c Ergothioneine (ERG) and mycothiol (MSH) are two low-molecular-weight thiols synthesized by mycobacteria. The role of MSH has been extensively investigated in mycobacteria; however, little is known about the role of ERG in mycobacterial physiology. In this study, quantification of ERG at various points in the growth cycle of Mycobacterium smegmatis revealed that a significant portion of ERG is found in the culture media, suggesting that it is actively secreted. A mutant of M. smegmatis lacking egtD (MSMEG_6247) was unable to synthesize ERG, confirming its role in ERG biosynthesis. Deletion of egtD from wild-type M. smegmatis and an MSH-deficient mutant did not affect their susceptibility to antibiotics tested in this study. The ERG-and MSH-deficient double mutant was significantly more sensitive to peroxide than either of the single mutants lacking either ERG or MSH, suggesting that both thiols play a role in protecting M. smegmatis against oxidative stress and that ERG is able to partly compensate for the loss of MSH. G lutathione (GSH) is a thiol known for its efficient detoxification of reactive oxygen species, reactive nitrogen species, and free radicals in eukaryotes. Mycobacteria do not synthesize GSH but produce two low-molecular-weight thiols,and ergothioneine (2-mercaptohistidine trimethylbetaine) (ERG) (4, 5). Four genes are involved in MSH biosynthesis in mycobacteria, namely, mshA, mshB, mshC, and mshD, and mutants harboring deletions in mshB, mshC, and mshD produce different levels of MSH due to the ability of other enzymes to partially compensate for their loss (6, 7). MSH-deficient mutants of Mycobacterium smegmatis show increased sensitivity to oxidative stress, alkylating agents, and a range of antibiotics, including erythromycin, azithromycin, vancomycin, penicillin G, streptomycin, and rifampin, but exhibit increased resistance to isoniazid (INH) and ethionamide (ETH) (8, 9). The MSH-deficient ⌬mshA mutant of Mycobacterium tuberculosis requires catalase during in vitro growth, implicating MSH in detoxifying reactive oxygen species (10).ERG biosynthetic genes (egtA, egtB, egtC, egtD, and egtE) were recently identified in M. smegmatis (11). Although several lines of evidence support the cytoprotective and antioxidative role of ERG in eukaryotes (12), bacteria (13), and, recently, fungi (14), nothing is known of its role in mycobacteria. ERG has also been implicated in modulating the immune response (15) and in the inhibition of metalloenzymes, preventing the copper-induced oxidation of DNA and protein due to its metal-chelating properties (16,17). Eukaryotes obtain ERG from their diet, and its accumulation in cells is dependent on the activity of a highly specific transporter, OCTN1, since the zwitterionic nature of ERG prevents it from crossing the plasma membrane (18,19). In an M. smegmatis ⌬mshA mutant, which is MSH deficient, the levels of ERG and the organic hydroperoxide resistance (Ohr) protein are elevated, suggesting that ERG may partly compensate for the loss of MSH (20). This may exp...
The high acquisition rate of drug resistance by necessitates the ongoing search for new drugs to be incorporated in the tuberculosis (TB) regimen. Compounds used for the treatment of other diseases have the potential to be repurposed for the treatment of TB. In this study, a high-throughput screening of compounds against thiol-deficient strains and subsequent validation with thiol-deficient strains revealed that and mutants had increased susceptibility to azaguanine (Aza) and sulfaguanidine (Su); and mutants had increased susceptibility to bacitracin (Ba); and, , and mutants had increased susceptibility to fusaric acid (Fu). Further analyses revealed that some of these compounds were able to modulate the levels of thiols and oxidative stress in This study reports the activities of Aza, Su, Fu, and Ba against and provides a rationale for further investigations.
Mycobacterium tuberculosis (Mtb) is an obligate human pathogen killing millions of people annually. Treatment for tuberculosis is lengthy and complicated, involving multiple drugs and often resulting in serious side effects and non-compliance. Mtb has developed numerous complex mechanisms enabling it to not only survive but replicate inside professional phagocytes. These mechanisms include, among others, overcoming the phagosome maturation process, inhibiting the acidification of the phagosome and inhibiting apoptosis. Within the past decade, technologies have been developed that enable a more accurate understanding of Mtb physiology within its intracellular niche, paving the way for more clinically relevant drug-development programmes. Here we review the molecular biology of Mtb pathogenesis offering a unique perspective on the use and development of therapies that target Mtb during its intracellular life stage.
Screening of a GSK-proprietary library against intracellular Mycobacterium tuberculosis identified 1, a thioalkylbenzoxazole hit. Biological profiling and mutant analysis revealed that this compound is a prodrug that is bioactivated by the mycobacterial enzyme MymA. A hit-expansion program including design, synthesis, and profiling of a defined set of analogues with optimized drug-like properties led to the identification of an emerging lead compound, displaying potency against intracellular bacteria in the low micromolar range, high in vitro solubility and permeability, and excellent microsomal stability.
BackgroundThree low molecular weight thiols are synthesized by Mycobacterium tuberculosis (M.tb), namely ergothioneine (ERG), mycothiol (MSH) and gamma-glutamylcysteine (GGC). They are able to counteract reactive oxygen species (ROS) and/or reactive nitrogen species (RNS). In addition, the production of ERG is elevated in the MSH-deficient M.tb mutant, while the production of MSH is elevated in the ERG-deficient mutants. Furthermore, the production of GGC is elevated in the MSH-deficient mutant and the ERG-deficient mutants. The propensity of one thiol to be elevated in the absence of the other prompted further investigations into their interplay in M.tb.MethodsTo achieve that, we generated two M.tb mutants that are unable to produce ERG nor MSH but are able to produce a moderate (ΔegtD-mshA) or significantly high (ΔegtB-mshA) amount of GGC relative to the wild-type strain. In addition, we generated an M.tb mutant that is unable to produce GGC nor MSH but is able to produce a significantly low level of ERG (ΔegtA-mshA) relative to the wild-type strain. The susceptibilities of these mutants to various in vitro and ex vivo stress conditions were investigated and compared.ResultsThe ΔegtA-mshA mutant was the most susceptible to cellular stress relative to its parent single mutant strains (ΔegtA and ∆mshA) and the other double mutants. In addition, it displayed a growth-defect in vitro, in mouse and human macrophages suggesting; that the complete inhibition of ERG, MSH and GGC biosynthesis is deleterious for the growth of M.tb.ConclusionsThis study indicates that ERG, MSH and GGC are able to compensate for each other to maximize the protection and ensure the fitness of M.tb. This study therefore suggests that the most effective strategy to target thiol biosynthesis for anti-tuberculosis drug development would be the simultaneous inhibition of the biosynthesis of ERG, MSH and GGC.
Mycothiol (MSH) and ergothioneine (ERG) are thiols able to compensate for each other to protect mycobacteria against oxidative stress. Gamma-glutamylcysteine (GGC), another thiol and an intermediate in ERG biosynthesis has detoxification abilities. Five enzymes are involved in ERG biosynthesis, namely EgtA, EgtB, EgtC, EgtD and EgtE. The role of these enzymes in the production of ERG had been unclear. On the other hand, the enzyme MshA is known to be essential for MSH biosynthesis. In this manuscript, we describe the raw data of the generation and characterization of Mycobacterium tuberculosis (M.tb) mutants harbouring a deletion of the gene coding for each of these enzymes, and the raw data of the phenotypic characterization of the obtained thiol-deficient M.tb mutants. High throughput screening (HTS) of off-patent drugs and natural compounds revealed few compounds that displayed a higher activity against the thiol-deficient mutants relative to the wild-type strain. The mode of action of these drugs was further investigated. Raw data displaying these results are described here.
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