Interactions between copper homeostasis and the fungal cell wall affect copper stress resistance
Fig 5
Cbi1 depletion effects chitin architecture during copper deficiency and host stress.
(A)-(D) The WT, cbi1Δ mutant, and cbi1ΔC-WT complemented strains were incubated for 24h in YPD+ 10 uM CuSO4 (copper sufficiency) or YPD +250 μM BCS (copper deficiency), harvested, and double stained with CFW and WGA-Alexa 488. Stained cells were analyzed using a FACS Canto A Analyzer, and data were analyzed using Flow Jo. Representative data indicating percentage of total cells staining as “High WGA” (increased exposed chitin/chitosan) after incubation in copper-sufficiency (A) or copper-deficiency (C). Quantification of three independent experiments for copper-sufficiency (B) or copper-deficiency (D) conditions. A 2-way ANOVA was performed from log transformed data using GraphPad Prism. (E)-(F) Cell wall analysis was performed for the WT, cbi1Δ mutant, and cbi1ΔC-WT complemented strains after incubation for 24h in CO2-independent medium at 37°C. Cells were analyzed microscopically using WGA-Alexa 488 staining (E), or by FACS analysis for High WGA cells per above (F). (G) Macrophage activation assay upon infection with copper sufficient or deficient WT, cbi1Δ and cbi1ΔC-WT complemented cells. BMMs were harvested from A/J mice and co-incubated with Cn cells at an MOI of 10:1 (Cn:BMMs), followed by an ELISA-based quantification of TNF-α (pg) in the supernatant. Presented is the mean +/- SEM of 5 independent experiments. A 2-way ANOVA was performed from log transformed data using GraphPad Prism. (I-J) Five-fold serial dilutiuons of indicated strains were incubated on YPD medium supplemented with indicated amounts of BCS (copper starvation) (I) or CuSO4 (toxic copper stress) (J). Plates were incubated at 30°C for 2-6d.