Journal: Frontiers in Microbiology

Article Title: Chemical disinfection of Encephalitozoon cuniculi : toward evidence-based infection control guidelines

doi: 10.3389/fmicb.2025.1744472

Figure Lengend Snippet: Fluorescent microscopy analysis of Vero cells infected with E. cuniculi spores exposed to various disinfectant regimens. (a) Merged images of PI and Calcofluor White M2R double-staining across different disinfectant treatment groups. (b) Quantitative analysis of fluorescence intensity derived from Calcofluor White M2R staining in each group. Fluorescence intensity was quantified using ImageJ software by measuring the mean fluorescence intensity of Calcofluor White M2R-stained signals from each image captured in the UV channel. Three independent imaging fields were analyzed per group. The control group consisted of cells infected with untreated microsporidia. Statistical analysis was performed using one-way ANOVA, with the mean fluorescence intensity of each treatment group compared to that of the control group. The symbol **** ( p < 0.0001) denotes statistically significant differences between each disinfectant-treated group and the untreated control group. Groups showing statistically significant differences from the control are accordingly indicated in the figure.

Article Snippet: Briefly, samples were fixed with 4% paraformaldehyde for 20 min, washed three times with PBS, and permeabilized with 0.05% Triton X-100 for 25 min. After another PBS wash, the samples were stained with PI (HY-D0815, MCE China) and Calcofluor White (HY-D0367, MCE China) at final concentrations of 10 μg/mL and 1 μg/mL, respectively, and incubated at room temperature for 20 min in the dark ( ).

Techniques: Microscopy, Infection, Double Staining, Fluorescence, Derivative Assay, Staining, Software, Imaging, Control

Journal: Frontiers in Microbiology

Article Title: Chemical disinfection of Encephalitozoon cuniculi : toward evidence-based infection control guidelines

doi: 10.3389/fmicb.2025.1744472

Figure Lengend Snippet: Representative fluorescence micrographs of mouse kidney sections dual-stained with Calcofluor White (CFW) and propidium iodide (PI). The untreated control showed intense staining, the EtOH and Ag groups displayed scattered patches, and all other disinfection groups (1-h rapid-acting or 5-h long-acting) showed absent staining. Images were taken at 200 × magnification (scale bar: 100 μm).

Article Snippet: Briefly, samples were fixed with 4% paraformaldehyde for 20 min, washed three times with PBS, and permeabilized with 0.05% Triton X-100 for 25 min. After another PBS wash, the samples were stained with PI (HY-D0815, MCE China) and Calcofluor White (HY-D0367, MCE China) at final concentrations of 10 μg/mL and 1 μg/mL, respectively, and incubated at room temperature for 20 min in the dark ( ).

Techniques: Fluorescence, Staining, Control

Journal: bioRxiv

Article Title: Toxic eburicol accumulation drives the antifungal activity of azoles against Aspergillus fumigatus

doi: 10.1101/2024.03.02.582832

Figure Lengend Snippet: (A) Ergosterol biosynthesis pathways in A. fumigatus ( Af, top pathway) and S. cerevisiae ( Sc, bottom pathway). In A. fumigatus , C24-methylation of lanosterol by sterol C24-methyltransferase ( Af Erg6A) is favored over C14-methylation. In yeasts, lanosterol is the favored substrate of sterol C14-demethylase ( Sc Erg11). Both pathways converge with the formation of fecosterol ( 10 ), which is then further processed to ergosterol ( 14 ). (B) Proposed alternative sterol biosynthesis pathway upon inhibition of sterol C14-demethylase (AfCyp51A/B and Sc Erg11). In yeast, lanosterol is converted to 14-methylcholesta-8,24-dien-3β-ol ( 17 ), which in turn is converted to 14-methylergosta-8,24(28)-dien-3β-ol ( 15 ). In A. fumigatus , eburicol ( 2 ) is directly converted to 14-methylergosta-8,24(28)-dien-3β-ol ( 15 ). 14-methylergosta-8,24(28)-dien-3β-ol ( 15 ) is then converted to the 14-methylergosta-8,24(28)-dien-3β,6α-diol ( 16 ) which is considered a “toxic diol”. (A and B) Ergosterol biosynthesis enzymes in A. fumigatus ( Af ) and S. cerevisiae ( Sc ): sterol C24-methyltransferase ( Af Erg6A, Af Erg6B and Sc Erg6), sterol C14-demethylase ( Af Cyp51A/B, Sc Erg11), sterol C14-reductase ( Af Erg24A/B, Sc Erg24), sterol C4-demethylase complex ( Af Erg25A/B, Sc Erg25/26/27), sterol C8-isomerase ( Af Erg2, Sc Erg2), sterol C22-desturase ( Af Erg5, Sc Erg5), sterol C5-desaturase ( Af Erg3A/B/C, Sc Erg3), sterol C24 reductase ( Af Erg4A/B, Sc Erg4). Sterols: lanosterol, ( 2 ) eburicol, ( 3 ) 4,4-dimethylergosta-8,14,24(28)-trien-3β-ol, ( 4 ) 4,4-dimethylergosta-8,24(28)-dien-3β-ol, ( 5 ) 4-methylergosta-8,24(28)-dien-3β-ol, ( 6 ) 4,4-dimethylcholesta-8,14,24-trien-3β-ol, ( 7 ) 4,4-dimethylcholesta-8,24-dien-3β-ol, ( 8 ) 4-methylcholesta-8,24-dien-3β-ol, ( 9 ) zymosterol, ( 10 ) fecosterol, ( 11 ) episterol, ( 12 ) ergosta-7,22,24(28)-trien-3β-ol, ( 13 ) ergosta-5,7,22,24(28)-tetraen-3β-ol, ( 14 ) ergosterol, ( 15 ) 14-methylergosta-8,24(28)-dien-3β-ol, ( 16 ) 14-methylergosta-8,24(28)-dien-3β,6α-diol, and ( 17 ) 14-methylcholesta-8,24-dien-3β-ol. (C) Conidia of A. fumigatus wild type and two Candida species ( C. albicans ATCC14053 and C. glabrata ATCC2950) were inoculated in Sabouraud medium and incubated at 37 °C. After 9.5 h of incubation, the samples were either fixed and stored at 4 °C (control) or, after the medium was supplemented with 3 µg ml - voriconazole (+Vori), further incubated at 37 °C. After 15 h additional incubation, the voriconazole-exposed hyphae and yeasts were also fixed. Samples were then stained with calcofluor white and analyzed with a confocal laser scanning microscope. Depicted are representative images of z-stack projections of optical stacks of the calcofluor white fluorescence covering the entire hyphae in focus. Upper panel, voriconazole-treated hyphae; lower panel, controls. Bars represent 10 μm.

Article Snippet: Calcofluor white (Fluorescent brightener 28; #ICNA0215806705) was obtained from VWR International (Radnor, PA, USA).

Techniques: Methylation, Inhibition, Incubation, Staining, Laser-Scanning Microscopy, Fluorescence

Journal: bioRxiv

Article Title: Toxic eburicol accumulation drives the antifungal activity of azoles against Aspergillus fumigatus

doi: 10.1101/2024.03.02.582832

Figure Lengend Snippet: (A) In a series of 10-fold dilutions derived from a starting suspension of 5 × 10 conidia ml - of wild type (wt), the conditional mutant erg6A tetOn , and the deletion mutant Δ erg6B , aliquots of 3 µl were spotted onto AMM agar plates. AMM was supplemented with 20 μg ml −1 doxycycline when indicated. Agar plates were incubated at 37 °C, representative photos were taken after 28 h. (B) 1.5 × 10 conidia of the indicated strains were spotted on AMM agar supplemented with the indicated amount of doxycycline. The plates were then incubated at 37 °C, representative photos were taken after 40 h. (C and D) Conidia of the erg6A tetOn and cyp51A tetOn Δ cyp51B mutants which express mitochondria-targeted GFP (D) or not (C) were inoculated in Sabouraud medium supplemented with 20 µg mL - doxycycline and incubated at 37 °C. After 6 h, doxycycline was depleted by washing the wells three times with Sabouraud medium without doxycycline. The hyphae were then incubated in Sabouraud medium without doxycycline at 37 °C for an additional 40 h. (C) Hyphae were stained with calcofluor white and analyzed with a confocal microscope. Depicted are representative images of z-stack projections of optical stacks of the calcofluor white fluorescence covering the entire hyphae in focus. The right images show magnifications of the framed sections in the left images. Bars represent 50 μm and are applicable to all images in the respective panel. (D, column graph) At the indicated time points after doxycycline depletion, the viability of the hyphae of the indicated strains expressing mitochondria-targeted GFP was analyzed with time-lapse spinning disc confocal microscopy. The bars indicate the percentage of hyphal compartments with evident mitochondrial dynamics (viable compartments). Data points represent the means of five technical replicates for each timepoint, with an average of approx. 80 analyzed compartments per strain for each timepoint. The error bars indicate standard deviations based on the five technical replicates. (D, lower panel) Representative overlay images of the bright field channel and of z-stack projections of optical stacks of the GFP fluorescence covering the entire hyphae in focus. Depicted are exemplary images of a viable hypha with tubular mitochondrial morphology (also showing mitochondrial dynamics in time-lapse microscopy; left image) and of hyphae with fragmented mitochondria (showing no mitochondrial dynamics in time-lapse microscopy; middle image) or with no or cytosolic GFP signal (right image) which were considered to be dead. Bars represent 50 μm and are applicable to all respective subpanels. (E) 1 × 10 conidia of the indicated strains were spread on Sabouraud agar plates. Agar was supplemented with the indicated amount of doxycycline to achieve a different induction of the conditional promoters. Voriconazole Etest strips were applied. The plates were incubated at 37 °C and representative photos were taken after 42 h.

Article Snippet: Calcofluor white (Fluorescent brightener 28; #ICNA0215806705) was obtained from VWR International (Radnor, PA, USA).

Techniques: Derivative Assay, Suspension, Mutagenesis, Incubation, Staining, Microscopy, Fluorescence, Expressing, Confocal Microscopy, Time-lapse Microscopy

Journal: bioRxiv

Article Title: Toxic eburicol accumulation drives the antifungal activity of azoles against Aspergillus fumigatus

doi: 10.1101/2024.03.02.582832

Figure Lengend Snippet: (A) 1.5 × 10 conidia of wild type (wt) or of the conditional squalene epoxidase ( erg9 tetOn ) or of the squalene synthase ( erg1 tetOn ) mutants were spotted on AMM agar supplemented with the indicated amount of doxycycline. The plates were then incubated at 37 °C, representative photos were taken after 40 h. (B) Conidia of the indicated strains were inoculated in Sabouraud medium supplemented with 20 µg ml - doxycycline and incubated at 37 °C. After 6 h, doxycycline was depleted by washing the wells three times with Sabouraud medium without doxycycline. The hyphae were then incubated in Sabouraud medium without doxycycline at 37 °C for an additional 40 h and subsequently stained with calcofluor white and analyzed with a confocal microscope. Depicted are representative images of z-stack projections of optical stacks of the calcofluor white fluorescence covering the entire hyphae in focus. The lower images show magnifications of the framed sections in the upper images. Bars represent 50 μm and are applicable to all images in the respective panel. (C) Conidia of wild-type expressing mitochondria-targeted GFP were inoculated in Sabouraud medium. After 8 h incubation at 37 °C, medium supplemented with 8 µg ml - terbinafine (upper panel) or with 4 µg ml - voriconazole (lower panel). After an additional 16 h incubation at 37 °C, hyphae were analyzed with a fluorescence microscope. Fluorescence signals were analyzed sequentially, first the GFP signal was recorded followed by staining with calcofluor white and recording of the calcofluor white signal. Depicted are representative images of bright-field (left) and z-stack projections of optical stacks of the calcofluor white fluorescence (middle) and GFP fluorescence (right) after deconvolution, that cover the entire hyphae in focus. The bar represents 50 µm and is applicable to all images. (E) 1 × 10 conidia of the indicated strains were spread on Sabouraud agar plates. Agar was supplemented with the indicated amount of doxycycline to achieve a different induction of the conditional promoters. Voriconazole Etest strips were applied. The plates were incubated at 37 °C and representative photos were taken after 42 h.

Article Snippet: Calcofluor white (Fluorescent brightener 28; #ICNA0215806705) was obtained from VWR International (Radnor, PA, USA).

Techniques: Incubation, Staining, Microscopy, Fluorescence, Expressing

Journal: bioRxiv

Article Title: Toxic eburicol accumulation drives the antifungal activity of azoles against Aspergillus fumigatus

doi: 10.1101/2024.03.02.582832

Figure Lengend Snippet: (B) 1 × 10 conidia of wild type (wt) and the indicated strains were spread on Sabouraud agar plates. When indicated, agar was supplemented with 25 μg ml −1 doxycycline. Voriconazole Etest strips were applied. The plates were incubated at 37 °C and representative photos were taken after 42 h. (A) Conidia of wild type and the conditional sterol C5-desaturase mutant ( erg3A tetOn Δ erg3B Δ erg3C ) were inoculated in Sabouraud medium. After 10 h, the samples were either directly analyzed (controls without azole) or the medium was supplemented with 4 µg ml - voriconazole (+Vori). The voriconazole-exposed hyphae were then incubated at 37 °C for another 5 h. The untreated hyphae (upper images per indicated strain) and the voriconazole-treated hyphae (lower images per indicated strain) were stained with calcofluor white and analyzed with a confocal microscope. Depicted are representative images of z-stack projections of optical stacks of the calcofluor white fluorescence covering the entire hyphae in focus. The right panels show magnifications of the framed sections in the left panels. Bars represent 50 μm and are applicable to all images in the respective panel.

Article Snippet: Calcofluor white (Fluorescent brightener 28; #ICNA0215806705) was obtained from VWR International (Radnor, PA, USA).

Techniques: Incubation, Mutagenesis, Staining, Microscopy, Fluorescence