G3: Genes|Genomes|Genetics Early Online, published on December 12, 2017 as doi:10.1534/g3.117.300444
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Had1 is Required for Cell Wall Integrity and Fungal Virulence in
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Cryptococcus neoformans
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Won-Hee Jung1, Ye-Eun Son1, Sang-Hun Oh2, Ci Fu3, Hye Shin Kim2, Jin-Hwan Kwak2,
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Maria E. Cardenas3, Joseph Heitman3, and Hee-Soo Park1*
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School of Food Science and Biotechnology, Institute of Agricultural Science and
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Technology, Kyungpook National University, Daegu, Republic of Korea; 2 School of Life
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Science, Handong Global University, Pohang, Republic of Korea; 3 Department of
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Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North
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Carolina, USA
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*
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Hee-Soo Park
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School of Food Science and Biotechnology
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80 Daehak-ro, Buk-gu, Daegu, 41566, South Korea
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Tel: +82-53-950-5751
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Fax: +82-53-950-6750
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Email:
[email protected]
Corresponding Author:
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Running title: The role of Had1 in C. neoformans. 1
© The Author(s) 2013. Published by the Genetics Society of America.
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ABSTRACT
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Calcineurin modulates environmental stress survival and virulence of the human fungal
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pathogen Cryptococcus neoformans. Previously, we identified 44 putative calcineurin
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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,
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which is one of the putative calcineurin substrates and belongs to a ubiquitously
31
conserved haloacid dehalogenase β-phosphoglucomutase protein super-family. Growth
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of the had1∆ mutant was found to be compromised at 38C or higher. In addition, the
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had1∆ mutant exhibited increased sensitivity to cell wall perturbing agents including
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Congo Red, and Calcofluor White, and to an ER stress inducer DTT. Virulence studies
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revealed that the had1 mutation results in attenuated virulence compared to the wild
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type strain in a murine inhalation infection model. Genetic epistasis analysis revealed
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that Had1 and the zinc finger transcription factor Crz1 play roles in parallel pathways
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that orchestrate stress survival and fungal virulence. Overall, our results demonstrate
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that Had1 is a key regulator of thermotolerance, cell wall integrity, and virulence of C.
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neoformans.
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INTRODUCTION
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Calcineurin is a highly conserved serine/threonine protein phosphatase, which is
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activated by Ca2+/calmodulin in eukaryotic organisms, and plays diverse roles in
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controlling gene expression and cellular processes (STEWART et al. 1982; RUSNAK AND
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MERTZ 2000; ARAMBURU et al. 2004). Increased intracellular Ca2+ levels in response to
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internal or external cues bind to calmodulin, and in turn, the Ca2+-calmodulin complex
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interacts with the calcineurin catalytic subunit and activates the phosphatase (CRABTREE
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2001). Activated calcineurin dephosphorylates target proteins that then orchestrate the
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response and adaptation to stress (KISSINGER et al. 1995; ARAMBURU et al. 2000). A key
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substrate of calcineurin is NFAT (nuclear factor of activated T cell), a transcription factor
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that regulates the expression of genes which are associated with T cell activation, and
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the development of the nervous and cardiac systems (NORTHROP et al. 1994; FESKE et
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al. 2003; HOGAN et al. 2003; ARAMBURU et al. 2004). The calcineurin signaling pathway
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is inhibited by the immunosuppressive and antifungal compounds FK506 and
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cyclosporin A (CsA), which bind to FK506-binding protein 12 (FKBP12) and cyclophilin A,
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respectively, forming stable complexes. The FK506-FKBP12 and cyclosporine A-
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cyclophilin A complexes interact with calcineurin and thereby inhibit the phosphatase
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activity (O'KEEFE et al. 1992; SCHREIBER AND CRABTREE 1992; SIEKIERKA AND SIGAL 1992;
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SIGAL AND DUMONT 1992; KISSINGER et al. 1995).
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While calcineurin is highly conserved in eukaryotes, the functions and biological
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roles regulated by calcineurin are distinct in mammals and fungi (STEINBACH et al.
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2007b). In the model yeast Saccharomyces cerevisiae, calcineurin is essential for stress
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responses, bud emergence, and cell cycle regulation (MIYAKAWA AND MIZUNUMA 2007). In 3
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yeast, Crz1 (calcineurin responsive zinc finger 1) is a prominent calcineurin substrate
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that transcriptionally regulates mRNA expression of target genes associated with cell-
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wall synthesis, ion transport, and vesicle transport (YOSHIMOTO et al. 2002; CYERT 2003).
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In the major pathogenic fungi, including Candida albicans and Aspergillus fumigatus,
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the calcineurin signaling pathway plays crucial roles in virulence and stress responses
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(BADER et al. 2003; BLANKENSHIP et al. 2003; STEINBACH et al. 2006; STEINBACH et al.
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2007a; STEINBACH et al. 2007b; LEE et al. 2013). Loss of calcineurin causes increased
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cell sensitivity to stresses including high temperature, salt, cell wall stress, and also
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results in attenuated or loss of virulence in human pathogenic fungi. Therefore,
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calcineurin is considered a target for anti-fungal drug discovery and in fact, the
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immunosuppressants FK506 and cyclosporine A show anti-fungal activity (BRIZUELA et
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al. 1991; ODOM et al. 1997a). A series of studies also demonstrated that the Crz1
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orthologs are prominent calcineurin downstream targets in pathogenic fungi (ONYEWU et
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al. 2004; CRAMER et al. 2008; SCHUMACHER et al. 2008; SORIANI et al. 2008; MIYAZAKI et
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al. 2010).
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Cryptococcus neoformans is a ubiquitous fungus that is widespread in the
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environment including associations with trees, soil, and bird guano (MONARI et al. 1999;
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IDNURM et al. 2005; CHANG et al. 2014). This fungus is a basidiomycetous fungus and
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both its spores and desiccated yeast cells serve as infectious propagules (HUNG
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SCHREIBER 1992; KWON-CHUNG et al. 2014). C. neoformans is an opportunistic
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pathogenic fungus that causes meningoencephalitis in patients who have weakened
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immune systems, including HIV/AIDS and organ transplant patients, leading to high
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mortality (KIDD et al. 2004; PARK et al. 2009). In this human pathogenic fungus, 4
AND
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calcineurin is a key virulence factor that is required for adaptation to stressful host
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environments including elevated temperature (ODOM et al. 1997b; FOX et al. 2001). In
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addition, calcineurin is essential for stress adaptation, sexual reproduction, and
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virulence of C. neoformans (FOX et al. 2001; DANIELSEN et al. 2013). During thermal and
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other stress conditions, calcineurin co-localizes with components of P-bodies/stress
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granules (KOZUBOWSKI et al. 2011a; KOZUBOWSKI et al. 2011b) (PBs/SGs) which consist
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of aggregates of RNA binding proteins, mRNA decay machinery, and translation
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initiation factors (BUCHAN AND PARKER 2009; MITCHELL et al. 2013; BUCHAN 2014).
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Recently, our phosphoproteomic analysis identified forty-four putative calcineurin
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targets in C. neoformans (PARK et al. 2016). We demonstrated that the Crz1 ortholog is
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a bona fide calcineurin target in C. neoformans. Under thermal stress conditions,
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calcineurin controls the transcriptional activity and nuclear translocation of Crz1 through
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dephosphorylation (PARK et al. 2016). In addition, we proposed that several RNA
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binding proteins including Pbp1 (PAB1-binding protein 1), Puf4 (PUmilio-homology
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domain Family 4), and Lhp1 are potential calcineurin targets. Pbp1 is involved in fungal
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virulence and sexual reproduction (PARK et al. 2016). Both Puf4 and Lhp1 are required
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for heat stress survival (PARK et al. 2016). Employing epistasis analyses, we
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demonstrated that two downstream branches of the calcineurin pathway govern cell
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viability at high temperature, sexual reproduction, and fungal virulence (PARK et al.
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2016).
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Although we characterized several calcineurin targets, including Crz1 and RNA
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binding proteins, in our previous study (PARK et al. 2016), many putative targets await
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characterization. To further characterize the remaining calcineurin targets revealed by 5
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the above study, we examined stress responses of 13 putative calcineurin-target
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mutants from a systematic deletion mutant library of C. neoformans (LIU et al. 2008).
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Among them, the CNAG_01744∆ mutant showed increased sensitivity to cell wall stress
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in comparison to the WT. Based on BLAST analysis, CNAG_01744 shares considerable
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homology with the S. cerevisiae Had1 protein. Had1 contains a HAD domain (Haloacid
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dehalogenase proteins) and is a member of the beta-phosphoglucomutase family of
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proteins that is highly conserved in most organisms (BURROUGHS et al. 2006;
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KUZNETSOVA et al. 2015). In yeast, several beta-phosphoglucomutase family proteins,
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including two 2-deoxyglucose-6-phosphatase (Dog1 and Dog2) and two glycerol-1-
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phosphate phospohydrolases (Rhr2/Gpp1 and Hor2/Gpp2), are well characterized and
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are required for cellular responses to environmental stresses such as osmotic and
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oxidative stresses (RANDEZ-GIL et al. 1995a; RANDEZ-GIL et al. 1995b; NORBECK et al.
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1996; KUZNETSOVA et al. 2015). Given the relevance of haloacid dehalogenase in stress
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responses, we were prompted to characterize the role of Had1 in the calcineurin
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pathway functions in C. neoformans. Our results demonstrate that loss of Had1 causes
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increased sensitivity to thermal or cell wall stresses and attenuated virulence. To dissect
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the link between Had1 and Crz1 in calcineurin-related functions, we tested the impact of
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the had1∆ crz1∆ double mutation. The results show that Crz1 and Had1 play additive
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roles in thermoresistance and virulence. However, whether Had1 is a direct calcineurin
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substrate remains to be established.
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MATERIAL AND METHODS
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Strains, media and culture conditions. Fungal strains used in this study are listed in 6
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Table 1. Liquid and solid yeast extract-peptone-dextrose (YPD, Difco, Sparks, MD, USA)
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media were used for general cultures of C. neoformans. To assay thermo-tolerance,
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fungal cells grown overnight at 30 C were ten-fold diluted, spotted on YPD plates, and
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then cultured at distinct temperatures (30, 37, 38, and 39C). To examine other stresses
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susceptibility, 2.5 to 5 μL of cultured cells grown in liquid YPD medium overnight were
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ten-fold serially diluted and spotted on YPD medium containing the indicated
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concentration of the following compounds; Congo red (CR, Sigma, St. Louis, MO, USA)
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and sodium dodecyl sulfate (SDS, Fisher, Fair Lawn, NJ, USA) for membrane
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destabilizing stress, dithiothreitol (DTT, Sigma) for reducing stress, calcofluor white
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(Sigma) for chitin synthesis inhibition that results in cell wall stress, NaCl (Fisher) and
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KCl (Fisher) for salt stresses. Fungal cells were incubated at 30C and photographed
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post-treatment from day 2 to day 3.
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Generation of mutant strains. The oligonucleotides used in this study are listed in
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Table 2. To generate deletion mutant, gene deletion cassettes were generated using a
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double-joint PCR (DJ-PCR) as described (YU et al. 2004). The 5’ and 3’-flanking region
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of the HAD1 gene were amplified using primer pairs JOHE42780;JOHE42782 and
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JOHE42781;JOHE42783, respectively, from the C. neoformans serotype A H99
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(PERFECT et al. 1993; JANBON et al. 2014) genomic DNA as a template. The selectable
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markers, NAT or NEO (FRASER et al. 2003), were amplified with the primer pair
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JOHE40706;JOHE40707 by using pAI3 and pJAF1, respectively. The final deletion
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cassettes were generated by means of DJ-PCR performed using primer pair
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JOHE42784;JOHE42785 and the 5’ and 3’-franking regions and markers as templates. 7
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The amplified gene deletion cassettes were purified, combined with 0.6 μm gold
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microcarrier beads (Bio-Rad) using the QIAquick Gel Extraction kit (Qiagen), and then
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introduced into the wild type (H99 or KN99a) mutant strains using the biolistic
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transformation methods (DAVIDSON et al. 2002).
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To generate the double deletion mutants, 5’ and 3’-flanking regions for CRZ1
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(JOHE41391;JOHE41393 and JOHE41392;JOHE41394) were amplified. The NAT
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marker was used for the disruption cassettes. After the fusion by DJ-PCR, crz1
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disruption constructs were amplified using JOHE41395;JOHE41396, and introduced
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into HPC24 (had1∆ mutant). Multiple stable transformants were isolated from
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independent experiments, and were selected on YPD medium containing nourseothricin
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sulfate or G418, and then confirmed by diagnostic PCR for the 5’ and 3’ junctions
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followed by restriction enzyme digestion.
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To express the Had1-FLAG fusion protein, the HAD1 gene region including its
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predicted promoter but lacking its termination codon was amplified using the primers
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JOHE42786 and JOHE42787. The PCR product was then digested with NotI and
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cloned into pHP2 (PARK et al. 2016), which contains a 4x FLAG tag, the HOG1
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terminator, and the hygromycin B-resistant gene. The resulting plasmid pHSP1 was
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then introduced into the recipient had1∆ strains. Multiple transformants were selected
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on YPD medium containing hygromycin B (Sigma), and then confirmed by PCR and
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Western blot analyses.
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Had1 mobility assay. Had1 mobility assay was conducted as described previously
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(PARK et al. 2016). The strains expressing Had1-FLAG was grown in YPD at 25°C to an 8
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optical density OD600 0.6–0.8 and the cultures were grown at 25°C or shifted from 25°C
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to 37°C for 1 hour with or without FK506 (2 μg/ml). Cells were collected and disrupted in
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lysis buffer (50 mM Tris-HCl pH = 7.5, 150 NaCl, 0.5mM EDTA, 0.5% Triton X-100
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supplemented with protease inhibitor tablet (Roche) and phosphatase inhibitor cocktails
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(Thermo) using a bead beater for 10 cycles (60 sec homogenization with 60 sec rest).
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Cell lysates were centrifuged for 15 min at 14.000×g, the supernatant was recovered
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and protein concentration was determined by employing the Bio-Rad Bradford reagent.
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The supernatant was subjected to SDS-PAGE and transferred to PVDF membranes
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(Bio-Rad). For western blot analysis, we employed mouse monoclonal anti-FLAG M2
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antibodies (Sigma), anti-mouse antibody conjugated to horseradish peroxidase
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(Thermo), and ECL western blotting detection reagent (GE healthcare).
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Virulence assay. Cryptococcus strains were cultured overnight in liquid YPD medium at
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30C. The resulting fungal cells were collected, washed with sterile PBS, counted with a
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hemocytometer, and the final density was adjusted to 1107 CFU/mL. Six- to seven-
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week-old female BALB/c mice were purchased from the Daehan BioLink Co., Ltd.,
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Korea and used for infection and fungal burden assays. Intranasal infection was
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performed as previously described (COX et al. 2000). Fourteen mice were anesthetized,
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and infected intranasally with 5 105 CFU in a volume of 50 μL as previously described
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(COX et al. 2000).
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For survival tests, groups of ten mice were used. Survival was monitored daily,
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and moribund mice were sacrificed with CO2. Survival curve were generated using the
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Kaplan-Meier method by the Prism 4.0 (GraphPad software) and statistical significance 9
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(p values) assessed with the log-rank test.
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For fungal burden assays, infected mice (three to four) were sacrificed by
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exposure to CO2. Lung and brain tissues were isolated, placed in saline, and then
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homogenized. The suspension was serially diluted with saline, plated onto YPD agar
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supplemented with antibiotics (kanamycin, ampicillin, and spectromycin), and incubated
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at 30C for 3 days. Colony counts were performed and adjusted to reflect the total lung
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or brain colony-forming unit. To assess statistically significant differences between the
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samples, statistical analysis was performed using the Student’s t-test.
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Ethics statement. Animal care and all experiments were conducted in accordance with
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the ethical guidelines of the Ethics Review Committee for Animal Experimentation
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(ERCAE) of Handong Global University (HGU). The HGU ERCAE approved the entire
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vertebrate animal protocol (protocol #HGU-20160616-009).
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Data availability. All the strains and plasmids used in this study are available upon
225
request. The authors state that all data necessary for confirming the conclusions
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presented in the article are represented fully within the article.
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RESULTS
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CNAG_01744 encodes a haloacid dehalogenase-like hydrolase protein (Had1)
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Previously, we performed phosphoproteomic analyses and identified 44 putative
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calcineurin targets. Among these targets, we generated deletion mutants for eight, and
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characterized these as authentic calcineurin targets (PARK et al. 2016). In the present 10
233
study, for 13 additional putative calcineurin targets identified by the above
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phosphoproteomic analysis mutants were available in a systematic deletion mutant
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library of C. neoformans (LIU et al. 2008), (Figure 1A-B) and these were selected and
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analyzed for calcineurin-related phenotypes. Because calcineurin is essential for
237
responses to stress, these mutants were subjected to phenotypic analyses under
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several of stress conditions. The CNAG_01744∆ and CNAG_03841∆ mutants exhibited
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increased sensitivity to cell wall stress (SDS and Congo red) as compared to the WT
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(Figure 1C). CNAG_03841 encodes a hypothetical protein and CNAG_01744 encodes
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a haloacid dehalogenase-like hydrolase protein. In this study, we focused on
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characterizing the functions of CNAG_01744.
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The protein sequence of the CNAG_01744 ORF was blasted against the S.
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cerevisiae S288 genome database and found that this ORF bears identity to six
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haloacid
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Furthermore, in the C. neoformans genome blast searches revealed a total of four
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ORFs that feature HAD (Haloacid dehalogenase proteins) domains including,
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CNAG_01744, CNAG_06122, CNAG_06132, and CNAG_06698 (Figure S1B).
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CNAG_01744 showed the highest homology (Score 110, identity 32%) to the S.
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cerevisiae Gpp2. However, we found that CNAG_06122 shows much higher homology
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(Score 119, identity 33%) to S. cerevisiae Gpp2 (a glycerol-1-phosphatase, which is
252
induced in response to osmotic and oxidative stress) than CNAG_01744. Therefore,
253
CNAG_01744 was named Had1 instead of Gpp2.
dehalogenase
β-phosphoglucomutase
family
proteins
(Figure
S1A).
254 255
Had1
is
essential
for
appropriate
stress 11
response
and
may
function
256
independently of calcineurin
257
To test the functions of Had1, we generated the had1 deletion (had1∆) mutant
258
and complemented strains, and examined their phenotypes under a variety of
259
temperature conditions. As mentioned above, we confirmed that the had1∆ mutant
260
exhibited increased sensitivity to high temperatures when compared to the WT strain
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(Figure 2A). To further examine the role of Had1, we tested the growth phenotypes of
262
had1 mutants on solid media containing various cell stressors agents including Congo
263
red (CR), calcofluor white (CFW), SDS, DTT, and KCl. As shown Figure 2B, the had1∆
264
mutant showed increased sensitivity to cell wall perturbing agents, in particular to SDS,
265
as compared to the WT strain, suggesting that Had1 may be involved in controlling
266
stress response.
267
Because
Had1
is
a
potential
calcineurin
target
identified
from the
268
phosphoproteomics results (PARK et al. 2016), we examined the migration of Had1
269
under both heat or high salt osmotic stress conditions. Cultures of yeast cells
270
expressing FLAG tagged Had1 were cultured at 25C and shifted or not to either 37C
271
for 1 hr, or to media containing 1 M NaCl and incubated at 25C for 1hr. For both stress
272
conditions cultures were treated or not with FK506 for 1 hr. Interestingly, The Had1-
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FLAG protein displayed reduced gel migration when isolated from cells shifted from
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25C to 37C or from cells exposed to high salt osmotic stress, as compared to that
275
isolated from cultures grown at 25C (Figure S2A). However, FK506 did not affect the
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gel migration of Had1 under either stress condition (Figure S2). These results suggest
277
that the stress-induced posttranslational modification resulting in altered Had1 gel
278
mobility is independent of calcineurin, but do not rule out the possibility that Had1 is a 12
279
substrate of calcineurin that cannot be detected by gel mobility shift assay.
280 281
Had1 and Crz1 play an additive role in stress response and virulence
282
Previously, our study demonstrated that calcineurin regulates high temperature
283
growth and virulence via transcriptional and post-transcriptional processes (ODOM et al.
284
1997b; CRUZ et al. 2001). Among the characterized calcineurin targets, Crz1 is the only
285
known target involved in cell wall integrity. To examine whether Crz1 and Had1 function
286
in a single pathway or in parallel pathways within the signaling cascades governing cell
287
wall integrity, we generated had1 crz1 double deletion mutants and examined their
288
phenotypes during stress responses. As shown Figure 3, the had1∆ crz1∆ double
289
mutant strains exhibited increased sensitivity to various stresses compared to either the
290
crz∆1 or had1∆ single deletion mutant. These results demonstrate that Had1 and Crz1
291
play an additive role in thermotolerance and cell wall stresses, and suggest they
292
operate in parallel pathways.
293 294
Had1 is involved in fungal pathogenicity
295
Because of hyper-susceptibility to heat and other stress conditions, we
296
hypothesized that Had1 would be required for pathogenicity of C. neoformans. To
297
examine virulence of these mutants, we conducted virulence animal studies using a
298
murine inhalation model. As shown in Figure 4A, the had1∆ mutant exhibited
299
attenuated virulence compared to the WT strains. Virulence was restored to nearly WT
300
levels when the had1∆ mutant was complemented by reintroduction of the wild-type
301
HAD1 gene in the had1∆ + HAD1 strain. The had1∆ crz1∆ double mutant strains were 13
302
more attenuated in virulence as compared with either the had1∆ or the crz1∆ single
303
mutant (Figure 4A). These results demonstrate that Had1 significantly contributes to C.
304
neoformans virulence.
305
To further examine the virulence defect conferred by the had1∆ mutation, fungal
306
burden in the lungs and brain of mice infected with WT and had1∆ mutant strains was
307
analyzed at day 14 post infection (Figure 4B). The cna1∆ mutant exhibited an
308
undetectable fungal burden in both the lung and brain tissues. By comparison, the
309
had1∆ and crz1∆ single mutant strains showed reduced fungal burden in the lung
310
compared with the WT strain. Moreover, the had1∆ crz1∆ double mutant strains
311
exhibited significantly reduced fungal burden in the lung compared with either single
312
deletion mutant strain. While we recovered WT and mutant strains from the lung, we did
313
not recover any yeast cells from brain tissue 14 days after infection for both WT and
314
mutant strains (data not shown). Collectively, these findings demonstrate that Had1 is
315
required for full virulence and that Had1 and Crz1 function in parallel pathways
316
controlling virulence.
317 318
DISCUSSION
319
Given that calcineurin is a key pathway regulating stress responses, mating, and
320
fungal virulence, identification and characterization of calcineurin targets is crucial for
321
understanding the calcineurin signaling network (ARAMBURU et al. 2004; STEINBACH et al.
322
2006; GOLDMAN et al. 2014). In a previous study, we identified Had1 as a potential
323
target of calcineurin in C. neoformans (PARK et al. 2016). In the present study, although
324
we were unable to confirm that Had1 is a calcineurin target, had1 mutation resulted in 14
325
hypersensitivity to thermal and other stresses and attenuated virulence, suggesting that
326
Had1 is important for stress responses and virulence. Moreover, double deletion mutant
327
analysis suggests that Had1 and Crz1 function in parallel pathways controlling fungal
328
pathogenicity and cell wall stress responses (Figure 5). However, we could not
329
determine if Had1 functions dependently or independently of calcineurin. The
330
preliminary results of our Had1 gel mobility analysis showed that while both thermal and
331
osmolarity stresses seem to affect Had1 mobility, in both cases, this is not altered by
332
FK506, and thus it seems to be independent of calcineurin (Figure S2). Our previous
333
phosphoproteomic screen revealed only one calcineurin-dependent phosphorylation site
334
in Had1 (PARK et al. 2016). Therefore, it is likely that this single site could not be
335
detected by our mobility shift assay (Figure S2), which is more suitable to detect
336
multiple phosphorylation sites on a protein. Thus, the present study does not rule out
337
the possibility that Had1 function is controlled by calcineurin and testing this model will
338
require more sensitive assays and further analysis.
339
Haloacid dehalogenase (HAD)-like hydrolases are large superfamilies of
340
enzymes that exhibit phosphatase, ATPase, phosphonatase and phosphomutase
341
activity, and are conserved in both prokaryotic and eukaryotic organisms (BURROUGHS
342
et al. 2006; KUZNETSOVA et al. 2015). Forty-five genes encoding HAD-like hydrolases
343
are found in the yeast genome and 15 of their protein products have been biochemically
344
characterized for this enzymatic activity (RANDEZ-GIL et al. 1995a; RANDEZ-GIL et al.
345
1995b; NORBECK et al. 1996; KUZNETSOVA et al. 2015). Among these, the beta-
346
phosphoglucomutase family proteins, such as Dog1, Dog2, Gpp1 and Gpp2, are well
347
characterized in yeast. These proteins are required for cellular responses to 15
348
environmental stresses. The expression of Gpp1, Gpp2, and Dog2 is induced by
349
environmental stresses, including osmotic or oxidative stresses, and by glucose
350
starvation (TSUJIMOTO et al. 2000; PAHLMAN et al. 2001). Interestingly, the expression of
351
these genes is controlled by either the HOG (High Osmolality Glycerol) or the Snf1
352
kinase pathways (TSUJIMOTO et al. 2000; PAHLMAN et al. 2001). In addition, loss of both
353
the GPP1 and GPP2 genes results in hyper sensitivity to oxidative stress (PAHLMAN et al.
354
2001). However, gpp1Δ, gpp2Δ, and dog2Δ single mutants did not show an increased
355
susceptibility to osmotic or oxidative stresses. Our study establishes that Had1 is
356
required for proper responses to heat and osmotic stresses, and this finding is in accord
357
with previous studies suggesting this function. Previous microarray analyses shown that
358
the expression of HAD1 (formerly GPP1) was induced in response to osmotic stress
359
and this microarray data was confirmed by our qRT-PCR analysis (Figure S3A). Ko et
360
al. also demonstrated that induction of HAD1 in response to osmotic stress was
361
decreased in hog1 and ssk1 mutants (KO et al. 2009), suggesting that the expression of
362
HAD1 is controlled by the stress-activated Hog1 signaling pathway in C. neoformans
363
(KO et al. 2009). These results suggest that the regulatory mechanism of HAD1 mRNA
364
expression in response to osmotic stress is conserved in S. cerevisiae and C.
365
neoformans. We demonstrate here that upon thermal stress, the expression of HAD1
366
slightly increases (Figure S3B). To examine whether the HAD1 expression is regulated
367
by the calcineurin-Crz1 pathway, we searched our published transcriptome analysis
368
(CHOW et al. 2017) and found that the HAD1 expression is independently regulated of
369
calcineurin or Crz1 under thermal stress.
370
Previously our phosphoproteomic screen revealed that Had1 is a potential 16
371
calcineurin
substrate
in
C.
neoformans.
372
phosphorylated peptide (RRAS376QSGQAGVTLDAFRR) in Had1 was increased more
373
than two-fold in abundance in the calcineurin cna1Δ mutant compared to wild-type cells
374
(PARK et al. 2016). In addition, the Had1 protein contains two predicted calcineurin
375
substrate docking sites PxIxIT motifs (P[^PG][IVLF][^PG]) (PARK et al. 2016). However,
376
as we discussed earlier in this section, whether Had1 is a bona fide calcineurin target
377
remains to be characterized. Previous studies identified 13 phosphorylation sites
378
located in the Had1 C-terminal region and some of these residues were proposed to be
379
phosphorylated by the PKA signaling cascade in C. neoformans (SELVAN et al. 2014;
380
GEDDES et al. 2016). These results indicate that phosphorylation of Had1 is important
381
for Had1 activation and the detailed mechanisms await further characterization to better
382
understand Had1 roles in stress responses and environmental adaptation. Based on the
383
transcriptomic (KO et al. 2009; CHOW et al. 2017) and phosphoproteomic (SELVAN et al.
384
2014; GEDDES et al. 2016; PARK et al. 2016) studies from our lab and other labs, we
385
propose that the expression of HAD1 mRNA is induced by the HOG signaling pathway
386
in response to environmental stresses (KO et al. 2009), and in turn phosphorylation of
387
the Had1 protein may be regulated by the PKA signaling cascade (GEDDES et al. 2016),
388
and possibly by the calcineurin pathway (PARK et al. 2016). Activated Had1 is required
389
for appropriate stress responses; however, the detailed mechanisms controlling Had1
390
function should be further studied to better understand the roles of the (HAD)-like
391
hydrolase superfamily in stress responses, environmental adaptation, and virulence.
392 393
ACKNOWLEDGEMENTS 17
The
single
calcineurin-dependent
394
We thank Anna Averette and Shelly Clancey for technical support and Hiten Madhani
395
and the Fungal Genetics Stock Center for making the gene deletion collection available.
396
This research by HSP was supported by Kyungpook National University Research Fund,
397
2016. The work at Duke University was supported by NIH/NIAID R01 AI50438-10 to JH
398
and MEC, AI112595-03 to JH, and NIH/NCI R01 CA154499 to MEC.
399 400
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598 599 600 601 24
602
Table 1. Cryptococcus neoformans strains used in this study Strains H99 HP235 HP242 HPC23 HPC24 HPC27 HPC28 HPC29 HPC30 HPC25 HPC26 KN99 HP243
Relevant genotype MATalpha, Wild type MATalpha crz1∆::NEO MATalpha cna1∆::NEO MATalpha had1∆::NEO MATalpha had1∆::NEO MATalpha had1∆::NEO HAD1_4xFLAG::HYG MATalpha had1∆::NEO HAD1_4xFLAG::HYG MATalpha crz1∆::NAT had1∆::NEO MATalpha crz1∆::NAT had1∆::NEO MATa had1∆::NAT MATa had1∆::NAT MATa, Wild type MATa cna1∆::NEO
603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 25
References (PERFECT et al. 1980) (PARK et al. 2016) (PARK et al. 2016) This study This study This study This study This study This study This study This study (NIELSEN et al. 2003) (PARK et al. 2016)
626
627 628 629 630
Table 2. Oligonucleotides used in this study Name
Sequence (5’-3’)a, b, c
Purpose
JOHE40706
GTAAAACGACGGCCAG
NAT, NEO, HYG markers (M13F)
JOHE40707
CAGGAAACAGCTATGAC
JOHE42780
GCAGGTGAGCAGTTGTGGCAAG
NAT, NEO, HYG markers (M13R) HAD1 disruption (5F)
JOHE42781
GACATCCAAATCCCACAATGCC TCACTGGCCGTCGTTTTAC TTTGGATAATTACTTGGGGGTCTATG
HAD1 disruption (3R)
JOHE42782 JOHE42783
CATGGTCATAGCTGTTTCCTG AATGTGTTAAATGTAGCGATAGGC
HAD1 disruption with marker (3F)
JOHE42784
GACGGCTCGTTACTGTGTTAGATTG
HAD1 disruption (NF_Nested)
JOHE42785
CAAGATCCCAGTGTCGTGGAG
HAD1 disruption (NR_Nested)
JOHE42786
aatt
GCGGCCGC CTATTGTGAGCTACTGGCCTGGTG
5’ HAD1 with promoter and NotI
JOHE42787
aatt
GCGGCCGC CTCGTCCTGAGACATTTCGCCTTG
3’ HAD1 with NotI
JOHE41391
GGTACTCACAACTGAGCCAGCAG
CRZ1 disruption (5F)
JOHE41392
TCATCGTCGTCGAAGTCGAGGC
CRZ1 disruption (3R)
JOHE41393
TCACTGGCCGTCGTTTTAC GTGGATTATAGGGGTGACTGATAGA
CRZ1 disruption with marker (5R)
JOHE41394
CATGGTCATAGCTGTTTCCTG CGATGGTCATAGGGCGCTGTGAG
CRZ1 disruption with marker (3F)
JOHE41395
GGTTCGTTAGTCGGGTCAACTG
CRZ1 disruption (NF)
JOHE41396
TTAGGGGAGGTTGGGATCGG
CRZ1 disruption (NR)
a
Underlined sequence is homologous to vector or cassette sequence. b Lowercase indicates linker sequence. c Bold sequence denotes restriction site sequence.
.
631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 26
HAD1 disruption with marker (5R)
649
Figure legends
650 651
Figure 1. Phenotypes of the potential calcineurin target mutants exposed to
652
various stresses. (A) Targets in the Venn diagram labelled red were characterized in
653
the previous study (PARK et al. 2016). Targets labelled blue were either deleted in our
654
lab or obtained from a C. neoformans deletion mutant library (LIU et al. 2008) and tested
655
in this study. Cna1 (green) is the calcineurin A catalytic subunit. (B) Functional
656
categories ascribed to the potential calcineurin targets tested in this study. (C) Spot
657
dilution assays with WT (H99), cna1Δ (HP242), and 13 mutants from a systematic gene
658
deletion library were performed under several stress conditions as indicated. Strain
659
cultures were incubated overnight, serially diluted 10-fold, and plated on YPD medium
660
without or with Congo red (CR) and sodium dodecyl sulfate (SDS). Cells were incubated
661
for 2 to 3 days at 30°C, or 37°C as indicated, and all cultures containing stressor
662
compounds (SDS and CR) were incubated at 30°C.
663 664
Figure 2. Deletion of had1 results in hypersensitivity to various stresses. Spot
665
dilution assays with WT (H99), cna1Δ (HP242) and had1Δ mutants (HPC23 and
666
HPC24), and had1∆ + HAD1 complemented strains (HPC27 and HPC28) were
667
performed under several stress conditions as indicated. (A) Cell cultures in liquid YPD
668
media were grown overnight at 30°C and serially diluted 10-fold. Equal aliquots were
669
spotted on solid YPD medium supplemented or not with the stressor compounds as
670
indicated in panel B. (A) For thermotolerance, cells were incubated on solid medium for
671
2 days at 30°C, 37°C, or 38°C as indicated. (B) For cell wall, ER, or osmolarity stress,
672
the solid YPD culture media was supplemented with Congo red (CR), calcofluor white
673
(CFW), dithiothreitol (DTT), sodium dodecyl sulfate (SDS), or KCl at the indicated
674
concentrations and the cultures were incubated at 30°C. Results shown are
675
representative of two independent experimental replicates.
676 677
Figure 3. Had1 and Crz1 orchestrate for stress response. Stress tolerances of WT
678
(H99), the single cna1Δ (HP242), crz1Δ (HP235), and had1Δ (HPC24) mutants, and the 27
679
had1∆ crz1Δ + HAD1 complemented strains (HPC29 and HPC30). Cells were grown
680
overnight at 30°C, serially diluted 10-fold, and plated on YPD medium. Plates were
681
incubated for 2 days at 30°C, 37°C, 38°C, or 39°C as indicated.
682 683
Figure 4. Had1 is required for virulence. (A) Virulence was tested for the WT (H99),
684
cna1Δ (HP242), crz1Δ (HP235), had1Δ (HPC24), had1∆ + HAD1 (HPC28) and had1∆
685
crz1Δ (HPC29 and HPC30) deletion mutant and complemented strains. Yeasts cells
686
were grown overnight in YPD liquid medium at 30°C, collected, washed with PBS, and 5
687
× 105 cells were inoculated into female BALB/c mice via intranasal instillation. Animal
688
survival was monitored for 60 days post infection. (B) Mice (n = 3~4) were inoculated
689
intranasally with 5 × 105 CFU of the indicated strains. Lungs were isolated at 14 days
690
post-infection. Results are expressed as mean colony forming unit (CFU) per lung ±
691
standard error of the mean. ND: not detected.
692 693
Figure 5. The calcineurin-Crz1 pathway and Had1 control growth at high
694
temperature, stress response, and virulence via parallel pathways. The calcineurin
695
signaling network acts via Crz1 to control Cryptococcus thermotolerance, cell wall
696
integrity, and virulence. Had1 also contributes to these functions in a pathway parallel to
697
Crz1 and possibly regulated by calcineurin. Dotted line arrow and question mark denote
698
a pathway step not as yet well defined as it remains to be determined whether Had1 is
699
an authentic calcineurin target.
700 701 702 703 704 705 706 707 708 28
709
Supplemental Figure List
710 711
Figure S1. Haloacid dehalogenase proteins in C. neoformans and S. cerevisiae. (A)
712
Domain architecture of beta-phosphoglucomutase family proteins in C. neoformans and
713
S. cerevisiae. (B) A phylogenetic tree of HAD proteins in C. neoformans and S.
714
cerevisiae was generated by MEGA 5 software (http://www.megasoftware.net/) using
715
the alignment data from ClustalW2. The tree results were submitted to iTOL
716
(http://itol.embl.de/) to generate the figure.
717 718
Figure S2. Had1-FLAG gel migration mobility under stress conditions. (A) Had1
719
mobility is altered under the thermal stress conditions. Cultures of the had1Δ HAD1-
720
4xFLAG complemented strain (HPC27) in YPD medium were grown to log phase at
721
25°C or shifted from 25°C to 37°C for 1 hour in the presence or absence of 2 μg/mL
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FK506. (B) Cultures of HPC27 were grown to log phase at 25°C as indicated in panel A,
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or shifted to YPD medium containing 1 M NaCl with or without 2 μg/mL FK506 and
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incubation was continued at 25°C for 1 hour. Whole cell protein extracts were prepared
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and analyzed by western blot as indicated in the materials and methods section.
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Figure S3. Expression of HAD1. Expression of HAD1 in WT (H99) strain under
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osmotic (A) or thermal (B) stresses. RNA was isolated from WT strain (H99) and the
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expression of HAD1 was assessed by real-time PCR. Error bars depict the standard
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errors from the mean of three independent experiments.
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