bioRxiv preprint doi: https://doi.org/10.1101/356774; this version posted June 28, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 The monothiol glutaredoxin Grx4 interacts with the Cryptococcus iron regulator Cir1 and 2 regulates iron homeostasis and virulence in the Cryptococcus neoformans 3 Rodgoun Attariana,1,2, Guanggan Hua,1, Melissa Caza1, Eddy Sánchez-León, Daniel Croll3, 4 Eunsoo Do4, Horacio Bach1,$, Tricia Missall5, %, Jennifer Lodge6, Won Hee Jung4, and James W. 5 Kronstad1,2,# a 6 These authors contributed equally to the work and should be considered co-first authors 1 7 Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada; 8 2Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, 9 Canada. 10 3Laboratory of Evolutionary Genetics, Institute of Biology, University of Neuchâtel, CH-2000 11 Neuchâtel, Switzerland 12 4Department of Systems Biotechnology, Chung-Ang University, Anseong 17546, Korea 13 5Department of Biochemistry, Saint Louis University School of Medicine, 1755 South Grand 14 Blvd. St. Louis, Missouri, USA 15 6Department of Molecular Microbiology, Washington University School of Medicine, Box 16 8230660 South Euclid Avenue, St. Louis, Missouri, USA 63110 17 # Corresponding author: James W. Kronstad, [email protected] 18 Present addresses: 19 $Division of Infectious Diseases, Faculty of Medicine, University of British Columbia, 410- 20 2660 Oak Street, Jack Bell Research Centre, Vancouver, BC 21 %The Department of Dermatology, Saint Louis University School of Medicine, 1755 South 22 Grand Blvd. St. Louis, Missouri, USA 23 24 Running title: Monothiol glutaredoxin and fungal iron homeostasis 25 Key words: Cryptococcosis, transcriptome, capsule, melanin, nuclear localization bioRxiv preprint doi: https://doi.org/10.1101/356774; this version posted June 28, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 26 Abstract 27 The acquisition of iron and the maintenance of iron homeostasis are important aspects of 28 the virulence in the pathogenic fungus Cryptococcus neoformans. In this study, we identified 29 the monothiol glutaredoxin Grx4 as a binding partner of Cir1, a master regulator of iron- 30 responsive genes and virulence factor elaboration in C. neoformans. Monothiol glutaredoxins 31 are important regulators of iron homeostasis because of their conserved roles in [2Fe-2S] cluster 32 sensing and trafficking. We confirmed that Grx4 binds Cir1 and demonstrated that iron 33 repletion promotes the relocalization of Grx4 from the nucleus to the cytoplasm. Nuclear 34 retention is partially dependent on Cir1 and also influenced by treatment with the proteasome 35 inhibitor bortezomib. Cir1 remains in the nucleus in both iron replete and iron limiting 36 conditions. We also found that a grx4Δ mutant displayed iron-related phenotypes similar to 37 those of a cir1Δ mutant, including poor growth upon iron deprivation. Importantly, a grx4Δ 38 mutant was avirulent in mice, a phenotype consistent with observed defects in the key virulence 39 determinants, capsule and melanin, and poor growth at 37°C. A comparative transcriptome 40 analysis of a grx4Δ mutant and the WT strain in low iron and iron-replete conditions confirmed 41 a central role for Grx4 in iron homeostasis. Dysregulation of iron-related metabolism was 42 consistent with grx4Δ mutant phenotypes related to oxidative stress, mitochondrial function, and 43 DNA repair. Overall, the phenotypes of the grx4Δ mutant and the RNA-Seq analysis support 44 the hypothesis that Grx4 functions as a sensor of iron levels, in part through an interaction with 45 Cir1, to extensively regulate iron homeostasis and contribute to virulence. 46 47 Importance 48 49 Fungal pathogens cause life-threatening diseases in humans, particularly in 50 immunocompromised people, and there is a tremendous need for a greater understanding of 51 pathogenesis to support new therapies. One prominent fungal pathogen, Cryptococcus 52 neoformans, causes menigitis in people suffering from HIV/AIDS. In the current study, we 53 focused on characterizing mechanisms by which C. neoformans senses iron availability because 54 iron is both a key nutrient and a signal for proliferation of the pathogen in vertebrate hosts. 55 Specifically, we characterized a monothiol glutaredoxin protein Grx4 that functions as a sensor 2 bioRxiv preprint doi: https://doi.org/10.1101/356774; this version posted June 28, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 56 of iron, and that interacts with regulatory factors to control the ability of C. neoformans to cause 57 disease. Grx4 regulates key virulence factors and a mutant is unable to cause disease in a mouse 58 model of cryptococcosis. Overall, our study provides new insights into nutrient sensing and the 59 role of iron in the pathogenesis of fungal diseases. 60 61 62 63 64 Introduction 65 Cryptococcus neoformans is an opportunistic pathogen that causes life-threatening 66 meningoencephalitis in immunocompromised people including those with HIV/AIDS (1-3). 67 Despite the use of highly active antiretroviral therapy (HAART), there are still ~200,000 cases 68 of cryptococcal meningoencephalitis per year, and the fungus is responsible for 15% of all 69 AIDS-related deaths (4). This burden of disease underlines the urgent need to understand the 70 mechanisms of fungus proliferation in vertebrate hosts as a foundation for identifying new drug 71 and vaccine targets. 72 As with other pathogenic microbes, iron sensing and acquisition are important aspects of 73 virulence for C. neoformans (5-8). Iron is important for C. neoformans both as a nutrient and as 74 a signal to regulate the expression of the main virulence factor of the fungus, the polysaccharide 75 capsule (5, 9). The abilities of C. neoformans to grow at the host body temperature of 37°C and 76 to deposit melanin in the cell wall are also crucial for virulence (2, 6). To cause disease, the 77 fungus must overcome nutritional immunity in which verterbrate hosts withhold iron to suppress 78 pathogen growth (5, 11). C. neoformans employs various iron regulators and uptake 79 mechanisms that contribute to virulence. These include heme uptake pathways as well as high 80 and low affinity iron uptake systems (5, 12, 13). The use of heme as an iron source depends on 81 an exported mannoprotein Cig1 and a cell surface reductase Fre2 (13, 14). High affinity uptake 82 involves reduction of ferric iron (Fe3+) to the ferrous form (Fe2+) by cell surface reductases, with 83 subsequent transport by a permease (Cft1) and ferroxidase (Cfo1) complex in the plasma 84 membrane (5, 12, 14). The expression of these and other iron-related functions in C. 85 neoformans is controlled by a GATA-type transcription factor Cir1 (cryptococcal iron regulator 86 1) and additional transcription factors including HapX (15, 16). Cir1 also integrates iron 87 sensing and the regulation of iron uptake functions with the elaboration of virulence factors in C. 3 bioRxiv preprint doi: https://doi.org/10.1101/356774; this version posted June 28, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 88 neoformans (15). 89 Other fungi also use GATA-type transcriptional repressors with similar to Cir1 to 90 regulate the expression of iron-responsive genes including Schizosaccharomyces pombe (Fep1), 91 Aspergillus sp. (SreA), Neurospora crassa (SRE), and Ustilago maydis (Urbs1) (17-21). These 92 transcription factors are characterized by one or two zinc finger motifs for DNA binding, and 93 these flank a region containing four conserved cysteine residues. In contrast, the regulators of 94 iron homeostasis in Saccharomyces cerevisiae, Aft1 and Aft2, are transcriptional activators (22, 95 23). 96 The mechanisms by which iron-responsive transcription factors in fungi sense 97 intracellular iron levels and regulate iron homeostasis are best understood in S. cerevisiae and S. 98 pombe (23-26). In these fungi, the transcription factors interact with monothiol glutaredoxins 99 (GRXs) that participate in iron sensing and regulation (19, 24, 28-33). Monothiol GRXs are 100 glutathione (GSH)-dependent proteins with a cysteine-glycine-phenylalanine-serine (CGFS) 101 motif at the active site. These proteins are found in both prokaryotes and eukaryotes, and have 102 emerged as key players in cellular redox and iron homeostasis (24, 35, 36). Recent studies in S. 103 cerevisiae and S. pombe demonstrated essential roles for CGFS GRXs in intracellular iron 104 homeostasis, iron trafficking and the maturation of [2Fe-2S] cluster proteins, and have 105 established the proteins as novel [2Fe-2S] cluster-binding regulatory partners for transcription 106 factors including Fep1 and Aft1 (24, 25, 27-33). 107 In this study, we demonstrate that the monothiol glutaredoxin Grx4 of C. neoformans 108 interacts with Cir1. We also present evidence that Grx4 is involved in virulence and the 109 maintenance of iron homeostasis. In particular, mutants lacking GRX4 are defective for growth 110 at the host temperature of 37°C and upon iron limitation. Along with defects in other virulence 111 factors such as capsule and melanin, these findings account for the loss of virulence for the 112 grx4Δ mutant in a murine model of cryptococcosis.