Journal: mBio

Article Title: Role of an Expanded Inositol Transporter Repertoire in Cryptococcus neoformans Sexual Reproduction and Virulence

doi: 10.1128/mBio.00084-10

Figure Lengend Snippet: Phylogenetic analysis of myo -inositol transporter (Itr) proteins in C. neoformans and C. gattii . (A) Potential myo -inositol transporters from H99, JEC21, WM276, and R265 were identified using BLASTn and BLASTp analyses. The phylogram was generated using ClustalX 2.0.1 and viewed with the TreeView program. Two human sodium-dependent myo -inositol transporters (HsSMIT1 and HsSMIT2) served as outgroup controls. *, genes with closely related duplicated paralogs. (B) ITR gene locations in the genomes of JEC21 and H99. All ITR homologs in JEC21 and H99 were labeled at the appropriate locations on the 14 chromosomes of the genomes. (C) Subgroup 3 ITR genes of H99 have similar intron locations. Transcripts of ITR3 , ITR3A , ITR3B , and ITR3C are shown in black, and lines indicate introns in each gene. nt, nucleotides.

Article Snippet: All sequences for C. neoformans var. grubii and C. gattii were obtained from the Broad Institute ( http://www.broadinstitute.org/ ), and sequences for C. neoformans var. neoformans were obtained from the TIGR database ( http://www.tigr.org/ ).

Techniques: Generated, Labeling

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: Journal of Fungi

Article Title: Base Excision Repair AP-Endonucleases-Like Genes Modulate DNA Damage Response and Virulence of the Human Pathogen Cryptococcus neoformans

doi: 10.3390/jof7020133

Figure Lengend Snippet: Comparative analysis of Apn deduced amino acid sequences. APN1 -like (CNAG_05468) and APN2 -like (CNAG_04268) gene sequences from C. neoformans were used as query for Blastp analyses. ( A ) Alignment results of Apn1 amino acid sequences from C. albicans , S. pombe , S. cerevisiae and E. coli , highlighting the cover and identity with the C. neoformans Apn1-like amino acid sequence. ( B ) Alignment results of Apn2 sequences from S. pombe , H. sapiens , C. albicans , S. cerevisiae and E. coli , highlighting the cover and identity with the C. neoformans Apn2-like deduced amino acid sequences. NCBI accession numbers are depicted in each protein representation. Triangles represent key-amino acids for the catalytic sites; Ident: percentage of amino acid identity provided by Blastp analysis on NCBI. EEP: exonuclease-endonuclease-phosphatase. zf-GRF: zinc finger domain.

Article Snippet: The supernatant was removed and 2.5 × 10 5 yeast cells of C. neoformans (Multiplicity of Infection; MOI = 5) were added to each well in 100 μL of RPMI medium supplemented with 10% FBS and the 18B7 mAb (kindly donated by Dr. Arturo Casadevall, Johns Hopkins University, Baltimore, MD, USA) at 10 μg/mL.

Techniques: Sequencing

Journal: Journal of Fungi

Article Title: Base Excision Repair AP-Endonucleases-Like Genes Modulate DNA Damage Response and Virulence of the Human Pathogen Cryptococcus neoformans

doi: 10.3390/jof7020133

Figure Lengend Snippet: Disruption of Apn1- and Apn2-like genes induced C. neoformans susceptibility to UV radiation. ( A ) Ten-fold serial dilutions of C. neoformans WT, mutant and reconstituted strains of yeasts were exposed to 120, 240 and 480 J/m 2 doses of UV radiation (254 nm) and grown at 30 °C, for 48 h. The results are representative of at least three independent experiments. ( B ) Cell viability (%) after UV irradiation. C. neoformans 5 × 10 2 cells were plated on YPD agar and exposed to UV radiation at 120, 240 and 480 J/m 2 at 254 nm. The plates were incubated at 30 °C for 48 h, protected from light, for colony forming unit (CFU) counting. Graph represents the percentage of viable cells normalized by the percentage of viable control cells. The WT H99 strain was used as control for statistical comparison. One-way analysis of variance (ANOVA) with Dunnett’s post-test was used to compare the means of results from three independent experiments. Error bars represent standard errors of the mean. **** p < 0.0001.

Article Snippet: The supernatant was removed and 2.5 × 10 5 yeast cells of C. neoformans (Multiplicity of Infection; MOI = 5) were added to each well in 100 μL of RPMI medium supplemented with 10% FBS and the 18B7 mAb (kindly donated by Dr. Arturo Casadevall, Johns Hopkins University, Baltimore, MD, USA) at 10 μg/mL.

Techniques: Disruption, Mutagenesis, Irradiation, Incubation, Control, Comparison

Journal: Journal of Fungi

Article Title: Base Excision Repair AP-Endonucleases-Like Genes Modulate DNA Damage Response and Virulence of the Human Pathogen Cryptococcus neoformans

doi: 10.3390/jof7020133

Figure Lengend Snippet: The ablation of the predicted Apn1 and Apn2 proteins sensitized C. neoformans yeasts to the exposure to Zeocin. ( A ) Cell viability of the WT and mutant strains after incubation with different concentrations of Zeocin. Yeasts from WT and mutant strains were cultured in liquid YPD containing 0.04%, 0.08%, 0.12% or 0.16% Zeocin for 1 h, at 30 °C, 150 rpm. Cells were then PBS-washed and plated on YPD agar for CFU counting. Percent of viable cells was calculated from the CFU counting. Data are presented as mean of three replicates. Statistical test: One-way ANOVA with Dunnett’s post-test, * p < 0.05. ( B ) Electrophoretic analysis in 1% agarose gel stained with 0.5 µg/mL EtBr of C . neoformans strains DNA after exposure to 0.08% Zeocin for 1 h. Unexposed mutant and H99 strains presented DNA with no fragmentation. Genomic DNA from the unexposed H99 strain was used as control. The result is representative of three independent experiments.

Article Snippet: The supernatant was removed and 2.5 × 10 5 yeast cells of C. neoformans (Multiplicity of Infection; MOI = 5) were added to each well in 100 μL of RPMI medium supplemented with 10% FBS and the 18B7 mAb (kindly donated by Dr. Arturo Casadevall, Johns Hopkins University, Baltimore, MD, USA) at 10 μg/mL.

Techniques: Mutagenesis, Incubation, Cell Culture, Agarose Gel Electrophoresis, Staining, Control

Journal: Journal of Fungi

Article Title: Base Excision Repair AP-Endonucleases-Like Genes Modulate DNA Damage Response and Virulence of the Human Pathogen Cryptococcus neoformans

doi: 10.3390/jof7020133

Figure Lengend Snippet: Methoxy-amine (MX) enhances the effects of H 2 O 2 on the viability of the C . neoformans apn 1Δ apn 2Δ mutant strain yeasts. Cell survival of C . neoformans WT and mutant strains for the combined exposure to MX and H 2 O 2 Strains were cultured in liquid YPD containing 0.417 mg/mL MX and ( A ) 3 or ( B ) 5 mM H 2 O 2 , for 1 h, at 30 °C and 150 rpm. Cells incubated only in YPD containing 3 or 5 mM H 2 O 2 were used as comparison. The graph represents the percent of survival of each strain exposed to each condition described compared to the control condition (YPD medium). Two-way ANOVA with Sidak’s multiple-comparisons test was used to compare the means of results from three independent experiments. Error bars represent standard errors of the mean. **** p < 0.0001; *** p < 0.001.

Article Snippet: The supernatant was removed and 2.5 × 10 5 yeast cells of C. neoformans (Multiplicity of Infection; MOI = 5) were added to each well in 100 μL of RPMI medium supplemented with 10% FBS and the 18B7 mAb (kindly donated by Dr. Arturo Casadevall, Johns Hopkins University, Baltimore, MD, USA) at 10 μg/mL.

Techniques: Mutagenesis, Cell Culture, Incubation, Comparison, Control

Journal: Journal of Fungi

Article Title: Base Excision Repair AP-Endonucleases-Like Genes Modulate DNA Damage Response and Virulence of the Human Pathogen Cryptococcus neoformans

doi: 10.3390/jof7020133

Figure Lengend Snippet: The disruption of the Apn2-like gene of C. neoformans impairs melanin production. C. neoformans yeasts were incubated in melanin-inducing minimal medium with 1 mM l -DOPA at 37 °C, 150 rpm, protected from light. Melanin production was visually assessed by the medium color change from translucent to brown and photo-documented at 72 and 144 h. The result is representative of three independent experiments.

Article Snippet: The supernatant was removed and 2.5 × 10 5 yeast cells of C. neoformans (Multiplicity of Infection; MOI = 5) were added to each well in 100 μL of RPMI medium supplemented with 10% FBS and the 18B7 mAb (kindly donated by Dr. Arturo Casadevall, Johns Hopkins University, Baltimore, MD, USA) at 10 μg/mL.

Techniques: Disruption, Incubation

Journal: Journal of Fungi

Article Title: Base Excision Repair AP-Endonucleases-Like Genes Modulate DNA Damage Response and Virulence of the Human Pathogen Cryptococcus neoformans

doi: 10.3390/jof7020133

Figure Lengend Snippet: Apn1- and Apn2-like genes are required for melanin production by C. neoformans . ( A ) Viability of C. neoformans cells after exposure to methoxy-amine (MX). Cells of the WT and mutant strains were incubated in liquid YPD medium for 1 h, at 30 °C, and 150 rpm in the absence (control) or in the presence of MX (0, 0.417, 0.83 or 1.67 mg/mL). Cells were then PBS-washed and the number of CFUs was assessed. The graph represents the percent of survival of each strain exposed to MX compared to the control condition. The wild-type H99 was used as a control group for statistical comparison. One-way ANOVA with Dunnett post-test was used to compare the means of results from three independent experiments. Error bars represent standard errors of the mean. **** p < 0.0001; *** p < 0.001; * p < 0.05. ( B ) Melanin production after exposure of yeast cells to MX. Cells from each strain were incubated with 0.417 mg/mL MX for 1 h, at 37 °C, PBS-washed, diluted and inoculated for melanin production analyses. Visual detection was performed daily and photo-documented after 72, 96 and 144 h of growth. Results are representative of three independent experiments. ( C ) Cell viability of C. neoformans strains after MX exposure and induction of melanin synthesis. Aliquots of cell suspensions were collected from the melanin assay plates after 144 h of incubation, and viable colonies were assessed by CFU counting. One-way ANOVA with Dunnett’s post-test was used to compare the means of results from three independent experiments.

Article Snippet: The supernatant was removed and 2.5 × 10 5 yeast cells of C. neoformans (Multiplicity of Infection; MOI = 5) were added to each well in 100 μL of RPMI medium supplemented with 10% FBS and the 18B7 mAb (kindly donated by Dr. Arturo Casadevall, Johns Hopkins University, Baltimore, MD, USA) at 10 μg/mL.

Techniques: Mutagenesis, Incubation, Control, Comparison

Journal: Journal of Fungi

Article Title: Base Excision Repair AP-Endonucleases-Like Genes Modulate DNA Damage Response and Virulence of the Human Pathogen Cryptococcus neoformans

doi: 10.3390/jof7020133

Figure Lengend Snippet: The deletion of APN-like genes results in C. neoformans reduced phagocytosis and survival in macrophages in vitro. Bone marrow-derived macrophages (BMDMs) were co-cultured for 2 h or 24 h with previously opsonized fungal cells from the different strains of C. neoformans . Each strain was inoculated into three wells of a 96-well polystyrene plate containing macrophages at a MOI of 5. ( A ) Percentage of phagocytosis. Data are presented as mean ±95% C.I. ( n = 3 experiments, *** p < 0.001). ( B ) Phagocytic index. ( C ) CFU counts of C. neoformans strains recovered from bone marrow-derived macrophages (BMDMs) after 2 ( D ) or 24 h of co-incubation. Error bars represent standard errors of the mean of three biological replicate experiments. The wild-type H99 was used as a control group for statistical comparison. Statistical test: One-way ANOVA with Dunnett’s post-test, ** p < 0.01.

Article Snippet: The supernatant was removed and 2.5 × 10 5 yeast cells of C. neoformans (Multiplicity of Infection; MOI = 5) were added to each well in 100 μL of RPMI medium supplemented with 10% FBS and the 18B7 mAb (kindly donated by Dr. Arturo Casadevall, Johns Hopkins University, Baltimore, MD, USA) at 10 μg/mL.

Techniques: In Vitro, Derivative Assay, Cell Culture, Incubation, Control, Comparison