Journal: The Cell Surface

Article Title: Disruption of protein rhamnosylation affects the Sporothrix schenckii- host interaction

doi: 10.1016/j.tcsw.2021.100058

Figure Lengend Snippet: Strains used in this work.

Article Snippet: HSS20 , Sporothrix schenckii , 1099–18 ATCC MYA 4821 transformed with pBGgHg- RmlD , This work.

Techniques: Transformation Assay

Journal: The Cell Surface

Article Title: Disruption of protein rhamnosylation affects the Sporothrix schenckii- host interaction

doi: 10.1016/j.tcsw.2021.100058

Figure Lengend Snippet: Analysis of RmlD expression and binary vector insertional events . In A, RT-qPCR reactions amplifying a 243 bp fragment of RmlD open reading frame were used to assess gene expression. In B, analysis of the binary vector insertional events by qPCR, amplifying the same fragment described in A. In both cases, the amplification of the gene encoding for the ribosomal protein L6 data was used for data normalization. Data are means ± SD of three independent experiments performed in duplicates. * P < 0.05 when compared to the WT strain. WT, strain 1099–18 ATCC MYA 4821. Strains HSS29 and HSS30 were transformed with pBGgHg; while HSS20-HSS25 with pBGgHg- RmlD .

Article Snippet: HSS20 , Sporothrix schenckii , 1099–18 ATCC MYA 4821 transformed with pBGgHg- RmlD , This work.

Techniques: Expressing, Plasmid Preparation, Quantitative RT-PCR, Amplification, Transformation Assay

Journal: The Cell Surface

Article Title: Disruption of protein rhamnosylation affects the Sporothrix schenckii- host interaction

doi: 10.1016/j.tcsw.2021.100058

Figure Lengend Snippet: Doubling times of Sporothrix schenckii wild-type, control, and RmlD -silenced strains. For hyphae, cells were grown in YPD, pH 4.5, and the biomass dry weight determined every 2 h. For yeast-like cells, these were grown in YPD, pH 7.8, and cells quantified in a hemocytometer. From the generated growth curves, the doubling time was calculated for each strain. WT, strain 1099–18 ATCC MYA 4821. Strains HSS29 and HSS30 were transformed with pBGgHg; while HSS20-HSS25 with pBGgHg- RmlD .

Article Snippet: HSS20 , Sporothrix schenckii , 1099–18 ATCC MYA 4821 transformed with pBGgHg- RmlD , This work.

Techniques: Generated, Transformation Assay

Journal: The Cell Surface

Article Title: Disruption of protein rhamnosylation affects the Sporothrix schenckii- host interaction

doi: 10.1016/j.tcsw.2021.100058

Figure Lengend Snippet: The RmlD silencing in Sporothrix schenckii affects the rhamnose content in both N -linked and O -linked glycans from the cell wall. The N -linked (A) or O -linked (B) glycans, were trimmed from the cell wall by incubating yeast-like cells with either endoglycosidase H or sodium hydroxide, respectively, the oligosaccharides acid hydrolyzed and monosaccharide content analyzed by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection. Data are means ± SD of three independent experiments performed in duplicates. * P < 0.05 when compared to WT or control cells. † P < 0.05 when compared to WT or control cells, or HSS20-HSS22 strains. WT, strain 1099–18 ATCC MYA 4821. Strains HSS29 and HSS30 were transformed with pBGgHg; while HSS20-HSS25 with pBGgHg- RmlD .

Article Snippet: HSS20 , Sporothrix schenckii , 1099–18 ATCC MYA 4821 transformed with pBGgHg- RmlD , This work.

Techniques: Chromatography, Transformation Assay

Journal: The Cell Surface

Article Title: Disruption of protein rhamnosylation affects the Sporothrix schenckii- host interaction

doi: 10.1016/j.tcsw.2021.100058

Figure Lengend Snippet: Cell wall chitin and β-1,3-glucan labeling in Sporothrix schenckii wild-type, control, and RmlD -silenced strains. Yeast-like cells were labeled with fluorescein isothiocyanate conjugated-wheat germ agglutinin for chitin staining (A) or IgG Fc-Dectin-1 chimera for β-1,3-glucan staining (B) as described in Materials and methods, inspected under fluorescence microscopy, and the fluorescence of 300 cells randomly selected was calculated. Data are means ± SD of three independent experiments performed in duplicates. * P < 0.05 when compared to the WT strain. WT, strain 1099–18 ATCC MYA 4821. Strains HSS29 and HSS30 were transformed with pBGgHg; while HSS20-HSS25 with pBGgHg- RmlD .

Article Snippet: HSS20 , Sporothrix schenckii , 1099–18 ATCC MYA 4821 transformed with pBGgHg- RmlD , This work.

Techniques: Labeling, Staining, Fluorescence, Microscopy, Transformation Assay

Journal: The Cell Surface

Article Title: Disruption of protein rhamnosylation affects the Sporothrix schenckii- host interaction

doi: 10.1016/j.tcsw.2021.100058

Figure Lengend Snippet: Cytokine stimulation by human peripheral blood mononuclear cells. Yeast-like cells and human peripheral blood mononuclear cells (PBMCs) were coincubated, the supernatants were collected and the concentration of TNFα, IL-6, IL1β, and IL-10 was measured by ELISA. Data are means ± SD obtained with samples from eight donors, each assayed in duplicate wells. * P < 0.05 when compared to WT cells. † P < 0.05 when compared to cells under no treatment from the same strain. No treatment, PBMCs preincubated with 5 μg mL −1 polymyxin B; Control anti-TLR2, PBMCs preincubated with 5 μg mL −1 polymyxin B and 10 μg mL −1 IgG 2 aκ; Control anti-TLR4, PBMCs preincubated with 5 μg mL −1 polymyxin B and 10 μg mL −1 IgG 1 ; + anti-TLR2, PBMCs preincubated with 5 μg mL −1 polymyxin B and 10 μg mL −1 anti-TLR2; + anti-TLR4, preincubated with 5 μg mL −1 polymyxin B and 10 μg mL −1 anti-TLR4; + laminarin, preincubated with 5 μg mL −1 polymyxin B and 200 μg mL −1 laminarin. Control, mock reactions were no fungal cells were included. WT, strain 1099–18 ATCC MYA 4821. Strains HSS29 and HSS30 were transformed with pBGgHg; while HSS20-HSS25 with pBGgHg- RmlD .

Article Snippet: HSS20 , Sporothrix schenckii , 1099–18 ATCC MYA 4821 transformed with pBGgHg- RmlD , This work.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Transformation Assay

Journal: The Cell Surface

Article Title: Disruption of protein rhamnosylation affects the Sporothrix schenckii- host interaction

doi: 10.1016/j.tcsw.2021.100058

Figure Lengend Snippet: Phagocytosis of RmlD -silenced S. schenckii by human monocyte-derived macrophages. In A, Acridine Orange-labeled yeast-like cells were coincubated with human monocyte-derived macrophages at a macrophage-yeast ratio 1:6, for 2 h at 37 °C and 5% (v/v) CO 2 . The macrophages were gated by FACS and 50,000 cells were counted per sample. Macrophages interacting with at least one fluorescent yeast cell were included as counted events. Control, macrophages interacting with no yeast-like cells. * P < 0.05 when compared to WT strain. In B, similar experiments to those described in panel A were performed but with human cells previously preincubated with 5 μg mL −1 polymyxin B and 10 μg mL −1 anti-TLR2 (+anti-TLR2), with 5 μg mL −1 polymyxin B and 200 μg mL −1 L-rhamnose (+Rhamnose), 5 μg mL −1 polymyxin B and 10 μg mL −1 anti-TLR4 (+anti-TLR4), or with 5 μg mL −1 polymyxin B and 200 μg mL −1 laminarin (+Laminarin). 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. Control, macrophages interacting with no yeast-like cells. * P < 0.05 when compared to the no treatment condition of the same strain. † P < 0.05 when compared to the no treatment condition of the same strains and other strains under the same condition. ‡ P < 0.05 when compared to WT strain under the same condition. For both panels, data represent means ± SD from six donors assayed by duplicate. WT, strain 1099–18 ATCC MYA 4821. Strains HSS29 and HSS30 were transformed with pBGgHg; while HSS20-HSS25 with pBGgHg- RmlD . (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: HSS20 , Sporothrix schenckii , 1099–18 ATCC MYA 4821 transformed with pBGgHg- RmlD , This work.

Techniques: Derivative Assay, Labeling, Transformation Assay

Journal: The Cell Surface

Article Title: Disruption of protein rhamnosylation affects the Sporothrix schenckii- host interaction

doi: 10.1016/j.tcsw.2021.100058

Figure Lengend Snippet: Mortality of Galleria mellonella larvae inoculated with RmlD -silenced Sporothrix schenckii . Groups containing 30 larvae were inoculated with 1 × 10 5 yeast-like cells in 10 μL of PBS and survival recorded daily for 15 days. Data are shown in Kaplan–Meier plots. The statistical analysis showed no differences among the WT, HSS29, and HSS30 strains ( P = 0.68), but the silenced strains generated survival curves with significantly increased median survival times ( P < 0.05). When compared among them, no differences were observed in the curves generated with strains HSS20, HSS21, and HSS22 ( P = 0.76), nor with those generated with strains HSS23, HSS24, and HSS25 ( P = 0.88). WT, strain 1099–18 ATCC MYA 4821. Strains HSS29 and HSS30 were transformed with pBGgHg; while HSS20-HSS25 with pBGgHg- RmlD .

Article Snippet: HSS20 , Sporothrix schenckii , 1099–18 ATCC MYA 4821 transformed with pBGgHg- RmlD , This work.

Techniques: Generated, Transformation Assay

Journal: The Cell Surface

Article Title: Disruption of protein rhamnosylation affects the Sporothrix schenckii- host interaction

doi: 10.1016/j.tcsw.2021.100058

Figure Lengend Snippet: Strains used in this work.

Article Snippet: 1099–18 ATCC MYA 4821 , Sporothrix schenckii , Wild-type , ( ) .

Techniques: Transformation Assay

Journal: The Cell Surface

Article Title: Disruption of protein rhamnosylation affects the Sporothrix schenckii- host interaction

doi: 10.1016/j.tcsw.2021.100058

Figure Lengend Snippet: Analysis of RmlD expression and binary vector insertional events . In A, RT-qPCR reactions amplifying a 243 bp fragment of RmlD open reading frame were used to assess gene expression. In B, analysis of the binary vector insertional events by qPCR, amplifying the same fragment described in A. In both cases, the amplification of the gene encoding for the ribosomal protein L6 data was used for data normalization. Data are means ± SD of three independent experiments performed in duplicates. * P < 0.05 when compared to the WT strain. WT, strain 1099–18 ATCC MYA 4821. Strains HSS29 and HSS30 were transformed with pBGgHg; while HSS20-HSS25 with pBGgHg- RmlD .

Article Snippet: 1099–18 ATCC MYA 4821 , Sporothrix schenckii , Wild-type , ( ) .

Techniques: Expressing, Plasmid Preparation, Quantitative RT-PCR, Amplification, Transformation Assay

Journal: The Cell Surface

Article Title: Disruption of protein rhamnosylation affects the Sporothrix schenckii- host interaction

doi: 10.1016/j.tcsw.2021.100058

Figure Lengend Snippet: Doubling times of Sporothrix schenckii wild-type, control, and RmlD -silenced strains. For hyphae, cells were grown in YPD, pH 4.5, and the biomass dry weight determined every 2 h. For yeast-like cells, these were grown in YPD, pH 7.8, and cells quantified in a hemocytometer. From the generated growth curves, the doubling time was calculated for each strain. WT, strain 1099–18 ATCC MYA 4821. Strains HSS29 and HSS30 were transformed with pBGgHg; while HSS20-HSS25 with pBGgHg- RmlD .

Article Snippet: 1099–18 ATCC MYA 4821 , Sporothrix schenckii , Wild-type , ( ) .

Techniques: Generated, Transformation Assay

Journal: The Cell Surface

Article Title: Disruption of protein rhamnosylation affects the Sporothrix schenckii- host interaction

doi: 10.1016/j.tcsw.2021.100058

Figure Lengend Snippet: The RmlD silencing in Sporothrix schenckii affects the rhamnose content in both N -linked and O -linked glycans from the cell wall. The N -linked (A) or O -linked (B) glycans, were trimmed from the cell wall by incubating yeast-like cells with either endoglycosidase H or sodium hydroxide, respectively, the oligosaccharides acid hydrolyzed and monosaccharide content analyzed by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection. Data are means ± SD of three independent experiments performed in duplicates. * P < 0.05 when compared to WT or control cells. † P < 0.05 when compared to WT or control cells, or HSS20-HSS22 strains. WT, strain 1099–18 ATCC MYA 4821. Strains HSS29 and HSS30 were transformed with pBGgHg; while HSS20-HSS25 with pBGgHg- RmlD .

Article Snippet: 1099–18 ATCC MYA 4821 , Sporothrix schenckii , Wild-type , ( ) .

Techniques: Chromatography, Transformation Assay

Journal: The Cell Surface

Article Title: Disruption of protein rhamnosylation affects the Sporothrix schenckii- host interaction

doi: 10.1016/j.tcsw.2021.100058

Figure Lengend Snippet: Cell wall chitin and β-1,3-glucan labeling in Sporothrix schenckii wild-type, control, and RmlD -silenced strains. Yeast-like cells were labeled with fluorescein isothiocyanate conjugated-wheat germ agglutinin for chitin staining (A) or IgG Fc-Dectin-1 chimera for β-1,3-glucan staining (B) as described in Materials and methods, inspected under fluorescence microscopy, and the fluorescence of 300 cells randomly selected was calculated. Data are means ± SD of three independent experiments performed in duplicates. * P < 0.05 when compared to the WT strain. WT, strain 1099–18 ATCC MYA 4821. Strains HSS29 and HSS30 were transformed with pBGgHg; while HSS20-HSS25 with pBGgHg- RmlD .

Article Snippet: 1099–18 ATCC MYA 4821 , Sporothrix schenckii , Wild-type , ( ) .

Techniques: Labeling, Staining, Fluorescence, Microscopy, Transformation Assay

Journal: The Cell Surface

Article Title: Disruption of protein rhamnosylation affects the Sporothrix schenckii- host interaction

doi: 10.1016/j.tcsw.2021.100058

Figure Lengend Snippet: Cytokine stimulation by human peripheral blood mononuclear cells. Yeast-like cells and human peripheral blood mononuclear cells (PBMCs) were coincubated, the supernatants were collected and the concentration of TNFα, IL-6, IL1β, and IL-10 was measured by ELISA. Data are means ± SD obtained with samples from eight donors, each assayed in duplicate wells. * P < 0.05 when compared to WT cells. † P < 0.05 when compared to cells under no treatment from the same strain. No treatment, PBMCs preincubated with 5 μg mL −1 polymyxin B; Control anti-TLR2, PBMCs preincubated with 5 μg mL −1 polymyxin B and 10 μg mL −1 IgG 2 aκ; Control anti-TLR4, PBMCs preincubated with 5 μg mL −1 polymyxin B and 10 μg mL −1 IgG 1 ; + anti-TLR2, PBMCs preincubated with 5 μg mL −1 polymyxin B and 10 μg mL −1 anti-TLR2; + anti-TLR4, preincubated with 5 μg mL −1 polymyxin B and 10 μg mL −1 anti-TLR4; + laminarin, preincubated with 5 μg mL −1 polymyxin B and 200 μg mL −1 laminarin. Control, mock reactions were no fungal cells were included. WT, strain 1099–18 ATCC MYA 4821. Strains HSS29 and HSS30 were transformed with pBGgHg; while HSS20-HSS25 with pBGgHg- RmlD .

Article Snippet: 1099–18 ATCC MYA 4821 , Sporothrix schenckii , Wild-type , ( ) .

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Transformation Assay

Journal: The Cell Surface

Article Title: Disruption of protein rhamnosylation affects the Sporothrix schenckii- host interaction

doi: 10.1016/j.tcsw.2021.100058

Figure Lengend Snippet: Phagocytosis of RmlD -silenced S. schenckii by human monocyte-derived macrophages. In A, Acridine Orange-labeled yeast-like cells were coincubated with human monocyte-derived macrophages at a macrophage-yeast ratio 1:6, for 2 h at 37 °C and 5% (v/v) CO 2 . The macrophages were gated by FACS and 50,000 cells were counted per sample. Macrophages interacting with at least one fluorescent yeast cell were included as counted events. Control, macrophages interacting with no yeast-like cells. * P < 0.05 when compared to WT strain. In B, similar experiments to those described in panel A were performed but with human cells previously preincubated with 5 μg mL −1 polymyxin B and 10 μg mL −1 anti-TLR2 (+anti-TLR2), with 5 μg mL −1 polymyxin B and 200 μg mL −1 L-rhamnose (+Rhamnose), 5 μg mL −1 polymyxin B and 10 μg mL −1 anti-TLR4 (+anti-TLR4), or with 5 μg mL −1 polymyxin B and 200 μg mL −1 laminarin (+Laminarin). 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. Control, macrophages interacting with no yeast-like cells. * P < 0.05 when compared to the no treatment condition of the same strain. † P < 0.05 when compared to the no treatment condition of the same strains and other strains under the same condition. ‡ P < 0.05 when compared to WT strain under the same condition. For both panels, data represent means ± SD from six donors assayed by duplicate. WT, strain 1099–18 ATCC MYA 4821. Strains HSS29 and HSS30 were transformed with pBGgHg; while HSS20-HSS25 with pBGgHg- RmlD . (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: 1099–18 ATCC MYA 4821 , Sporothrix schenckii , Wild-type , ( ) .

Techniques: Derivative Assay, Labeling, Transformation Assay

Journal: The Cell Surface

Article Title: Disruption of protein rhamnosylation affects the Sporothrix schenckii- host interaction

doi: 10.1016/j.tcsw.2021.100058

Figure Lengend Snippet: Mortality of Galleria mellonella larvae inoculated with RmlD -silenced Sporothrix schenckii . Groups containing 30 larvae were inoculated with 1 × 10 5 yeast-like cells in 10 μL of PBS and survival recorded daily for 15 days. Data are shown in Kaplan–Meier plots. The statistical analysis showed no differences among the WT, HSS29, and HSS30 strains ( P = 0.68), but the silenced strains generated survival curves with significantly increased median survival times ( P < 0.05). When compared among them, no differences were observed in the curves generated with strains HSS20, HSS21, and HSS22 ( P = 0.76), nor with those generated with strains HSS23, HSS24, and HSS25 ( P = 0.88). WT, strain 1099–18 ATCC MYA 4821. Strains HSS29 and HSS30 were transformed with pBGgHg; while HSS20-HSS25 with pBGgHg- RmlD .

Article Snippet: 1099–18 ATCC MYA 4821 , Sporothrix schenckii , Wild-type , ( ) .

Techniques: Generated, Transformation Assay