Journal: PLoS ONE

Article Title: Cryptococcus neoformans Is Internalized by Receptor-Mediated or ‘Triggered’ Phagocytosis, Dependent on Actin Recruitment

doi: 10.1371/journal.pone.0089250

Figure Lengend Snippet: Quantification of the attachment to macrophages and the internalization of C. neoformans yeast cells from capsular (H99 and B3501) and acapsular (CAP67 and CAP 59) strains, in the presence of the actin polymerization inhibitors cytochalasin D or latrunculin B (A and B) or the microtubule stabilizers nocodazole or paclitaxel (C and D). Graphs show normalized mean values and standard deviation from three experiments. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: We used the following Cryptococcus neoformans strains: the capsular wild type strains H99 used for the genome project (ATCC 208821, serotype A,) and B3501 (serotype D); and the acapsular mutants CAP59 (NE367), derived from H99 , and CAP 67 (ATCC 52817), derived from B3501 .

Techniques: Standard Deviation

Journal: PLoS ONE

Article Title: Cryptococcus neoformans Is Internalized by Receptor-Mediated or ‘Triggered’ Phagocytosis, Dependent on Actin Recruitment

doi: 10.1371/journal.pone.0089250

Figure Lengend Snippet: Quantification of the internalization (A) and the attachment (B) to macrophages of C. neoformans yeast cells from capsular (H99 and B3501) and acapsular (CAP67 and CAP 59) strains, in the absence of cytoskeletal inhibitors or in the presence of cytochalasin D and nocodazole. The metabolic viability of C. neoformans strains H99 and CAP59 was measured using the FUN®-1 dye (C) and the metabolic viability of macrophages was measured by MTS/PMS (D) after incubation with cytoskeletal inhibitors for 2 h. Yeast cells fixed with 70% ethanol, and macrophages with 4% formaldehyde, were used as a positive control for the loss of cell viability in each method. Graphs show normalized mean values and standard deviation from three experiments (A–B) and mean and standard deviation from absolute values of fluorescence intensity (C) and absorbance (D).*p<0.05; **p<0.01; ***p<0.001.

Article Snippet: We used the following Cryptococcus neoformans strains: the capsular wild type strains H99 used for the genome project (ATCC 208821, serotype A,) and B3501 (serotype D); and the acapsular mutants CAP59 (NE367), derived from H99 , and CAP 67 (ATCC 52817), derived from B3501 .

Techniques: Incubation, Positive Control, Standard Deviation, Fluorescence

Journal: PLoS ONE

Article Title: Cryptococcus neoformans Is Internalized by Receptor-Mediated or ‘Triggered’ Phagocytosis, Dependent on Actin Recruitment

doi: 10.1371/journal.pone.0089250

Figure Lengend Snippet: Scanning electron microscopy of membrane extracted macrophages interacting with C. neoformans strains H99 (A) and CAP59 (B and C), showing cytoskeletal filaments associated with yeasts in untreated samples (A–B). After 5 µm nocodazole treatment (C) the area surrounding yeast cells appeared mostly devoid of cytoskeletal components but association with yeast still occurred (inset in C). Scale bars, 2 µm.

Article Snippet: We used the following Cryptococcus neoformans strains: the capsular wild type strains H99 used for the genome project (ATCC 208821, serotype A,) and B3501 (serotype D); and the acapsular mutants CAP59 (NE367), derived from H99 , and CAP 67 (ATCC 52817), derived from B3501 .

Techniques: Electron Microscopy, Membrane

Journal: PLoS ONE

Article Title: Cryptococcus neoformans Is Internalized by Receptor-Mediated or ‘Triggered’ Phagocytosis, Dependent on Actin Recruitment

doi: 10.1371/journal.pone.0089250

Figure Lengend Snippet: Confocal laser scanning microscopy (z-stack series of confocal planes) of interacting macrophages and C. neoformans yeast cells from strains H99 (A and B) and CAP59 (C and D). Internalized yeasts identified by DIC (arrows in A and C) can be visualized in the context of host cell actin (red) and microtubule (green) cytoskeletons (B and D). Host cell DNA is labeled with DAPI (blue, indicated by the letter ‘N’) and yeast is labeled with calcofluor (blue, indicated by arrows). Actin, but not tubulin, is recruited to sites of yeast internalization. Scale bars, 5 µm.

Article Snippet: We used the following Cryptococcus neoformans strains: the capsular wild type strains H99 used for the genome project (ATCC 208821, serotype A,) and B3501 (serotype D); and the acapsular mutants CAP59 (NE367), derived from H99 , and CAP 67 (ATCC 52817), derived from B3501 .

Techniques: Confocal Laser Scanning Microscopy, Labeling

Journal: PLoS ONE

Article Title: Cryptococcus neoformans Is Internalized by Receptor-Mediated or ‘Triggered’ Phagocytosis, Dependent on Actin Recruitment

doi: 10.1371/journal.pone.0089250

Figure Lengend Snippet: Confocal laser scanning microscopy of C. neoformans capsular strain H99 interacting with macrophages (single confocal plane). DIC showing internalized yeasts (arrows); and confocal images showing actin filaments (red), microtubules (green), yeast (blue) and host DNA (blue, indicated by ‘n’). Actin is recruited to the site of phagocytosis in untreated cells (A), and actin recruitment was inhibited by 0.5 µM cytochalasin D (B). In contrast, treatment with 5 µM nocodazole (C) or with a combination of nocodazole and cytochalasin D (D) did not inhibit actin recruitment to the phagosome area. Scale bars, 5 µm.

Article Snippet: We used the following Cryptococcus neoformans strains: the capsular wild type strains H99 used for the genome project (ATCC 208821, serotype A,) and B3501 (serotype D); and the acapsular mutants CAP59 (NE367), derived from H99 , and CAP 67 (ATCC 52817), derived from B3501 .

Techniques: Confocal Laser Scanning Microscopy

Journal: PLoS ONE

Article Title: Cryptococcus neoformans Is Internalized by Receptor-Mediated or ‘Triggered’ Phagocytosis, Dependent on Actin Recruitment

doi: 10.1371/journal.pone.0089250

Figure Lengend Snippet: Scanning electron microscopy of C. neoformans capsular strain H99 (A–E) and acapsular strain CAP59 (F–G) interacting with peritoneal macrophages. Improved preservation of macrophage membranes was obtained with routine SEM fixation (A–B; F–G), although post-fixation in the presence of sucrose provided better capsule preservation and allowed visualization of direct interactions between the capsule and host cell membranes, prior to internalization (C–E). Both trigger-like (arrow in A and F) and zipper-like (arrow-head in B and G) uptake structures were observed. Scale bars, 1 µm (A–C; F–G) and 0.5 µm (D–E).

Article Snippet: We used the following Cryptococcus neoformans strains: the capsular wild type strains H99 used for the genome project (ATCC 208821, serotype A,) and B3501 (serotype D); and the acapsular mutants CAP59 (NE367), derived from H99 , and CAP 67 (ATCC 52817), derived from B3501 .

Techniques: Electron Microscopy, Preserving

Journal: PLoS ONE

Article Title: Cryptococcus neoformans Is Internalized by Receptor-Mediated or ‘Triggered’ Phagocytosis, Dependent on Actin Recruitment

doi: 10.1371/journal.pone.0089250

Figure Lengend Snippet: Quantification of the uptake mechanisms during C. neoformans -macrophage interaction.

Article Snippet: We used the following Cryptococcus neoformans strains: the capsular wild type strains H99 used for the genome project (ATCC 208821, serotype A,) and B3501 (serotype D); and the acapsular mutants CAP59 (NE367), derived from H99 , and CAP 67 (ATCC 52817), derived from B3501 .

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: A Xylosylphosphotransferase of Cryptococcus neoformans Acts in Protein O -Glycan Synthesis *

doi: 10.1074/jbc.M111.262162

Figure Lengend Snippet: C. neoformans strains used in these studies

Article Snippet: These results were consistent with our identification of sequences homologous to the serotype A XPT1 (locus CNAG_04860 , from the C. neoformans var. grubii H99 data base maintained by the Broad Institute) in the genomes of serotypes B (locus CNBG_5687 ; from the C. gattii R265 data base maintained by the Broad Institute) and D (locus {"type":"entrez-protein","attrs":{"text":"CNJ02890","term_id":"812677313","term_text":"CNJ02890"}} CNJ02890 ; from the C. neoformans var. neoformans JEC21 data base maintained by TIGR).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: A Xylosylphosphotransferase of Cryptococcus neoformans Acts in Protein O -Glycan Synthesis *

doi: 10.1074/jbc.M111.262162

Figure Lengend Snippet: Xylosyltransferase activities in C. neoformans. Total membranes prepared from wild-type KN99α (XPT1), mutant (xpt1Δ), or episomally-complemented mutant (xpt1Δ pXPT1 and xpt1Δ pXPT1-HA) strains were assayed with UDP-[14C]Xyl and α-1,3-Man2 in the absence (−) or presence (+) of MnCl2, as described under “Experimental Procedures.” An autoradiograph of the products resolved by TLC is shown; no signal was detected in other regions of the plate beyond minor amounts of free Xyl. The filled triangle indicates the product of the manganese-dependent xylosyltransferase activity, and the open triangle indicates the product of the unrelated xylosyltransferase Cxt1p (see “Results”). No signal was detected in the absence of the α-1,3-Man2 acceptor (not shown).

Article Snippet: These results were consistent with our identification of sequences homologous to the serotype A XPT1 (locus CNAG_04860 , from the C. neoformans var. grubii H99 data base maintained by the Broad Institute) in the genomes of serotypes B (locus CNBG_5687 ; from the C. gattii R265 data base maintained by the Broad Institute) and D (locus {"type":"entrez-protein","attrs":{"text":"CNJ02890","term_id":"812677313","term_text":"CNJ02890"}} CNJ02890 ; from the C. neoformans var. neoformans JEC21 data base maintained by TIGR).

Techniques: Mutagenesis, Autoradiography, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: A Xylosylphosphotransferase of Cryptococcus neoformans Acts in Protein O -Glycan Synthesis *

doi: 10.1074/jbc.M111.262162

Figure Lengend Snippet: Expression of XPT1 from serotypes A and D similarly complements the serotype A xpt1Δ mutant. Total membranes prepared from serotype A wild-type (XPT1), serotype A xpt1Δ (xpt1Δ), serotype A xpt1Δ cells expressing XPT1 from the same serotype (xpt1Δ pXPT1), and serotype A xpt1Δ cells expressing XPT1 from serotype D (xpt1Δ pXPT1 (D)) were assayed with UDP-[14C]Xyl and α-1,3-Man2 in the absence (−) or presence (+) of MnCl2, as described under “Experimental Procedures.” An autoradiograph of the products resolved by TLC is shown; no signal was detected in other regions of the plate beyond minor amounts of free Xyl or in the absence of the α-1,3-Man2 acceptor (not shown). The filled triangle indicates the product of the manganese-dependent xylosyltransferase activity, and the open triangle indicates the product of the unrelated xylosyltransferase Cxt1p (see “Results”).

Article Snippet: These results were consistent with our identification of sequences homologous to the serotype A XPT1 (locus CNAG_04860 , from the C. neoformans var. grubii H99 data base maintained by the Broad Institute) in the genomes of serotypes B (locus CNBG_5687 ; from the C. gattii R265 data base maintained by the Broad Institute) and D (locus {"type":"entrez-protein","attrs":{"text":"CNJ02890","term_id":"812677313","term_text":"CNJ02890"}} CNJ02890 ; from the C. neoformans var. neoformans JEC21 data base maintained by TIGR).

Techniques: Expressing, Mutagenesis, Autoradiography, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: A Xylosylphosphotransferase of Cryptococcus neoformans Acts in Protein O -Glycan Synthesis *

doi: 10.1074/jbc.M111.262162

Figure Lengend Snippet: Protein glycosylation studies of xpt1Δ mutant. Total membranes prepared from the indicated strains were assayed with UDP-[14C]Xyl in the presence (+) or absence (−) of MnCl2 as indicated; some samples were subsequently mock-treated (m) or treated with protease (+) as indicated. Panel A shows manganese stimulation of radiolabel incorporation. Panel B shows protease degradation of assay products. Panel C shows Xpt1p dependence of radiolabel incorporation. Autoradiographs of dried SDS-PAGE gels are shown. Molecular weight standards (in kDa) are indicated at the right.

Article Snippet: These results were consistent with our identification of sequences homologous to the serotype A XPT1 (locus CNAG_04860 , from the C. neoformans var. grubii H99 data base maintained by the Broad Institute) in the genomes of serotypes B (locus CNBG_5687 ; from the C. gattii R265 data base maintained by the Broad Institute) and D (locus {"type":"entrez-protein","attrs":{"text":"CNJ02890","term_id":"812677313","term_text":"CNJ02890"}} CNJ02890 ; from the C. neoformans var. neoformans JEC21 data base maintained by TIGR).

Techniques: Glycoproteomics, Mutagenesis, Degradation Assay, SDS Page, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: A Xylosylphosphotransferase of Cryptococcus neoformans Acts in Protein O -Glycan Synthesis *

doi: 10.1074/jbc.M111.262162

Figure Lengend Snippet: The profiles of major O-linked glycans released from wild-type and xpt1Δ mutant proteins are similar. Full MS scans are shown for glycans released from equivalent amounts of protein harvested from wild-type KN99α (panel A) and the KN99α xpt1Δ mutant (panel B). Mann and Xyl-Mann glycans dominate the O-linked glycan profile. Asterisks denote the presence of a poly-hexose contaminant of unknown origin (non-reduced). 2+ denotes double-charged ion species; circle, Man; star, Xyl.

Article Snippet: These results were consistent with our identification of sequences homologous to the serotype A XPT1 (locus CNAG_04860 , from the C. neoformans var. grubii H99 data base maintained by the Broad Institute) in the genomes of serotypes B (locus CNBG_5687 ; from the C. gattii R265 data base maintained by the Broad Institute) and D (locus {"type":"entrez-protein","attrs":{"text":"CNJ02890","term_id":"812677313","term_text":"CNJ02890"}} CNJ02890 ; from the C. neoformans var. neoformans JEC21 data base maintained by TIGR).

Techniques: Mutagenesis, Glycoproteomics

Journal: The Journal of Biological Chemistry

Article Title: A Xylosylphosphotransferase of Cryptococcus neoformans Acts in Protein O -Glycan Synthesis *

doi: 10.1074/jbc.M111.262162

Figure Lengend Snippet: Filtered TIM scans demonstrate Xyl-P-Man2 and Xyl-P-Man3 in O-linked glycans from wild-type, xpt1Δ mutant, and XPT1 overexpressing strains. Total ion mapping scans were filtered for the production of MS2 fragments that indicate the presence of Xyl-P motifs (panel A, m/z = 573 for Xyl-P-Man2; panel B, m/z = 777 for Xyl-P-Man3) in the indicated strains. MS2 is inherently more sensitive than full MS for ion-trap instruments, yet the Xyl-P modified glycans are detected as only minor deflections above base line in the xpt1Δ O-linked glycan preparations. The signal traces for KN99α in the left panels and for xpt1Δ pXPT1 in the right panels are displaced upward on the y axis by 5% of full scale for clarity.

Article Snippet: These results were consistent with our identification of sequences homologous to the serotype A XPT1 (locus CNAG_04860 , from the C. neoformans var. grubii H99 data base maintained by the Broad Institute) in the genomes of serotypes B (locus CNBG_5687 ; from the C. gattii R265 data base maintained by the Broad Institute) and D (locus {"type":"entrez-protein","attrs":{"text":"CNJ02890","term_id":"812677313","term_text":"CNJ02890"}} CNJ02890 ; from the C. neoformans var. neoformans JEC21 data base maintained by TIGR).

Techniques: Mutagenesis, Modification, Glycoproteomics

Journal: The Journal of Biological Chemistry

Article Title: A Xylosylphosphotransferase of Cryptococcus neoformans Acts in Protein O -Glycan Synthesis *

doi: 10.1074/jbc.M111.262162

Figure Lengend Snippet: Xpt1p overexpression yields parallel increases in enzyme activity and O-glycan modifications. Panel A, total membranes were prepared from serotype A wild-type cells or serotype A xpt1Δ cells episomally expressing XPT1 from the same serotype; the latter were cultured either with G418 to maintain the expression plasmid or without it to allow plasmid loss as indicated. Xpt1p activity was assayed with UDP-[14C]Xyl and α-1,3-Man2 in the absence (−) or presence (+) of MnCl2, as described under “Experimental Procedures.” An autoradiograph of the products resolved by TLC is shown; no signal was detected in other regions of the plate beyond minor amounts of free Xyl or in the absence of the α-1,3-Man2 acceptor (not shown). The filled triangle indicates the product of the manganese-dependent xylosyltransferase activity, and the open triangle indicates the product of the unrelated xylosyltransferase Cxt1P (see “Results”). Panel B, shown are Xpt1p products for the strains indicated, normalized to wild-type. Black bars, radiolabel in Xyl-P-Man2 (filled triangle) made in vitro in the reactions shown in panel A, quantified using a BIOSCAN System 200 Imaging scanner; white bars, Xyl-P-Man2 in protein O-glycans, derived from the abundance of MS2 fragments of m/z = 573; gray bars, Xyl-P-Man3 in protein O-glycans, derived from the abundance of MS2 fragments of m/z = 777.

Article Snippet: These results were consistent with our identification of sequences homologous to the serotype A XPT1 (locus CNAG_04860 , from the C. neoformans var. grubii H99 data base maintained by the Broad Institute) in the genomes of serotypes B (locus CNBG_5687 ; from the C. gattii R265 data base maintained by the Broad Institute) and D (locus {"type":"entrez-protein","attrs":{"text":"CNJ02890","term_id":"812677313","term_text":"CNJ02890"}} CNJ02890 ; from the C. neoformans var. neoformans JEC21 data base maintained by TIGR).

Techniques: Over Expression, Activity Assay, Glycoproteomics, Expressing, Cell Culture, Plasmid Preparation, Autoradiography, In Vitro, Imaging, Derivative Assay

Journal: Glycobiology

Article Title: Cryptococcus neoformans UGT1 encodes a UDP-Galactose/UDP-GalNAc transporter

doi: 10.1093/glycob/cww078

Figure Lengend Snippet: C. neoformans strains used in these studies

Article Snippet: UGT1 was identified by BLASTP searches against C. neoformans predicted proteins (Broad Institute; C. neoformans var. grubii H99 database) using known UDP-galactose transporters from Schizosaccharomyces pombe ( {"type":"entrez-protein","attrs":{"text":"NP_588041","term_id":"19075541","term_text":"NP_588041"}} NP_588041 ), Arabidopsis thaliana ( {"type":"entrez-protein","attrs":{"text":"NP_565158.1","term_id":"18411611","term_text":"NP_565158.1"}} NP_565158.1 ), Caenorhabditis elegans ( {"type":"entrez-protein","attrs":{"text":"NP_001255676.1","term_id":"392901327","term_text":"NP_001255676.1"}} NP_001255676.1 ) and Homo sapiens ( {"type":"entrez-nucleotide","attrs":{"text":"NC_000023.11","term_id":"568815575","term_text":"NC_000023.11"}} NC_000023.11 ).

Techniques:

Journal: Glycobiology

Article Title: Cryptococcus neoformans UGT1 encodes a UDP-Galactose/UDP-GalNAc transporter

doi: 10.1093/glycob/cww078

Figure Lengend Snippet: Topology of C. neoformans Ugt1 as predicted by TMHMM server v 2.0, showing 10 putative transmembrane domains and long N- and C-terminal cytosolic tails. Arrowheads indicate the new N-terminus for each N-terminal truncation and the terminal residue of the single C-terminal truncation (C1); see text and Table III for details.

Article Snippet: UGT1 was identified by BLASTP searches against C. neoformans predicted proteins (Broad Institute; C. neoformans var. grubii H99 database) using known UDP-galactose transporters from Schizosaccharomyces pombe ( {"type":"entrez-protein","attrs":{"text":"NP_588041","term_id":"19075541","term_text":"NP_588041"}} NP_588041 ), Arabidopsis thaliana ( {"type":"entrez-protein","attrs":{"text":"NP_565158.1","term_id":"18411611","term_text":"NP_565158.1"}} NP_565158.1 ), Caenorhabditis elegans ( {"type":"entrez-protein","attrs":{"text":"NP_001255676.1","term_id":"392901327","term_text":"NP_001255676.1"}} NP_001255676.1 ) and Homo sapiens ( {"type":"entrez-nucleotide","attrs":{"text":"NC_000023.11","term_id":"568815575","term_text":"NC_000023.11"}} NC_000023.11 ).

Techniques: Residue

Journal: Glycobiology

Article Title: Cryptococcus neoformans UGT1 encodes a UDP-Galactose/UDP-GalNAc transporter

doi: 10.1093/glycob/cww078

Figure Lengend Snippet: Cells lacking Ugt1 are more efficiently phagocytosed and killed by THP-1 cells than wild-type C. neoformans. (A) Phagocytic index (engulfed fungi/100 host cells) of strains grown in YPD (−/+ opsonization) or in inducing media (+ opsonization). (B) Survival of YPD-grown, opsonized fungi after internalization by THP-1 cells. Data are representative of three independent experiments performed with n = 3 (*, P < 0.01; **, P < 0.001).

Article Snippet: UGT1 was identified by BLASTP searches against C. neoformans predicted proteins (Broad Institute; C. neoformans var. grubii H99 database) using known UDP-galactose transporters from Schizosaccharomyces pombe ( {"type":"entrez-protein","attrs":{"text":"NP_588041","term_id":"19075541","term_text":"NP_588041"}} NP_588041 ), Arabidopsis thaliana ( {"type":"entrez-protein","attrs":{"text":"NP_565158.1","term_id":"18411611","term_text":"NP_565158.1"}} NP_565158.1 ), Caenorhabditis elegans ( {"type":"entrez-protein","attrs":{"text":"NP_001255676.1","term_id":"392901327","term_text":"NP_001255676.1"}} NP_001255676.1 ) and Homo sapiens ( {"type":"entrez-nucleotide","attrs":{"text":"NC_000023.11","term_id":"568815575","term_text":"NC_000023.11"}} NC_000023.11 ).

Techniques:

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antimicrobial Octapeptin C4 Analogues Active against Cryptococcus Species

doi: 10.1128/AAC.00986-17

Figure Lengend Snippet: Antifungal MICs of PMB, PME, and octapeptin C4 as determined by broth microdilution assays against common fungal pathogens

Article Snippet: A consistent increase in the MIC values was observed from 1.56 to 6.5 μg/ml in the H99 and ATCC 90113 strains, indicating that the Dab residues were all equally important for the antifungal activity of the octapeptins ( ).

Techniques:

Journal: Genetics

Article Title: The TOR Pathway Plays Pleiotropic Roles in Growth and Stress Responses of the Fungal Pathogen Cryptococcus neoformans

doi: 10.1534/genetics.119.302191

Figure Lengend Snippet: TOR1 is required for viability in Cryptococcus neoformans. (A) Maximum likelihood tree of the identified Tor1 and Tlk1 proteins in S. cerevisiae (Sc), C. albicans (Ca), A. fumigatus (Af), C. neoformans (Cn), M. sympodialis (Ms), and U. maydis (Um). (B) The domain structure of C. neoformans TOR1 and TLK1. (C) Alignment of FKBP12-rapamycin binding domains of S. cerevisiae TOR1 (ScTOR1), Tor2 (ScTOR2), C. neoformans TOR1 (CnTOR1), and TLK1 (CnTLK1). Multiple sequence alignment is by ClustalW alignment using MacVector software. The position of the serine residue required for inhibition by rapamycin is in the box. (D) WT, PCTR4:TOR1 (YSB3176 and YSB3178), and PCTR4:TLK1 (YSB3043 and YSB3044) strains were spotted on YPD or YNB medium containing 200 μM BCS or 25 μM CuSO4. The plates were incubated at 30° and photographed daily for 5 days. This spot assay was repeated more than three times, and one representative image was shown here. (E) Schematic representation of the TOR1 gene before and after the targeted replacement. Position of primers used for confirmation are marked by arrows. (F) Δ indicates the NAT resistant heterozygote tor1Δ/TOR1 mutant generated. Random basidiospores (150) were dissected from TOR1/tor1Δ, and 14 that germinated were grown on four medium types for genetic analysis; 12 strains showed a NAT-sensitive phenotype and independent segregation of the other genetic markers. Conversely, spores #7 and #8 yielded colonies with irregular segregation, and hence were subjected to further molecular characterization reported in (G). (G) Molecular characterization of the four markers that segregate during meiosis (NAT, URA5, ADE2, MAT) in NATR progeny #7 and #8 obtained from the sporulation of the heterozygous TOR1/tor1Δ. The parental TOR1/tor1Δ and the diploid AI187 were included as control. The asterisk (*) indicates the mutated tor1Δ and ura5 alleles.

Article Snippet: The reference genome sequence of C. neoformans H99 and annotation data were downloaded from the Broad Institute ftp server (archive.broadinstitute.org).

Techniques: Binding Assay, Sequencing, Software, Residue, Inhibition, Incubation, Spot Test, Mutagenesis, Generated, Control

Journal: Genetics

Article Title: The TOR Pathway Plays Pleiotropic Roles in Growth and Stress Responses of the Fungal Pathogen Cryptococcus neoformans

doi: 10.1534/genetics.119.302191

Figure Lengend Snippet: Tor1 is the target of rapamycin in C. neoformans. (A) WT (H99) and PCTR4:TOR1 (YSB3176) strains were cultured in liquid YPD medium overnight at 30°, 10-fold serially diluted, and spotted on YNB agar medium containing either 25 μM CuSO4 or 200 μM BCS with or without 7 ng/ml rapamycin. This spot assay was repeated more than three times and one representative image was shown here. (B and D) The following strains were used: TOR1 (haploid WT H99), TOR1/TOR1 (diploid AI187), and TOR1/tor1Δ (YSB3450) for (B); WT (H99), TOR1oe (YSB3144), TLK1oe (YSB3118), and tlk1Δ (YSB3153) for (D). Each strain was cultured in liquid YPD medium overnight at 30°, 10-fold serially diluted, and spotted on YPD medium containing 10 ng/ml rapamycin. The plates were further incubated at 30° and photographed daily for 5 days. This spot assay was repeated more than three times and one representative image is shown here. (C) Quantitative RT-PCR analysis of TOR1 expression in WT, TOR1oe (YSB3144), and rapamycin-treated (3 ng/ml) WT cells. Three independent biological experiments with three technical replicates were performed. Error bars indicate SEM. Statistical significance of the differences was determined by one-way ANOVA with Bonferroni’s multiple-comparison test (***P < 0.0001).

Article Snippet: The reference genome sequence of C. neoformans H99 and annotation data were downloaded from the Broad Institute ftp server (archive.broadinstitute.org).

Techniques: Cell Culture, Spot Test, Incubation, Quantitative RT-PCR, Expressing, Comparison

Journal: Genetics

Article Title: The TOR Pathway Plays Pleiotropic Roles in Growth and Stress Responses of the Fungal Pathogen Cryptococcus neoformans

doi: 10.1534/genetics.119.302191

Figure Lengend Snippet: Tor1 negatively regulates the thermotolerance of C. neoformans. (A) To test thermotolerance, cells were cultured at 30° for 16 hr. Each culture was serially diluted and spotted onto YPD medium with or without 10 ng/ml rapamycin. Strains were then incubated at 30, 37, and 39°. The plates were photographed daily for 4 days. This spot assay was repeated more than three times, and one representative image is shown here. (B) Analysis of HSF1, HSP90, HSP104, and ERG11 expression levels under high temperature conditions in WT (H99), TOR1oe (YSB3144), and rapamycin-treated (3 ng/ml) WT strains. The cDNA was synthesized from the total RNA extracted from cells exposed to temperature upshift (from 30 to 37° or 39°) for 30 min. The specific primers for the amplification of HSF1, HSP90, HSP104, ERG11, and ACT1 are described in Table S2. Three independent biological experiments with three technical replicates were performed. Error bars indicate SEM. Statistical significance of the differences was determined by one-way ANOVA with Bonferroni’s multiple-comparison test (* P < 0.05, ** P < 0.001, *** P < 0.0001; ns: not significant). (C) WT cells (H99) and TOR1oe strains were grown to midlogarithmic phase and exposed to 10 ng/ml rapamycin for the indicated time. Mpk1 phosphorylation levels were monitored using anti-P-p44/p42 antibody. The blot was stripped and used for detection of Hog1 with a polyclonal anti-Hog1 antibody as a loading control. This western blot analysis was repeated twice, and one representative result is shown here. (D) The proposed Tor1 regulatory mechanism for thermotolerance of C. neoformans. Tor1 negatively regulates thermotolerance in an Hsf1-independent and Sch9 and Mpk1-dependent manner.

Article Snippet: The reference genome sequence of C. neoformans H99 and annotation data were downloaded from the Broad Institute ftp server (archive.broadinstitute.org).

Techniques: Cell Culture, Incubation, Spot Test, Expressing, Synthesized, Amplification, Comparison, Phospho-proteomics, Control, Western Blot

Journal: Genetics

Article Title: The TOR Pathway Plays Pleiotropic Roles in Growth and Stress Responses of the Fungal Pathogen Cryptococcus neoformans

doi: 10.1534/genetics.119.302191

Figure Lengend Snippet: Tor1 negatively regulates the C. neoformans DNA damage response. (A) WT (H99), TOR1oe (YSB3144), and rad53Δ (YSB3785) strains were cultured in liquid YPD medium overnight at 30°, 10-fold serially diluted, and spotted on YPD medium containing 0.01% MMS or 90 mM HU, or 3 μg/ml bleomycin with and without 5 ng/ml rapamycin. This spot assay was repeated more than three times, and one representative image is shown here. (B) Expression levels of putative Rad53-dependent genes were verified by qRT-PCR analysis using cDNA of the WT, TOR1oe, and rapamycin (3 ng/ml)-treated WT strains with and without MMS treatment. Three independent biological experiments with three technical replicates were performed. Error bars indicate SEM. Statistical significance of the differences were determined by one-way ANOVA with Bonferroni’s multiple-comparison test (* P < 0.05, ** P < 0.001, *** P < 0.0001). (C) Phosphorylation of Rad53 was monitored using western blot with anti-FLAG antibody. The strains were grown to midlogarithmic phase, and then treated with MMS (0.02%) or rapamycin for 90 min. The cell extracts were incubated at 30° for 1 hr with and without λ-phosphatase and phosphatase inhibitor. This experiment was repeated twice, and one representative result is shown here.

Article Snippet: The reference genome sequence of C. neoformans H99 and annotation data were downloaded from the Broad Institute ftp server (archive.broadinstitute.org).

Techniques: Cell Culture, Spot Test, Expressing, Quantitative RT-PCR, Comparison, Phospho-proteomics, Western Blot, Incubation

Journal: Genetics

Article Title: The TOR Pathway Plays Pleiotropic Roles in Growth and Stress Responses of the Fungal Pathogen Cryptococcus neoformans

doi: 10.1534/genetics.119.302191

Figure Lengend Snippet: Tor1 localizes to the vacuolar membrane in C. neoformans. (A) Schematic diagram of construction of the Tor1-3xGFP strain. (B) Strains [WT H99 strain (haploid), TOR1oe (YSB3144), AI187 (diploid strain), TOR1/TOR1-loxP-NEOr-loxP-3xGFP (YSB4869), and TOR1/TOR1-3xGFP (YSB4870)] were spotted on YPD medium containing rapamycin. The plates were then incubated at 30° and photographed daily for 5 days. This spot assay was repeated more than three times, and one representative image was shown here. (C and D) Tor1-3xGFP cellular localization was monitored by fluorescence microscopy. (C) To activate the Cre-Lox system, the TOR1/tor1-3xGFP strain (YSB4869) was incubated in YPG (galactose) medium for 3 hr. (D) The Tor1-3xGFP strain was grown to mid-log phase and treated with MMS or further incubated at 39° for the indicated times. FM4-64 and Hoechst staining were used to stain the vacuole membrane and nucleus, respectively. Bar, 10 μm.

Article Snippet: The reference genome sequence of C. neoformans H99 and annotation data were downloaded from the Broad Institute ftp server (archive.broadinstitute.org).

Techniques: Membrane, Incubation, Spot Test, Fluorescence, Microscopy, Staining

Journal: Genetics

Article Title: The TOR Pathway Plays Pleiotropic Roles in Growth and Stress Responses of the Fungal Pathogen Cryptococcus neoformans

doi: 10.1534/genetics.119.302191

Figure Lengend Snippet: Tor1 is involved in C. neoformans actin cytoskeleton organization. (A) Strains [WT H99 strain, TOR1oe (YSB3144 and YSB3147)] were spotted on YPD medium containing rapamycin (15 ng/ml) and/or LatA (Latrunculin A; actin cytoskeleton inhibitor, 1 μg/ml). The plates were incubated at 30° and photographed daily for 7 days. This spot assay was repeated more than three times, and one representative image is shown here. (B) Distribution of polymerized actin upon rapamycin treatment in WT and TOR1oe WT and TOR1oe strains were grown to midlogarithmic phase at 30°. The cells were exposed to 10 ng/ml rapamycin for 1 hr. Then, cells were fixed, stained with rhodamine phalloidin, and visualized by fluorescence microscopy. Bar, 10 μm. (C) Rapamycin treatment significantly perturb the actin cytoskeleton. The graph shows the actin distribution in rapamycin treatment in WT and TOR1oe strains. (n; counted number of cells)

Article Snippet: The reference genome sequence of C. neoformans H99 and annotation data were downloaded from the Broad Institute ftp server (archive.broadinstitute.org).

Techniques: Incubation, Spot Test, Staining, Fluorescence, Microscopy

Journal: Genetics

Article Title: The TOR Pathway Plays Pleiotropic Roles in Growth and Stress Responses of the Fungal Pathogen Cryptococcus neoformans

doi: 10.1534/genetics.119.302191

Figure Lengend Snippet: The TOR pathway has a functional relationship with the signaling pathway modulating thermotolerance and DNA damage response, as well as the HOG MAPK pathway in C. neoformans. (A) Phenome heat map for transcription factor/kinase mutants exhibiting altered rapamycin-susceptibility. Red and blue circles in the heat map represent susceptibility and tolerance, respectively, against rapamycin, temperature stress, and DNA damage stress. The level of susceptibility and tolerance are distinguished by the sizes of circle and gradients of red or blue color. The heat map data for the thermal stress and DNA damage response of transcription factor/kinase mutants were retrieved from previous papers (Jung et al. 2015; Lee et al. 2016). (B) C. neoformans strains were grown overnight at 30° in liquid YPD medium. The 10-fold serially diluted cells (1–104 dilutions) were spotted on YPD plates containing rapamycin (10 ng/ml), and the plates were incubated at 30° and photographed daily for 5 days. [Strains: WT H99, tco1∆ (YSB278), tco2∆ (YSB281), ssk1∆ (YSB261), ssk2∆ (YSB264), pbs2∆ (YSB123), hog1∆ (YSB64), atf1∆ (YSB676), and tco1∆ tco2∆ (YSB324)]. This spot assay was repeated more than three times, and one representative image was shown here. (C) WT cells (H99) and TOR1oe strains were grown to midlogarithmic phase, and exposed to 10 ng/ml rapamycin for the indicated time. Hog1 phosphorylation levels were monitored using anti-P-p38 antibody. The blot was stripped and used for detection of Hog1 with a polyclonal anti-Hog1 antibody as a loading control This western blot analysis was repeated twice, and one representative result is shown here. HU, hydroxylurea; MMS, methyl methanesulphonate.

Article Snippet: The reference genome sequence of C. neoformans H99 and annotation data were downloaded from the Broad Institute ftp server (archive.broadinstitute.org).

Techniques: Functional Assay, Incubation, Spot Test, Phospho-proteomics, Control, Western Blot