Flo8 contributes to iron adaptation and modulates pyridoxal 5′-phosphate homeostasis in Candida glabrata

Candida glabrata is an important opportunistic fungal pathogen responsible for severe invasive infections. Iron homeostasis is essential for its survival and pathogenicity across diverse host niches. In C. glabrata , intracellular iron distribution is supported by the mitochondrial transporter Atm1, which exports iron–sulfur cluster precursors to the cytosol. Atm1 function depends on glutathione, whose metabolism is closely linked to pyridoxal 5′-phosphate (PLP), the active form of vitamin B6 maintained by pyridoxal 5′-phosphate homeostasis protein (PLPHP). However, the transcriptional regulation of this metabolic network and its contribution to iron adaptation remain poorly understood. Flo8 is a conserved transcription factor previously recognized as a regulator of virulence-associated phenotypes in C. glabrata , but its role in iron adaptation remains unknown, and its broader involvement in metabolic regulation has not been explored. In C. glabrata , deletion of FLO8 impaired growth under both iron-limited and iron-excess conditions, which was alleviated by FLO8 complementation. ChIP-seq analysis identified a candidate Flo8-associated region in the promoter of CAGL0F04367g , which encodes PLPHP. RT-qPCR showed reduced PLPHP expression and increased expression of the iron-responsive genes FTR1 and AFT1 in the FLO8 deletion mutant, whereas ATM1 expression showed no significant change. Untargeted metabolomics revealed decreased PLP and glutathione levels alongside increased pyridoxal (PL) and pyridoxine (PN) levels. Targeted LC–MS/MS analysis further supported the reductions in PLP and glutathione levels, with both metabolites showing recovery following FLO8 complementation. Our findings reveal a previously unrecognized role of Flo8 in iron adaptation in C. glabrata , supporting a working model linking Flo8-associated transcriptional regulation with PLP homeostasis and glutathione metabolism, with Atm1-associated Fe–S processes potentially connecting these metabolic changes with iron adaptation. Not applicable.