Wor1‐regulated ferroxidases contribute to pigment formation in opaque cells of Candida albicans

Candida albicans is a harmless commensal resident in the human gut and a prevalent opportunistic pathogen. A key part of its commensalism and pathogenesis is its ability to switch between different morphological forms, including white‐to‐opaque switching. The Wor1 protein was previously identified as a master regulator of white‐to‐opaque switching in mating type locus (MTL) homozygous cells. The mechanisms by which the dark color of the opaque colonies is controlled and the pimpled surface of opaque cells is formed remain unknown. Candida albicans produces melanin pigment in vitro and during infection. However, the molecular mechanism underlying the regulation of melanin production is unclear. In this study, we demonstrated that ferroxidases (Fets) function as pigment multicopper oxidases and regulate the production of dark‐pigmented melanin in opaque cells. The FET genes presented distinct regulation patterns in response to different extracellular stimuli. In YPD (1% yeast extract, 2% peptone and 2% dextrose)‐rich medium, four of the five FET genes were up‐regulated by Wor1, especially at the human body temperature of 37 °C. In minimal medium with low ammonium concentrations, all five FET genes were up‐regulated by Wor1. However, at high ammonium concentrations, some FET genes were down‐regulated by Wor1. Wor1‐up‐regulated Fets contributed to dark pigment formation in opaque colonies, but not to the elongated shape of these opaque cells. Increased melanin externalization was associated with the pimpled surface of the opaque cells. Melanized C. albicans cells were more resistant to fungal clearance. Deletion of the five FET genes completely blocked melanin production in opaque cells and resulted in the generation of white elongated ‘opaque’ cells. In addition, the up‐regulated Fets are important for defense against oxidant attacks. The functional diversity of Fets may reflect the multiple strategies of C. albicans to rapidly adapt to diverse host niches. In Candida albicans opaque cells, the master regulator Wor1 up‐regulates Fets, which function as pigment multicopper oxidases and promote the production of dark‐pigmented melanin. Increased melanin externalization is associated with the pimpled surface and dark color of opaque cells. The melanized C. albicans cells were more resistant to fungal clearance, including bone marrow‐derived macrophage phagocytosis and oxidative killing.