Generation and characterization of thiol-deficient Mycobacterium tuberculosis mutants

Abstract

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.

GlnA3Mtis able to glutamylate spermine but it is not essential for the detoxification of spermine inMycobacterium tuberculosis

“…As GlnA3 Sc plays a fundamental role in the survival of S. coelicolor at high spermine concentrations (16), we decided to employ a genetic approach to interrogate whether the same is true in M. tuberculosis . To this end, we constructed an unmarked, in-frame glnA3 Mt -deletion mutant of M. tuberculosis , as described in Materials and Methods (36, 37). The ΔglnA3 Mt ( Δrv1878 ) mutant was identified by PCR, and validated by Southern blotting and targeted genome sequencing.…”

“…This was achieved as previously described (36, 37). Briefly, approximately 2500 bp fragments upstream (US) and downstream (DS) rv1878 ( glnA3 ) were amplified using pfu high fidelity GC rich target polymerase (Agilent) primers listed in Table 3.…”

“…The resulting construct p2NIL-A3-US/DS-G17, was used to transform M. tuberculosis , and the mutant was generated through two subsequent homologous recombination events as previously described (36). The mutant was screened by PCR (primers in Table 3), confirmed by Southern blotting as previously described (37) and by targeted genome sequencing (58). The mutant was complemented as previously described (37), using the pMVhsp60 integrative vector (59), where glnA3 was cloned downstream the heat shock promoter (P hsp60 ), to maintain constitutive expression at 37°C (Table 3).…”

The role of thiols in antioxidant systems

“…However, ESH-producing microbes often also synthesize other LMW thiols, such as GSH and MSH that assist with the maintenance of the cellular redox homeostasis [222]. For instance, Mycobacterium tuberculosis contains MSH, ESH and γ-Glu-Cys, which appear to be all able to compensate for each other, and ensure survival and fitness under oxidative stress conditions [223].…”

The biology of ergothioneine, an antioxidant nutraceutical

“…Although it is possible that the role of ERG lies simply in being an antioxidant capable of mopping up hydroxyl radicals and other reactive oxygen species, especially in prokaryotes (36,66,93,254,255,(258)(259)(260)552,553) , the roles of most other vitamins involve interaction with proteins, often as cofactors. This is also true for mycothiol (73,(554)(555)(556) , though that molecule can also serve as a signal and nutrient resource (557) .…”