Disulfiram induces redox imbalance and perturbations in central glucose catabolism and metal homeostasis to inhibit the growth of Staphylococcus aureus

Abstract

Disulfiram (Antabuse^®) is a prescription alcohol sobriety aid that has shown repurposing potential as an antibacterial drug for infections due to Gram-positive bacteria. In this investigation, we sought to define the principal mechanisms that disulfiram operates as a growth inhibitor of Staphylococcus aureus using differential transcriptomic, metabolomic, bioenergetic, and phenotypic growth analyses. The RNA-seq transcriptome analysis revealed that disulfiram induces oxidative stress, redox imbalance, metal acquisition, and the biosynthesis of pantothenate, coenzyme A, thiamine, menaquinone, siderophores/metallophores, and bacillithiol. The metabolomic analysis indicated that disulfiram depletes coenzyme A and attenuates the catabolism of glucose, pyruvate, and NADH. Conversely, disulfiram appeared to up-regulate arginine catabolism for ATP production and accelerate citrate consumption that was attributed to induction of siderophore biosynthesis (i.e., staphyloferrin). The bioenergetic studies further revealed that the primary metabolite of disulfiram (i.e., diethyldithiocarbamate) is likely involved in the mechanism of action as an inhibitor of oxidative phosphorylation and chelating agent of iron and other metals. In the final analysis, disulfiram inhibits the growth of S. aureus by inducing perturbations in central glucose catabolism and redox imbalance (e.g., oxidative stress). Moreover, the chelation of metal ions and antagonism of the respiratory chain by diethyldithiocarbamate are believed to contribute to the inhibition of cell replication.

Fig. 1

Chemistry of disulfiram (DSF) and its primary metabolite diethyldithiocarbamate (DDTC). Thiophilic substances (RSH) cleave DSF to form RS-DDTC conjugates and DDTC that can chelate metals to form M[DDTC]₂ and M[DDTC]₃ complexes. Pelleted cultures of S. aureus treated with DSF and DDTC exhibit similar discoloration as evidence of M[DDTC]_n formation.

Fig. 2

Transcriptional effects of DSF on genes governing glycolysis, pantothenate/CoASH biosynthesis, and associated pathways in S. aureus. (a) Schematic representation of up-regulated (red) and down-regulated (blue) genes. The KEGG pathway analysis revealed that DSF up-regulates the biosynthesis of coenzyme A (CoASH) and thiamine pyrophosphate (TPP). Conversely, DSF down-regulated the genes governing glycolysis and acetyl-coenzyme A (CoASAc) formation. (b) Mass spectroscopy revealed that DSF and CoASH react to form the mix disulfide CoAS-DDTC at physiological pH.

Fig. 3

Changes in the metabolome of S. aureus induced by DSF and DDTC. (a) Differential analysis of S. aureus JE2 lysates revealed that DSF and DDTC alters the cellular levels of metabolites involved in energy metabolism (e.g., NADH) and redox homeostasis (e.g., BSH). (b) HPLC-based metabolomics profiling indicates that DSF and DDTC alters the procurement or utilization of amino acids in S. aureus. Values represent the log₂FC calculated from the vehicle control (*p ≤ 0.05).

Fig. 4

Transcriptional effects of DSF on genes governing the citric acid cycle, oxidative phosphorylation, and associated pathways. (a) Schematic representation of up-regulated (red) and down-regulated (blue) genes. The KEGG pathway analysis revealed that DSF induces arginine catabolism and staphyloferrin biosynthesis from citrate, glutamate, and ornithine. The transcriptomic data further indicated that DSF decelerates citric acid cycle and oxidative phosphorylation activity. (b) Results of the bioenergetic studies in S. aureus JE2 depicted as percent baseline of mean vehicle control values. The graphs show that DDTC is a more effective inhibitor of the OCR (left) and ECAR (middle) in S. aureus than DSF or the uncoupler standard CCCP. Moreover, DSF and DDTC reduced the basal cell membrane potential in S. aureus but to a lesser degree compared to CCCP (right).

Fig. 5

Transcriptional effects of DSF on genes governing cell wall, menaquinone, and phenylalanine biosynthesis in S. aureus. Schematic representation of up-regulated (red) and down-regulated (blue) genes. The KEGG pathway analysis indicates that DSF increases menaquinone biosynthesis and attenuates peptidoglycan and phenylalanine synthesis.

Fig. 6

Effects of DSF and DDTC on the growth of wildtype (wt) JE2 in comparison with transposon mutants. (a) Growth phenotype analysis indicates that transposon mutants with defects in citrate (ΔgltA), thiamine phosphate (ΔthiE), and bacillithiol (ΔbshA) production were more susceptible to DSF while the staphyloferrin B-deficient mutant JE2 ΔsbnC was more sensitive to DDTC. (b) Growth curve of untreated versus DSF-treated (4x MIC*) wt JE2 and ΔbshA mutant treated. (c) Differential analysis using wt JE2 labeled with the oxidant-sensitive H₂DCFDA probe indicates that DSF and H₂O₂ elicit oxidative stress while 1x MIC DDTC reduced the cellular oxidation state.