Ergothioneine Is a Secreted Antioxidant in Mycobacterium smegmatis

Abstract

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 evolutionary history of the genes involved in the biosynthesis of the antioxidant ergothioneine

“…Previous studies have shown that EgtB and EgtD are key enzymes in the production of ergothioneine (Seebeck, 2010;Sao Emani et al, 2013). From our original dataset of 2509 prokaryotic genomes (Additional file 1-A), 438 were found to contain a putative ortholog of egtB and egtD (Additional file 1-B).…”

“…Because of their importance in the synthesis of ergothioneine (Seebeck, 2010;Sao Emani et al, 2013), the EgtB and EgtD phylogenies will be discussed in tandem.…”

Regulation of Ergothioneine Biosynthesis and Its Effect on Mycobacterium tuberculosis Growth and Infectivity

“…During infection, the mutant was only slightly attenuated (half-log) at 120 h, an unexpected result because of the reputation of EGT as an antioxidant. Perhaps MSH provides a compensatory effect in M. tuberculosis in the absence of EGT ex vivo; however, current work does not show increased levels of MSH in the absence of EGT (11,42). The pknD:Tn mutant showed no difference in viability when infecting J774A.1 macrophages (13), further supporting the notion that EGT levels have a limited effect on the survival of M. tuberculosis in murine macrophages (43).…”

“…We have found that similarly to M. smegmatis EgtD is essential in the production of EGT in M. tuberculosis (42). These findings along with the commitment of histidine to the pathway suggested EgtD to be a likely candidate for regulation in EGT biosynthesis.…”

“…As a result, we believe that EGT does not provide protection against peroxides during in vitro growth of M. tuberculosis. Studies examining the survival of M. smegmatis ⌬egtD when challenged with cumene hydroperoxide or tert-butyl hydroperoxide found the mutant to be similar to wild type (42), which additionally suggests little to no involvement of EGT in the elimination of peroxides in mycobacteria.…”