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The role of ergothioneine in the physiology and pathogenesis of Mycobacterium tuberculosis. Richard-Greenblatt, Melissa

Abstract

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is capable of synthesizing the small sulfur compound ergothioneine (EGT). The physiological role and regulation of EGT biosynthesis in Mtb is unknown. However, mammalian cells deficient in EGT demonstrate augmented oxidative stress and cell death, suggesting that EGT is an antioxidant. Through this work, we identified the Mtb EGT biosynthesis genes and characterized rv3701c (egtD) to encode for a histidine methyltransferase that forms the EGT biosynthetic pathway intermediate, hercynine (N-α-trimethylhistidine). Construction of a knockout (ΔegtD) demonstrated the methyltransferase to be essential for EGT biosynthesis in Mtb. We investigated the role of EGT in protecting Mtb during macrophage infection and observed a small but significant difference in the ex vivo growth and survival of the ΔegtD strain relative to H37Rv wildtype and the complement strains 120 h post-infection of murine macrophages. However, there was no difference in survival of the ΔegtD mutant in human THP-1 cells over this time period suggesting an alternative role for EGT in Mtb physiology and pathogenesis. Upon further analysis we found EgtD to be phosphorylated by and interact with the Mtb Serine/Threonine Protein Kinase PknD. Phosphorylation of EgtD both in vitro and inside Escherichia coli identified phosphorylation at site Thr-213, permitting us to generate an EgtD phosphomimetic and phosphoablative mutant strains with our ΔegtD mutant. In vitro analysis, demonstrated phosphorylated EgtD to produce significantly lower quantities of methylated histidine relative to the non-phosphorylated form. Further quantification of intracellular EGT levels in the Mtb EgtD phosphomutants and Mtb PknD transposon mutant identified PknD to negatively regulate EGT biosynthesis. From these results, we identified that EGT biosynthesis is up-regulated in response to nutrient starvation. Under these conditions, the Mtb ΔegtD mutant was unable to maintain viability compared to its parental wildtype and complement strains. As starvation induces a non-replicative state in Mtb, these findings indicate that EGT plays a role in mediating persistent infection or disease latency. Further metabolic analysis identified Mtb intracellular EGT levels to be directly correlated with carbon source type and availability, suggesting a role for EGT in long-term energy storage.

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