microplate Alamar blue assay versus BACTEC 460 system for High-Throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrob Agents Chemother. 1997;41:1004–9. 10.1128/AAC.41.5.1004. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR42] 42.Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and Memory-Efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009;10:R25. 10.1186/gb-2009-10-3-r25. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR43] 43.Anders S, Pyl PT, Huber W. HTSeq—a python framework to work with High-Throughput sequencing data. Bioinformatics. 2015;31:166–9. 10.1093/bioinformatics/btu638. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR44] 44.Robinson MD, McCarthy DJ, Smyth GK, edgeR. A bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010;26:139–40. 10.1093/bioinformatics/btp616. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR45] 45.Olivieri P, Crack JC, Lehmann A, Le Brun NE, Leimkühler S. CyaY and TusA regulate ISC- and SUF-Mediated l -Cysteine desulfurase activity. RSC Chem Biol. 2024;5:1165–76. 10.1039/D4CB00225C. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Zinc pyrithione impairs iron-sulfur cluster biogenesis in Mycobacterium tuberculosis

Lu Wang

Xuelin Wang

Lingling Xu

Bin Wang

Deqian Ji

Yangxue Ye

Yu Lu

Abstract

Background

Methods

Results

Conclusions

Supplementary Information

Introduction

Results

ZnPT exerts potent copper-dependent bactericidal activity against both drug-susceptible and drug-resistant Mtb

Table 1.

Fig. 1.

Table 2.

ZnPT impacts the SUF iron-sulfur biogenesis and metabolic activities in Mtb

Fig. 2.

Fig. 3.

ZnPT inhibits the activity of cysteine desulfurase and Fe-S enzymes in energy metabolism

Fig. 4.

ZnPT induces remodeling of energy metabolism characterized by impaired activity of Fe-S cluster enzymes

Fig. 5.

ZnPT collapses proton motive force and inhibits ATP synthesis

Fig. 6.

Discussion

Fig. 7.

Conclusions

Materials and methods

Strains and culture conditions

Drugs and compounds

Minimum inhibitory concentration determination

Time-kill kinetics assay

Intracellular antituberculosis activity in J774A.1 macrophages

Intracellular metal ion concentrations

RNA extraction

RNA sequencing and analysis

RT-qPCR analysis

Enzyme activity assays

Metabolomic profiling

Proton motive force

ATP assay

Supplementary Information

Acknowledgements

Clinical trial number

Authors’ contributions

Funding

Data availability

Declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Footnotes

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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