Schematic model of the biological activities of <i>A. fumigatus</i> TcsC.

<p>Schematic model of the biological activities of <i>A. fumigatus</i> TcsC.</p

The impact of cAMP and light on the fluffy growth phenotype.

<p>Drop dilution assays were performed on AMM plates (supplemented with ammonium). The plates were supplemented or treated as indicated and incubated in incubator. When indicated plates were incubated under white light produced by an LED light source. Pictures were taken after 48 h at 37°C.</p

The Δ<i>tcsC</i> mutant is sensitive to hyperosmotic stress and resistant to fludioxonil.

<p>Drop dilution assays were performed on AMM plates (supplemented with ammonium). Panel A: control; B: 1.2 M sorbitol; C: 1 M KCl; D: 100 µg/ml congo red; E: 1 µg/ml fludioxonil. The depicted colonies were obtained after 48 h at 37°C. Top: AfS35; middle: Δ<i>tcsC</i>; bottom: complemented strain.</p

Infection of immuno-compromized mice.

<p>Intranasal infection of cortisone-acetate treated mice infected with 1×10⁶ conidia of the Δ<i>tcsC</i> mutant (n = 20), the parental strain AfS35 (n = 20) and the complemented strain (n = 20). Controls received PBS only. Survival of mice is shown over time.</p

Impact of fludioxonil on <i>A. fumigatus</i> germ tubes.

<p>Conidia of the Δ<i>tcsC</i> mutant (panels A, C, E, G) and its parental strain AfS35 (panels B, D, F, H) were seeded on glass cover slips and incubated overnight in AMM at 30°C. The resulting germ tubes were treated with 1 µg/ml fludioxonil for 2 h (A, B), 4 h (C, D) and 6 h (E–H) at 37°C. A DAPI staining is shown in panels G and H. Arrows indicate lysed cells that lack intracellular nuclei and are associated with amorphous extracellular material. All bars represent 10 µm.</p

Dur31 mediates both spermidine assimilation and histatin 5 sensitivity.

<p>(A) Growth curve for the wild type (Wt), the <i>dur31</i>Δ/Δ mutant and the <i>dur31</i>Δ/Δ::<i>DUR31</i> complemented strain with spermidine as sole carbon source. Fungal overnight SD cultures were washed once in water and adjusted to an OD₆₀₀ of 0.1 in yeast nitrogen base (YNB) medium supplemented with 100 µg ml⁻¹ spermidine. Growth was monitored in an ELISA reader at 37°C for 50 h. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002592#s2" target="_blank">Results</a> are the mean ± SEM of two independent experiments, each performed in triplicate. (B) A <i>dur31</i>Δ/Δ mutant is more tolerant towards histatin 5. Fungal overnight YPD cultures were washed twice in water and adjusted to 10⁶ cells ml⁻¹ in 10 mM NaPB. Cells were then incubated with 15 µM histatin 5 for 1 h at 30°C and shaking (300 rpm). Dilutions were plated on YPD agar plates and incubated at 30°C for 2 d for determination of colony forming units. The percentage cell death was determined compared with untreated cells. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002592#s2" target="_blank">Results</a> are the mean ± SD of three independent experiments, each performed in duplicate. *P≤0.05 compared with the wild type (Wt). (C) Differential interference contrast (DIC) and fluorescent microscopic images showing uptake of FITC-histatin 5 (30 µM) by wild type (Wt), <i>dur31</i>Δ/Δ mutant and <i>dur31</i>Δ/Δ::<i>DUR31</i> complemented cells after 15 min co-incubation at 30°C. The <i>dur31</i>Δ/Δ mutant translocates FITC-histatin 5 less efficiently than the Wt and revertant. Scale bar: 10 µM. (D) Quantification of the FITC-histatin 5 mean fluorescent intensity of at least 80 cells per strain. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002592#s2" target="_blank">Results</a> are the mean ± SD of two independent experiments. ***P<0.001 compared with the wild type and <i>dur31</i>Δ/Δ::<i>DUR31</i> complemented strain.</p

The role of TcsC in the stress-induced developmental program leading to a fluffy growth phenotype.

<p>Drop dilution assays were performed on AMM plates (supplemented with ammonium). Panel A: 1% oxygen; B: 2% oxygen; C: 2 mM farnesol; D: 200 µM farnesol; E: 2 mM farnesol; F: 100 mM MgSO₄; G: 100 mM CaCl₂; H: 100 mM MgSO₄; I: 50 mM CaCl₂; J: 500 mM CaCl₂. Side views of colonies from C and D are shown in panels E and F. The depicted colonies were photographed after 48 h at 37°C. AfS35 (top/left); Δ<i>tcsC</i> (middle); complemented strain (bottom/right).</p

Dur31 is required for filamentous colony formation.

<p>(A) <i>dur31</i>Δ/Δ displays defective microcolony formation. For induction of hyphal microcolonies, fungal cells were grown overnight to stationary phase in YPD medium, washed twice with PBS, and resuspended in PBS. 100 cells per strain were inoculated into 500 µl RPMI medium per well of a 24-well cell culture plate and incubated at 37°C for 24 hours in presence of 5% CO₂. Representative pictures are shown. Scale bar: 100 µm. (B) Quantification of wild type (Wt), <i>dur31</i>Δ/Δ mutant and <i>dur31</i>Δ/Δ::<i>DUR31</i> revertant microcolony dimensions after incubation of fungal cells in RPMI medium at 37°C for 24 hours in presence of 5% CO₂. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002592#s2" target="_blank">Results</a> are the mean ± SD of two independent experiments, each performed in quadruplicate. ***P<0.001 compared with the wild type and <i>dur31</i>Δ/Δ::<i>DUR31</i> complemented strain. (C) Analysis of <i>C. albicans</i> wild type (Wt) and <i>dur31</i>Δ/Δ mutant filamentation by plating 50 cells per strain on solid water agar supplemented with either 10% fetal bovine serum or 5% RPMI, or by plating cells on solid Spider or SLAD medium. For embedding approximately 50 cells per strain were added to molten YPS (YP-saccharose) agar. The agar was allowed to solidify and plates were subsequently incubated for 5 days at 25°C. RPMI agar and SLAD agar plates were incubated for 4 days, serum agar plates for 2 days, and Spider agar plates for 10 days at 37°C. Experiments were performed twice in duplicate. Representative pictures are shown. Scale bar: 100 µm.</p

The role of TcsC in the phosphorylation of SakA.

<p>Protein extracts of resting conidia (RC)(panel A) and germlings (panel B) were analyzed by immunoblot using specific antibodies to phosphorylated SakA and as a loading control mitochondrial MnSOD. Extracts were prepared from germlings treated with 10 µg/ml fludioxonil and 1.2 M sorbitol for 2 and 20 min, respectively. A: parental strain AfS35, B: Δ<i>tcsC</i> mutant, C: complemented mutant.</p

Growth of the Δ<i>tcsC</i> mutant.

<p>Colonies of the AfS35 wild type and the Δ<i>tcsC</i> mutant grown for 72 h on AMM plates are shown in panels A/C and B/D, respectively. Magnifications of the edge of the colonies are depicted in panels C and D. Note the reduced number of extending hyphae in the mutant. Panel E: Quantification of the radial growth of AfS35 (black), Δ<i>tcsC</i> mutant (white) and complemented mutant colonies (gray) on AMM plates after 48 h and 96 h at 37°C or 48°C. Panel F: Quantification of the radial growth after 96 h of AfS35 (black), Δ<i>tcsC</i> mutant (white) and complemented mutant colonies (gray) on AMM plates supplemented with 1.4 M NaNO3 or 0.2 M ammonium tartrate at 37°C. The experiments shown in panels E and F were done in triplicate. Standard deviations are indicated. Student’s <i>t</i>-test: *p<0.005; **p<0.001.</p