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The human solute carrier 22A (SLC22A) family consists of 23 members, representing one of the largest families in the human SLC superfamily. Despite their pharmacological and physiological importance in the absorption and disposition of a range of solutes, eight SLC22A family members remain classified as orphans. In this study, we used a multifaceted approach to identify ligands of orphan SLC22A15. Ligands of SLC22A15 were proposed based on phylogenetic analysis and comparative modeling. The putative ligands were then confirmed by metabolomic screening and uptake assays in SLC22A15 transfected HEK293 cells. Metabolomic studies and transporter assays revealed that SLC22A15 prefers zwitterionic compounds over cations and anions. We identified eight zwitterions, including ergothioneine, carnitine, carnosine, gabapentin, as well as four cations, including MPP+ , thiamine, and cimetidine, as substrates of SLC22A15. Carnosine was a specific substrate of SLC22A15 among the transporters in the SLC22A family. SLC22A15 transport of several substrates was sodium-dependent and exhibited a higher Km for ergothioneine, carnitine, and carnosine compared to previously identified transporters for these ligands. This is the first study to characterize the function of SLC22A15. Our studies demonstrate that SLC22A15 may play an important role in determining the systemic and tissue levels of ergothioneine, carnosine, and other zwitterions.

We show that Mycobacterium smegmatis mutants disrupted in mscR, coding for a dual function S-nitrosomycothiol reductase and formaldehyde dehydrogenase, and mshC, coding for a mycothiol ligase and lacking mycothiol (MSH), are more susceptible to S-nitrosoglutathione (GSNO) and aldehydes than wild type. MSH is a cofactor for MscR, and both mshC and mscR are induced by GSNO and aldehydes. We also show that a mutant disrupted in egtA, coding for a γ-glutamyl cysteine synthetase and lacking in ergothioneine, is sensitive to nitrosative stress but not to aldehydes. In addition, we find that MSH and S-nitrosomycothiol reductase are required for normal biofilm formation in M. smegmatis, suggesting potential new therapeutic pathways to target to inhibit or disrupt biofilm formation. © 2016 IUBMB Life, 68(8):621-628, 2016.

Ergothioneine, a histidine derivative, is concentrated in conidia of ascomycetous fungi. To investigate the function of ergothioneine, we crossed the wild type Neurospora crassa (Egt(+)) and an ergothioneine non-producer (Egt(-), Δegt-1, a knockout in NCU04343.5) and used the Egt(+) and Egt(-) progeny strains for phenotypic analyses. Compared to the Egt(+) strains, Egt(-) strains had a 59% reduction in the number of conidia produced on Vogel's agar. After storage of Egt(+) and Egt(-) conidia at 97% and 52% relative humidity (RH) for a time course to either 17 or 98 days, respectively, Egt(-) strains had a 23% and a 18% reduction in life expectancy at 97% and 52% RH, respectively, compared to the Egt(+) strains. Based on a Cu(II) reduction assay with the chelator bathocuproinedisulfonic acid disodium salt, ergothioneine accounts for 38% and 33% of water-soluble antioxidant capacity in N. crassa conidia from seven and 20 day-old cultures, respectively. In contrast, ergothioneine did not account for significant (α=0.05) anti-oxidant capacity in mycelia, which have lower concentrations of ergothioneine than conidia. The data are consistent with the hypothesis that ergothioneine has an antioxidant function in vivo. In contrast, experiments on the spontaneous mutation rate in Egt(+) and Egt(-) strains and on the effects of 254 nm UV light on mutation rate and conidial viability do not support the hypothesis that ergothioneine protects DNA in vivo.

Ergothioneine, a histidine derivative, is concentrated in conidia of ascomycetous fungi. To investigate the function of ergothioneine, we crossed the wild type Neurospora crassa (Egt(+)) and an ergothioneine non-producer (Egt(-), Δegt-1, a knockout in NCU04343.5) and used the Egt(+) and Egt(-) progeny strains for phenotypic analyses. Compared to the Egt(+) strains, Egt(-) strains had a 59% reduction in the number of conidia produced on Vogel's agar. After storage of Egt(+) and Egt(-) conidia at 97% and 52% relative humidity (RH) for a time course to either 17 or 98 days, respectively, Egt(-) strains had a 23% and a 18% reduction in life expectancy at 97% and 52% RH, respectively, compared to the Egt(+) strains. Based on a Cu(II) reduction assay with the chelator bathocuproinedisulfonic acid disodium salt, ergothioneine accounts for 38% and 33% of water-soluble antioxidant capacity in N. crassa conidia from seven and 20 day-old cultures, respectively. In contrast, ergothioneine did not account for significant (α=0.05) anti-oxidant capacity in mycelia, which have lower concentrations of ergothioneine than conidia. The data are consistent with the hypothesis that ergothioneine has an antioxidant function in vivo. In contrast, experiments on the spontaneous mutation rate in Egt(+) and Egt(-) strains and on the effects of 254 nm UV light on mutation rate and conidial viability do not support the hypothesis that ergothioneine protects DNA in vivo.