An Affinity-based Depletion Strategy for Evaluating the Effects of Ergothioneine on Bacterial Physiology

Ergothioneine (EGT) is a thiol-based antioxidant synthesized by certain fungal and bacterial species that is prevalent in the human diet. Recently, an EGT-specific transporter, EgtUV, was discovered in bacteria that are incapable of EGT biosynthesis, including the gastric pathogen Helicobacter pylori. However, EGT is naturally abundant in the complex media required to culture H. pylori and many other host-associated microbes, complicating efforts to understand how this small molecule influences microbial physiology. Using the solute-binding domain of H. pylori EgtUV, we generated an EGT-chelating resin that depletes EGT from nutrient-rich media. We determined that wild-type H. pylori requires EGT to outcompete a transporter-deficient strain in vitro. Furthermore, EGT induces expression of outer-membrane proteins that regulate intracellular EGT content upstream of the inner-membrane-localized EgtUV transporter. Our work establishes a method for tuning exposure to an abundant antioxidant in vitro, enabling future studies of EGT in diverse bacterial strains and microbial communities. We investigated transcriptomic changes in WT H. pylori PMSS1 grown in EGT-depleted (EgtU-WT-resin-treated) vs. EGT-replete (EgtU-Y390A-resin-treated) culture media in biological triplicate to assess the effects of EGT on gene expression. We first used our EgtUWT- or EgtUY390A-resin to treat H. pylori culture media (Brucella Broth + 10% (v/v) heat-inactivated fetal bovine serum (FBS); "BB10") and verified EGT depletion from EgtUWT-treated BB10 using liquid-chromatography mass-spectrometry (LC-MS). WT PMSS1 H. pylori were then cultured in EgtU-WT-resin-treated and EgtU-Y390A-resin-treated BB10, and samples were collected at 8 h, 21 h, and 24 h for RNA extraction and transcriptomic analysis (RNA-seq).