The monothiol glutaredoxin Grx4 interacts with the Cryptococcus iron regulator Cirl and regulates iron homeostasis and virulence in the Cryptococcus neoformans

The acquisition of iron and the maintenance of iron homeostasis are important aspects of the virulence in the pathogenic fungus Cryptococcus neoformans. In this study, we identified the monothiol glutaredoxin Grx4 as a binding partner of Cir1, a master regulator of iron-responsive genes and virulence factor elaboration in C. neoformans. Monothiol glutaredoxins are important regulators of iron homeostasis because of their conserved roles in [2Fe-2S] cluster sensing and trafficking. We confirmed that Grx4 binds Cir1 and demonstrated that iron repletion promotes the relocalization of Grx4 from the nucleus to the cytoplasm. Nuclear retention is partially dependent on Cir1 and also influenced by treatment with the proteasome inhibitor bortezomib. Cir1 remains in the nucleus in both iron replete and iron limiting conditions. We also found that a grx4 Δ mutant displayed iron-related phenotypes similar to those of a cir1 Δ mutant, including poor growth upon iron deprivation. Importantly, a grx4 Δ mutant was avirulent in mice, a phenotype consistent with observed defects in the key virulence determinants, capsule and melanin, and poor growth at 37°C. A comparative transcriptome analysis of a grx4 Δ mutant and the WT strain in low iron and iron-replete conditions confirmed a central role for Grx4 in iron homeostasis. Dysregulation of iron-related metabolism was consistent with grx4 Δ mutant phenotypes related to oxidative stress, mitochondrial function, and DNA repair. Overall, the phenotypes of the grx4 Δ mutant and the RNA-Seq analysis support the hypothesis that Grx4 functions as a sensor of iron levels, in part through an interaction with Cir1, to extensively regulate iron homeostasis and contribute to virulence.