Journal: PLoS ONE

Article Title: Chromosomal Polymorphism in the Sporothrix schenckii Complex

doi: 10.1371/journal.pone.0086819

Figure Lengend Snippet: A: The average minimum genome size for different Sporothrix species was estimated by summing all the chromosomal bands detected after pulsed field gel electrophoresis. B: Comparison of genome sizes estimated from genome sequencing (blue) and estimated from the sum of chromosomal bands (red) for isolates ATCC MYA-4821 ( S. schenckii ) and ATCC MYA-4823 ( S. brasiliensis ); the differences in size estimated with the different methodologies are indicated on the right.

Article Snippet: ATCC MYA-4821 , CBS 132984, Ss323 , S. schenckii , Human , USA , , KF574470 , JQ070112 , This study.

Techniques: Pulsed-Field Gel, Electrophoresis, Sequencing

Journal: PLoS ONE

Article Title: Chromosomal Polymorphism in the Sporothrix schenckii Complex

doi: 10.1371/journal.pone.0086819

Figure Lengend Snippet: Strains, species, origin, and GenBank accession numbers (CAL and ITS) for the Sporothrix spp. isolates used in this study.

Article Snippet: The haploid genome sizes of S. schenckii (ATCC MYA-4821) and S. brasiliensis (ATCC MYA-4823), estimated from DNA sequencing, were 32.5 Mb and 33.4 Mb, respectively (Marcus de Melo Teixeira and Maria Sueli Soares Felipe, personal communication).

Techniques:

Journal: PLoS ONE

Article Title: Chromosomal Polymorphism in the Sporothrix schenckii Complex

doi: 10.1371/journal.pone.0086819

Figure Lengend Snippet: Each species is grouped inside a colored rectangle. Red: S. schenckii ; Yellow: S. brasiliensis ; Blue: S. mexicana ; Purple: S. luriei . The sizes of chromosomal bands (Mb) are shown on the left. Σ: The minimum genome sizes estimated by summing the chromosomal bands (Mb) are shown at the bottom.

Article Snippet: The haploid genome sizes of S. schenckii (ATCC MYA-4821) and S. brasiliensis (ATCC MYA-4823), estimated from DNA sequencing, were 32.5 Mb and 33.4 Mb, respectively (Marcus de Melo Teixeira and Maria Sueli Soares Felipe, personal communication).

Techniques:

Journal: PLoS ONE

Article Title: Chromosomal Polymorphism in the Sporothrix schenckii Complex

doi: 10.1371/journal.pone.0086819

Figure Lengend Snippet: The S. schenckii and S. brasiliensis isolates are indicated at the top. Each grey box represents a chromosomal band with the indicated size (Mb, left ). The probes that hybridized to each band are color-coded ( bottom ). β-tubulin (β-Tub), calmodulin (Calm), catalase (Cata), chitin synthase 1 (CHS1), internal transcribed spacer (ITS), Pho85 cyclin-dependent kinase (Pho85), protein kinase C Ss-2 (PKC), G protein α subunit (GProt), and topoisomerase II (Topo).

Article Snippet: The haploid genome sizes of S. schenckii (ATCC MYA-4821) and S. brasiliensis (ATCC MYA-4823), estimated from DNA sequencing, were 32.5 Mb and 33.4 Mb, respectively (Marcus de Melo Teixeira and Maria Sueli Soares Felipe, personal communication).

Techniques:

Journal: PLoS ONE

Article Title: Chromosomal Polymorphism in the Sporothrix schenckii Complex

doi: 10.1371/journal.pone.0086819

Figure Lengend Snippet: A: The average minimum genome size for different Sporothrix species was estimated by summing all the chromosomal bands detected after pulsed field gel electrophoresis. B: Comparison of genome sizes estimated from genome sequencing (blue) and estimated from the sum of chromosomal bands (red) for isolates ATCC MYA-4821 ( S. schenckii ) and ATCC MYA-4823 ( S. brasiliensis ); the differences in size estimated with the different methodologies are indicated on the right.

Article Snippet: The haploid genome sizes of S. schenckii (ATCC MYA-4821) and S. brasiliensis (ATCC MYA-4823), estimated from DNA sequencing, were 32.5 Mb and 33.4 Mb, respectively (Marcus de Melo Teixeira and Maria Sueli Soares Felipe, personal communication).

Techniques: Pulsed-Field Gel, Electrophoresis, Sequencing

Journal: Journal of Fungi

Article Title: Silencing of ROT2 , the Encoding Gene of the Endoplasmic Reticulum Glucosidase II, Affects the Cell Wall and the Sporothrix schenckii –Host Interaction

doi: 10.3390/jof8111220

Figure Lengend Snippet: ROT2 expression and analysis of binary vector insertional events in Sporothrix schenckii wild-type and mutant strains. In ( A ), total RNA was isolated from the different mutant strains and used in RT-qPCR reactions to amplify a specific part of the ROT2 open reading frame. In ( B ), genomic DNA was isolated from the different strains and used in qPCR reactions that amplified the same DNA fragment used for gene expression analysis. In both cases, the amplification of the gene encoding the ribosomal protein L6 was used for data normalization. Data are means ± SD of three independent experiments performed in duplicates. * p < 0.05 when compared to the WT, HSS31, or HSS32 strains.

Article Snippet: S. schenckii ATCC MYA-4821 [ ] was used to generate the silenced strains analyzed in this work and is referred to as the wild-type (WT) strain.

Techniques: Expressing, Plasmid Preparation, Mutagenesis, Isolation, Quantitative RT-PCR, Amplification

Journal: Journal of Fungi

Article Title: Silencing of ROT2 , the Encoding Gene of the Endoplasmic Reticulum Glucosidase II, Affects the Cell Wall and the Sporothrix schenckii –Host Interaction

doi: 10.3390/jof8111220

Figure Lengend Snippet: Detection of glucose in the Sporothrix schenckii cell wall N -linked glycan core. The N -linked glycans were trimmed from the yeast-like cell wall by incubating with 25 U of endoglycosidase H, the released N -linked glycans were filtrated in a 3 kDa-exclusion filtration unit, and the eluted material was hydrolyzed with recombinant α-glucosidase and analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection. The arrow indicated the elution time of glucose. ( A ) A sample from the WT strain; ( B ) a sample from the ROT2 -silenced mutant strain HSS34; ( C ) a sample from the ROT2 -silenced mutant strain HSS38. Chromatograms are representative of experiments performed three times per duplicate.

Article Snippet: S. schenckii ATCC MYA-4821 [ ] was used to generate the silenced strains analyzed in this work and is referred to as the wild-type (WT) strain.

Techniques: Filtration, Recombinant, Chromatography, Mutagenesis

Journal: Journal of Fungi

Article Title: Silencing of ROT2 , the Encoding Gene of the Endoplasmic Reticulum Glucosidase II, Affects the Cell Wall and the Sporothrix schenckii –Host Interaction

doi: 10.3390/jof8111220

Figure Lengend Snippet: Cell wall analysis of Sporothrix schenckii wild-type, control, and ROT2 -silenced mutant strains. In ( A ), the cell wall was isolated from yeast-like cells and acid hydrolyzed, and monosaccharides were identified and quantified by high-performance anion-exchange chromatography with pulsed amperometric detection. In ( B ), yeast-like cells were incubated with either fluorescein isothiocyanate (FITC) conjugated wheat germ agglutinin or IgG Fc-Dectin-1 chimera and anti-Fc IgG-FITC for chitin or β-1,3-glucan labeling, respectively, and the fluorescence associated to 300 cells was estimated. Data were normalized to the fluorescence observed in heat-killed cells, which corresponds to 100% in both cases. Data are represented as mean ± SD of three independent experiments performed in duplicates. * p < 0.05 when compared to WT, HSS31, or HSS32 cells.

Article Snippet: S. schenckii ATCC MYA-4821 [ ] was used to generate the silenced strains analyzed in this work and is referred to as the wild-type (WT) strain.

Techniques: Mutagenesis, Isolation, Chromatography, Incubation, Labeling, Fluorescence

Journal: Journal of Fungi

Article Title: Silencing of ROT2 , the Encoding Gene of the Endoplasmic Reticulum Glucosidase II, Affects the Cell Wall and the Sporothrix schenckii –Host Interaction

doi: 10.3390/jof8111220

Figure Lengend Snippet: Cell wall protein content, porosity, and ability to bind Alcian blue of Sporothrix schenckii wild-type, control, and ROT2 -silenced strains.

Article Snippet: S. schenckii ATCC MYA-4821 [ ] was used to generate the silenced strains analyzed in this work and is referred to as the wild-type (WT) strain.

Techniques:

Journal: Journal of Fungi

Article Title: Silencing of ROT2 , the Encoding Gene of the Endoplasmic Reticulum Glucosidase II, Affects the Cell Wall and the Sporothrix schenckii –Host Interaction

doi: 10.3390/jof8111220

Figure Lengend Snippet: Cytokine stimulation by human peripheral blood mononuclear cells stimulated with Sporothrix schenckii wild-type, control, and ROT2 -silenced strains. Human peripheral blood mononuclear cells were preincubated for 60 min with 200 μg mL −1 laminarin or 10 μg mL −1 of any of the following antibodies: anti-mannose receptor, anti-CR3 receptor, anti-TLR2, or anti-TLR4; then, they were coincubated with yeast-like cells for 24 h. In all cases, the interactions were centrifuged, supernatants collected, and cytokines quantified by ELISA. None refers to the system where human cells were preincubated with PBS and then challenged with the fungal cells. Ab, antibody. Control refers to wells where the human cells were preincubated only with PBS. Results are the media ± standard deviation from data generated with samples from eight donors, analyzed by duplicate. * p < 0.05, when compared with the cytokine level stimulated by WT strain. † p < 0.05, when compared with the same strain with no preincubation step.

Article Snippet: S. schenckii ATCC MYA-4821 [ ] was used to generate the silenced strains analyzed in this work and is referred to as the wild-type (WT) strain.

Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation, Generated

Journal: Journal of Fungi

Article Title: Silencing of ROT2 , the Encoding Gene of the Endoplasmic Reticulum Glucosidase II, Affects the Cell Wall and the Sporothrix schenckii –Host Interaction

doi: 10.3390/jof8111220

Figure Lengend Snippet: Phagocytosis of Sporothrix schenckii wild-type, control, and ROT2 -silenced strains by human monocyte-derived macrophages. In ( A ), yeast-like cells were labeled with Acridine Orange, coincubated with human monocyte-derived macrophages at a macrophage–fungus ratio of 1:6, for 2 h at 37 °C and 5% ( v / v ) CO 2 . Human cells were gated by FACS, and 50,000 events, defined as a human cell interacting with at least one fluorescent fungal cell, were counted per sample. Control, macrophages interacting with no yeast-like cells. * p < 0.05 when compared to WT, HSS31, or HSS32 strains. † p < 0.05 when compared with HSS33, HSS34, or HSSS35 strains. In ( B ), experiments described in panel A were performed, but macrophages were previously preincubated with 10 μg mL −1 of any of the following antibodies: anti-mannose receptor, anti-CR3, anti-TLR2, or anti-TLR4. Alternatively, the human cells were preincubated with 200 μg mL −1 laminarin. In all cases, the interactions were performed in the presence of 5 μg mL −1 polymyxin B. No treatment refers to cells preincubated only with 5 μg mL −1 polymyxin B. Results correspond to cells in the late stage of phagocytosis. For all cases, 100% corresponds to the system with no treatment, and the absolute values were similar to those shown in panel A. CR3, complement receptor 3. * p < 0.05 when compared to the no treatment condition of the same strain. For both panels, data represent means ± SD from six donors assayed by duplicate.

Article Snippet: S. schenckii ATCC MYA-4821 [ ] was used to generate the silenced strains analyzed in this work and is referred to as the wild-type (WT) strain.

Techniques: Derivative Assay, Labeling

Journal: Journal of Fungi

Article Title: Silencing of ROT2 , the Encoding Gene of the Endoplasmic Reticulum Glucosidase II, Affects the Cell Wall and the Sporothrix schenckii –Host Interaction

doi: 10.3390/jof8111220

Figure Lengend Snippet: Galleria mellonella killing curves by Sporothrix schenckii wild-type, control, and ROT2 -silenced strains. Animal groups contained 30 larvae and were inoculated with 1 × 10 5 yeast-like cells of the different strains, and survival was recorded daily for two weeks. Data are shown in Kaplan–Meier plots. The statistical analysis showed no differences among the WT, HSS31, and HSS32 strains ( p = 0.57), among HSS33, HSS34, and HSS35 ( p = 0.88), or among HSS36, HSS37, and HSS38 ( p = 0.79). However, the two groups of silenced strains showed curves statistically different when compared between them or with the control group (in black lines; p < 0.05, for both cases).

Article Snippet: S. schenckii ATCC MYA-4821 [ ] was used to generate the silenced strains analyzed in this work and is referred to as the wild-type (WT) strain.

Techniques:

Journal: Journal of Fungi

Article Title: Silencing of ROT2 , the Encoding Gene of the Endoplasmic Reticulum Glucosidase II, Affects the Cell Wall and the Sporothrix schenckii –Host Interaction

doi: 10.3390/jof8111220

Figure Lengend Snippet: Cytotoxicity and immunological parameters of Galleria mellonella larvae infected with different strains of S. schenckii . G. mellonella larvae were infected with the different fungal strains as described in the Materials and Methods section and incubated for 24 h at 37 °C before animal decapitation and hemolymph collection. The cell-free hemolymph was used to measure lactate dehydrogenase activity, an indicator of cytotoxicity. The 100% cytotoxicity was the enzyme activity measured in lysate hemocytes. The other three parameters were quantified in anticoagulated total hemolymph. PBS, animal group inoculated only with PBS. Data represent means ± SD from ten animals per group, assayed by duplicate. * p < 0.05 when compared to wild-type (WT) strain. † p < 0.05 when compared to strains HSS33, HSS34, or HSS35.

Article Snippet: S. schenckii ATCC MYA-4821 [ ] was used to generate the silenced strains analyzed in this work and is referred to as the wild-type (WT) strain.

Techniques: Infection, Incubation, Activity Assay