Defects in intracellular trafficking of fungal cell wall synthases lead to aberrant host immune recognition
Fig 4
Cell wall components are altered in the mar1Δ cell wall.
(A) The mar1Δ cell wall has increased chitin and chitosan staining by flow cytometry, but mar1Δ staining for α-glucans, β-(1,3)-glucan, and mannoproteins is limited above baseline. WT and mar1Δ cultures were incubated for 16–18 hours at 37°C in TC medium, fixed, labeled, and analyzed by flow cytometry. WGA was used to stain exposed chitin and CFW was used to stain total chitin. EY was used to stain chitosan. An MOPC-104E antibody with an anti-mouse AlexaFluor 488 secondary antibody was used to label α-glucan. An Fc-Dectin-1 fusion protein coupled with an anti-human AlexaFluor 488 secondary antibody was used to label β-(1,3)-glucan. Concanavalin A conjugated to AlexaFluor 488 was used to label mannoproteins. Relevant events were gated in the FSC/SSC plots and are represented as histograms with mean fluorescence on the x-axis and cell counts on the y-axis. Unstained cells were sorted as controls to determined positive labeling. (B) The mar1Δ cell wall has decreased glucan and mannan. Cells were incubated for 16–18 hours at 37°C in TC medium, followed by cell wall isolation. Cell wall carbohydrate levels were quantified using high performance anion-exchange chromatography with pulse ampherometric detection (HPAEC-PAD). All data represent means of results from 3 independent cell wall preparations for each strain. *, p < 0.05; **, p < 0.01 as determined by one-way ANOVA with Tukey’s multiple comparisons test. All other comparisons, not significant (C) Cell wall genes are differentially regulated in mar1Δ. A concentration of 107 cells/ml in 25 ml YPD (30°C) or TC (37°C) were incubated for 1.5 hours, followed by RNA extraction and cDNA synthesis. Expression of cell wall biosynthesis genes was determined by real-time PCR. Fold change was calculated relative to WT YPD levels and normalized to the expression of an internal control. Data represent means of results from 2 independent C. neoformans cultures and RNA extractions per strain. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 as determined by two-way ANOVA with Tukey’s multiple comparisons test.