G3: Genes|Genomes|Genetics Early Online, published on December 12, 2017 as doi:10.1534/g3.117.300444 1 Had1 is Required for Cell Wall Integrity and Fungal Virulence in 2 Cryptococcus neoformans 3 4 Won-Hee Jung1, Ye-Eun Son1, Sang-Hun Oh2, Ci Fu3, Hye Shin Kim2, Jin-Hwan Kwak2, 5 Maria E. Cardenas3, Joseph Heitman3, and Hee-Soo Park1* 6 7 8 1 School of Food Science and Biotechnology, Institute of Agricultural Science and 9 Technology, Kyungpook National University, Daegu, Republic of Korea; 2 School of Life 10 Science, Handong Global University, Pohang, Republic of Korea; 3 Department of 11 Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North 12 Carolina, USA 13 14 15 16 * Corresponding Author: 17 Hee-Soo Park 18 School of Food Science and Biotechnology 19 80 Daehak-ro, Buk-gu, Daegu, 41566, South Korea 20 Tel: +82-53-950-5751 21 Fax: +82-53-950-6750 22 Email: [email protected] 23 24 Running title: The role of Had1 in C. neoformans. 1 © The Author(s) 2013. Published by the Genetics Society of America. 25 ABSTRACT 26 Calcineurin modulates environmental stress survival and virulence of the human fungal 27 pathogen Cryptococcus neoformans. Previously, we identified 44 putative calcineurin 28 substrates and proposed that the calcineurin pathway is branched to regulate targets 29 including Crz1, Pbp1, and Puf4 in C. neoformans. In this study we characterized Had1, 30 which is one of the putative calcineurin substrates and belongs to a ubiquitously 31 conserved haloacid dehalogenase β-phosphoglucomutase protein super-family. Growth 32 of the had1∆ mutant was found to be compromised at 38C or higher. In addition, the 33 had1∆ mutant exhibited increased sensitivity to cell wall perturbing agents including 34 Congo Red, and Calcofluor White, and to an ER stress inducer DTT. Virulence studies 35 revealed that the had1 mutation results in attenuated virulence compared to the wild 36 type strain in a murine inhalation infection model. Genetic epistasis analysis revealed 37 that Had1 and the zinc finger transcription factor Crz1 play roles in parallel pathways 38 that orchestrate stress survival and fungal virulence. Overall, our results demonstrate 39 that Had1 is a key regulator of thermotolerance, cell wall integrity, and virulence of C. 40 neoformans. 41 42 43 44 45 46 47 48 2 49 INTRODUCTION 50 Calcineurin is a highly conserved serine/threonine protein phosphatase, which is 51 activated by Ca2+/calmodulin in eukaryotic organisms, and plays diverse roles in 52 controlling gene expression and cellular processes (STEWART et al. 1982; RUSNAK AND 2+ 53 MERTZ 2000; ARAMBURU et al. 2004). Increased intracellular Ca levels in response to 54 internal or external cues bind to calmodulin, and in turn, the Ca2+-calmodulin complex 55 interacts with the calcineurin catalytic subunit and activates the phosphatase (CRABTREE 56 2001). Activated calcineurin dephosphorylates target proteins that then orchestrate the 57 response and adaptation to stress (KISSINGER et al. 1995; ARAMBURU et al. 2000). A key 58 substrate of calcineurin is NFAT (nuclear factor of activated T cell), a transcription factor 59 that regulates the expression of genes which are associated with T cell activation, and 60 the development of the nervous and cardiac systems (NORTHROP et al. 1994; FESKE et 61 al. 2003; HOGAN et al. 2003; ARAMBURU et al. 2004). The calcineurin signaling pathway 62 is inhibited by the immunosuppressive and antifungal compounds FK506 and 63 cyclosporin A (CsA), which bind to FK506-binding protein 12 (FKBP12) and cyclophilin A, 64 respectively, forming stable complexes. The FK506-FKBP12 and cyclosporine A- 65 cyclophilin A complexes interact with calcineurin and thereby inhibit the phosphatase 66 activity (O'KEEFE et al. 1992; SCHREIBER AND CRABTREE 1992; SIEKIERKA AND SIGAL 1992; 67 SIGAL AND DUMONT 1992; KISSINGER et al. 1995). 68 While calcineurin is highly conserved in eukaryotes, the functions and biological 69 roles regulated by calcineurin are distinct in mammals and fungi (STEINBACH et al. 70 2007b). In the model yeast Saccharomyces cerevisiae, calcineurin is essential for stress 71 responses, bud emergence, and cell cycle regulation (MIYAKAWA AND MIZUNUMA 2007). In 3 72 yeast, Crz1 (calcineurin responsive zinc finger 1) is a prominent calcineurin substrate 73 that transcriptionally regulates mRNA expression of target genes associated with cell- 74 wall synthesis, ion transport, and vesicle transport (YOSHIMOTO et al. 2002; CYERT 2003). 75 In the major pathogenic fungi, including Candida albicans and Aspergillus fumigatus, 76 the calcineurin signaling pathway plays crucial roles in virulence and stress responses 77 (BADER et al. 2003; BLANKENSHIP et al. 2003; STEINBACH et al. 2006; STEINBACH et al. 78 2007a; STEINBACH et al. 2007b; LEE et al. 2013). Loss of calcineurin causes increased 79 cell sensitivity to stresses including high temperature, salt, cell wall stress, and also 80 results in attenuated or loss of virulence in human pathogenic fungi. Therefore, 81 calcineurin is considered a target for anti-fungal drug discovery and in fact, the 82 immunosuppressants FK506 and cyclosporine A show anti-fungal activity (BRIZUELA et 83 al. 1991; ODOM et al. 1997a). A series of studies also demonstrated that the Crz1 84 orthologs are prominent calcineurin downstream targets in pathogenic fungi (ONYEWU et 85 al. 2004; CRAMER et al. 2008; SCHUMACHER et al. 2008; SORIANI et al. 2008; MIYAZAKI et 86 al. 2010). 87 Cryptococcus neoformans is a ubiquitous fungus that is widespread in the 88 environment including associations with trees, soil, and bird guano (MONARI et al. 1999; 89 IDNURM et al. 2005; CHANG et al. 2014). This fungus is a basidiomycetous fungus and 90 both its spores and desiccated yeast cells serve as infectious propagules (HUNG AND 91 SCHREIBER 1992; KWON-CHUNG et al. 2014). C. neoformans is an opportunistic 92 pathogenic fungus that causes meningoencephalitis in patients who have weakened 93 immune systems, including HIV/AIDS and organ transplant patients, leading to high 94 mortality (KIDD et al. 2004; PARK et al. 2009). In this human pathogenic fungus, 4 95 calcineurin is a key virulence factor that is required for adaptation to stressful host 96 environments including elevated temperature (ODOM et al. 1997b; FOX et al. 2001). In 97 addition, calcineurin is essential for stress adaptation, sexual reproduction, and 98 virulence of C. neoformans (FOX et al. 2001; DANIELSEN et al. 2013). During thermal and 99 other stress conditions, calcineurin co-localizes with components of P-bodies/stress 100 granules (KOZUBOWSKI et al. 2011a; KOZUBOWSKI et al. 2011b) (PBs/SGs) which consist 101 of aggregates of RNA binding proteins, mRNA decay machinery, and translation 102 initiation factors (BUCHAN AND PARKER 2009; MITCHELL et al. 2013; BUCHAN 2014). 103 Recently, our phosphoproteomic analysis identified forty-four putative calcineurin 104 targets in C. neoformans (PARK et al. 2016). We demonstrated that the Crz1 ortholog is 105 a bona fide calcineurin target in C. neoformans. Under thermal stress conditions, 106 calcineurin controls the transcriptional activity and nuclear translocation of Crz1 through 107 dephosphorylation (PARK et al. 2016). In addition, we proposed that several RNA 108 binding proteins including Pbp1 (PAB1-binding protein 1), Puf4 (PUmilio-homology 109 domain Family 4), and Lhp1 are potential calcineurin targets. Pbp1 is involved in fungal 110 virulence and sexual reproduction (PARK et al. 2016). Both Puf4 and Lhp1 are required 111 for heat stress survival (PARK et al. 2016). Employing epistasis analyses, we 112 demonstrated that two downstream branches of the calcineurin pathway govern cell 113 viability at high temperature, sexual reproduction, and fungal virulence (PARK et al. 114 2016). 115 Although we characterized several calcineurin targets, including Crz1 and RNA 116 binding proteins, in our previous study (PARK et al. 2016), many putative targets await 117 characterization. To further characterize the remaining calcineurin targets revealed by 5 118 the above study, we examined stress responses of 13 putative calcineurin-target 119 mutants from a systematic deletion mutant library of C. neoformans (LIU et al. 2008). 120 Among them, the CNAG_01744∆ mutant showed increased sensitivity to cell wall stress 121 in comparison to the WT. Based on BLAST analysis, CNAG_01744 shares considerable 122 homology with the S. cerevisiae Had1 protein. Had1 contains a HAD domain (Haloacid 123 dehalogenase proteins) and is a member of the beta-phosphoglucomutase family of 124 proteins that is highly conserved in most organisms (BURROUGHS et al. 2006; 125 KUZNETSOVA et al. 2015). In yeast, several beta-phosphoglucomutase family proteins, 126 including two 2-deoxyglucose-6-phosphatase (Dog1 and Dog2) and two glycerol-1- 127 phosphate phospohydrolases (Rhr2/Gpp1 and Hor2/Gpp2), are well characterized and 128 are required for cellular responses to environmental stresses such as osmotic and 129 oxidative stresses (RANDEZ-GIL et al. 1995a; RANDEZ-GIL et al. 1995b; NORBECK et al. 130 1996; KUZNETSOVA et al. 2015). Given the relevance of haloacid dehalogenase in stress 131 responses, we were prompted to characterize the role of Had1 in the calcineurin 132 pathway functions in C. neoformans. Our results demonstrate that loss of Had1 causes 133 increased sensitivity to thermal or cell wall stresses and attenuated virulence. To dissect 134 the link between Had1 and Crz1 in calcineurin-related functions, we tested the impact of 135 the had1∆ crz1∆ double mutation. The results show that Crz1 and Had1 play additive 136 roles in thermoresistance and virulence. However, whether Had1 is a direct calcineurin 137 substrate remains to be established. 138 139 MATERIAL AND METHODS 140 Strains, media and culture conditions. Fungal strains used in this study are listed in 6 141 Table 1. Liquid and solid yeast extract-peptone-dextrose (YPD, Difco, Sparks, MD, USA) 142 media were used for general cultures of C. neoformans. To assay thermo-tolerance, 143 fungal cells grown overnight at 30 C were ten-fold diluted, spotted on YPD plates, and 144 then cultured at distinct temperatures (30, 37, 38, and 39C).