Fig 1.
Growth morphology of Δryp1 mutants.
(A) C. posadasii Δryp1 strains (1563.4 and 1563.7) exhibit a growth-limiting phenotype, in comparison to WT and an ectopic transformant. (B) Plugs of 6 mm were transferred from freshly growing plates to 2X GYE plates and incubated at room temperature, to measure colony radial growth. Colony diameters were measured on days 8, 14 and 20. Four independent Δryp1 strains (1563.4, 1563.6, 1563.7 and 1563.14), as well as the WT parental strain and an ectopic transformed strain (1563.1) were tested.
Fig 2.
C. posadasii Δryp1 does not produce normal spherules in vitro.
(A, B) Spherules of WT, a Δryp1 mutant and an ectopic transformed strain, grown in Converse medium at 37°C with 20% CO2 and aliquots analyzed during in vitro spherulation. (A) Images at 24 h show that while the WT and ectopic strains are rounding up to form early spherules, Δryp1 does not form round structures. At 96 h, the WT and ectopic strains produced mature spherules, while the Δryp1 exhibited limited polar hyphal growth. (B) Images stained with lactophenol blue at 120 h show mature spherules produced by WT and polar hyphal growth by Δryp1. Black bars represent 10 μm. (C) Percentage of spore viabilities for WT and ectopic strains, and Δryp1 mutants (1563.7 and 1563.10) were determined based on the growth on 2X GYE plates at 37°C and 25°C.
Fig 3.
Ryp1 regulates distinct sets of genes in spherules and hyphae.
(A) Heatmap showing differential expression of the 4175 genes described in the text. Genes are grouped by expression pattern (significantly up, down, or neutral in each of the three contrasts) with different classes labeled in the color bar to the right of the heatmap. The position of genes of interest in the heatmap are indicated to the right of the color bar. (B) Venn diagram of genes observed to be morphology-regulated (differentially expressed in WTSph/WTHy), Ryp1-dependent in spherulation conditions (differentially expressed in WTSph/Δryp1Sph), or Ryp1-dependent in hyphal conditions (differentially expressed in WTHy/Δryp1Hy). (C and D) Scatter plots of the differentially expressed genes comparing the WT spherule-enrichment (right) or hyphal-enrichment (left) to ryp1-depletion (top) or ryp1-enrichment (bottom) in (C) spherules or (D) hyphae. Genes in the scatter plots are colored by expression pattern, as in the color bar of (A).
Fig 4.
Infection of mice with C. posadasii Δryp1.
Survival results for C57BL/6 mice (N = 8 mice/group) infected intranasally with four independent Δryp1 strains, 1563.7 (circle), 1563.10 (square), 1563.14 (inverted triangle) and 1563.15 (triangle); one strain with the ryp1 deletion cassette integrated ectopically into WT, 1563.1 (half-filled circle); and WT (half-filled square). The Δryp1 strains were infected at between 525 and 1133 viable arthroconidia, while 50 viable arthroconidia of the ectopic stain and WT were used.
Fig 5.
Vaccination of mice with C. posadasii Δryp1 mutant.
Protection results for C57BL/6 mice vaccinated with either Δcps1, Δryp1 or a S. cerevisiae culture supernatant (Control). C57BL/6 mice (N = 8 mice/group) were injected either by intraperitoneal (IP) or subcutaneous (SC) injection twice 2 weeks apart with Δcps1, Δryp1, or Control. Vaccinated mice were challenged with a lethal dose of WT (90 spores) and total lung CFUs were determined 14 days post-infection. Δcps1 significantly protects mice from C. posadasii infection compared to Control (P<0.004 both comparisons) while Δryp1 shows no protection (P = 0.99 vs. control). Bar equals geometric mean.