The Monothiol Glutaredoxin Grx4 Regulates Iron Homeostasis and Virulence in Cryptococcus neoformans

Abstract

The acquisition of iron and the maintenance of iron homeostasis are important aspects of virulence for the pathogenic fungus Cryptococcus neoformans. In this study, we characterized the role of the monothiol glutaredoxin Grx4 in iron homeostasis and virulence in C. neoformans. Monothiol glutaredoxins are important regulators of iron homeostasis because of their conserved roles in [2Fe-2S] cluster sensing and trafficking. We initially identified Grx4 as a binding partner of Cir1, a master regulator of iron-responsive genes and virulence factor elaboration in C. neoformans. We confirmed that Grx4 binds Cir1 and demonstrated that iron repletion promotes the relocalization of Grx4 from the nucleus to the cytoplasm. We also found that a grx4 mutant lacking the GRX domain displayed iron-related phenotypes similar to those of a cir1 Delta mutant, including poor growth upon iron deprivation. Importantly, the grx4 mutant was avirulent in mice, a phenotype consistent with observed defects in the key virulence determinants, capsule and melanin, and poor growth at 37 degrees C. A comparative transcriptome analysis of the grx4 mutant and the WT strain under low-iron and iron-replete conditions confirmed a central role for Grx4 in iron homeostasis. Dysregulation of iron-related metabolism was consistent with grx4 mutant phenotypes related to oxidative stress, mitochondrial function, and DNA repair. Overall, the phenotypes of the grx4 mutant lacking the GRX domain and the transcriptome sequencing (RNA-Seq) analysis of the mutant support the hypothesis that Grx4 functions as an iron sensor, in part through an interaction with Cir1, to extensively regulate iron homeostasis. IMPORTANCE Fungal pathogens cause life-threatening diseases in humans, particularly in immunocompromised people, and there is a tremendous need for a greater understanding of pathogenesis to support new therapies. One prominent fungal pathogen, Cryptococcus neoformans, causes meningitis in people suffering from HIV/AIDS. In the present study, we focused on characterizing mechanisms by which C. neoformans senses iron availability because iron is both a signal and a key nutrient for proliferation of the pathogen in vertebrate hosts. Specifically, we characterized a monothiol glutaredoxin protein, Grx4, that functions as a sensor of iron availability and interacts with regulatory factors to control the ability of C. neoformans to cause disease. Grx4 regulates key virulence factors, and a mutant is unable to cause disease in a mouse model of cryptococcosis. Overall, our study provides new insights into nutrient sensing and the role of iron in the pathogenesis of fungal diseases.