The Neurospora crassa mutant NcΔEgt-1 identifies an ergothioneine biosynthetic gene and demonstrates that ergothioneine enhances conidial survival and protects against peroxide toxicity during conidial germination

Ergothioneine (EGT) is a histidine derivative with sulfur on the imidazole ring and a trimethylated amine; it is postulated to have an antioxidant function. Although EGT apparently is only produced by fungi and some prokaryotes, it is acquired by animals and plants from the environment, and is concentrated in animal tissues in cells with an EGT transporter. Monobromobimane derivatives of EGT allowed conclusive identification of EGT by LC/MS and the quantification of EGT in Colletotrichum graminicola and Neurospora crassa conidia and mycelia. EGT concentrations were significantly (α=0.05) higher in conidia than in mycelia, with approximately 17X and 5X more in C. graminicola and N. crassa, respectively. The first EGT biosynthetic gene in a fungus was identified by quantifying EGT in N. crassa wild type and knockouts in putative homologs of actinomycete EGT biosynthetic genes. NcΔEgt-1, a strain with a knockout in gene NCU04343, does not produce EGT, in contrast to the wild type. To determine the effects of EGT in vivo, we compared NcΔEgt-1 to the wild type. NcΔEgt-1 is not pleiotropically affected in rate of hyphal elongation in Vogel’s medium either with or without ammonium nitrate and in the rate of germination of macroconidia on Vogel’s medium. The superoxide-producer menadione had indistinguishable effects on conidial germination between the two strains. Cupric sulfate also had indistinguishable effects on conidial germination and on hyphal growth between the two strains. In contrast, germination of NcΔEgt-1 conidia was significantly more sensitive to tert-butyl hydroperoxide than the wild type; germination of 50% (GI₅₀) of the NcΔEgt-1 conidia was prevented at 2.7mM tert-butyl hydroperoxide whereas the GI₅₀ for the wild type was 4.7mM tert-butyl hydroperoxide, or at a 1.7X greater concentration. In the presence of tert-butyl hydroperoxide and the fluorescent reactive oxygen species indicator 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate, significantly (P=0.0002) more NcΔEgt-1 conidia fluoresced than wild type conidia, indicating that EGT quenched peroxides in vivo. While five to 21-day-old conidia of both strains germinated 100%, NcΔEgt-1 conidia had significantly (P<0.001) diminished longevity. Linear regression analysis indicates that germination of the wild type declined to 50% in 35days, in comparison to 25days for the NcΔEgt-1, which is equivalent to a 29% reduction in conidial life span in the NcEgt-1 deletion strain. Consequently, the data indicate that endogenous EGT helps protect conidia during the quiescent period between conidiogenesis and germination, and that EGT helps protect conidia during the germination process from the toxicity of peroxide but not from superoxide or Cu²⁺. Based on an in silico analysis, we postulate that NcEgt-1 was acquired early in the mycota lineage as a fusion of two adjacent prokaryotic genes, that was then lost in the Saccharomycotina, and that NcEgt-1 catalyzes the first two steps of EGT biosynthesis from histidine to hercynine to hercynylcysteine sulfoxide.