Fig 1.
HFP-TEM of the cell wall of the yeast parasitic phase of S. schenckii (MYA4820) and S. brasiliensis (MYA4823) cultivated for 3, 7 or 10 days in YPD broth, pH 7.8.
The TEM images show a cell wall double layer of S. schenckii and its shedding over time in culture (A) and a thicker and extensive microfibrillar outer layer of S. brasiliensis interconnecting yeast cells (B).
Fig 2.
HFP-TEM of the cell wall of the yeast parasitic phase of S. schenckii MYA4822.
S. schenckii yeast cells of the clinical isolate MYA 4822 were grown for 7 days in YPD broth. The TEM images show a cell wall double layer (A) and the complete shedding of a cell wall layer (B), similarly to the process observed in other S. schenckii clinical isolates (Fig 1A and S1 Fig).
Table 1.
Inner layer cell wall thickness of Sporothrix spp. yeast cells.
Table 2.
Cell wall polysaccharide and amino acid content of S. schenckii and S. brasiliensis yeast cells at different days of growth.
Table 3.
Cell wall carbohydrate content in Sporothrix spp. yeast cell grown either 4 and 10 days in YPD broth.
Fig 3.
Flow cytometry analysis of S. schenckii (MYA4820 10 d) cells and cell wall fractions isolated in a sucrose gradient.
(A) Discontinuous sucrose gradient fractionation of S. schenckii (MYA4820) after 10 days in culture (yeast phase). (B) Unstained whole cells (10 days culture) were analyzed to compensate for autofluorescence and to locate the cell populations. (C) Gradient fractions were analyzed by size and complexity (forward scatter vs. side scatter) and by ConA-Alexa Fluor 594 labeling. The red histogram represents the fluorescence pattern of unstained whole cells, and the blue histogram represents the fluorescence pattern for the labeled ConA-Alexa Fluor 594 gradient fraction; (D) flow cytometry analysis of Triton X-100-permeabilized sucrose gradient fractions stained with propidium iodide (PI) to assess the number of cells (via DNA labeling) present in the sample. Size vs. PI staining is plotted. PI fluorescence was used to detect nuclei (Cells P1 and P2) and nucleic acid contaminated cellular debris (CW particles).
Fig 4.
HFP-TEM of the cell wall of S. brasiliensis yeast parasitic phase.
(A, B) HFP-TEM images at two magnifications show details of long cell wall fibrils (~400 nm) in S. brasiliensis (10 days in culture) interconnecting the yeast cells. In (B) the fibrils form a physical bridge between two S. brasiliensis yeast cells. Scale bars are indicated in each panel.
Fig 5.
Fluorescence microscopy of Sporothrix spp. 4 days yeast cells labeled with ConA and CFW.
S. schenckii (A to C) and S. brasiliensis (D to F) yeast cells were grown for 4 days in YPD broth (exponential growth phase). The yeast cells were labeled with Calcofluor White (B and E) and Con A-Texas Red (C and F). Differential interference contrast (DIC) images of S. schenckii and S. brasiliensis are shown in panels A and D, respectively. The labeled yeast cells were visualized in a Zeiss Confocal Microscope (63X magnification).
Fig 6.
Fluorescence microscopy of S. brasiliensis 7 days yeast cells labeled with ConA, WGA and CFW.
Yeast cells were grown for 7 days in YPD broth (stationary growth phase) and labeled with Con A-Texas Red (A), Calcofluor White (B), and WGA-FITC (C), and the images were overlaid (D). The labeled yeast cells were visualized in a Zeiss Confocal Microscope (63X magnification).
Fig 7.
Infrared spectroscopy of the alkali-insoluble cell wall fraction of S. schenckii (MYA 4820) and S. brasiliensis (MYA 4823).
Infrared spectra of S. schenckii (A) and S. brasiliensis (B) cell wall showing the peaks at energies corresponding to the cell wall polysaccharides chitin (1560 and 1640 cm-1; blue arrows) and β-glucan (890 cm-1; purple arrow). The presence of β-glucans (1→3 and 1→6) are evidenced by peaks at 1156, 1076, and 1041 cm-1 (green arrows).
Fig 8.
1H-NMR of the rhamnomannan fraction isolated from S. schenckii and S. brasiliensis yeast cells.
1H-MNR spectra showing the signals corresponding to the α-L-Rha 1→3 α-D-Manp structure (A and B). The H1 region of the rhamnomannans of S. schenckii (C) and S. brasiliensis (D) is enlarged.
Table 4.
S. schenckii and S. brasiliensis rhamnomannan 13C-NMR signals, yeast phase.
Fig 9.
HFP-TEM images showing the typical rosette-like glycogen alpha-particles in Sporothrix sp. yeast cells.
TEM images show rosette-like glycogen alpha-particles organized close to the plasma membrane and cell wall of (A and B) S. schenckii (MYA4820) and (C and D) S. brasiliensis (MYA4823). The arrowheads indicate rosette-like glycogen alpha-particles.
Fig 10.
Panel illustrating the uptake of Sporothrix spp. by hMac.
Optical microscopy images showing the differences in uptake by human monocyte-derived macrophages of Sporothrix sp. yeast cells cultivated for 4 or 10 days in YPD broth. S. schenckii is shown at 4 days (A) and 10 days (B). S. brasiliensis is shown at 4 days (C) and 10 days (D). (E) hMac uninfected control. The slides were stained with an Instant Prov Kit (Newprov) to determine the internalization of yeast cells.
Fig 11.
Uptake by human monocyte-derived macrophages (hMac) of S. schenckii (MYA 4820) and S. brasiliensis (MYA4823) yeast cells cultivated for 4 and 10 days in YPD broth.
The uptake of S. schenckii and S. brasiliensis yeast cells (4 and 10 days) are reported as the mean values of three experiments performed in duplicate with their standard deviation (SD). Unpaired t-test: (a) p ≥ 0.0001 S. brasiliensis compared with S. schenckii– 4 days yeast cells; (B) p ≥ 0.0001 S. brasiliensis compared with S. schenckii– 10 days yeast cells.
Fig 12.
A new hypothetical cell wall model for the dimorphic pathogenic fungi S. schenckii and S. brasiliensis.
This model is based on biochemical and structural analyses (IR, 1H-NMR and 13C-NMR spectroscopy) as well as HPF-TEM and fluorescence microscopy with ConA-Texas Red, WGA-FITC and CFW, which indicated the presence of a peptido-rhamnomannan component localized on the outermost fibrillary layer (green); chitin (brown) and β-glucans 1→3 (blue) and 1→6 (pink) β-glucans at the innermost layer; and rosette-like glycogen alpha-particles organized close to the plasma membrane and cell wall. α-Glucans, whose presence has been described in other dimorphic pathogenic fungi, were absent. The presence of rhamnomannan polymers and the absence of α-glucans (based on IR spectra) on the cell surface support the proposition of a new model for the thermodimorphic fungi of the genus Sporothrix (Fig 10). Other models proposed for pathogenic dimorphic fungi [reviewed by 23] differ significantly with the particular biochemical and structural characteristics observed for the cell wall of S. schenckii and S. brasiliensis.