Functional characterization of PMT2, encoding a protein-O-mannosyltransferase, in the human pathogen Cryptococcus neoformans.

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5 June 2014 Fungal Genetics and Biology xxx (2014) xxx–xxx 1

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Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi 6 7

Functional characterization of PMT2, encoding a protein-O-mannosyltransferase, in the human pathogen Cryptococcus neoformans

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Medical Mycology Research Center, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260-8673, Japan Department of Applied Material and Life Science, College of Engineering, Kanto Gakuin University, Mutsuura-higashi 1-50-1, Kanazawa-ku, Yokohama 236-8501, Japan c Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin–Madison, WI 53706, USA d Department of Medical Microbiology & Immunology, School of Medicine and Public Health, University of Wisconsin–Madison, WI 53706, USA b

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Kiminori Shimizu a, Yumi Imanishi a,b,⇑, Akio Toh-e a, Jun Uno a, Hiroji Chibana a, Christina M. Hull c,d, Susumu Kawamoto a

i n f o

Article history: Received 18 December 2013 Accepted 18 May 2014 Available online xxxx

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Keywords: Agrobacterium tumefaciens-mediated transformation Cryptococcus gattii Cryptococcus neoformans Diazonium blue B Glycosylation

a b s t r a c t Diazobenzoic acid B (DBB), also known as diazonium blue B or fast blue B, can be used to distinguish basidiomycetous yeasts from ascomycetes. This chemical has long been used for the taxonomic study of yeast species at the phylum level, but the mechanism underlying the DBB staining remains unknown. To identify molecular targets of DBB staining, we isolated Agrobacterium tumefaciens-mediated insertional mutants of Cryptococcus neoformans, a basidiomycetous pathogenic yeast, which were negative to DBB staining. In one of these mutants, we found that the PMT2 gene, encoding a protein-O-mannosyltransferase, was interrupted by a T-DNA insertion. A complete gene knockout of the PMT2 gene revealed that the gene was responsible for DBB staining in C. neoformans, suggesting that one of the targets of Pmt2-mediated glycosylation is responsible for interacting with DBB. We also determined that Cryptococcus gattii, a close relative of C. neoformans, was not stained by DBB when the PMT2 gene was deleted. Our finding suggests that the protein-O-mannosylation by the PMT2 gene product is required for DBB staining in Cryptococcus species in general. We also showed that glycosylation in Cryptococcus by Pmt2 plays important roles in controlling cell size, resistance to high temperature and osmolarity, capsule formation, sexual reproduction, and virulence. Ó 2014 Published by Elsevier Inc.

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1. Introduction

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The most well-known phyla in the fungal kingdom are Ascomycota and Basidiomycota, which are also referred as the subkingdom Dikarya (James et al., 2006). Both groups contain two major morpho-types; unicellular yeasts and multicellular filamentous fungi. Unicellular yeasts in general have few morphological features to distinguish ascomycetes from basidiomycetes, so that a number of different biochemical methods have been developed to distinguish between them. Traditionally, a diazonium blue B (DBB) staining test has been used to determine yeast taxonomy (Kurtzman et al., 2011a,b). DBB staining has been verified and developed by using various basidiomycetous species, and all 1414 proposed yeast species have

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⇑ Corresponding author at: Department of Applied Material and Life Science, College of Engineering, Kanto Gakuin University, Mutsuura-higashi 1-50-1, Kanazawa-ku, Yokohama 236-8501, Japan. E-mail address: [email protected] (Y. Imanishi).

been evaluated for DBB staining. In every case, staining occurred only in basidiomycete fungi. (Hagler and Ahearn, 1981; Kurtzman et al., 2011a; Oblack and Rhodes, 1983; van der Walt and HopsuHavu, 1976). Other characteristics, such as activity of extracellular urease or DNase, cell wall composition (b-glucanase sensitivity), GC content (30–60% for ascomycetes, 50–70% for basidiomycetes) have been used to try to classify ascomycetous and basidiomycetous yeasts, but nothing other than DBB staining has been shown to fully separate these two phyla (Kurtzman et al., 2011a). Given the specificity of DBB staining for basidiomycetes, we hypothesized that DBB staining could be used as a tool to determine molecular differences between basidiomycetous and ascomycetous yeasts. Using the basidiomycete yeast Cryptococcus neoformans, we carried out a screen for mutants that could not be stained with DBB. C. neoformans is a common pathogen that infects humans and other animals (Casadevall and Perfect, 1998). Because of its clinical relevance, molecular genetic techniques such as transformation and gene knockouts have been developed (Idnurm and Williamson, 2011), and genome sequence information is publically

http://dx.doi.org/10.1016/j.fgb.2014.05.007 1087-1845/Ó 2014 Published by Elsevier Inc.

Please cite this article in press as: Shimizu, K., et al. Functional characterization of PMT2, encoding a protein-O-mannosyltransferase, in the human pathogen Cryptococcus neoformans. Fungal Genet. Biol. (2014), http://dx.doi.org/10.1016/j.fgb.2014.05.007

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available (Loftus et al., 2005). In addition, Agrobacterium tumefaciens-mediated transformation (AtMT) has been used for classical genetic studies of C. neoformans (Walton et al., 2005). A screen of a library of T-DNA-insertion mutants in C. neoformans led us to identify a non-staining mutant that harbored an insertion in the PMT2 gene. Subsequent deletion analysis and characterization confirmed that PMT2, which encodes a putative dolichyl-phosphate-mannose-protein mannosyltransferase, is required for DBB staining. To confirm the broader applicability of DBB stainingdependence on PMT2 activity, we also deleted the CgPMT2 gene of another Cryptococcus yeast, C. gattii, and showed a defect in DBB staining. This role for PMT2 was surprising because the PMT2 homolog in ascomycetes does not play a role in DBB staining, indicating that Pmt2 (or one of its substrates) confers DBB staining specificity in basidiomycetes. The discovery of this novel role for PMT2 and/or its targets lays the groundwork for determining the role of protein glycosylation in establishing fundamental differences among diverse fungi. We also present here that the Pmt2 has important roles for capsule formation, cell wall synthesis, mating response, and virulence.

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2. Materials and methods

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2.1. Fungal strains and growth conditions

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Fungal strains used in this study are listed in Table 1. The strains were grown and maintained on solid YPD (1% yeast extract, 2% polypeptone, 2% glucose, 2% agar) or YNB (0.17% yeast nitrogen base w/o amino acid and ammonium sulfate (Difco), 0.5% ammonium sulfate, 2% glucose, 2% agar) at 25 °C unless stated otherwise. For induction of capsule, diluted Sabouraud medium buffered with 50 mM MOPS (pH 7.3) was used (Zaragoza and Casadevall, 2004).

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2.2. A. tumefaciens-mediated transformation and gene identification

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The C. neoformans wild type strain JEC21 was transformed by A. tumefaciens-mediated transformation (AtMT) as described by

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Walton et al. (2005). A. tumefaciens strain EHA105 carrying pPZPHYG2 (Walton et al., 2005) was used to introduce a hygromycin B resistant cassette into JEC21. The location of the T-DNA insertion was identified by TAIL PCR (Liu and Huang, 1998) followed by sequence analysis of the PCR fragments, and the obtained sequences were compared to the C. neoformans genome database using the BLASTn algorithm (Altschul et al., 1990).

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2.3. Diazobenzoic acid B staining

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A C. neoformans var. neoformans mutant library created by an Agrobacterium-mediated T-DNA insertional mutagenesis was grown on a YPD plate with 200 lg/ml hygromycin B at 30 °C for 2–3 days. The library was diluted so that approximately 100 colonies were grown on each plate. Five ml of diazobenzoic acid solution (1 mg/ml in 0.25 M Tris–HCl, pH 7.5) were overlaid onto the plate, incubated at room temperature for 1 min, and unstained colonies were transferred to a fresh YPD plate. C. neoformans var. neoformans or C. gattii strains were grown in a 3 ml liquid YPD liquid medium at 30 °C with shaking at 200 rpm overnight. The cells were collected by centrifugation and washed twice with 0.25 M Tris–HCl (pH 7.5). The cells were then resuspended in a 100 ll 0.25 M Tris–HCl (pH 7.5) and mixed with a 100 ll of diazobenzoic acid solution (1 mg/ml in 0.25 M Tris–HCl, pH 7.5), vortexed and kept on ice for 1 min. The cell suspension was then centrifuged to pellet the cells and the supernatant was removed, washed twice with 0.25 M Tris–HCl (pH 7.5), and resuspended in a 50 ll 0.25 M Tris–HCl (pH 7.5). The resulting cell suspension was spotted onto a filter paper, and photographed.

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2.4. Fungal strain and plasmid construction

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The pmt2D and Cgpmt2D strains were constructed by replacing the PMT2 open reading frame (ORF) with either a nourseothricin (NAT) or neomycin (NEO) resistance gene cassette. Knockout cassettes were made by overlap PCR using the oligos listed in Table S1 by the method described by Davidson et al. (2000a). The

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Table 1 Strains used in this study. Strain

Genotype

Source

Cryptococcus neoformans JEC20 JEC21 TAD1 CKIS30 TLHM15 TLHM20 AtMT-DBB14 DDBB14-4 DBB14-R1-29 DBB14-R2-1 DBB14-R2-2 DBB14R-1 DBB14R-PMT1 DBB14R-PMT4 DBB14NEOa DBB14NATa JEC20NEOa JEC21NATa SW6 SW12 PMT1-R1-2 PMT4-R1-1

Wild type, serotype D, MATa Wild type, serotype D, MATa ura5D, MATa ura5D, MATa cku70D::NEO, ura5D, MATa cnlig4D::NAT, MATa pmt2::HYG2, MATa pmt2D::NEO, cnlig4D::NAT, MATa pmt2D::NEO, ura5D, MATa pmt2D::NEO, MATa pmt2D::NEO, MATa pmt2D::NEO + PMT2::URA5, ura5D, MATa pmt2D::NEO + PMT1::URA5, ura5D, MATa pmt2D::NEO + PMT4::URA5, ura5D, MATa pmt2D::NEO, MATa pmt2D::NAT, MATa NEO, MATa NAT, MATa pmt1D::URA5, MATa pmt4D::URA5, MATa pmt1D::URA5, ura5D, MATa pmt4D::URA5, ura5D, MATa

Kwon-Chung et al. (1992) Kwon-Chung et al. (1992) Drivinya et al. (2004) This study, progeny of TAD1 JEC20 Li et al. (2010) Shimizu et al. (2010) This study, T-DNA insertional mutant This study, pmt2D::NEO in TLHM20 This study, progeny of DDBB14-4 CKIS30 This study, progeny of DDBB14-4 CKIS30 This study, progeny of DDBB14-4 CKIS30 This study, DBB14-R1-29 transformed with pKIS421 This study, DBB14-R1-29 transformed with pKIS425 This study, DBB14-R1-29 transformed with pKIS423 This study, pmt2D::NEO in JEC20 This study, pmt2D::NAT in JEC21 This study, NEO in JEC20 This study, NAT in JEC21 Willger et al. (2009) Willger et al. (2009) This study, progeny of SW6 and CKIS30 This study, progeny of SW12 and CKIS30

C. gattii C762 C762-PMT2 C762-PMT2FOA C762-PMT2R-B C762-PMT2R-D

Wild type, MATa Cgpmt2D::NAT, MATa Cgpmt2D::NAT, ura5, MATa Cgpmt2D::NAT + CgPMT2::URA5, ura5, MATa Cgpmt2D::NAT + PMT2::URA5, ura5, MATa

=PNG18, Campbell et al. (2005) This study, DCgpmt2::NAT in C762 This study, uracil auxotrophic mutant of C762-PMT2 This study, C762-PMT2FOA transformed with pKIS428 This study, C762-PMT2FOA transformed with pKIS421


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amplified pmt2D::NEO or Cgpmt2D::NAT deletion cassettes were introduced into the C. neoformans TLHM20 (Shimizu et al., 2010) or C. gattii C762 (Campbell et al., 2005) using biolistic transformation (Toffaletti et al., 1993). Positive clones were identified by PCR and confirmed by Southern blot analysis. Strains with desired genotypes were constructed by sexual crosses. For isolation of basidiospores from crosses, strains were mix-inoculated on hayinfusion (HI) agar medium (Tanaka et al., 1999) and incubated at 24 °C in the dark until basidiospores were produced. Single basidiospores were isolated individually using a micromanipulator (SINGER MSM system, Singer instruments, UK) and transferred to fresh YPD plates. For complementation experiments, the C. neoformans PMT1, PMT2, and PMT4, and C. gattii CgPMT2 genes were PCR amplified with primers listed in Table S1. The PMT2 and CgPMT2 fragment was digested with BamHI and EcoRV, PMT1 fragment was digested by BamHI, and the PMT4 fragment was digested with BamHI and ScaI, respectively, and cloned into BamHI/HpaI site of pCH304 (C.M. Hull, unpublished), where the cloned gene fragments can be overexpressed under the control of GPD (glyceraldehyde3-phosphate dehydrogenase) promoter. The plasmid pCH304 harbors a URA5 marker gene and CnTel sequence allowing autonomous replication in C. neoformans. Plasmids carrying PMT1, PMT2, PMT4 or CgPMT2 were designated as pKIS425, pKIS421, pKIS423, and pKIS428, respectively. These plasmids were introduced into cryptococcal cells as described previously (Davidson et al., 2000b). C762-PMT2FOA was a spontaneous uracil auxotrophic mutant of C762-PMT2 selected on a YNB plate containing 5-fluoroorotic acid (Drivinya et al., 2004).

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2.5. Real-time PCR

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Total RNA extraction and cDNA synthesis were performed using Trizol reagent (Life Technologies, Carlsbad, CA) or ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan) following the provided instructions. The cDNA was used as a template in a real-time PCR (RT-PCR) using THUNDERBIRD qPCR MixSYBR (Toyobo) according to the manufacturer’s recommendations. The Applied Biosystems 7300 real-time PCR detection system (Life Technologies) was used to detect and quantify the PCR products. Each set of PCR included a triplicate of each target gene. The data were normalized to ACT1 (encoding actin) expression levels in each set of PCRs.

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2.6. Sexual reproduction and fusion assay

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Wild type and mutant strains on YPD plates were resuspended in a 100 ll sterile water and adjusted to equal concentrations. An equal volume of each cell suspension was mixed and spotted onto solid HI plates. The plates were incubated in the dark at 24 °C for 3 days, and development was assessed using light microscopy. Cell Q5 fusion efficiency was measured as previously described by Bahn et al. (2004) with some modifications. Cells were adjusted to 107 cells/ml, and an equal volume of each strains were mixed. Five ml of the cell mixture was spotted onto HI medium, and the plate was incubated in the dark at 24 °C for 24 h. The spots were scraped and resuspended in 1 ml of distilled H2O, and the cell suspension was serially diluted and plated onto YPD medium containing nourseothricin and G418. The number of colonies on each plate was determined after 2 days of incubation at 30 °C.

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2.7. Virulence assay

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The animal study in this report was approved by the institutional animal care and use committee, Chiba University (DOU25296). The silkworm infection experiment was performed as

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described previously (Matsumoto et al., 2012). Fifth-instar larvae Q6 of the silkworm, Bombyx mori (HuYo TukubaNe), were purchased from Ehime Sanshu, Ehime, Japan, and maintained in disposable plastic containers at 25 °C. The larvae were fed an artificial diet (Silkmate; Katakura Industries, Tokyo, Japan) supplemented with 1% glucose for 1 day before performing the infection experiments. Yeast suspensions (1.5 107 CFU/0.05 ml saline) were injected into the haemolymph of the larvae through the dorsal surface using a 27-gauge needle. The injected larvae were maintained without feed at 25 °C and monitored twice daily. Ten larvae were used for infection of each strain to assess the virulence of C. neoformans. Three larvae were infected as described above for each strain, and haemolymph was collected from the larvae through a cut on the first proleg at 3 days after injection, to determine the capsule thickness and cell size of C. neoformans in the silkworm haemolymph. The haemolymph was mixed with the India ink and 30 cells were randomly chosen from each, and the cell diameters and capsule sizes were measured under the microscope.

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3. Results

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3.1. Identification of DBB negative strains and their insertion sites

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Out of ca. 10,000 random T-DNA insertional mutants, we identified two colonies (named DBB14 and DBB48) that were not stained by DBB. By a sexual cross with a wild type strain of the opposite mating type (JEC20), we obtained progeny of these mutants. The hygromycin B resistant progeny out of DBB14 and JEC20 crosses were all DBB negative, but those produced by DBB48 and JEC20 mating were not. We concluded that the hygromycin B resistant gene cassette within the T-DNA co-segregated with the DBB staining negative phenotype in DBB14, but not in DBB48. Thus, we further analyzed the location of T-DNA insertion in DBB14. By TAIL-PCR, we successfully amplified the T-DNA and the adjacent genomic region of DBB14. We sequenced the resulting PCR fragment and determined that the T-DNA was inserted into the locus tagged as CNJ01930 (Fig. 1A). The junction between the C. neoformans genome and the T-DNA was determined as shown in Fig. 1B. The predicted amino acid sequence of CNJ01930 contains a highly conserved motif of the dolichyl-phosphate-mannose-protein mannosyltransferase and was the most similar to the PMT2 of Saccharomyces cerevisiae. We designated the CNJ01930 as PMT2 and further analyzed its function.

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3.2. PMT2, but not PMT1, or PMT4, is required for DBB staining in C. neoformans

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We constructed targeted deletion strains of the entire PMT2 open reading frame. Multiple independent knockout strains (6 in total) were evaluated by PCR and Southern blot and found to harbor no copies of PMT2 (Fig. 1C and data not shown). We were surprised to find that pmt2D strains were viable because Willger et al. (2009) disrupted PMT2 with a marker gene after amino acid position 468 and found the resulting strains to be inviable. We further confirmed the pmt2D genotype in our deletion strains by backcrossing independent knockouts and rescreening segregants for deletion of the PMT2 open reading frame. In all cases, the resulting pmt2D strains were viable. In addition all of the pmt2D segregants tested were negative for DBB staining (Fig. 1C and data not shown). Positive staining was restored by re-introducing a wild type copy of the PMT2 gene into the disruptants, indicating that PMT2 is required for DBB staining in C. neoformans (Fig. 2A). Fungal species are known to have three classes of the dolichylphosphate-mannose-protein mannosyltransferases, called Pmt1,

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Fig. 1. Identification of the gene responsible for diazobenzoic acid B (DBB) staining in Cryptococcus neoformans. (A) Schematic diagram of T-DNA insertion in CNJ01930 (PMT2). The coding sequence (closed arrows) was interrupted by 6 introns. The T-DNA insertion point was at the border of the 5th intron and the 6th exon. The right border (RB) and the left border (LB) direct repeats are indicated, but only RB was inserted to the genome and the LB sequence was truncated. The direction of the hygromycin B resistant gene (hyg2, open arrow) is also indicated. (B) Sequences of the T-DNA junction sites. Sequences of both junctions between T-DNA (upper case letters) and the C. neoformans genome (lower case letters) are indicated. Positions of the 5th intron and the 6th intron (arrows), micro-homology region (enclosed letters) and the missing nucleotides (10 bases, gray) are also indicated. (C) The PMT2 gene (hatched box) was replaced with the neomycin resistant cassette (NEO). The replacement and reconstitution of the PMT2 gene was confirmed by the Southern blot analysis. Total genomic DNA was digested with PstI and the upstream portion of the PMT2 coding sequence (shaded box) was used as a probe and the presence of each fragment (6.1 kb for the wild type, 2.7 kb for the pmt2D::NEO, and 2.7 kb and 9.1 kb for proper integration of the plasmid pKIS421 into the target locus in the pmt2D::NEO + PMT2 strain.

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Pmt2, and Pmt4 (Girrbach and Strahl, 2003). These proteins are responsible for O-glycosylation and have different substrate specificities. In C. neoformans, genes encoding Pmt1 and Pmt4 homologues have been identified and analyzed for their involvement in morphology, sexual reproduction, and virulence by Willger et al. (2009), but their roles in DBB staining were unknown. DBB was applied to the cells of pmt1D or pmt4D mutant strains (provided from S.D. Willger), and we found that neither PMT1 nor PMT4 are required for DBB staining in C. neoformans (Fig. 2B). Because the PMT gene products share target proteins for Oglycosylation in other fungi, we tested whether overexpression of PMT1 or PMT4 can complement the DBB staining defect in a pmt2D strain. PMT1 and PMT4 were each introduced into the PMT2 disruptant. Neither PMT1 nor PMT4 were able to restore the DBB staining deficiency (Fig. 2C). Thus, the PMT proteins, even though structurally similar and responsible for O-glycosylation as a group, function differently for DBB staining in C. neoformans. These data

indicate specificity of DBB staining for the Pmt2 family member and its corresponding substrates.

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3.3. pmt1D pmt2D and pmt2D pmt4D mutants are inviable

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The Pmt proteins are predicted to function in heterodimeric complexes with one another. Willger et al. (2009), indicated that PMT1 and PMT4 were not essential for viability, but disruption of both together (pmt1 pmt4) lead to synthetic lethality. To investigate whether combinations of deletions in PMT2 and PMT1 or PMT4 exhibited synthetic lethality, we crossed a pmt2D strain with pmt1D and pmt4D strains, respectively. We isolated individual basidiospores by microdissection, and tested the genotypes of the resulting progeny by growth on selective media (PMT2 was replaced by NEO, and PMT1 and PMT4 were replaced by URA5). The genotypes were further confirmed by PCR (Fig. 3A and B). Neither pmt1D pmt2D nor pmt2D pmt4D strains were identified in

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WT pmt2::T-DNA pmt2 Δ ::NEO pmt2 Δ ::NEO+PMT2

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PMT1 expression

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pmt2 Δ ::NEO

pmt2 Δ +PMT2

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pmt2 Δ +PMT4

pmt4 Δ ::URA5

pmt2 Δ +vector

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PMT4 expression

pmt2 Δ +PMT1 pmt2 Δ +PMT2

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pmt2 Δ +PMT4 pmt2 Δ +vector

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Fig. 2. Diazobenzoic acid B (DBB) staining in PMT mutants of Cryptococcus neoformans. (A) DBB staining assay for the wild type (WT), T-DNA insertion mutant (pmt2::T-DNA), target disruptant (pmt2D::NEO) and reconstituted strain (pmt2D::NEO + PMT2). (B) DBB staining assay for the wild type (WT), T-DNA insertion mutant (pmt2::T-DNA), PMT2 targeted deletion (pmt2D::NEO), PMT1 targeted deletion (pmt1D::URA5) and PMT4 targeted deletion (pmt4D::URA5). (C) DBB staining assay for the PMT2 targeted disruptant overexpressing PMT1 (pmt2D + PMT1), pmt2D strain overexpressing PMT2 (pmt2D + PMT2), pmt2D strain overexpressing PMT4 (pmt2D + PMT4) or pmt2D strain having an empty plasmid (pmt2D + vector). (D) Quantitative RT-PCR analysis of the PMT1, PMT2, and PMT4 genes. RT-PCR was performed triplicate for each reaction. The expression value of each gene was determined by the threshold cycle method, and the value of ACT1 (encoding actin) was used to normalize the expression level of each gene.

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progeny from these crosses (Fig. 3C and D). The probabilities (P) of the v2 values calculated on the null hypothesis (the segregation ratios were 1:1:1:1), were less than 0.05, indicating that the frequency of appearance for progenies of respective genotypes rejects the hypothesis. These results suggest that the mutations in both PMT1 and PMT2, or in both PMT2 and PMT4 are synthetically lethal. These findings are consistent with the idea that functional O-glycosylation requires at least two members from each class of mannosyltransferases to form a functional complex.

3.4. The PMT2 gene is essential for DBB staining in C. gattii

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C. gattii is a close relative of C. neoformans and is also a causal agent of cryptococcosis. To investigate if the PMT2 gene in C. gattii (CgPMT2) is also responsible for DBB staining of the fungus, we constructed a pmt2D strain by replacing the PMT2 ORF with NAT (Cgpmt2D; Fig. 4A). As was seen in C. neoformans, the Cgpmt2D strain was not able to be stained by DBB (Fig. 4B), and staining was restored by introducing a wild type copy of CgPMT2

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Fig. 3. (A) 14 Individual progenies were subjected to colony PCR to determine the PMT1 (upper panel) and PMT2 alleles (lower panel). Position of the respective wild-type and knockout alleles are indicated at right. Parental strains PMT1-R1-2 (pmt1D) and DBB14-R1-29 (pmt2D) were used as controls. (B) Summary of the genotypes identified by PCR (panel A and data not shown) with respect to PMT alleles. (C) 20 Individual progeny were subjected to colony PCR to determine the PMT2 (upper panel) and PMT4 alleles (lower panel). Position of the respective wild-type and knockout alleles are indicated at right. Parental strains DBB14-R1-29 (pmt2D) and PMT4-R1-1 (pmt4D) were used as controls. (D) Summary of the genotypes identified by PCR (panel C and data not shown) with respect to PMT alleles.


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Fig. 4. Gene disruption of CgPMT2 gene in Cryptococcus gattii. (A) Schematic structure of C. gattii protein-O-mannosyltransferase gene (CgPMT2) alleles of the wild type (WT) and disruptant (Cgpmt2D). The CgPMT2 gene of WT was replaced by a nourseothricin resistant gene cassette (NAT) in the deletion strain. (B) Gene knockout was confirmed by PCR with primers outside the deletion construct for 8 randomly chosen transformants (lower panel). Positions of the respective wild-type (4.9 kb) and knockout (4.1 kb) alleles are indicated at right. Those transformants were stained with DBB, and those having 4.1 kb PCR fragments remained unstained (lanes 2, 3 and 7). (C) The C. neoformans PMT2 gene or the C. gattii CgPMT2 gene was introduced into the Cgpmt2D strain (C762-PMT2FOA), and stained by DBB. The C. neoformans PMT2 gene as well as the CgPMT2 gene complemented the deficiency for DBB staining of the Cgpmt2D strain.

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(Fig. 4C). Staining of the Cgpmt2D strain was also restored by introducing the PMT2 genes of C. neoformans (Fig. 4C), indicating that the functions of those genes are highly conserved between the two species.

3.5. The PMT2 gene is required for normal morphology, capsule formation, and sexual reproduction

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Because the C. neoformans PMT1 and PMT4 genes have been shown to function in normal cell growth, melanin production, sexual reproduction, and growth under stress conditions, we tested whether the PMT2 gene also contributes to these processes. When grown in the diluted Sabouraud medium buffered with 50 mM MOPS (pH 7.3), the pmt2D strain was substantially larger in yeast cell size (4.5 ± 0.73 lm diameter for the wild type and 11.2 ± 1.54 lm for the pmt2D, Fig. 5A and B) and appeared to produce thicker capsules compared to the wild type strain, although the difference was not statistically significant (Fig. 5C). Total cell size (yeast and capsule together) was also significantly larger in the pmt2D strain compared to the wild type (Fig. 5D). Wild type phenotypes were restored by introducing a wild type copy of the PMT2 gene (Fig. 5A–D). A difference in melanin production was not discernable between pmt2D and the wild type strains (data not shown). Furthermore, the pmt2D strain failed to grow at 37 °C (Fig. 6), and shifting the plate after three days from 37 °C to 30 °C for continued incubation did not restore growth (data not shown). The pmt2D strain showed slower growth on YPD plates containing high concentrations (0.7 M) of NaCl or KCl (Fig. 6), but small colonies were detected when incubated longer (7 days, data not shown). High concentrations of sorbitol (1.2 M) also slowed the growth of the pmt2D strain (Fig. 6), but the effect was not as severe as seen by NaCl or KCl. The pmt2D strain failed to grow in the presence of 0.01% SDS (Fig. 6). In unilateral crosses with the wild-type strains (JEC20 or JEC21) of opposite mating types, both MATa and MATa pmt2D mutants (DBB14-R2-2 and DBB14-R2-1) showed filamentation defects on HI medium relative to the wild type crosses (Fig. 7A). A bilateral cross of two pmt2D strains of opposite mating types (DBB14-R21 DBB14-R2-2, see Table 1) revealed a complete loss of reproductive structures (Fig. 7), suggesting that sexual reproduction in C. neoformans requires PMT2. To test whether the defects observed with pmt2D mutants result from defects in cell fusion, efficiency of cell fusion was determined with strains with different drug resistant marker genes (NAT and NEO). pmt2D mutants were less efficient in cell fusion than the wild-type strain, but still bore fusants (approximately 5% of the wild-type mating, see Fig. 7B), indicating that the deletion of PMT2 results in a mating defect. Finally, the bilateral cross (pmt2D pmt2D) yielded no fusion products (Fig. 7B), suggesting that the PMT2 is essential for the mating process, which initiates sexual development.

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3.6. The pmt2D mutant is hypervirulent in a silkworm model of infection

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To assess the virulence of the pmt2D mutant strain, we used a silkworm model of infection. Because the pmt2D strain cannot survive at 37 °C, but grows as well as the wild type strain at 25 °C, we surmised that silkworms would provide host infection information that could not be garnered from a mouse model. We injected silkworms with wild type (WT), pmt2D, and pmt2D + PMT2 strains and assessed worm survival. The WT-injected silkworms survived until the end of the experiment (10 days), but the pmt2D-injected silkworms were killed all within 8 days (Fig. 8A). The survival phenotype was recovered by the reintroduction of the wild type copy of PMT2 (Fig. 8A). Cells recovered from the haemolymph of the silkworms at the endpoint of the experiment were observed under the microscope. The cell diameter and total cell size (including capsules) were significantly different between WT and pmt2D and between pmt2D and pmt2D + PMT2 (Fig. 8B and D). The capsule thickness of the pmt2D strain was significantly greater than those of the WT or pmt2D + PMT2 (Fig. 8C; P < 0.0001).

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Fig. 5. Phenotypes of the pmt2D strains of Cryptococcus neoformans. (A) India ink staining of the wild type (WT), PMT2 deletion strain (pmt2D) and PMT2 reconstituted strain (pmt2 + PMT2). Bar = 10 lm. (B) Yeast diameter, (C) capsule thickness and (D) total cell size (yeast and capsule together) of the wild type (WT), PMT2 deletion strain (pmt2D) and PMT2 reconstituted strain (pmt2D + PMT2). Indicate significant difference by the unpaired t-test (P < 0.001).

Fig. 6. Growth of the pmt2D in the various conditions. The WT, pmt2D and pmt2D + PMT2 strains were adjusted to 106 CFU/ml and diluted by 10-fold serial dilutions down to 103 CFU/ml. Ten ll of each dilution was spotted onto YPD plates and the plates were incubated at the indicated temperature for 2–3 days. The serial dilutions were also spotted on to YPD plates supplemented with 0.7 M KCl, 0.7 M NaCl, 1.2 M Sorbitol or 0.01% SDS, and the plates were incubated at 30 °C for 3 days.

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4. Discussion

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DBB has been used for yeast taxonomy for over 50 years: basidiomycetous yeasts are stained by this chemical, and ascomycetous ones are not. Because of its simplicity and reliability, all of the described yeast species have been tested for their reactions to this compound and classified accordingly (Kurtzman et al., 2011a). Despite its wide usage, the molecular and biochemical basis for DBB staining has not been elucidated, even though the reaction might reflect a fundamental difference between basidio- and ascomycete biology. To uncover the mechanism for DBB staining, we isolated a mutant strain negative for DBB staining from a T-DNA insertional mutant library created by AtMT. This technique has been used for isolation of mutants in C. neoformans for a variety of characteristics including iron acquisition (Hu et al., 2013), filamentation (Fu et al., 2011) and virulence (Idnurm et al., 2004).

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We identified a gene encoding a dolichyl-phosphate-mannoseprotein mannosyltransferase. This enzyme is involved in protein mannosylation, and fungi are considered to possess three classes of the protein-O-mannosyltransferases, Pmt1, Pmt2, and Pmt4 (Girrbach and Strahl, 2003). Previously, Willger et al. (2009) reported that the PMT2 gene was essential and could not be disrupted in C. neoformans. Despite this prior finding, we have shown in this study that the complete deletion of the gene does not result in lethality and results in a DBB staining negative phenotype. In Ustilago maydis, a basidiomycetous corn smut fungus, the PMT2 gene was reported to be essential (Fernández-Álvarez et al., 2009). In a fission yeast Schizosaccharomyces pombe, Willer et al. (2005) reported that the oma2+ (PMT2 homolog) is essential, but Tanaka et al. (2005) showed that the ogm2+ (PMT2 homolog) can be deleted with no apparent phenotype. In the filamentous fungus Aspergillus nidulans, three protein-O-mannosyltransferase genes, pmtA, pmtB and pmtC, coding for PMT1, PMT2 and PMT4,


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Fig. 7. Mating responses of the pmt2D strain. (A) Edges of the colonies (right panels) and colonies themselves (left panels) were photographed after 72 h incubation on a hey cube infusion (HI) agar plate in the dark. Wild type (WT) MATa, JEC20, MATa, JEC21, pmt2D MATa, DBB14-R2-2, MATa, DBB14-R2-1, were used for the crosses, respectively. (B) Cell fusion efficiency was determined by counting the number of appeared colonies after 24 h incubation on a HI plate in the dark. WT MATa, JEC20NEOa, WT MATa, JEC21NATa, pmt2D MATa, DBB14NEOa, pmt2D MATa, DBB14NATa, were used for the assay. Indicate the significant difference by the unpaired t test (P < 0.001), and Indicate the not-significant difference by the unpaired t test (P > 0.05).

Fig. 8. Virulence of the Cryptococcus neoformans pmt2D strain. Yeast suspensions (1.5 107 CFU/0.05 ml saline) were injected into the haemolymph of the silkworm. (A) Survival time post inoculation was determined. Indicate significant difference by the Log-rank test (P < 0.001). (B)–(D), yeast cells were recovered from the silkworm haemolymph and their morphology was determined. (B) Yeast diameter, (C) capsule thickness and (D) total cell size (yeast and capsule together) of the wild type (WT), PMT2 deletion strain (pmt2D) and PMT2 reconstituted strain (pmt2D + PMT2). Indicate significant difference by the unpaired t test (P < 0.001).

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respectively, were knocked out without affecting viability (Goto et al., 2009), but in the human pathogen Aspergillus fumigatus, PMT2 was shown to be essential (Mouyna et al., 2010). Even in the same or close organisms, the involvement of PMTs in viability differs depending on the growth environments or strain backgrounds, which may not be always detectable. These differences may also be reflected by the nature of the gene disruptions in each case. Insertions into open reading frames (vs. complete deletions of ORFs) can result in phenotypes as a consequence of the production of partial protein products in ORF-containing strains. These products could act as dominant-negative partners, essentially preventing interacting proteins from forming functional complexes and causing extreme effects, such as lethality.

Protein-O-mannosylation in yeasts and filamentous fungi has been shown to be involved in a number of different cellular processes by affecting protein stability, localization, and secretion (reviewed by Strahl-Bolsinger et al., 1999; Ernst and Prill, 2001; Goto, 2007; Lommel and Strahl, 2009). In this study, we showed that the capsule of C. neoformans became thicker when the PMT2 gene was deleted (Fig. 3B), suggesting that the Pmt2 protein in C. neoformans has a negative effect in capsule formation, even though Pmt2 functions to mannosylate target proteins. Capsule of C. neoformans is known to be composed of glucuronoxylomannan and galactoxylomannan (which comprise over 90% of the total capsule mass) and mannoproteins (Doering, 2009). It has been shown that the protein-O-mannosylation and N-glycosylation machineries


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compete for target proteins (Ecker et al., 2003). In C. neoformans, some proteins have been identified to be glycosylated and mannosylated (Levitz and Specht, 2006), and approximately 50% of the secreted proteins were mannosylated (Mansour et al., 2002). Willger et al. (2009) indicated that in C. neoformans, Pmt4 has a positive association with capsule production while Pmt1 does not. Thus, irregular composition of glycosylation on proteins that may be transported extracellularly might result in the unusually larger capsule development in a pmt2D strain. Fernández-Álvarez et al. (2012) has identified a target protein of PMT4 which is involved in virulence of a corn smut fungus U. maydis. A proteomic comparison in a wild type and PMT mutants of C. neoformans may provide an insight for the involvement of PMTs in capsule production and other phenotypes. In S. cerevisiae, a precursor of mutant a-factor, a mating pheromone, was progressively O-mannosylated and degraded by endoplasmic reticulum-associated degradation (ERAD, Harty et al., 2001; Nakatsukasa et al., 2004). This fail-safe mechanism for ERAD to mannosylate aberrant proteins was only catalyzed by Pmt2 (Harty et al., 2001). The biogenesis pathway of the mating pheromones of C. neoformans has not yet been reported even though their structures have been suggested (Davidson et al., 2000b). Involvement of Pmt2 in the quality control of proper pheromone production may account for our observation: a bilateral cross of two pmt2D strains completely lost the formation of reproductive structures. Our observations also indicated that the simultaneous inactivation of PMT2 and PMT1 or PMT4 was synthetically lethal in C. neoformans. In humans, it has been suggested that POMT1 (homologue of Pmt4) and POMT2 (homologue of Pmt2) form heterodimers (Akasaka-Manya et al., 2006). In S. cerevisiae, Pmt1 and Pmt2 form a heterodimeric protein complex, which exhibits a protein-O-mannosylation activity, but Pmt4 family members act as a homomeric complex (Girrbach and Strahl, 2003). In C. neoformans, any two of the three PMT genes seem to be essential, suggesting that at least one heterodimeric complex of Pmt proteins is required for its viability. Our observation for the virulence assay using silkworm model was surprising, because the PMT2 gene function seems to be important for growth in stressful conditions such as high osmolality or higher temperature. Interestingly, however, the pmt2D cells recovered from the silkworm haemolymph possessed significantly thicker capsules than the WT or the reconstituted strains, and the pmt2D strain produced thicker capsules than the control strains (Fig. 5C). One possibility is that the immune system of the silkworm could have cleared normal cells or cells with thinner capsules, but the pmt2D cells that were too large or that have very thick capsules were able to escape from the host immune system and subsequently kill the silkworms. We also showed the involvement of the PMT2 gene in DBB staining in C. gattii, another causal agent of cryptococcosis. At present, no other basidiomycetous species have been tested for whether the PMT2 gene is necessary for DBB staining, but future studies with different members of the basidiomycetous yeasts will answer this question, and provide additional pathways that distinguish fundamental biological differences between ascomycetes and basidiomycetes.

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5. Conclusion

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In this study, we isolated a mutant of the basidiomycetous yeast C. neoformans which was not stained by DBB, and identified a gene (PMT2) responsible for DBB staining. Deletion of the PMT2 gene in C. gattii also resulted in abolition of DBB staining. In C. neoformans the deletion mutant of PMT2 exhibited pleiotropic phenotypes:

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pmt2D strains produced larger cells and thicker capsules, were sensitive to osmotic stress, failed to carry out sexual development, and showed increased virulence in a silkworm model of infection. By investigating the basic phenomenon of DBB staining, we succeeded in identifying previously unknown roles for PMT2 in diverse and critical cellular processes. Our findings will facilitate future studies of the roles of the targets of PMT2 glycosylation in the basic biology of Cryptococcus.

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6. Uncited reference

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Kwon-Chung et al. (2002).

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Acknowledgments

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This study was partly supported by Grant-in-Aid for Scientific Research (No. 08035956) from the Ministry of Education, Science, Sports and Culture of Japan and Hokuto Foundation for Biological Science to KS. This work was in part conducted at the University of Wisconsin, Madison under the Chiba University Institutional Program for Young Researcher Overseas Visits supported by the Japan Society for the Promotion of Science. We are grateful to Dee Carter for C. gattii strains.

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Appendix A. Supplementary material

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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fgb.2014.05.007.

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Cryptococcus neoformans: historical curiosity to modern pathogen.

Systematic functional analysis of kinases in the fungal pathogen Cryptococcus neoformans.

Targeted Genome Editing via CRISPR in the Pathogen Cryptococcus neoformans.

Elucidation of the calcineurin-Crz1 stress response transcriptional network in the human fungal pathogen Cryptococcus neoformans.

Mitochondrial Protein Nfu1 Influences Homeostasis of Essential Metals in the Human Fungal Pathogen Cryptococcus neoformans.

The ER stress response and host temperature adaptation in the human fungal pathogen Cryptococcus neoformans.

Characterization of the antigenicity of Cpl1, a surface protein of Cryptococcus neoformans var. neoformans.

Systematic functional profiling of transcription factor networks in Cryptococcus neoformans.

Biolistic transformation of a fluorescent tagged gene into the opportunistic fungal pathogen Cryptococcus neoformans.

A novel bZIP protein, Gsb1, is required for oxidative stress response, mating, and virulence in the human pathogen Cryptococcus neoformans.

The ecology of Cryptococcus neoformans.

Population genomics and the evolution of virulence in the fungal pathogen Cryptococcus neoformans.

Role of ferric reductases in iron acquisition and virulence in the fungal pathogen Cryptococcus neoformans.

Molecular mechanisms of hypoxic responses via unique roles of Ras1, Cdc24 and Ptp3 in a human fungal pathogen Cryptococcus neoformans.

The Cryptococcus neoformans transcriptome at the site of human meningitis.

Balancing stability and flexibility within the genome of the pathogen Cryptococcus neoformans.

The epidemiology of cryptococcosis and the characterization of Cryptococcus neoformans isolated in a Brazilian University Hospital.

Galactoxylomannans of Cryptococcus neoformans.

Cryptococcus neoformans and Cryptococcus gattii, the etiologic agents of cryptococcosis.

Auxotrophic mutants of Cryptococcus neoformans.

ALL2, a Homologue of ALL1, Has a Distinct Role in Regulating pH Homeostasis in the Pathogen Cryptococcus neoformans.

Cryptococcus neoformans var gattii.

The tools for virulence of Cryptococcus neoformans.

Heterogeneity of phenol oxidases in Cryptococcus neoformans.