Fig 1.
Kinetics of germination of S. boydii in various conditions.
(A) Conidia isolated from 5-, 9- and 14-day-old cultures on yeast peptone dextrose (YPD) agar were incubated in YPD liquid medium over 8 h at 37°C. (B) Conidia isolated from 9-day-old cultures on Malt or YPD agar were incubated in Malt or YPD liquid media over 8 h at 37°C. (C) Conidia isolated from 9-day-old cultures on YPD agar were incubated in YPD liquid medium for 16 h at 20°C, 25°C or 37°C (200X).
Fig 2.
The life cycle of S. boydii under scanning electron microscopy.
After release, conidia (A) germinate (B) and the hyphal part of germ tubes elongates (C) until a first branch emerges near the mother cell (D). Both hyphae grow and more branching sites appear on filaments at the subapical region of the articles (E) until the mother cell can no more be distinguished (F). Arrows indicate sites of first branching, and later branching are indicated by arrowheads. All cultures were performed in YPD broth with incubation at 37°C for 6h (B), 8h (C), 10 h (D and E) or 24 h (F). Bars: 1 μm in A, B and C; 0.5 μm in D; and 5 μm in E and F.
Fig 3.
Cell wall modifications during germination of S. boydii under transmission electron microscopy.
(A) hyphal cell wall; (B) conidial cell wall; and (C) cell wall of a germinating conidium. (D) Enlarged part of (C) highlighting the cell wall structural modifications during germination, particularly the electron dense outer layer being less continuous at the surface of the hyphal part of germ tubes. Bars: 0.2 μm in A and B; 1 μm in C; and 0.5 μm in D.
Fig 4.
Surface charge modifications during germination of S. boydii by ferritin labeling and zeta potential measurements.
TEM images of germ tubes labeled with cationized ferritin (A), germ tubes treated with neuraminidase prior cationized ferritin labeling (B) or germ tubes incubated with native ferritin (C). (D) Comparison of the surface electrostatic charge of resting and germinated conidia calculated from the electrophoretic mobility of 10 000 cells using Zetasizer Nano ZS (P = 0.0005). H: hyphal part of germ tube; MC: mother cell of germ tube. Bars: 1 μm.
Fig 5.
Fluorescence labeling of S. boydii surface carbohydrates with FITC-conjugated lectins.
Germ tubes after labeling with concanavalin A (A and C) or wheat germ agglutinin (B and D) lectins. The same fields are presented under fluorescence (A and B) and phase contrast microscopy (C and D) respectively. Arrows indicate mother cells.
Fig 6.
Gold labeling of cell wall mannan groups in S. boydii germ tubes.
Germ tubes labeled with gold-conjugated concanavalin A (Con A; 5-nm gold particles) showing higher affinity of gold particles to the hyphal part (H) of germ tubes compared to the mother cell (MC) under transmission electron microscopy. Arrow indicates the limit of the outer cell wall layer of the mother cell. Bar: 0.5 μm.
Fig 7.
High resolution imaging and chemical force spectroscopy analysis of S. boydii resting conidia and germ tubes.
AFM amplitude images of a resting (A) or germinated (B) S. boydii conidium. (C) Left, scheme for chemical functionalization of AFM tips. Gold-coated tips were modified with CH3-terminated alkanethiols or OH-terminated alkanethiols. (C) Right, histograms of hydrophobic adhesion forces measured on the surface of a resting conidium (1.8 ± 0.3 nN, in red) and the hyphal part of a germinated conidium (0.85 ± 0.15 nN, in blue).
Table 1.
GPI-anchored proteins identified in conidial and/or germ tube extracts.
Table 2.
Sequence analysis of the identified GPI-anchored proteins.